BACTERIOLOGY 
AND 
MYCOLOGY OF FOODS 
BY 
FRED WILBUR TANNER, M.S., Ph.D. 
/(t 
Associate in Bacteriology, University of Illinois 
FIRST EDITION 
NEW YORK 
JOHN WILEY & SONS, Inc. 
London: CHAPMAN & HALL, Limited 
1919 Copyright, 1919 
BY 
FRED WILBUR TANNER 
PRESS OF 
BRAUNWORTH & CO. 
BOOK MANUFACTURERS 
BROOKLYN. N. Y. PREFACE 
The present volume is an outgrowth of a course in food microbiology 
at the University of Illinois. This course has been developed for those 
who wish to fit themselves for food control, food chemists and for those 
students in household science who possess a sufficient fundamental 
training in chemistry. A thorough training in chemistry is essential 
for intelligent work in microbiology. 
The aim of the volume is to present the methods of analysis along 
with sufficient discussion based on the literature of the subject, to 
show the history and " make-up " of these methods. An intelligent 
use of them would be difficult without this. The field of food micro- 
biology is a broad one and the author has drawn freely from the reported 
investigations of others. Many of these several investigators are 
experts in their fields, having devoted a lifetime to the study of cer- 
tain subjects. Some of the methods of analysis are given in the language 
of their originators, since, by doing this, the personal touch of the 
author may be retained. No apology is made for the rather liberal 
quotations from the different " Standard Methods " of the American 
Public Health Association. These have been worked out by com- 
mittees composed of men who are experts and their opinions may 
be given considerable weight. 
Numerous references are given at the end of each chapter. Much 
of the data presented in the chapter has been taken from them; in most 
instances direct reference is made to them. It is believed that such 
a feature will render the book more useful to both the practitioner 
and the student. No serious attempt has been made to list all of the 
literature on the different subjects which have been treated in this 
book for this must obviously come from the abstract literature. As 
the data in this field are accumulating very rapidly, a working reference 
library is essential. 
The author will appreciate helpful suggestions and criticisms from 
those who use this book. Only by the help of them may the errors and 
crudities in the first edition of any book be corrected for later editions. 
III IV 
PREFACE 
It is a pleasure to acknowledge the kindness of various individuals 
and organizations for assistance. To the Cornell Agricultural Experi- 
ment station the author is indebted for permission to use the colored 
plates in the Chapter on Eggs, which were prepared by Professor E. D. 
Benjamin; to the various supply houses for permission to use illus- 
trations of apparatus; to Bausch & Lomb Optical Company and the 
Spencer Lens Company for permission to use parts of their manuals 
on the " Use and Care of the Microscope "; to the American Public 
Health Association for permission to use parts of the Standard Methods 
for the Examination of Sewage and Water, Milk, and Air, finally, to 
all those various instructors and friends with whom the author has 
come in contact and who have contributed some of the ideas reflected 
in the pages of this book. 
University of Illinois, 
Urbana, Illinois, 
August, 1918. TABLE OF CONTENTS 
CHAPTER 
PAGE 
Bacteriological Apparatus, General Technique, Enumeration of Bacteria, Anae- 
robic Methods, The Microscope and Microscopic Methods 1 
CHAPTER II 
Media and Their Preparation, Bouillon Media, Agar Media, Gelatin Media, 
Special Media, Potato and Starch Media, Synthetic Media, Blood and 
Blood Serum Media, Milk Media 39 
CHAPTER III 
Staining Technique. Microscopic Methods, Theory and Methods of Staining.. 76 
CHAPTER IV 
Classification and Description of Bacteria. Classification of Bacteria, His- 
torical Life Cycles, Descriptive Chart of the Society of American Bac- 
teriologists 93 
CHAPTER V 
Sterilization and Disinfection. Dry Heat, Moist Heat, Filtration, Light Dis- 
infection, Inorganic Compounds, Organic Compounds, Standardization of 
Disinfectants i 121 
CHAPTER VI 
Proteins and Carbohydrates. Classification of Carbohydrates, Fats 182 
CHAPTER VII 
Yeasts and Molds. Fungi, Morphology of Molds, Classification of Molds, 
Classification of Yeasts, Methods of Study of Yeasts, Compressed Yeast, 
Yeasts in Bread Making, Methods for the Examination of Bread and 
Yeasts 202 
V VI 
CONTENTS 
CHAPTER VIII 
PAGE 
Intestinal Bacteria. The Stomach, Bacteria in Intestines and Feces, The 
Intestinal Flora, Methods for Determining Bacteria in Feces, Examination 
of Feces for Cholera Vibrio, Isolation of B. Dysenteric from Feces, Exami- 
nation of Feces for Tubercle Bacilli, Isolation of Yeasts from Feces 228 
CHAPTER IX 
Bacterial Examination of Air. Standard Methods 264 
CHAPTER X 
I 
Water Hygiene. The Chemical Analysis, Bacteriological Analysis, Classifica- 
tion of Water Bacteria, Indicators of Pollution, Test for B. Coli Group, 
Presumptive and Confirmed Tests, Treasury Department Standard 
Detection of Pathogenic Bacteria in WTater, Self Improvement of Streams, 
Treatment of Water, Bacteriology of Ice 268 
CHAPTER XI 
Milk and Milk Products. Composition of Milk, Chemical Examinatien of Milk, 
Bacterial Examination of Milk, Factors Influencing Bacterial Content of 
Milk, Sanitary Significance of Bacterial Count, Milk Standards, Standard 
Plate and Microscopic Methods for Determining Bacteria in Milk, Leuco- 
cytes in Milk, Pathogenic Bacteria in Milk, Pasteurization, Bacteriology 
of Butter, Pathogenic Bacteria in Butter, Cheese, Methods of Examination, 
Pathogenic Bacteria in Cheese, Condensed Milk, Ice Cream, Pathogenic 
Bacteria in Ice Cream 363 
CHAPTER XII 
Bacteriology of Eggs. Composition of Eggs, Commercial Grading of Eggs, 
Factors Governing Bacterial Content of Eggs, Methods for the Bacterial 
Examination of Eggs 451 
CHAPTER XIII 
Meat and Meat Products. Origin of Bacteria in Meat, Methods of Examina- 
tion of Meat, Bacteriology and Examination of Oysters 479 
CHAPTER XIV 
Food Preservation. Methods, Examination of Canned Foods, Examination 
of Tomato Products, Food Poisoning 494 
CHAPTER XV 
Epidemiology. Characteristics of Epidemics, Methods for Investigation, 
Case Cards 540 
Appendix 553 
Index 581 BACTERIOLOGY AND MYCOLOGY OF FOODS 
CHAPTER I 
BACTERIOLOGICAL APPARATUS 
The apparatus which is required in a bacteriological laboratory is 
similar in many respects to that used in a chemical laboratory. Since 
chemical methods are often used for studying bacteria, it is evident 
that much the same apparatus may be used. There are several types, 
however, which may receive special consideration, since a proper con- 
struction and type is essential for the best results in bacteriology. 
Fermentation Tubes. There are several types of fermentation 
tubes any of which may be used provided they hold a sufficient excess 
of the medium over the sample under examination. Standard Methods 
for the Examination of Water and Sewage, 1917, recommends that the 
fermentation tubes used in water analysis should hold four times as 
much medium as sample. All fermentation tubes are constructed with 
a closed arm which retains the gases that are given off in bacterial 
metabolism. There are three general types now in use by bacteriolo- 
gists, Brown's, Smith's (1890), and Durham's (1898). 
The Brown fermentation tube was devised for use in connection 
with water analysis and is quite similar to Smith's. The Smith tube 
is made both with and without glass feet. Those without the glass feet 
require a special rack but have the advantage over those with feet that 
they do not fall over so easily. A convenient rack for holding Smith 
tubes has been devised by Bain (Ill. State Water Survey Bull. 6, 31, 
1908). The Durham tubes are the most satisfactory of all since they 
are made from ordinary test tubes. The inner tube, according to Lee, 
should be cut off at an angle to allow the ingress more readily of motil 
bacteria. Hall (1914) also found this to be true. The efficiency of the 
Smith and Durham tubes was compared by Browne (1913) and Lee and 
Fegely (1914). These investigators found that in low dilutions the 2 
BACTERIOLOGICAL APPARATUS 
Durham tube was more efficient but with the higher dilutions the Smith 
tube gave more consistent results. Browne and Ackman (1917) and 
Browne (1917) have stated that the following factors cause variations 
in the amounts of gas produced in lactose peptone bile and lactose 
broth: (1) Temperature; (2) Time of incubation; (3) Initial reaction 
of the culture medium; (4) Length of inverted vial; (5) Source of bile; 
(6) Absorption of formed gas. 
Graves (1917) inverts the small inner tube over a wooden rod which 
■lifts the tube a distance from the bottom of the outer tube. It is claimed 
that certain advantages obtain in this procedure. 
A 
B 
C 
Fig. 1.-Types of Fermentation Tubes. 
A, Brown's; B, Smith's; C, Smith's, without foot; D, Graves' 
Modification of the Durham Fermentation Tube. 
D 
Petri Dishes. These should be about 10 cm. in diameter, thus 
having an area of about 65 sq. cm. Either glass or porous tops may be 
used. The porous tops devised by Hill prevent the spreading colonies 
which appear so often on Petri dishes in humid weather. Very often 
it is difficult to secure Petri dishes with flat bottoms. This is essential 
to an even distribution of bacteria over the plate and a uniform thick- 
ness of medium. 
Dilution Bottles. These may be of various types and shapes but 
should contain the amount of dilution water when about half full. This 
allows ample room for thorough shaking of the diluted sample. Bottles 3 
DILUTION BOTTLES 
with glass stoppers are greatly superior to those requiring cotton stoppers 
but these may be used if the cotton is rolled tightly or contains a cork 
stopper inside of it. Square bottles of the " Blake " type holding 
Fig. 2.-Types of'Petri Dishes and Other Dishes for Bacteriological Purposes. 
about 200 c.c. are useful and pack together well. If bottles are used for 
dilutions great care must be exercised in sterilization to prevent cracking. 
On account of this some laboratories use Florence flasks for holding the 
dilution water. The dilution bottles should be filled with decimal 
quantities of water. Where it is desired to obtain exact quantities 4 
BACTERIOLOGICAL APPARATUS 
Fig. 3.-Types of Containers and Holders for Sterilizing Petri Dishes, Pipettes and 
Other Apparatus. of dilution water, it may be sterilized in larger quantities and delivered 
under aseptic conditions in any required amount to the sterile dilution 
flask. Frost has shown that during sterilization the volume in dilution 
bottles may be changed and this might cause considerable error where 
accuracy is desired. 
Sample Bottles. A glass-stoppered bottle of any type may be used. 
The necks should be wrapped with tin foil before sterilization. Great 
care should be taken to secure bottles which possess tight stoppers. 
In general a bottle with a narrow neck is preferable since the possibility 
of contamination during analysis is greatly decreased. 
SAMPLE BOTTLES 
5 
A 
B 
C 
D 
E 
G 
rF 
H 
Fig. 4.-Types of Flasks for Bacteriological Work. 
~A, Koch's Culture Flask; B, Fernbach's Antitoxin Flask; C, Freudenreich's; D, Lister Culture 
Flask; E, Miquel Flask; F, Kolle Culture for Growing Bacteria in Large Numbers; G, Roux 
Flask; H, Piorowski's Flask. 
Flasks. Many of the flasks which are used for bacterial work are no 
different from those used in chemistry. Other flasks have been in- 
vented for special purposes and usually bear the name of the originator. 
A few are given in Fig. 4. 
Slides and Cover Glasses. Slides should be made of clear white 
glass and should be free from defects. Different sizes are available but 
those which are 2.5 X 7.7 mm. have widest use. 
Cover glasses are classified as No. 1, No. 2, and No. 3. The follow- 
ing thicknesses are characteristic of each number. No. 1, 2^0 to T|o in. 6 
BACTERIOLOGICAL APPAR ATUS 
or 0.13 to 0.17 mm. thick. No. 2, to TJo in. or 0.17 to 0.25 mm. 
thick. No. 3, to in. or 0.25 to 0.50 mm. thick. 
No. 2 cover glasses are the best for general use since they satisfy 
the demands of most objectives. No. 1, may be used with oil immersion 
objectives. The objectives of the different makers are standardized 
for the following thicknesses of cover glasses: 
Zeiss 15-20 mm. 
Leitz 17-20 mm. 
Bausch & Lomb 18-20 mm. 
Spencer Lens Co 18-20 mm. 
Cleaning of Apparatus. Clean apparatus is the first essential for 
successful laboratory work in bacteriology. All apparatus whether 
new or old should be carefully washed and rinsed before using. This 
removes any free acid or alkali, both of which are very detrimental to 
the growth of bacteria. Glassware should be washed as clean as pos- 
sible in tap water with laboratory brushes. These are now constructed 
in many shapes and sizes which enables one to reach the interior of most 
flasks. After this is done and if any dirt, etc., remains in the apparatus, 
it should be taken out by the cleaning solution. This is accomplished 
by soaking the apparatus, if glassware, in the cleaning solution which 
is contained in a lead bath. After it has stood for a time, it may be 
taken out, the cleaning solution allowed to run back into the original 
container. The clean.ng solution may be used again and should not be 
thrown away. 
The cleaning of slides and cover glasses deserves some special atten- 
tion. They should be boiled in dilute alkali to saponify any oil or 
grease which may be on them. After they have been washed in water 
they should be boiled in cleaning solution for twenty minutes. Then 
each one should be carefully rinsed in distilled water and stored in ether 
or dry, as preferred. 
Cleaning Solution. This has the following composition: 
Sodium or potassium chromate (K2Cr2O7) 6 gms. 
Concentrated sulphuric acid (H2SO4) 6 c.c. 
Water 100 c.c. 
K2Cr2O-+4H2SO4 = K2SO4+Cr2(SO4)3+4H2O+3O. 
This is a strong oxidizing reagent. Its action may be accelerated 
by heat. Care should be used in handling it since it is very destructive PLATINUM WIRES 
7 
to clothing, etc. The oxygen which is formed is available for the oxida- 
tion of any reduced substance. 
Platinum Wires. Platinum wires have extended use in bacteriology. 
Different sizes may be used but gauge No. 26 yields the best results. 
A heavier wire requires a longer time for cooling after it has been ster- 
ilized in the flame. A lighter wire may be too flexible. 
In many cases standardized loops are required which deliver the 
same amount of liquid. Instances are the standardization of disin- 
fectants and determination of bacteria in feces according to the Eberle- 
Klien method. St. John (1914) devised a "loop" which gave drops 
of from 0.019 to 0.021 gm. according to the angle at which it was taken 
from the fluid. Under more unfavorable circumstances the variation 
A 
B 
a 
Fig. 5.-Types of Holders for Inoculating Needles and Loops. 
A, Special Aluminum and Wooden Handle; B, Aluminum Rod; C, Glass Rod with Fused Wire. 
was from 0.017 to 0.023 gm. The size of the drop cariied by this loop 
is about 0.020 c.c. 
Baskets. These should be made of wire with a mesh small enough 
to retain most of the test tubes used in a laboratory. Any special 
shape or size may be made at the average tin shop. 
General Technique 
Isolation of Microorganisms. This necessitates the plating of 
bacteria in decimal dilutions on solid media. Agar or gelatin may 
be used but the latter gives more data. If the organism which is sought 
in pure culture is known to be mixed with other forms, greater care 
must be exercised. The plating of decimal dilutions is advisable, 
especially where little is known with regard to the number of cells which 
are present. All apparatus must be sterile. Where careful work is 8 
BACTERIOLOGICAL APPARATUS 
Fig. 6.-Various Types of Wire Baskets for the Bacteriological Laboratory. COUNTING APPARATUS 
9 
being done, the sample of food should be thoroughly agitated on a 
shaking machine with glass beads or broken glass. 
Fig. 7.-Wolffhuegel Counting Apparatus. 
A 
C 
D 
B 
E 
Fig. 8.-Types of Counting Devices. 
A, Petri Dish with the Bottom Divided into Squares; B, Lafar's Counting Plate; C and D, 
Roszahegyi's Counting Flask; E, Jeffer's Counting Plate. 
Pouring of Plates. A series of these should be poured with different 
concentrations of the sample. These may be obtained by thoroughly 
shaking a portion of the sample in sterile water and transferring a loop 10 
BACTERIOLOGICAL APPARATUS 
of this suspension to a second tube of sterile water. This procedure 
is repeated until several concentrations of the sample are secured. 
Portions of these are placed in sterile Petri dishes or under these condi- 
tions put into tubes of melted medium. These are then put into the 
Petri dishes. 
Isolation of Pure Cultures. A pure culture may be defined in 
different ways but in this relation it may be regarded as the descend- 
ants of a single cell. Each single cell on the Petri dishes poured in the 
above paragraph will have developed into a colony. In most cases this 
colony will contain a pure culture if the plate does not contain too many 
colonies. When there are but a few colonies on the plates, each one 
that is isolated from the others will be pure. If it is found to be a 
mixed culture the plating operation must be repeated. In fact in 
careful work it is essential to replate two or three times. This makes 
the probability of a pure culture more certain and also serves to " tune- 
up " or strengthen the organism. 
Fig. 9.-Bottcher's Counting Chamber. 
It. consists of an ordinary glass slide with a glass ring 20 mm. in diameter and 8 mm. high. 
The cover glass is ruled into 100 squares, 19 of which are numbered. 
The isolated colonies are picked by means of a sterile platinum 
wire. A small portion of the colony is removed to either an agar slant 
or broth tube. Before any extensive investigations are carried out 
with the organism, it should be studied in hanging drop and stained 
smear to demonstrate its purity. 
Transferring of Cultures. This procedure should be carefully 
carried out in a room with little dust. Sterile platinum wires should 
be used which should be sufficiently cooled. If a wire of too great 
diameter is used, it will retain its heat longer and there is then greater 
danger that the culture will be killed or altered. Also, if much trans- 
ferring is done at the same time there will be a tendency for some of 
the material to cake on the wire. This should be removed. A piece 
of emery paper or No. 00 sand paper will be found to be a valua- 
ble aid. 
Dilutions. These are essential in the quantitative enumeration of 
bacteria. The kind of containers to be used has been described above. ENUMERATION OF MICROORGANISMS 
11 
The dilution water in definite amounts may be put into them before 
sterilization but the quantity of water has been found to be altered 
during this procedure. To obviate this difficulty, the containers may be 
plugged and sterilized empty while the water may be sterilized in a larger 
bottle. This may then be transferred to the dilution bottles with a 
sterile pipette. 
Fig. 10.-Stewart's Counting Apparatus. 
The Petri dish is illuminated by oblique rays of light coming from an incandescent light. 
Enumeration of Bacteria 
Several methods have been devised for counting the number of 
bacteria which are present in any substance. For convenience they 
may be classified as follows: 
A. Plate cultures. 
(a) Petri dish cultures. 
(b) Frost's microscopic plate method. 
B. Direct microscopic method. 
(a) Breed's method. 
(b) Heemocytometer methods. 
The plate method is probably the oldest and involves the use of 
the Petri dish. Any transparent solid medium may be used in the 
Petri dish, but the dilution of sample introduced is important. Hill 
(1908) and Breed (1916) have both shown that over-crowded plates 12 
BACTERIOLOGICAL APPARATUS 
will not give accurate results. Breed, in studying plates made from 
milk, found that those having more than thirty and less than four 
hundred colonies gave satisfactory results. 
Frost's microscopic plate method and Breed's microscopic method 
have been described in the Chapter on Milk. These methods were 
Fig. 11.-Hsemocytometer with Pipettes in Case. 
originally divised for milk but undoubtedly could be adapted to other 
food substances. 
Haemocytometer. The hsemocytometer, which is used for counting 
leucocytes and erythrocytes, is now applied to the enumeration of 
bacteria in very heavily populated materials. 
b 
a 
Fig. 12.-Showing Hsemocytometer Chamber. 
The instrument consists of a glass slide upon which is fastened pieces 
of glass in such a way as to form a chamber. The outer glass is square 
with a round hole in the center. In this is put a round piece of glass 
of smaller diameter so that there is a shelf, moat and disk formed. 
The upper surface of the disk is ruled into squares. These rulings are 
of various kinds as indicated in Fig. 11, the Thoma ruling being as com- BLOOD COUNTS 
13 
mon as any. This Thoma ruling, however, is more suitable for counting 
red blood cells. 
The pipettes are two in number and are graduated to deliver differ- 
ent amounts. The pipette for erythrocytes delivers dilution of 
or 2^0! the pipette for leucocytes delivers a dilution of yo or 
The hannocytometer was originally devised for counting the blood 
corpuscles. From this it has been adapted to the enumeration of bac- 
teria in substances containing large numbers. It is best for a person 
not familiar with the technique to apply it to the counting of blood 
corpuscles before attempting to count bacteria. 
Procedure, Red Corpuscles. Collect a drop of blood on a clean 
slide and draw part of it up to the mark on the red corpuscle pipette 
(101). The blood may be secured by cutting the tip of the finger 
(across the lines) or by a syringe from a vein in the forearm. By 
drawing the blood to the 0.5 mark a dilution of 200 is secured; when 
drawn to the 1.0 mark the dilution is 1 to 100. This first measurement 
of blood is very important since a slight variation from the mark 
introduces a larger error in the final results. Should the blood be drawn 
up too far in the pipette it may be shaken down carefully to the mark. 
After this the diluting fluid should be drawn into the pipette up to the 
101 mark and the pipette thoroughly shaken. The blood is now suf- 
ficiently diluted for counting the red blood corpuscles. The contents 
of the end of the pipette below the bulb should be discarded since it is 
filled mostly with diluting fluid. A drop of the diluted blood is now 
placed on the disk of the counting chamber. This should be just suf- 
ficient to cover the disk after the cover glass has been put on. The cover 
glass should be carefully applied with a very slight sliding motion. 
This should be placed on the chamber as soon as possible in order to 
secure as even a distribution of corpuscles over the bottom of the 
chamber as possible. The chamber must be left on a level surface for 
about a half hour, after which the corpuscles will have settled to the 
bottom and will be resting on the ruled surface. The number of red 
corpuscles is now determined by putting the chamber under a micro- 
scope fitted with a mechanical stage. 
The small squares cover an area sq. mm. since they are mm. 
on a slide. The depth of the chamber from the bottom of the cover 
glass to the top of the disk is ro mm., hence the volume of liquid above 
a square is toXtoXto equals ttot mm. 4000 Xdilution (200) equals 
red corpuscles per cubic millimeter. A sufficient number of squares 
should be counted to give a fairly correct average per square. To 
accomplish this it is best to count about one hundred squares. By 14 
BACTERIOLOGICAL APPARATUS 
Thoma Ruling 
Neubauer Ruling 
Zappert Ruling 
Breuer Ruling 
Fuchs and Rosenthal Ruling 
Turk Ruling ' 
Biirker No. 1 Ruling 
Zappert-Ewing Ruling 
Fig. 13-Diagrams Showing the Commonest Ihvmocytometer Rulings. 
Zappert-Neubauer Ruling 
Bass Ruling (15x) BLOOD COUNTS 
15 
means of the mechanical stage the counting chamber should be moved 
carefully from square to square. It is useful to always count the cells 
when they lie on the lines, which are above and to the left. By fol- 
lowing some definite system the chance of counting the same corpuscle 
twice is eliminated. 
White Corpuscles. The blood should be drawn into the pipette 
up tlto the 0.5 mark after which the special diluting fluid for white 
corpuscles should be drawn up to the mark. This will give a dilution 
of A special diluting fluid which will destroy the red and leave 
the white corpuscles, must be used. The preparation of the disk of 
the counting chamber is identical with that for counting red corpuscles. 
A systematic procedure should be followed in going.over the slide in 
order not to count the same square twice. In a well-prepared mount, 
the number of cells per square should not vary very much from the 
average for 100 squares. 
MADE IN U.S.A,. 
MADE N U-S-A. 
Fig. 14.-Diluting Pipettes for Haemocytometer. 
A counting chamber of more recent construction has been made 
for the Arthur H. Thomas Co., Philadelphia, known as the Thoma- 
Levy hsemacytometer. This has certain advantages over the types 
of the older construction and is described as follows: 
" In the Levy construction a rectangular depression is cut into the slide itself 
extending across the entire width. In the middle of this depression is perma- 
nently fixed a rectangular strip of glass also extending entirely across the slide, 
and on this are the rulings. When the cover glass is placed in position on the 
slide itself the solution over the ruled areas is of the required depth. The Levy 
method of construction avoids the cemented cell and the attendant danger of 
its loosening by the drying out of the balsam cement, and the possibility of the 
loosening of the ruled counting surface is also greatly reduced by this construc- 
tion. The parallel form of cell greatly facilitates cleaning as compared with the 
circular type and the method of ruling used in the manufacture of these chambers 
provides a line with absolutely cleancut edges and of distinctly increased visi- 
bility when the chamber is filled with solution for-the count. This increase 
in visibility of the ruling greatly lessens the eye fatigue experienced in making 
repeated counts." 
The manufacturers claim for this counting chamber the following 
advantages: 
Increased visibility of the rulings when chamber is filled with solution. 16 
BACTERIOLOGICAL APPARATUS 
The new method of construction entirely avoids the cemented cell and the 
attendant danger of its loosening by the drying out of the balsam cement, 
and the possibility of the loosening of the ruled counting surface is also greatly 
reduced by this construction. 
The parallel form of cell first suggested by Biirker and used- in all Levy 
counting chambers provides a more uniform distribution of corpuscles over the 
ruled area and entirely removes the effect of atmospheric pressure upon the depth 
of the solution, a source of considerable error in the circular-form chambers. 
The parallel form of cell also greatly facilitates cleaning as compared with 
the circular type. 
Critical tests have shown the Levy rulings to be more accurate, both in the 
dimensions of the ruling and the depth of the cell, than any counting chambers 
of best European makes we have tested. All Levy counting chambers are 
Vertical longitudinal section of slide, with cover glass 
in position, showing new method of construction 
' J* > ' 'j. 1 ■ 
400 SG.imxi. 
xrom.deep. 
MADE BV 
V 
PHILADELPHIA,PA. 
U.S.N 
ArthurHThomasCo. 
PATENT APPU.FOR 
- 
iosq.mm. j 
mm. deep. 
MADE BV 
PHILADELPHIA.PA. 
ArthurHThomas Ga ' 
PATtMT Aepo FOR 
Fig. 15.-Thoma-Levy Counting Chamber, 
guaranteed to be well within such limit of error as may be later established by 
the U. S. Bureau of Standards for instruments of this type. 
Application of Haemocytometer to the Enumeration of Bacteria. 
Since the samples which are used in the ha?mocytometer are small, 
the bacteria must be present in large numbers. A slight error in 
making dilutions will cause a large error in the determination of the 
number of bacteria per cubic centimeter since the factor is large which 
converts the reading of the microscope into terms of cubic centimeters. 
It is also an easy matter to confuse organic matter with bacteria. This 
is especially true in the applications of this apparatus to the enumera- 
tion of bacteria in feces and tomato products. These are taken up in 
other places. 
Cleaning of Haemocytometer Apparatus. This is one of the most 
important essentials in the use of the apparatus. Pipettes should 
be cleaned by attaching them to a suction pump and drawing dilute 
alkali through them. After this alcohol and distilled water may be 
used. If they become stopped up a small wire may be run through, 17 
ANAEROBIC METHODS 
but this should be used with care since the bottom of the pipette may 
chip off. 
The counting chamber should be washed with dilute alkali or soap. 
It should be thoroughly rinsed in distilled water and wiped dry and 
should not be sterilized by heating because the cement which , holds 
the disk will be melted. 
Anaerobic Methods 
For the cultivation of those microorganisms which grow under 
anaerobic conditions, some special apparatus and methods are neces- 
sary to remove free oxygen. Many procedures have been proposed, 
a few of which will be given here. 
Boiling. Air is very soluble in water at room temperature. Wink- 
ler has found that 1 liter of water at 20° C. will dissolve about 9 mg. 
of oxygen. By boiling water for some time this dissolved oxygen may 
be driven off. Special methods are then necessary to prevent re- 
absorption of air. In liquid media where heating will cause no change, 
this may be done by a layer of sterile vaseline or ligroin. 
Displacement by an Inert Gas. For this purpose several gases, 
such as nitrogen, hydrogen, carbon dioxide, coal gas, etc., have been 
used. Hydrogen has had the widest application on account of its 
ease of preparation and lack of harmful effects on bacterial develop- 
ment. When these gases are used, they are bubbled through the 
medium and should be carefully washed by the proper solutions to 
render them pure. Gas wash bottles of the Bunsen or Friedrich types 
will allow satisfactory results. 
Absorption of Oxygen. For this purpose alkaline pyrogallol which 
has great affinity for oxygen is usually used. It becomes reduced 
to compounds which are not definitely known. In applying this 
chemical to the growth of anaerobes many devices have been used. 
Any one is quite satisfactory which prevents the ingress of more oxygen 
after the initial oxygen has been absorbed. 
Buchner's Method. In this method the culture tube is placed 
in a larger one which contains the alkaline pyrogallol. It has the 
disadvantage that if some special support is not used to keep the cul- 
ture tube above the surface of the pyrogallol, it is difficult to follow 
growth during incubation. These tubes may be prepared as follows: 
Introduce a few grams of pyrogallol into the bottom of a large 
test tube and place therein a support for the culture tube. This may 
be made from wood, cotton, glass, etc. After the culture tube has been 18 
BACTERIOLOGICAL APPARATUS 
placed on its support, introduce the NaOH by means of a pipette into 
the bottom of the tube and seal the tube immediately with a rubber 
stopper. 
Wright's Method. This is another absorption method and is 
different from the others only in method of procedure. It is especially 
adaptable to the isolation of anaerobic spore-forming bacteria. Decimal 
dilutions of the sample arc introduced into sterile litmus milk. About 
1 in. above the meniscus of the milk a tight cotton cylinder is forced 
upon which arc put about 2 gms. of pyrogallol. This is treated with 
.strong NaOH and the original plug replaced in the tube. This tube 
should then be heated for fifteen minutes at 80° to kill all vegetative 
bacteria. Later incubation will give the characteristic changes. 
Lentz's Method. This requires a glass plate 125 mm. square and 
cellulose absorbent rings. Before using the rings are soaked in pyrogal- 
lic acid and immediately before the culture is started in 1 per cent 
potassium hydroxide. The plate with the culture is inverted over the 
cellulose ring, being scaled to the glass with plasticine. 
Fig. 16.-Lentz's Anaerobic 
Apparatus. 
Fig. 17.-Kuster's Anaerobic 
Culture Apparatus. 
Kuster's Method. This depends upon the same principle as 
McLeod's and Lentz's methods. A glass absorption chamber is used 
having a small hole in the top through which is introduced the absorp- 
tion substance. The Petri dish containing the culture is inverted over 
this and sealed with plasticine. 
McLeod's Method. This method requires a porcelain dish con- 
sisting of two separate chambers for containing the pyrogallic acid and 
potassium hydroxide. The special Petri dish has its free edge turned 
inward and upward. Around the upper cover of the porcelain dish 
is a groove to contain plasticine which prevents the ingress of air. 
About 7 c.c. of a 10 to 20 per cent pyrogallic acid is run into the chamber 
marked A in Fig. 18. Into part B are put 7 c.c. of a 10 per cent potas- 
sium hydroxide. The Petri dish is then pressed down into the plas- ANAEROBIC METHODS 
19 
ticine. After this is done air is excluded and the potassium hydroxide 
and pyrogallic acid are mixed by tilting the dish. 
Zinsser's Method. The main apparatus involved in the applica- 
tion of this method is a set of circular glass dishes similar to a Petri 
dish except that they are much deeper and a larger space is left between 
the sides. The agar which has been inoculated with the sample is 
Fig. 18.-McLeod's Anaerobic Culture Apparatus. 
poured into the smaller of the dishes. This is allowed to harden and 
the excess of moisture is evaporated. Into the other dish, is placed 
a quantity of pyrogallol, over which the smaller dish is inverted. So- 
dium hydroxide is poured into the larger dish. While this is reacting 
with pyrogallol, an oil such as abolin is dropped into the same place. 
This successfully seals the chamber which is formed by the two dishes. 
Torrey's Method. This method was especially successful in the 
hands of Torrey for the isolation of B. bifidus and B. acidophilus. 
■A, 
0 
D 
-B 
Fig. 19.-Cross-section of Torrey's Anaerobic Chamber. 
A, Solid Medium Containing the Sample; B, Agar which has been Heavily Inoculated with 
B. subtilis or B. mesentericus; C and D, Petri Dishes which Form the Chamber. 
It is similar to the method devised by Zinsser. A Petri dish about 
10 cm. in diameter and about 2 cm. high is poured and inoculated with 
the sample under investigation. It is placed in an incubator to remove 
excess moisture and dry the sides of the dish. This is important since 
too much moisture will allow contamination with the culture which is 
used to absorb the oxygen. This dish after drying is inverted into 
another dish, containing agar heavily inoculated with Bacillus cereus, 
of at least 12 cm. in diameter. The agar in the lower plate forms a seal, 
and the strict aerobic organism Bacillus cereus will use up the free 
oxygen. This apparatus is placed in an incubator in a moist chamber 
to prevent evaporation of the agar in the larger dish. 20 
BACTERIOLOGICAL APPARATUS 
Smillie's Method. This method depends upon the catalytic action 
of platinized asbestos upon oxygen and hydrogen when they are brought 
into contact. The author claims that the method is well adapted to 
the study of strict anaerobes such as B. botulinus. The method adapt- 
able to cultures in test tubes is described as follows by the author: 
" Platinized asbestos is first prepared in the usual way, or it may be 
purchased from any laboratory supply house. A small mass of the 
catalyzer is firmly fixed at the end of a platinum wire by coiling the wire 
about it. The other end of the wire is inserted in the end of a short 
glass rod, and the rod is inserted into a No. 1 
one-hole rubber stopper. The apparatus is 
wrapped in a package and autoclaved. 
" The water of condensation is removed 
from a plain agar slant the tube inoculated, 
inverted, the cotton plug removed and the 
tube filled with hydrogen by means of a sterile 
capillary pipette. The hydrogen may be 
obtained from a Kipp generator, or more 
satisfactorily from a hydrogen tank. It should 
be passed through a series of wash bottles con- 
taining silver nitrate, sulphuric acid potassium 
permanganate, and lead acetate to remove all 
impurities. 
" After allowing the hydrogen to fill the 
inverted inoculated test tube, the platinized 
asbestos is heated for a few minutes in a free 
flame, the rubber stopper is inserted firmly into 
the inverted tube and the end of the tube 
dipped into melted paraffin. 
" The catalyzer glows for a second or 
two as the hydrogen and oxygen are actively 
united, and the water formed is deposited 
on the surface of the tube. The process 
is now complete and the tube is ready 
for incubation." Smillie states that this method is satisfactory 
for the usual anaerobe but does not remove all traces of oxygen. 
Another method had to be devised. This is described as follows: 
" Two lengths of nichrome wire No. 22, 6 cm. long, are separately 
fused into a glass tube so that they are insulated (see Fig. 20a) and 
the glass tube B closed at each end is passed through a one-hole rubber 
stopper C. To the lower end of the nichrome wire D thus completing 
Fig. 20.-Sketch of Anae- 
robic Culture Apparatus 
for the Cultivations of 
Obligate Anaerobes in 
Test Tubes According 
to Smillie's Method. SMILLIE'S ANAEROBIC METHOD 
21 
the circuit. In the coil of the fine nichrome wire is placed a small mass 
of platinized asbestos E. The apparatus is placed in a package and 
autoclaved." Smillie has applied the same method to the culture of 
large amounts of bacteria in Blake bottles. 
This method applied to the culture of bacteria in anaerobic jars is 
as follows: 
Smillie experienced some trouble in getting jars which would stand 
up under great vacuum. He finally adopted the ordinary specimen 
- Rubber Stopper. 
- Connecting rubber tube. 
Glass Bulb containing platinized asbestos. 
^2 mm. perforations. 
Fig. 21.-Detail of Platinized Asbestos Bulb for Anaerobic Jar According to Smillie's 
Method. 
jar about 30 cm. high and an inside diameter of 12.5 cm. In the cover 
of this were ground two holes into which were fitted No. 4 one-hole 
rubber stoppers, carrying a glass " angle " stop-cock. A rubber tube 
extending to the bottom of the jar was attached to one of these. To 
the other stop-cock, a glass bulb with perforations is attached by means 22 
BACTERK)L()GICAL APPARATUS 
of rubber tubing. This glass bulb is filled with platinized asbestos. 
The cultures are placed in a glass tumbler which is put into the jar. 
Fig. 21 taken from Smillie's original publication will give the details 
of the asbestos bulb. 
Exclusion of Oxygen 
Beattie's Method. Ordinary test tubes containing plain or car- 
bohydrate broths are used. Melted sterilized vaseline is poured into 
the tube until a plug about | in. long is formed. The broth and vase- 
line are then boiled for twenty to thirty minutes in order to expel any 
dissolved air and allowed to cool. These tubes may be kept for an 
Fig. 22.-Types of Novy Jars. 
indefinite time and are air tight. When they are to be inoculated they 
may be melted in a water bath at 55° C. The sample may be intro- 
duced by means of a sterile pipette. The vaseline may then be heated 
over a flame to reform the solid plug. The tube may then be placed 
in the incubator. In order to remove any portion of the culture, a 
sterile sealed pipette (capillary) is thrust through the plug. This may 
be broken by forcing the end against the bottom of the tube after which 
any portion of the culture may be removed. By melting the vaseline 
plug again the culture tube is sealed for further incubation. 
Novy's Method. The apparatus used in the application of this 
method consists of a large glass jar with a removable top (Fig. 22). 
The culture may be grown away from oxygen by using an inert gas 
vacuum or alkalin pyrogallol. The jar is constructed with a glass stop- 
cock which will allow the removal of air with a vacuum pump. This THE MICROSCOPE 
23 
may be combined with alkalin pyrogallol and give an atmosphere which 
is entirely free from oxygen. 
Petri Dish 10 cm diam. 
Annular Groove 
3mm. Deep x 
I mm. Wide. 
Rubber 
Stopper 
Air Outlet 
> 
Fig. 23.-Jones Anaerobic Culture Apparatus. Cross-section of the Stone Base with 
Petri Dish in Position. 
Bulloch's Method. This is very similar to Novy's and needs no 
special attention here. The air in the jar should be replaced by an 
inert gas. 
Jones' Method. This method 
allows the observation of growth 
and requires very little gas. It is 
described by the author as follows: 
" The apparatus consists of one-half 
of a Petri dish sealed with paraffin 
on a square stone or metal base 
provided for an inlet of the inert 
gas and outlet of air. The figure 
gives the general construction of the 
apparatus. 
The Microscope 
A compound microscope is 
necessary for a study of bacteria. 
The success which a student has 
in the pursuit of microbiology is 
often dependent upon thorough 
knowledge of the microscope. It is 
desirable to study the microscope 
with much care in order to under- 
stand its limitations and the sig- 
nificance of each part. It is much 
better to regard it as a delicate 
piece of mechanism through whose 
intelligent use an entire world of 
living organisms will be made visi- 
-Eye Lons 
- Diaphragm 
of the 
Ocular 
Field Lens 
- Draw Tube 
-Tube Length 160 m.m. 
Body Tube 
Draw Tube Diaphgram 
with Society Screw 
" Society Screw 
- Mount 
^Back Lons 
of the 
Objective 
Cover Glass. 
"Middle Lens 
- Front Lens 
Working Distance 
Slide7 
Object 
Fig. 24.-Showing Cross-section of a 
Microscope. 
(Courtesy of the Spencer Lens Company.) 24 
BACTERIOLOGICAL APPARATUS 
ble. The following pages have been prepared from a number of 
sources-.' Direct reference has been given in a few cases. Most of the 
data, however, have been taken with permission from microscope 
manuals which are furnished by the Bausch & Lomb Optical and 
Spencer Lens Companies. 
Don'ts 
Don't allow dust and dirt to settle on the microscope. 
Don't carry the microscope by the arm, unless the fine adjustment 
is protected. 
Don't use alcohol on the microscope. 
Don't expect toe great a range in the fine adjustment. 
Don't take the fine adjustment apart. 
Don't bring the objective into contact with the cover glass. 
Don't fail to focus up before turning the nosepiece unless you know 
the objectives are parfocal. 
Don't forget that high powers have short working distances. 
Don't focus down with the eye at the eyepiece. 
Don't fail to secure good even illumination. 
Don't drop the objectives and oculars. 
Don't try to take an objective apart. 
Don't try to clean the lens with a dirty cloth. 
Don't fail to clean oil from an immersion lens immediately after 
using. 
Don't try to work with an immersion lens when there are air bubbles 
in the oil. 
Don't use high powers when low ones will do. 
Don't use higher oculars than necessary. 
Don't expect a lens to work at its best unless used on a cover thick- 
ness, and with a tube length, for which it is corrected. 
Don't shut one eye. 
Don't get discouraged if desired results do not come immediately. 
The instrument should always be kept in a case, either the one 
in which it was received from the factory or one which is specially 
constructed to hold a series of them in the laboratory. When carry- 
ing a microscope or removing it from its case, grasp it by the pillar 
and not by any other part. 
Finger marks and stains should be carefully rubbed with soft cloth 
or lens paper. If this is not sufficient, a little ether, chloroform or 
xylol may be used. Alcohol should never be used since this will remove 
the lacquer. PARTS OF THE MICROSCOPE 
25 
Stage. This deserves little attention. It may be cleaned as above 
described. 
Inclination Joint. By means of this joint the body of the micro- 
scope may be inclined at an angle. Should the joint become so loose 
that the microscope will not remain at the desired angle, it may be 
tightened by tightening a nut. 
Fig. 25.-Showing the Different Parts of a Microscope. 
Course Adjustment. This is accomplished in one of two ways: 
The sliding tube and the rack and pinion. The former is found only 
on the cheaper microscopes, and is practically out of date. In it 
the body tube is made to slide inside of a sleeve which is fastened 
to the arm of the microscope. It is very hard to keep in order, because 
the exposed surfaces of the body tube and the inside of the sleeve 26 
BACTERIOLOGICAL APPARATUS 
become gummed and corroded from exposure to the atmosphere and 
reagents of the laboratory, and particularly from the perspiring or 
dirty hand of the operator in focusing. In this condition the tube 
works hard and unevenly, so that one is apt to force an objective irito 
the slide. The tube and sleeve can generally be cleaned by rubbing 
with a coarse towel after moistening with xylol. Do not grease the 
tube or sleeve because it gums and corrodes the tube, and is very 
unsightly. Should the tube be too loose in the sleeve remove the tube 
and press in the top edge of the sleeve on either side of the one or more 
slots running down from the top edge. 
All of the better microscopes are provided with the rack and pinion 
coarse adjustment. The bearings must necessarily fit very closely. 
Any foreign matter on them interferes seriously. Do not strain the 
teeth of the rack and pinion by forcing the bearings back and forth 
over one another when they are not clean. A little xylol or chloro- 
form rubbed on the surfaces will clean them. Do not use emery in 
any form. When the bearings are perfectly clean, oil them slightly 
with a good acid-free lubricant (paraffin oil or watch oil). If the bear- 
ings become so loose that the tube will not stay in place tighten the 
little screws at the back of the pinion box. All makers have a provision 
here for taking up lost motion and wear. Do not fill the teeth of the rack 
with paper paraffin or any other foreign substance. If anything should 
accumulate in these teeth clean it out. 
Fine Adjustment. The fine adjustment is necessarily of limited 
range and delicate in its mechanism. If, when looking into the eye- 
piece, no change of focus is noticed by turning the micrometer head or, 
if the micrometer head ceases to turn, the adjustment has reached its 
limit. Turn the micrometer back to bring the fine adjustment mid- 
way within its range. When the fine adjustment head stops do not 
force it. All of the better microscopes are made so that the head stops 
at both ends of the range and so that the micrometer threads cannot 
be removed from their bearings. Other microscopes do not have this 
safeguard, and great care should be exercised not to remove the thread. 
If by chance it should be removed, exercise great care in replacing it 
to see that the threads are started properly so that they do not " run." 
Do not force them if they run at all hard. If they are started to " run- 
ning " they must go to a machinist. On some microscopes these 
threads are " left-handed," which should be noted in trying to start 
the threads. 
In some microscopes a little steel pin is fitted loosely into the hollow 
end of the micrometer thread. Be careful to see that this little pin PARTS OF THE MICROSCOPE 
27 
is in its place before starting the micrometer thread. In some cases 
this pin even drops out of place before the micrometer thread is entirely 
out of its bearings and the defect is not noticed until the fine adjust- 
ment fails to respond. In such a case the top of the fine adjustment 
must be opened to secure the pin and put it in place. This ought to 
be done by the maker or experienced mechanic. 
In some cases, especially in microscopes (Continental type) where 
the prism is used in the fine adjustment, the lubricant in the prism 
becomes gummed so that the adjustment fails to respond promptly 
and then jumps. On the best microscopes provision is now made 
against this. The bearings should be thoroughly cleaned and oiled 
with paraffin oil or watch oil. This ought to be done by the manu- 
facturers, because the mechanism is so delicate that even though safely 
taken apart it would be put together and adjusted with great difficulty. 
All the modern microscopes have their fine adjustments so arranged 
that the fine adjustment ceases to work when the objective rests on 
the cover glass. This feature should be insisted upon. 
Draw Tube. This should work easily and smoothly and should 
be kept clean and dry. In sliding this draw tube, do so with a revolving 
motion. The draw tube should always be drawn out to give the proper 
focal length for which the microscope was standardized. All micro- 
scopes give best results if used with cover glasses of definite thick- 
nesses. 
Substage. This should be kept dry and clean to prevent cutting 
and rusting of the delicate bearings. Should the leaves of the iris 
diaphragm become rusted, they may be cleaned with xylol and oiled. 
When they become bent, an expert is necessary. 
Lenses. Absolute cleanliness is necessary with the lenses. Japa- 
nese lens paper of highest quality should be used for cleaning. It is 
best to use no other material on the lenses. Great care should be used 
in cleaning them since they are very easily marred and scratched. 
Objectives. If these become soiled rub with lens paper using 
a little chloroform if necessary. Hard rubbing must be avoided. 
The immersion objective should always be cleaned by wiping with 
lens paper. If the oil is old and tends to be hard, xylol may have to 
be used. 
All dry objectives are corrected for a definite thickness of the cover 
glass. (Bausch & Lomb, 0.18 mm.; Spencer Lens Co., 0.18 mm.; Zeiss, 
15 mm.; Leitz, 0.17 mm.) The objectives are corrected to a certain 
tube length, which is 160 mm. by most makers and 170 mm. by Leitz. 
Such tube lengths should always be used for which the objectives have 28 
BACTERIOLOGICAL APPARATUS 
been standardized. Variations in the length of this draw tube should 
be made when cover glasses of a different thickness are used. 
Numerical Aperture. N.A. = n sin u. 
n - the lowest refractive index that appears between the object 
and the front lens of the objective; 
u = half the angular aperture. 
The numerical aperture is important since it determines the resolv- 
ing power definition and illumination. It is especially important with 
reference to the resolving power. The resolving power is directly pro- 
portional to the numerical aperture. The higher the numerical aper- 
ture the greater the resolving power and the finer the detail. Bausch 
has pointed out the significance of the N.A. (numerical aperture) as 
follows: 
" If a very narrow central pencil is used for illumination, the finest 
detail that can be shown by a microscope, with high enough magni- 
fication, is equal to where X is the wave length of the light used 
for illumination. The wider the pencil used for illumination, the greater 
the resolving power, until a maximum is reached, when the width of 
the pencil is sufficient to fill the whole aperture of the objective. In this 
case the resolving power is twice as great, the finest detail that the 
objective can show being now equal to 2 N.A. This same limit is 
reached when a narrow pencil of greatest possible obliquity is used. 
For example, the wave length of the brightest part of the spectrum 
may be assumed to equal 0.00053 mm. Consequently an objective of 
N.A. equal to 1.00 will resolve two lines separated by a distance of 
equal 0 00053 with a narrow central illumination cone, and 
equal 0.000265, with a cone filling the whole aperture, or with a 
narrow oblique cone. 
" A 4 mm. 0.85 N.A. objective will resolve lines separated by dis- 
tance ranging between 0.00062 and 0.00031, dependent upon the aperture 
employed. For a 4 mm., 0.65 N.A. objective the limiting values are 
0.00081 and 0.000405. 
" The N.A. can also be expressed by the equation. 
N a _ _ effective aperture of back lens 
2f 2 X equivalent focus 
" Two objectives of the same equivalent focal length (E.F.) and the 
same N.A. should show the same illuminated area in the back lens, USE OF THE MICROSCOPE 
29 
when viewed without an eyepiece and illuminated with the widest 
cone of light they can take in. 
" The foregoing explanation shows the importance of the N-A. to 
the efficiency of an objective. 
" It is also evident that an objective cannot show its full efficiency 
if it is not used with a condenser of a N.A. large enough to fill the back 
of the objective with light." 
The depth of sharpness of an objective is in inverse ratio to the 
N.A. great penetration goes along with a low N.A. and low penetration 
but great resolving power with a high N.A. 
Illuminating Power. The brilliancy of the objective increases 
with the square of the numerical aperture of the objective. An objec- 
tive of 0.40 N.A. will give an image four times as brilliant as one of 
0.20 N.A., provided the magnification is the same and the full cone of 
the illumination is used in both cases. 
Magnifying Power. The magnifying power of an objective is in 
inverse ratio to its focal distance. An objective of 2 mm. focal distance 
will give, with the same ocular, a magnification eight times greater 
than one of 16 mm. focal distance. Numerical aperture and magnify- 
ing power are of little advantage if the definition is not good. 
Chromatic Aberration. This is due to the fact that a ray of white 
light passing from one medium to another of different refractive index 
at any angle other than 90° to the surface between them is refracted 
and dispersed into its component colors. 
Spherical Aberration. This is due to the fact that a spherical 
surface cannot bring a beam of light which passes through its vertex 
to the same focus as that of a beam of light passing through any other 
zone. 
Both aberrations are corrected by the use of different kinds of glass 
(crown and flint) combined as double and triple lenses in the objective. 
Neither can be corrected absolutely for all colors in an achromatic 
objective. Apochromatic objectives approach the ideally corrected 
objective almost to perfection. 
An objective can be tested for chromatic correction by using a 
narrow cone of oblique light and a coarse grating. Abbe's test plate is 
best. Diatoms are good. No stained object should be used. 
If the spherical correction is perfect (see next paragraph) and one 
side of a line passing through the center of the field shows a clear, 
narrow greenish yellow border, while the other side is fringed with a 
violet red (secondary colors) the objective is chromatically corrected. 
The colors shown in the higher power objectives are of a more primary 30 
BACTERIOLOGICAL APPARATUS 
character, i.e., nearer the yellow and blue. Apochromatic objectives 
show no color borders in this test. 
The spherical correction of an objective is perfected for a certain 
thickness of cover glass and a certain tube length, and is influenced 
greatly by any variation in either. This is especially true with the 
high-power dry objectives. The homogeneous immersion objectives 
are not sensitive to the variation in the cover thickness, because the 
immersion oil between the cover glass and the lens is of the same refrac- 
tive index as the glass. They must be used, however, with the proper 
tube length. In testing an objective for its spherical correction it is 
therefore very important to supply the proper thickness of cover and 
tube length. It is manifestly unfair to judge an objective on this 
point without complying with these conditions. The test for spherical 
correction can be made on the same object as used for the chromatic 
test. If the edges of the lines in the center of the field appear equally 
sharp and clear when illuminated by either a narrow central cone of 
light or a narrow oblique cone without having to change the fine adjust- 
ment the objective is spherically corrected. The color remnants men- 
tioned above will be clear and transparent, while if the lens is poorly 
corrected spherically, these borders will appear muddy ami turbid. 
Defects in spherical corrections can often be corrected by using cover 
glasses suitable to them, also by changing the tube length. The fact 
that the periphery of the field is not in focus at the same time as the 
center does not bespeak a lack of spherical correction, but a lack of 
flatness of field with which it is often confused. 
Flatness of field depends not only upon the objective itself, but upon 
the ocular and the cone of light used, whereas the spherical aberration 
is inherent in the objective itself. No field is absolutely flat. It is a 
desirable quality in a lens, but spherical and chromatic corrections 
should never be sacrificed for it. Some lenses appear to be " flatter " 
than they really are, because their corrections are so poor that little 
contrast is noticed between objects in the center of the field and 
at the edge. Narrow cones of light give a flatter field than wide 
ones. Thin objects are more critical tests for flatness of field than 
thick ones. 
Working distance is the free distance between the cover glass and 
the objective when the latter is focused. It decreases generally with 
increasing power and numerical aperture of the objective. Of two lenses 
with the same focal distance the one with the higher N.A. will have 
the shorter working distance. The working distance also depends 
on the mounting of the front lens. If the lens has a prominent mount- 31 
USE OF THE MICROSCOPE 
ing projecting beyond its surface the working distance is lessened 
thereby. 
Oculars. These have a simpler structure than the objectives. 
They serve as collective lenses, making all of the light constituting 
the image from the objective enter the eye of the observer. 
Illumination. Practically all microscopic studies are carried out 
by transmitted light, and some special attachment is necessary to direct 
this light along the optical axis of the microscope. For this purpose 
substage or Abbe condensers are used. This condenser allows the 
utilization of all light which enters the object space. Light is sent 
through the object at an angle great enough to fill the objective. 
The condenser is constructed to bring the rays of light to a focus 
above the upper surface of the uppermost lens. When the concave 
Fig. 26.-Showing Incidence of Light Rays in Condenser. (After Bausch.) 
mirror is used the rays of light are brought to a focus within the con- 
denser. The concave mirror acts as a lens and together with the con- 
verging effect of the lens in the condenser causes the light to converge 
more rapidly than that for which the microscope was standardized. 
Bausch has depicted this in Fig. 26. 
Bausch makes the following statement with regard to the use of 
the condenser: "In the use of the condenser with oil immersion 
objectives the custom prevails of using the condenser dry. It is well 
to point out, however, that both the condenser and the objective lose 
in their efficiency when the former is used dry, and for critical work 
the condenser should be in immersion contact with the slide. 
To make immersion contact between condenser and slide place 
a drop of oil on the top of condenser, drop the slide upon the stage, 
first turning the slips to one side. 
With immersion objectives the proper focusing of the condenser 
becomes a matter of nice distinction to obtain best results and can 32 
BACTERIOLOGICAL APPARATUS 
only be reliably accomplished by considerable practice and experience. 
To obtain best position: 
Use a 16-mm. (j in.) objective; focus upon the object; adjust con- 
denser until image of window-sash or flame is in the same plane with 
object. 
Practically all substages are provided with means for focusing 
the condenser. The rays of light should be focused sharply on the 
object. A little experience will indicate when the proper aperture is 
Fig. 27.-Showing Action of Different Parts of the Condenser. (After Bausch.) 
being used. The iris-diaphragm when used with dry lenses should 
not be more than half open. With the oil immersion objective, how- 
ever, the full aperture should be used. 
Final Hints 
" Sometimes the worker may have faithfully carried out all the 
directions heretofore given and been assured that his lenses possess the 
above-named qualities as they ought, yet be unable to obtain the desired 
results. He may be working with a water mount and dry objective 
become ' immersed ' in some water which has worked to the top of 
the cover glass. His objective may be dirty from a previous ' immer- 
sion ' or it may have some other dirt upon the front lens. The field 
may be covered with specks which revolve when the ocular is turned. 
The field may be dim or hazy, due to dirt on the back of the objec- 
tive or a film on the inner surfaces of the lenses of the ocular, or because 
of moisture settling on the lenses because they have just been brought 
from a cold into a warm room. He may see great streaks on his field, 
which are due to his own eyelashes, or he may see small slowly moving 
bodies floating across the field. With the exception of this last, the 
ailment has only to be mentioned to suggest the remedy. The muscae 
volintantes, as these last-named bodies are called, are little specks of 33 
MAGNIFICATION TABLES 
shreds in the vitreous humor of the eye which cannot be removed, but 
which can easily be disregarded. 
"In water mounts and fresh balsam mounts one is apt to find air 
bubbles. To be sure that the object is an air bubble, focus up with 
central light. The bright spot in the center will become clearer while 
the edge will become darker. With oblique light the bright spot 
will be thrown to one side. In studying water, blood or any fluid, 
always cover the drop with a cover glass. The objectives are cor- 
rected for rays passing through media with parallel surfaces. If such 
a mount is not kept horizontal, currents will be set up, due to gravita- 
tion, and they will be seen with a magnified velocity seemingly running 
up hill. 
" The fact that the microscope reverses every movement and magni- 
fies it may be mentioned again. 
" Beside any movement due to currents there is sometimes a pecu- 
liar indefinite to and fro movement of particles from one position to 
another. This is called Brownian movement. 
" In studying sections a true idea of the structure of the tissue 
can only be obtained by moving the slide about to bring different 
parts into the optical axis and by focusing with the fine adjustment to 
bring different levels, or optical planes, successively into view Where 
serial sections are used each section must be studied in relation to its 
neighbors. 
Table I 
MAGNIFICATION TABLE 
Bausch & Lomb Optical Co., Rochester, N. Y. 
Tube Length 160 mm. 
Image Distance 250 mm. 
Objec- 
tives, 
mm. 
Eyepieces. 
Objec- 
tives, 
Inch. 
Working 
Distance, 
mm. 
5x 
6 Ax 
7.5z 
lOz 
12.5z 
48.0 
10 
' 13 
15 
20 
25 
2 
53 
32.0 
20 
26 
30 
40 
50 
U 
38 
16.0 
50 
64 
75 
100 
125 
2 
3 
7.0 
8.0 
100 
130 
150 
200 
260 
1 
3 
1.6 
4.0 
215 
275 
320 
430 
560 
1 
6 
0.6 
4.0t 
215 
275 
320 
430 
560 
1 
6 
0.3 
3.0s 
285 
365 
420 
570 
740 
1 
8 
0.2 
1.90 
475 
610 
720 
950 
1260 
1 
1 2 
0.15 34 
BACTERIOLOGICAL APPARATUS 
" Sometimes sections which are freshly mounted in balsam appear 
cloudy and indistinct. This is because of failure to thoroughly dehy- 
drate the specimen before putting it into the balsam. But this brings 
us into the realm of laboratory technique, which is beyond the scope of 
this volume." 
Table II 
SPENCER LENS COMPANY MAGNIFICATION TABLE 
Tube Length, 160 mm. 
Objec- 
tives, 
min. 
Initial 
Magnifi- 
cation. 
Oculars. 
Objec- 
tives, 
in in. 
4z 
5.r 
6x 
8z 
lOz 
15z 
20z 
48 
2.2 
8 
11 
13 
18 
22 
27 
33 
44 
48 
40 
2.8 
11 
14 
17 
22 
28 
33 
42 
56 
40 
32 
4 
16 
20 
24 
32 
40 
48 
60 
80 
32 
30-22 
2-4.5 
4-9 
5-12 
8-19 
10-24 
15-35 
18-43 
20-48 
30-70 
30-22 
25.4 
6 
24 
30 
36 
48 
60 
72 
90 
120 
25.4 
16 
10 
40 
50 
60 
80 
100 
120 
150 
200 
16 
12 
15 
60 
75 
90 
120 
150 
180 
225 
300 
12 
8 
20 
80 
100 
120 
160 
200 
240 
300 
400 
8 
5 
36 
144 
180 
216 
288 
360 
432 
540 
720 
5 
4 
44 
176 
220 
264 
352 
440 
528 
660 
880 
4 
3 
60 
240 
300 
360 
480 
600 
720 
900 
1200 
3 
1.8 
95 
380 
475 
570 
760 
950 
1140 
1425 
1900 
1.8 
1.5 
109 
436 
545 
654 
872 
1090 
1308 
1635 
2180 
1.5 
Image Distance, 250 mm. 
When using the camera lucida with the paper lying on the table 
add 60 per cent to the above magnifications. 
Micrometry 
This has to do with the measurement of objects under the micro- 
scope. While there are several ways of doing this the micrometer 
eyepiece has the widest application. More accurate results are obtained 
because the real image is measured. Since this image has been magni- 
fied by the objective, the " micrometer eyepiece " must be standardized 
for each object. 
The Spencer Micrometer Eyepiece No. 425 is described as follows: 
" This micrometer eyepiece represents the highest type of pre- 
cision in construction and guarantees the greatest possible accuracy 
of measurement. Instead of the usual cross hairs, a finely ruled glass 
scale is used in its construction, each interval in the scale being exactly MICROMETRY 
35 
equivalent w one revolution of the screw which moves it. This system 
has important advantages, especially in the measurements of large 
objects. Unlike the filar micrometer, it does not require the moving 
of the index over the entire length of the object, as a fraction of one 
rotation of the screw is all that is necessary. 
" The scale is 5 mm. long and is divided into twenty spaces; each 
millimeter is marked by a line of double length. As the screw is made 
with a pitch of | mm., one revolution of the screw moves the scale 
1 mm., or one of the intervals. 
Fig. 28.-Spencer Lens Company's Micrometer Eyepiece. 
11 Upon the axis of the screw is placed an adjustable drum which 
may be set to any desired position. This is graduated with one hundred 
divisions, for reading of which an index pointer is fastened on top of 
the micrometer case. 
" In measuring the length of an object, the scale is moved until 
one of the millimeter lines coincides with the margin of the object 
under examination; as for example, line No. 0 in the sketch, then, 
holding the milled head of the screw fast, the drum is turned until 
the index stands at zero. Now turning the screw until the line' which 
the other edge of the drum added to the number of full | mm. divisions 
covered (nine in the sketch) gives the apparent length of the object 
-its real length depending on magnification used. 36 
BACTERIOLOGICAL APPARATUS 
11 With an ordinary filar micrometer it would have been necessary 
to move the cross hairs over the whole length of the object, which in the 
above instance would have required nine full revolutions besides the 
fractional turning of the drum, while with our screw micrometer it 
was only necessary to move the scale over a part of one space. 
"For measurements of less accuracy, this micrometer can also be 
used without turning the screw, simply using the subdivisions of the 
scale as in the ordinary micrometer." 
Zeiss Micrometer Ocular. This consists of a Ramsden ocular 
of about 20 mm. focal length. The glass plate, with a crossed as well 
Fig. 29.-Zeiss Micrometer Ocular. 
as a double line, is moved over the field by turning the micrometer 
screw. Each unit on the revolving drum corresponds to a move- 
ment of 0.01 mm. of the point marked. The absolute values of the 
units on the drum must be determined from a stage micrometer. 
Calibration of Micrometer Eyepiece. For this pull the draw tube 
to the proper length and focus the lines of an object micrometer sharply. 
This may be done with an ordinary ocular. Replace this with the 
micrometer eyepiece, and focus again so that the lines on both microm- 
eters are sharp. 
The object micrometer is made from a cover glass bearing a scale 
which is divided into a certain number of parts. These data are usually BIBLIOGRAPHY 
37 
given on one end of the object micrometer. By focusing the object 
micrometer with a definite combination of an objective and micrometer 
eyepiece at a definitely fixed tube length and comparing the two microm- 
eter scales, it is easy to find the actual length in the plane of the object 
which corresponds to one scale division of the eyepiece micrometer. 
BIBLIOGRAPHY 
Bausch, E. Use and Care of the Microscope. Bausch & Lomb Optical Co., 
Rochester, N. Y. 
Beattie, J. M. 1916. Simple and Inexpensive Methods for Fermentation 
Tests and for Obtaining Culture of Anaerobes. Brit. Med. Jour., 1916-1, 
756-7. 
Breed, R. S. and Dotterer, W. D. 1916. The Number of Colonies Allow- 
able on Satisfactory Agar Plates. Jour. Bact., 1, 321-31. 
Browne, W. W. 1913. A Comparative Study of the Smith Fermentation 
Tube and the Inverted Vial in the Determination of Sugar Fermentation. 
Am. Jour. Pub. Health., 3, 701-4. 
Browne, W. W. 1917. The Fallacy of Reading Accurately Gas Percentages 
in the Fermentation of Lactose Peptone Bile and Lactose Broth. Amer. 
Jour. Pub. Health, 7, 663; Jour. Bact., 2 (1917), 249-267. 
Buchner, H. 1888. Eine neue Methode zur Kultur anaerober Mikroorgan- 
ismen. Cent. Bakt., 4, 149-151. 
Carpenter, W. B. 1901. The Microscope and Its Revelations. 8th Edit., 
by W. H. Dallinger. 
Durham. 1898-1. A New Method for Demonstrating the Production of 
Gas by Bacteria. Brit. Med. Jour., 1387. 
Graves, K. D. 1917. An Improved Modification of the Durham Fermenta- 
tion Tube. Jour. Amer. Med. Assn., 69, 2102. 
Hall, I. C. 1914. An Improved Durham Fermentation Tube. Amer. Jour. 
Public Health, 4, 1173-1178. 
Hill, H. W. 1904. Porous Tops for Petri Dishes. Jour. Med. Res., 13, 93. 
Hill, H. W. 1908. The Mathematics of the Bacterial Count. Amer. Jour. 
Pub. Hyg., 18, 300-310. 
Jones, H. M. 1916. A Method of Anaerobic Plating Permitting Observation 
of Growth. Jour. Bact., 1, 339-341. 
Kuster. 1913. Ein einfacher Apparat zur anaeroben Ziichtung und eine 
Verrichtung zur einwandfreien. Entnahme von Untersuchungsmaterial 
aus der Tiefe von Korperhohlen. Cent. f. Bakt. Ref., 57, 269-271. 
Lee, R. E. and Fegely, W. H. An Experimental Study of the Relative 
Efficiencies of Certain Fermentation Tubes. Am. Jour. Pub. Health, 
4 (1914), 999-1005. 
Lentz, O. 1910. Ein neues Verfahren fur die Anaeroben. Cent. f. Bakt. 
abt., I, 53, 358-365. 38 
BACTERIOLOGICAL APPARATUS 
McLeod, J. E. 1913. A Method for Plate Culture of Anaerobic Bacteria. 
Jour. Path, and Bact., J, 454-57. 
Novy, F. G. 1893. Die Kultur anaerober Bakterien. Cent. Bakt., 14, 
581-600. 
Nowak. 1908. Le Bacille de Bang et sa Biologie. Am. Inst. Past., 22, 541. 
Smillie, W. G. 1917. New Anaerobic Methods. Jour. Exper. Med., 26, 
59-66. 
Smith, T. 1890. Das Gahrungskolbchen in der Bakteriologie. Cent. f. 
Bakt., 7, 50, 2-506. 
Spencer Lens Company. How to Use and Care for the Microscope. Buffalo, 
N. Y. 
Spitta, E. J. 1909. Microscopy. 2d Edit. 
Stewart, A. H. 1906. A New Colony Counter and Dissecting Microscope. 
Jour. Med. Res., 14, 423. 
Torrey, J. C. 1917. New Differential Plating Methods for B. bifidus (Lis- 
sier) and B. acidophilus (Moro). Jour. Bact., 2 (1917), 435-439. 
Wright. 1900. A Simple Method for Cultivating Anaerobic Bacteria. 
Jour. Boston Soc. Med. Sci., 5, 114. 
Zinsser, H. 1906. A Simple Method for the Plating of Anaerobic Organisms. 
Jour. Exper. Med., 8 (1906) 542. CHAPTER II 
MEDIA AND THEIR PREPARATION 
Bacteria are such minute organisms that their environment exerts 
great influence on their development. In the laboratory where it is 
desired to get as accurate information with regard to their life histories 
as possible, every attempt should be made to create an environment 
which simulates the natural one for the variety under examination. 
The bacteriologist calls this a' medium. The characteristics of a good 
medium are: 1. It must approach the isotonic solution for the bac- 
terial cells. 2. Must contain proper kinds and amounts of the essen- 
tial food substances such as: (a) Water concentration. (6) Nitrogen 
requirement, organic and inorganic, (c) Inorganic salts. 3. Should 
be used at optimum temperature for organism under study. 
All media should be made up according to standard methods. In 
America the Standard Methods for the Examination of Water and 
Sewage by a Committee of the American Public Health Association 
are taken as the fundamental basis upon which most official media 
are made. These may not be the ideal procedures but doubtless 
embody most of the essential points according to our present information. 
By following these standard methods the analyst is more certain of 
securing results which may be compared with those obtained in other 
laboratories, than if he adopted his own formulae. Some objections 
have been raised with regard to parts of these standard methods, 
but constructive criticism is necessary if they are to be brought to a cor- 
rect stage. 
Constituents of Media 
While there are many different substances which may be used 
for cultering bacteria, a certain few yield the best results and are 
therefore used at the present time in most of our common media. 
These substances furnish the optimum conditions for the average 
bacterium. For these bacteria, certain pathogenic forms for instance, 
which do not grow on the ordinary media, special media must be pre- 
39 40 
MEDIA AND THEIR PREPARATION 
pared which embody the essential food substance. Some of the patho- 
genic bacteria grow better if a little blood serum or hemoglobin is 
added to our common media. The following substances are in common 
use by bacteriologists for preparing media. 
Peptone. This is added to common culture media to satisfy the 
organic nitrogen requirement of the bacteria. The- peptones which 
are used in media are mixtures of the various cleavage products of 
the protein molecule. Important among these are the amino acids, 
which are precursors for many of the characteristic products of bac- 
terial activity. These must be hydrolyzed from the protein molecule. 
Many analyses of peptones are available in the literature, but as 
Banzhaf and Hirshleifer (1914) have pointed out, they may mean 
very little because in many cases the methods of analysis are not 
known. Generally, peptones consist of salts, among which are the 
phosphates, amino acids and proteoses. 
Witte's peptone has formerly been specified for all bacteriological 
media. Berry (1914-1915) and Conn (1915) have found that equally 
good results are secured with the domestic peptones as with Witte's. 
Berry secured her data by comparing colonies of B. typhi, B. coli and 
streptococcus on agar containing 2 per cent of the different peptones. 
Conn compared two samples of American-made peptone to Witte's 
when he studied the methods of milk analysis in New York City. The 
three peptones showed such slight difference in average that Conn 
regarded one as good as the other. 
Meat Extract. This is an important constituent of laboratory 
media. For bacteriological purposes it is usually secured in jars. Meat 
infusion formerly was used in place of meat extract. 
This is made by soaking 500 gms. of lean chopped beef in 500 c.c. 
of distilled water for twenty-four hours. By keeping this in the refrig- 
erator all bacterial changes are inhibited. At the end of this time it is 
filtered through cheese cloth or flannel and brought back to the original 
volume by adding distilled water. This meat infusion is used as the 
base to which the other constituents are added. Its chemical composi- 
tion is not far different from that of the commercial meat extract. 
Gage and Adams (1904), however, have shown that the chemical com- 
position is variable. The reaction and the organic solids are variable. 
This variation is probably due to the different grades of meat which are 
used. High amounts of fat and connective tissue will tend to give a 
product weaker in strength. The kind of beef might also explain the 
variation in chemical content. The variation is illustrated by the fol- 
lowing data which are taken from Bigelow and Cook (1908). COMPOSITION OF MEDIA 
41 
Table III 
COMPOSITION OF MEAT JUICES PREPARED IN THE LABORATORY 
(After Bigelow and Cook, 1908) 
Round beef cold pressed 
Chuck beef cold pressed. ... 
Round beef pressed at 60° C. 
Chuck beef pressed at 60° C. 
85.76 
86.85 
90.65 
91.90 
Water in Juice. 
1.53 
1.86 
1.36 
1.29 
Ash. 
0.12 
0.20 
0.15 
0.19 
Chlorine as Sodium 
Chloride in Ash. 
0.37 
0.31 
0.36 
0.29 
Phosphoric Acid. 
19 0 
61 0 
OSO 
LZZ 
Ether Extract. 
0.27 
0.32 
0.15 
0.20 
Acidity as Lactic 
■ Acid. 
2.08 
1.74 
1.16 
1.09 
Total Nitrogen. 
0.16 
0.29 
0.68 
0.12 
Nitrogen Insoluble. 
1.37 
0.98 
0.68 
0.41 
Nitrogen Coagulable. 
0.06 
0.07 
0.04 
0.07 
Protein Nitrogen. 
ISO 
100 
ll 0 
910 
Peptone Nitrogen. 
0.33 
0.29 
0.43 
0.27 
Amido Nitrogen. 
0.47 
1.03 
1.90 
0.40 
Undetermined. 
Many different grades of commercial meat extracts are on the market. 
Bacteriologists have seen fit to specify Liebig's as the " standard " 
for all bacteriological media. Bigelow and Cook (1908) describe its 
manufacture as follows: 
A first grade extract is prepared from beef alone and is usually sold in jars. 
An extract of the trimmed bones to which considerable meat adheres is also 
made. The trimmings include odds and ends of meats, muscles, tissue, bone, 
etc., and the product is a second-grade article. In preparing this extract, the 
bones are heated, not boiled, for thirty minutes to forty minutes and the liquor 
evaporated to the consistency of extract. The extract prepared from corned- 
beef liquor constitutes another second-grade product. This extract has a high 
content of nitrates and sodium chloride. In addition there is an extract pre- 
pared from pork and other meats sold under the general term of meat extract. 
Mixtures of various meat and bone extracts are often made. A fluid meat 
extract is usually a 50 per cent solution of solid meat extract. 
Horse meat is now used to some extent in the preparation of meat 
extracts. It has the special advantage of possessing less muscle sugar. 
The following analysis of Liebig's meat extract is given by Bigelow 
and Cook (1908). 
Moisture 21.14 
Mineral constituents: 
Total ash 21.03 
Chlorine as sodium chloride 3.11 
Total phosphoric acid 2.40 
Organic phosphoric acid 0.61 
Inorganic phosphoric acid 1.79 42 
MEDIA AND THEIR PREPARATION 
Acidity: 
N/10 NaOH 9.04 
As lactic acid 8.13 
Nitrogen as: 
Total 9.07 
Insoluble and coagulable 0.19 
Proteoses 2.01 
Peptones 2.68 
Total meat bases 3.82 
Creatin and creatinin 1.14 
Xanthin bases 0.03 
Meat bases other than 2.65 
Ammonia 0.37 
Ether extract 0.94 
U ndetermined 5.89 
Net weight 57.80 
Table IV 
PARTITION OF THE NITROGEN OF MEAT EXTRACTS 
(Cook, 1908) 
Percentage of Total Nitrogen. 
V ariety 
and 
Sample 
Total 
Nitrogen 
Per 
Cent. 
Total 
Creati- 
nine 
Nitrogen. 
Nitrogen 
in Phos- 
photung- 
stic Acid 
Filtrate. 
Nitrogen 
in Acid 
Alcohol 
Filtrate. 
Nitrogen 
in Tannin 
Salt 
Filtrate. 
Amino Nitrogen. 
Num- 
ber. 
Ammonia 
Nitrogen. 
Purine 
Nitrogen. 
F ormol 
Method. 
Van 
Slyke 
Method. 
1 
9.56 
2.62 
22.49 
3.35 
7.64 
78.04 
54.91 
10.94 
18.50 
2 
9.65 
2.49 
22.59 
3.52 
6.84 
78.45 
55.85 
10.94 
18.23 
3 
7.68 
1.56 
32.42 
2.86 
71 35 
100.00 
73.04 
10.63 
15.63 
4 
9.65 
29.01 
4.92 
49.74 
76.73 
59.37 
10.27* 
15.54 
5 
7.49 
27.50 
0.13 
89.19 
64.21 
9.53 
17.89 
* No correction for ammonia; no determination 
Gelatin. This was first used by Koch as a solid medium. Since 
that time it has become firmly established in the bacteriological labora- 
tory. It has the advantage over some other substances used in solid 
media of allowing a larger number of colonies but the great disadvantage 
that it is a bacterial food and may be liquefied. This often causes a 
loss of plates. 
Gelatin is made from collagen, a constituent of white fibrous tissue. 
Emmett and Gies (1907) explain the formation of gelatin from collagen COMPOSITION OF MEDIA 
43 
as being a rearrangement of the molecule. The composition of gelatin 
is indicated in Table V, which is taken from Mathews' Physiological 
Chemistry. 
Table V 
SHOWING THE AMINO ACID CONTENT OF GELATIN 
Glycocoll 16.50 
Alanine 0.80 
Valine 1.00 
Leucine 2.10 
Proline 5.20 
Phenylalanine 0.40 
Aspartic acid 0.56 
Glutamic acid 1.88 
Serine 0.40 
Cystine 0.00 
Tyrosine 0.00 
Arginine 7.62 
Histidine 0.40 
Lysine 2.75 
Ammonia 
Tryptophane 0.00 
The manufacture of gelatin has been described by Thiele (1914). 
He states that all varieties, whether used for bacteriological, photo- 
graphical or food purposes are made in about the same way. 
Agar. This according to Whittaker (1911) is derived from many 
plants, but principally from the genus " Gelidium " which grows 
on rocks along the coast of Japan. It is carefully cleaned by beating 
and washing. Finally it is dissolved and filtered to be latei dried in 
different shapes. Whittaker reports the composition of agar as shown 
in Table VI. Fellers (1916) found a remarkable uniformity upon anal- 
ysis of sixteen agars from very different sources. 
Table VI 
CHEMICAL COMPOSITION OF AGAR-AGAR 
(After Whittaker, 1911) 
Form. 
Quality. 
Moisture. 
Crude 
Fiber. 
Crude 
Protein 
NX6.25. 
Carbo- 
hydrate. 
Ash. 
Slender 
No. 1 
13.10 
0.47 
2.21 
80.34 
3.88 
Best 
14.39 
0.35 
1.49 
80.17 
3.60 
Powdered 
11.39 
2.62 
3.58 
74.91 
7.50 
Bar 
15.13 
0.72 
3.17 
77.11 
3.88 
Chemically agar is a carbohydrate, a hexosan of the cellulose group 
of the polysaccharides. It is not used by bacteria. Very few instances 
are on record where agar was decomposed by a bacterial enzyme (see 
Smith's Bacteria in Relation to Plant Diseases, 1905, p. 32). 
Reaction of Culture Media. Bacteria require an alkalin or neutral 
medium. This is an important factor in the growth of all microorgan- 44 
MEDIA AND THEIR PREPARATION 
isms. The reaction should be determined with phenolphthalein since 
this has been adopted as the standard indicator. Five cubic centi- 
meters of the media should be titrated and brought to the desired 
reaction means of standard NaOH or HC1. 
Recent work, however, indicates that this method does not yield 
satisfactory results. Clark and Lubs (1917) have pointed out that 
the hydrogen ion concentration must be determined more accurately 
either by means of the hydrogen electrode or special indicators. The 
former technique is too long and complicated to be applied to the 
preparation of ordinary media and it is probable that indicators will 
have to be used. These are mentioned in the appendix under indicators. 
Bouillon Media 
Nutrient Broth. This medium is used as a base to which the other 
constituents of media may be added. The following procedure for its 
preparation is recommended by the Laboratory Committee of the Ameri- 
can Public Health Association in Standard Methods for the Examina- 
tion of Water and Sewage, 1917, p. 95. 
1. Add 3 gms. of meat extract and 5 gms. of peptone to a liter of 
distilled water. 
2. Heat slowly on the steam bath to at least 65° C. 
3. Make up lost weight, titrate, and if the reaction is not already 
between -|-0.5 and +1 adjust to +1. 
4. Cool to 25° C. and filter * through paper until clear. 
5. Distribute in test tubes about 10 c.c. in each tube. 
6. Sterilize in the autoclave at 15 lbs. pressure (120° C.) for fifteen 
minutes after the pressure reaches 15 lbs. 
Carbohydrate Broths. These are made by adding to plain broth, 
as prepared above, 1 per cent of the sugar desired just before steriliza- 
tion. No attempts are necessary to remove muscle sugar since the 
constituents of broth are free from fermentable sugar. The reaction 
of carbohydrate broth may be neutral to phenolphthalein or adjusted 
to neutrality according to Standard Methods, 1917. Mudge (1917) 
has shown that during sterilization maltose and lactose are broken 
down and that sucrose and raffinose are not. The Arnold steam 
sterilizer was found to cause more hydrolysis than the autoclave. 
* The Sharples laboratory centrifuge is in use at the University of Illinois for 
the clearing of solid media. It gives satisfactory results and obviates the use of cot- 
ton and egg albumin. It is considered a valuable addition to the laboratory equip- 
ment. BOUILLON MEDIA 
45 
Hasseltine (1917) has confirmed these results and has emphasized 
the importance of this question in the differentiation of members 
of the B. coll group. The work of these investigators emphasizes the 
necessity of using some other method then heating for sterilizing media 
containing disaccharides and trisaccharides. 
Trypsin Broth (Gordon et al). Take some fresh bullock's heart 
free from fat and vessels, mince the meat very finely and weigh. To 
each | kilo add 1 liter of water and make faintly alkaline to litmus 
with 20 per cent KOH solution. Heat this slowly to 75° to 80° C. 
for five minutes. Cool to 37° C., and add 1 per cent of liquor trypsine 
comp., and keep it at 37° C. for 2| to 3 hours. When trypsinizing is 
finished, test for peptone with copper sulphate and KOH, then render 
slightly acid with glacial acetic acid, and bring slowly to the boil for 
one-fourth hour. Leave over night in a cool place, and siphon off the 
clear liquid in the morning. Make faintly alkaline to litmus, and 
sterilize in an autoclave at 118° C. for one hour on each of two days 
(if not to be used at once). 
Levene (1918) and Burling and Levene (1918) have stated that 
0.5 per cent lactose broth should be used in the presumptive test for 
coli bacteria in water analysis. With the higher carbohydrate con- 
tent of the broth, the limiting hydrogen ion concentration is reached 
more quickly. 
Nitrate Broth. This is a special broth containing nitrite-free 
nitrate. It may be prepared in different ways. Standard methods for 
the Examination of Water and Sewage, 1912, recommended a medium 
of the following composition: 
Tap water 1000 c.c. 
Witte's peptone 1 gm. 
Potassium nitrate (nitrite free) 0.2 gm. 
Another formula is as follows: 
Distilled water 1000 c.c. 
Leibig's meat extract 3 gms. 
Potassium nitrate c.p. KNO3 10 gms. 
The following has been used with some success: 
Distilled water , 1000 c.c. 
Leibig's meat extract 3 gms. 
Witte's peptone 10 gms. 
Potassium nitrate, KNO3 3 gms. 46 
MEDIA AND THEIR PREPARATION 
Breed (1915) has shown that by increasing the peptone content of 
nitrate broth to 5 gms. per liter more uniform results were obtained 
when fifty cultures of B. coli were tested for nitrate reduction. With 
soil bacteria 1 per cent peptone seemed to allow more constant results. 
He concludes that nitrate destruction must be tested for in media which 
allows a vigorous growth of the organisms. Suitable nitrate broths 
may have to be devised to fit each group. 
Neutral Red Broth (Houston, 1914). 
Peptone 10 gms. 
Salt, NaCl 5 gms. 
Plain broth. 1000 c.c. 
Add 2 c.c sterile aqueous 1 per cent solution of neutral red per liter. 
Make slightly alkaline with 20 per cent KOH. Tube and sterilize. 
Bile Salt Broth (MacConkey, 1900). 
Peptone 20 gms. 
Desiccated bile 5 gms. 
Dextrose (CeH^Oe) 5 gms. 
Distilled water 1000 c.c. 
Boil for twenty minutes and add sterile litmus solution to color. 
Filter and put in fermentation tubes. 
Vegetable Bouillon (Berthelot, 1917). This medium may be used 
as a substitute for meat infusion and is prepared as follows: 
Water 4000 c.c. 
Potato 300 gms. 
Carrots 150 gms. 
Turnips 150 gms. 
Peel the potatoes and wash the carrots and turnips. Cut them into 
small pieces and put in cold water. Boil for four hours, strain and make 
feebly alkalin. Heat in an autoclave at 120° C. for one-half hour 
and allow to stand over night in a refrigerator. Filter through paper. 
Chicken Broth (Besson, 1913). Five hundred grams of lean chicken 
meat are soaked in water and filtered. The filtrate is used in the 
same manner as beef broth. 
Giblet Broth (Besson, 1913). Liver, spleen, etc., may be used for 
making broth. Five hundred grams of the solid organs are soaked 
and filtered. The filtrate is used as in other media of this sort. LIQUID MEDIA 
47 
Glucose Formate Broth (Kitasato). 
Nutrient broth 1000 c.c. 
Glucose (CoH^Oe) 20 gms. 
O 
Z 
Sodium formate (H-C-O-Na) 4 gms. 
Tube and sterilize. 
Peptone Solution (Dunham). 
Witte's peptone 10 gms. 
Distilled water 1000 c.c. 
Reaction not adjusted. 
Carbonated Broth (Besson, 1913). Add CaCOg, 2 per cent to lac- 
tose, mannite glucose, etc., broth before distributing into test tubes. 
CaCOg is most frequently added to lactose broth. When an organ- 
ism which ferments lactose is grown in a CaCOg lactose broth, the acids 
formed act on the CaCOg giving off CO2. 
Serum Broth (Besson, 1913). This medium is made by adding 
to tubes of ordinary sterile broth one-half, one-third, or one-quarter 
their volume of blood serum or blood collected under aseptic conditions. 
It is best to incubate the tubes before using in order to be certain that 
they are sterile. 
Dextrose Potassium Phosphate Broth. 
Witte's peptone 5 gms. 
Dextrose (CrH^Og) 5 gms. 
Di-potassium hydrogen phosphate (K2HPO4). 5 gms. 
Heat with occasional stirring over steam for twenty minutes. 
Filter and cool to 20° C. Dilute to 1000 c.c. with distilled water. 
Tube and sterilize. (Standard Methods of Water Analysis, 1917, 
p. 210.) 
Tryptophane Broth. To 1000 c.c. distilled water add 5 gms. di- 
potassium phosphate, 0.3 gm. tryptophane, and 1 gm. peptone. Heat 
until the ingredients are thoroughly dissolved, tube and sterilize at 
15 lbs. for fifteen minutes. Some American peptones are standardized 
to contain a certain amount of tryptophane. If such peptone is used 
the tryptophane in the above formula may be omitted and the peptone 
increased to 5 gms. 48 
MEDIA AND THEIR PREPARATION 
Agar Media 
Nutrient Agar. 1. Add 3 gms. of beef extract, 5 gms. of peptone 
and 12 gms. of agar dried for one-half hour at 105° C. before weighing 
to 1000 c.c. of distilled water. Boil over a water bath or cook in an 
autoclave until all the constituents are thoroughly dissolved. Make up 
the loss of evaporation. 
2. Cool to about 45° to 50° C. and add 10 gms. of desiccated egg 
albumin per liter. Heat in the autoclave for thirty minutes. Make up 
the loss on evaporation and adjust reaction to 1 + . 
3. Filter through cotton or cloth until dear.* Tube and sterilize 
in the autoclave at 120° C. 
Carbohydrate Agar. Any carbohydrate agar which is desired may 
be made by adding 1 per cent of the carbohydrate to plain agar. For 
careful work, carbohydrate media prepared from di- or tri-saccharides 
should not be sterilized in the autoclave as has been shown by Mudge 
(1917) and Hasseltine (1917). 
Litmus Lactose Agar (Bendick, 1913). Bendick has devised the 
following for litmus lactose agar. 
Agar 15 gms. 
Beef extract 5 gms. 
Salt 10 gms. 
Peptone 10 gms. 
Lactose 20 gms. 
Calcium carbonate 4 gms. 
Litmus 100 gms. 
Distilled water 1000 c.c. 
The agar, beef extract, salt and peptone are dissolved in 1000 c.c. 
of water. This is cleared with egg and filtered. No adjusting of the 
reactions is necessary. This is put into flasks (250 c.c. in a 500-c.c. 
flask). One gram of CaCOs is added and the whole sterilized. 
Finally to each flask are added 25 c.c. of Kahlbaum's aqueous litmus 
and 5 gms. of lactose. Tube by pouring direct to the tubes from the 
flasks keeping the contents well mixed. 
Litmus Lactose Agar (Meyer). Meyer (1917) has shown that 
a litmus lactose agar prepared from 3 per cent agar gives plates which 
* The Sharples laboratory centrifuge will give a clear centrifugate in a short time 
with none of the usual trouble and expense connected with filtration through cotton 
or paper. AGAR MEDIA 
49 
are free from many of the objections incident to plates made from a 
lesser per cent of agar. The results obtained were approximately equiv- 
alent to Endo's medium agar. 
Trypagar (Gordon et al). Take a measured quantity of the trypsinized 
broth, add 2 per cent of agar fiber, and 0.125 gm. of calcium chloride 
per liter. Autoclave at 118° C. for three-fourths hour to dissolve the 
agar. Mix together in a saucepan; titrate with N/10 KOH to give an 
absolutely neutral reaction. Cool to 60° C., add white of two eggs 
beaten up with the crushed shells, autoclave again at 118° C. for seventy- 
five minutes (or in the steamer for two hours). Filter, add to the filtrate 
5 per cent of the sterile pea extract, and sterilize in the ordinary way. 
Neutral Red Bile Salt Agar (Savage, 1901). 
Sodium taurocholate 5 gms. 
Witte's peptone 20 gms. 
Distilled water 1000 c.c. 
Agar 20 gms. 
After filtration 10 gms. lactose and 5 c.c. of a 1 per cent solution 
of neutral red are added. 
Glucose Formate Agar (Kitasato). 
Nutrient agar 1000 c.c. 
Glucose 20 gms. 
Sodium formate 4 gms. 
Tube and sterilize. 
Soil Extract Agar (Conn, 1914). 
Agar 15 gms. 
Dextrose 1 gm. 
Soil extract 100 c.c. 
Water 900 c.c. 
Synthetic Agar. 
Dextrose 10 gms. 
MgSO4 0.2 gms. 
• K2HPO4 0.5 gm. 
Peptone 0.05 gm. 
Agar 20 gms. 
Distilled water 1000 c.c. 50 
MEDIA AND THEIR PREPARATION 
Hesse and Niedner's (1898) Agar. 
Agar-agar 12.5 
Nahrstoff Heyden 7.5 
Distilled water 980.0 
Claimed to be a suitable medium for the bacterial examination of 
water. 
Dolt's Agar (1908). 
Purified agar (3 per cent solution) 250 c.c. 
Asparagin 2.5 gms. 
NaaHPCU 0.5 gm. 
Distilled water 100 c.c. 
This medium was found to support good growth of B. coli in twenty- 
four hours. 
Neutral Red Agar. Add enough 0.5 per cent neutral red solution to 
agar containing 1 per cent dextrose to produce a clear red color. 
Corn Agar (After Barlow, 1912). 
Corn 1000 gms. 
Sucrose 80 gms. 
Agar 60 gms. 
Litmus 100 gms. 
Water 4000 gms. 
Stir the corn into the boiling water add to other materials, boil 
five minutes, heat in an autoclave and cool slowly, the heavy matter 
settles off. The clear supernatant agar may be drawn off and tubed. 
Agar for Ps. Radicicola (Moore). 
Di-potassium phosphate 1 gm. 
Magnesium sulphate 0.5 gm. 
Sucrose 10.0 gms. 
Agar 10.0 gms. 
Distilled water 1000 c.c. 
Bean Agar (Thom, 1910). Common white beans are heated in 
five volumes of water. Boiling is stopped just before the swelling 
of the cotyledons would rupture the seed coats. This gives a clear 
yellow solution which filters easily and contains sufficient nutrients to 
grow many species normally. Agar may be as desired. Since this 
medium is poor in available carbon it is often desirable to add carbo- 
hydrate for certain species. SPECIAL AGAR MEDIA 
51 
Sabouraud's Agar for Yeasts (after Anderson, 1917). 
Agar 20 gms. 
Peptone 10 gms. 
Glucose 40 gms. 
Water 1000 c.c. 
Mix these ingredients in the usual way and adjust the reaction to 
+ 2 per cent acid. Without further heating the sugar is added and the 
medium tubed in previously sterilized tubes. Anderson advises care 
in sterilizing since the high acidity of this medium may prevent solidi- 
fication of the agar. 
Agar Gelatin Medium (North 1909). 
Lean chopped beef or veal 500 gms. 
Agar 10 gms. 
Gelatin 20 gms. 
Peptone 20 gms. 
NaCl 5 gms. 
Distilled water 1000 c.c. 
Adjust neutral to phenolphthalein. North states that this medium 
is excellent for streptococci, pneumococci and diphtheria bacilli because 
it is soft and moist and can be used at 37° C. It is claimed to be of 
special value in keeping stock cultures. 
Hesse Medium. 
Agar 5 gms. 
Peptone 10 gms. 
Liebig's beef extract 5 gms. 
Sodium chloride 8.5 gms. 
Distilled water 1000 c.c. 
Agar is dissolved in 500 c.c. H2O. Other ingredients are dissolved 
in 500 c.c. H2O. Solutions are mixed, filtered and tubed. 
Hiss Plating Medium (1897). 
Agar 15 gms. 
Gelatin 15 gms. 
Liebig's meat extract 5 gms. 
NaCl 5 gms. 
Dextrose 10 gms. 
Distilled water 1000 c.c. 52 
MEDIA AND THEIR PREPARATION 
The agar is dissolved in 1000 c.c. H2O. When melted the extract, 
gelatin and salts are added. It is cleared and filtered. Dextrose is 
then added and it is tubed. 
Malachite Green Media (1906). 1. Three gms. Liebig's meat 
extract. 
2. Acidify this with 7.5 c.c. NHC1. 
3. Dissolve 30 gms. agar by boiling. 
4. Neutralize with 7 c.c. NNaOH until neutral to litmus. 
5. Add 5 c.c. NNa2CO3 and heat in an Arnold for several hours. 
6. Add 100 c.c. of 10 per cent sucrose solution. This may be stored 
in 100 c.c. quantities. 
7. Before use redissolve and to 100 c.c. add 2 to 2.9 c.c. of 2 per cent 
malachite green (trade mark Hochst 120). This should be made in 
sterile H2O and not boiled. 
8. Medium is smeared in Petri dishes. 
Russell's Medium (1911). " Enough 5 per cent aqueous solution of 
litmus (3 to 5 per cent) is added to plain agar (2 or 3 per cent), which 
usually has a reaction of about 0.8 per cent acid to phenolphthalein, 
to give it a distinct purple violet, color, the amount of litmus depending 
on the original color of the agar; dark requiring more than light, and 
the reaction is then adjusted by adding sodium hydrate until the mix- 
ture is neutral to litmus. Next, and last, 1 per cent of lactose and 
one-tenth of 1 per cent of glucose dissolved in a small amount of hot 
water is added and the medium tubed for slants. The sterilization is 
done in the Arnold and because of the danger of breaking down the 
lactose must not be carried too far; if the tubes are packed loosely in 
the sterilizer basket to allow good circulation of the steam, ten minutes 
on the first and fifteen on the second day has been time enough. The 
tubes are then slanted and stored in small quantities in a dark place." 
Krumwiede has substituted Andrade for the litmus and secured 
good results. 
Endo's Medium (after Kendall, 191112). 
I. Preparation of agar. 
(a) Prepare plain sugar free agar using 15 gms. agar per liter. 
(6) Adjust to a point just alkaline to litmus. 
(c) Place in flasks in 100 c.c. quantities and sterilize. ENDO'S MEDIUM 
53 
IL Preparation of the indicator. 
(a) Prepare a 10 per cent basic fuchsin solution in 96 per cent 
alcohol. This solution is fairly stable if kept away 
from the light. 
(6) Prepare a 10 per cent solution of chemically pure anhydrous 
sodium sulphite. (1 gm. in 10 c.c. water.) This solu- 
tion does not keep. 
(c) Add 1 c.c. of 11(a) to 10 c.c. of 11(b) and heat in the Arnold 
sterilizer for twenty minutes. The color of the solution 
should be nearly discharged. This solution must be 
prepared each day. It does not keep. 
III. Preparation of medium. 
(a) Add 1 gm. of c.c. lactose to 100 c.c. of the agar and place 
in the autoclave until melted and lactose is dissolved. 
(6) Add enough of 11(c) to impart a pink color (about 1 c.c.). 
(c) Pour into sterile Petri dishes and allow to harden. In the 
above medium Kendall has reduced the agar content 
to 1.5 per cent. The original formula of Endo called for 
a 3 per cent solution which was supposed to prevent 
diffusion of the acid and prodistinct colonies. 
Endo's Medium (Hygienic Laboratory Method). Hasseltine (1918) 
has described the preparation of this medium as follows: It consists 
of a 3 per cent agar which is titrated and corrected to +0.5 to phenol- 
phthalein, to which is added 3.7 c.c. of a 10 per cent solution of anhy- 
drous sodium carbonate. For convenience it is flasked, sterilized and 
stored in 200 c.c. quantities. When ready to use the following ingredi- 
ents are added to 200 c.c. of agar as follows: 
(a) Dissolve 2 gms. chemically pure lactose in 25 to 30 c.c. of distilled 
water, with the aid of gentle heat. 
(b) Dissolve 0.5 gm. of anhydrous sodium sulphite in 10 to 15 c.c. 
of distilled water. 
(c) To the sulphite solution add 1 c.c. of saturated solution of basic 
fuchsin in 95 per cent alcohol. 
Add the fuchsin sulphite solution to the lactose solution and then 
add the whole to the agar. Pour plates at once, and after hardening 
dry for fifteen minutes in the incubator. 
Teague (1918) has shown that a 10 per cent solution of sodium 
sulphite may be heated for twenty minutes at 15 lbs. with practically 54 
MEDIA AND THEIR PREPARATION 
no change. If this is then kept covered with a layer of petrolatum, it 
may be kept for three weeks at room temperature. " 
Endo's Medium (After Levine, 1918). Levine states the method 
of preparation as follows: 
Distilled water 1000 c.c. 
Peptone (Difco) 10 gms. 
Dipotassium phosphate 2-5 gms. 
Agar 15-30 gms. 
These ingredients should be boiled until dissolved and the loss by 
evaporation made up with distilled water. No adjustment of the 
reaction is necessary; neither is filtration necessary if the medium 
is to be used for streaked cultures. Store in quantities of 100 c.c. in 
Erlenmeyer flasks and when used add. the following materials: 
20 per cent lactose solution 1 gm. or 5.0 c.c. 
10 per cent alcoholic solution of basic fuchsin. 0.5 c.c. 
Freshly prepared sodium sulphite solution.... 2.5 c.c. 
Eosin Brilliant-Green Agar (Teague and Clurman, 1916). This 
medium was devised for the rapid isolation of B. typhi from stools 
and was prepared by these workers after the following method: 500 
gms. of chopped beef are placed in 1 liter of distilled water and kept in 
the icebox over night. The infusion is squeezed through cheese cloth, 
heated in the Arnold sterilizer and passed through filter paper. Witte's 
peptone (1 per cent), chemically pure sodium chloride (0.5 per cent) 
and agar (1.5 per cent) are added to the warm infusion, the peptone 
being rubbed into a paste in a little warm water. Then heat the 
flask of medium in the autoclave for thirty minutes at 120° C. Adjust 
the reaction to 1+ by adding 2N sodium hydroxide, cool to 55° C., 
clear and filter through cotton. Put into flasks in 100 c.c. quantities 
and sterilize at 120° C. for twenty minutes. 
When ready to use, melt the agar and add 1 per cent each of 
sucrose and lactose. To every 50 c.c. of agar add 1 c.c. of a 3 per cent 
solution of yellowish eosin. From the stock solution of brilliant green 
in 50 per cent alcohol, a | per cent solution is prepared in distilled water 
and 1 c.c. of this is added to each 50 c.c. of the agar. After the dyes 
are well distributed, pour into plates. 
Eosin-Methylene Blue Agar (Levine, 1918). This is a modification 
of Harris and Teague's medium and was found by Levine to allow GELATIN MEDIA 
55 
a close separation between B. coli and B. aerogenes. Levine describes 
its preparation as follows: 
Distilled water 1000 c.c. 
Peptone (Difco) 10 gms. 
Dipotassium phosphate 2 gms. 
Agar 15 gms. 
Boil the ingredients until dissolved and make up any loss to evapo- 
ration. Place measured quantities in flasks and sterilize at 15 lbs. for 
fifteen minutes. Just prior to use add to each 100 c.c. of the melted 
agar prepared as above, the following constituents: 
Sterile (20 per cent lactose) 1 gm. or 5 c.c. 
Aqueous (2 per cent) eosin (yellowish) solution. 2 c.c. 
Aqueous (2 per cent) methylene blue solution. 2 c.c. 
Pour the medium into dishes allow them to harden in the incubator 
and inoculate in the usual way. There should be no adjustment of the 
reaction and filtration of the medium is not necessary. 
Gelatin Media 
Nutrient Gelatin. 1. Add 3 gms. of beef extract and 5 gms. of pep- 
tone to 1000 c.c. of distilled water and add 100 gms. of gelatin dried for 
one-half hour at 105° C. before weighing. 
2. Heat slowly on a steam bath to 60° C. until the gelatin is dissolved. 
3. Make up lost weight, titrate, and if the reaction is not already 
between +0.5 and +1, adjust to +1. 
4. Filter through cloth and cotton until clear. 
5. Distribute in test tubes, 10 c.c. to each tube, or in larger contain- 
ers if desired. 
6. Sterilize in the autoclave at 15 lbs. (120° C.) for fifteen minutes 
after the pressure reaches 15 lbs. 
Whey Gelatin. Add 10 per cent to clarified whey. This medium 
is of value in studying'certain members of the lactic acid bacteria. 
Soil Extract Gelatin (Conn, 1914). 
Gelatin 100-150 gms. 
Dextrose 1 gm. 
Soil extract 100 c.c. 
Water 900 c.c. 56 
MEDIA AND THEIR PREPARATION 
Litmus Gelatin. This may be prepared from lactose gelatin the 
reaction of which must be neutral. Deposit 1 c.c. of sterile litmus 
solution in the sterile Petri dish with the sample and pour in the melted 
gelatin. Shake the dish until the litmus is evenly distributed through- 
out. Incubate at 20° C. This medium is of much value in studying 
the bacteria in milk. 
Hiss Medium (1902). 
Agar 15 gms. 
Gelatin 15 gms. 
Liebig's meat extract 5 gms. 
Sodium chloride 5 gms. 
Dextrose 10 gms. 
Distilled water 1 liter 
Adjust the reaction to 1 + . 
Raisin Gelatin (Besson). Make a decoction of raisins by grinding 
and stewing 50 to 100 gms. of raisins in 1000 gms. of H2O for one hour. 
Filter and add 100 gms. of gelatin and a pinch of sodium phosphate. 
Boil for two or three minutes. Neutralize and finish as usual. 
Wort Gelatin. 1. Measure out 900 c.c. of beer wort in sterile flask. 
2. Weigh out 100 gms. gelatin (10 per cent) and add it to the wort 
in the flask. 
3. Bubble live steam through for ten minutes to dissolve the gelatin. 
4. Cool to 60° C., clarify with egg. 
5. Filter and tube. 
Gelatin Agar (Besson). By mixing agar and gelatin a medium is 
obtained with a melting-point between that of either plain agar or plain 
gelatin. 
Peptone broth '. 1000 gms. 
Gelatin 80 gms. 
Agar 5 gms. 
Or 
Gelatin 50 gms. 
Agar 8 gms. 
Dissolve the gelatin in the broth, neutralize and then add the agar. 
Sterilization must not be accomplished above 115° C. (See North's 
Agar Gelatin Medium.) SPECIAL MEDIA 
57 
Special Media^ 
Peptone Sucrose Solution (Buchanan, 1909). 
Monobasic potassium phosphate (KH2PO4).. 2.0 gms. 
Magnesium sulphate (MgSO4-7H2O) 0.1 gms. 
Peptone 1.0 gms. 
Sucrose 20.0 gms. 
Distilled water 1000 c.c. 
Hay Infusion. Macerate 15-20 gms. of finely chopped hay or 
in 1000 gms. of H2O for one or two hours. Boil for a few minutes, 
filter, tube and sterilize at 115° C. The infusion which is sometimes 
a little acid may be neutralized in the usual way. 
Beer Wort (Eyre, 1903). 1. Weigh out 250 gms. malt and put into 
a flask. 
2. Add 1000 c.c. distilled water heated to 70° C. and close flask 
with rubber stopper. 
. 3. Place the mixture in a water bath at 60° C. and allow the mixture 
to stand for one hour. 
4. Strain through cheese cloth into clean flask and heat in an Arnold 
for thirty minutes. 
5. Filter through paper. 
6. Tube in 10 c.c. quantities and sterilize in an Arnold. 
Spronck's Peptone Yeast Extract. To 5 liters of H2O add 1000 
gms. commercial yeast, not brewers' yeast. Boil for twenty minutes 
with stirring, pour into cylindrical vessels and leave for twenty-four 
hours. Decant the cloudy liquid and add 5 gms. of NaCl and 10 gms. 
of peptone per liter. Neutralize exactly and then make alkaline to 
7 per cent with NNaOH. Filter through Chardin paper and pour 
into flasks. Sterilize at 115° C. 
Wine. Before sterilizing make neutral in the ordinary way. 
Prune Decoction. Macerate 50 gms. of prunes in 1 liter of distilled 
water and allow to stand in the ice chest until the next morning. Filter 
and tube for sterilization. 
Pea-flour Extract (Gorden et al., 1916). Take 100 gms. of pea flour 
and add 1 liter of distilled water with 100 gms. of salt. Mix and steam 
for one-half hour, stirring constantly. Allow to settle and filter, then 
sterilize and label " saline pea extract." This pea-flour extract should 
preferably be freshly made for each batch of agar. This medium was 
especially designed to replace Witte's peptone. 58 
MEDIA AND THEIR PREPARA DON 
Haricot Decoction. Macerate 50 to 60 gms. of white haricot beans 
in a liter of water for several hours in the cold. Boil for one-half hour. 
Pour on a<coarse sieve, collect the liquid, and add to it 1 per cent of 
salt, 2 per cent of sugar and a pinch of sodium bicarbonate. Boil 
and filter through paper. Tube and sterilize at 115° C. 
This medium is used by Maze to cultivate the bacteria in nodules 
of leguminous plants. 
Malt Extract (Besson). 1. Grind up 100 gms. malt and add 1000 
gms. of H2O. 
2. Heat to 56° C. for one hour; the starch is converted into maltose 
by the diastase and a true beer wort obtained 
3. Boil, filter through Chardin paper. 
4. Tube, sterilize at 115° C. 
Urine (Besson). 1. Boil some recently passed urine. 
2. If the reaction is markedly alkaline after boiling, add a little 
tartaric acid solution, testing the reaction with litmus paper. 
3. Filter, tube and sterilize at 155° C. The composition of urine 
is altered by this proceeding, the urea in solution being decomposed 
at boiling temperature. It is better to sterilize through a Chamber- 
land bougie. 
Capaldi's Egg Medium. A few loopfuls of egg yolk are added to 
a tube of liquefied agar, previously cooled to 45 to 47° C. 
Dorset's Egg Medium. Eggs are broken into a flask and the 
yolks broken with a needle or glass rod. The flask is shaken until 
the yolks and whites are thoroughly mixed. Foam formation should be 
avoided. Distribute in culture tubes and sterilize in a Koch inspis- 
sator or autoclave in the same manner as blood serum. 
Egg-Meat Mixture (Rettger, 1906). A. One-half pound of lean 
chopped beef is stirred up in 250 c.c. of water, and after neutralizing 
the meat acids with sodium carbonate, the mixture is heated in an 
Arnold sterilizer for thirty minutes, with occasional stirring. It is then 
set away in a cold place for several hours after which the fatty scum 
is removed. 
B. The whites of three eggs are mixed with 250 c.c. of water, and 
after neutralizing, the albumen is coagulated, with occasional stirring, 
by heating in the Arnold sterilizer for thirty minutes. 
A and B are then mixed and introduced into a liter flask, along 
with 2.5 gms. (0.5 per cent) of powdered calcium carbonate. The flask 
is plugged with cotton and sterilized for thirty minutes, at 110 to 112° C. 
(autoclave). SPECIAL MEDIA 
59 
Redfield's (1912) Medium. 
Distilled water 1000 c.c. 
Peptone 300 gms. 
Potassium chloride (KC1) 74 gms. 
Add the peptone to 700 c.c. of hot water. Stir until it is in solution 
and cool. Allow to 'stand over night in the icebox. Filter and put in 
test tubes. 
Ox-bile Medium. Dissolve 1 per cent lactose and 1 per cent pep- 
tone in fresh ox-bile, filter and place in fermentation tubes. Sterilize 
in an Arnold on three successive days. 
Decoction of Dried Fruits. 1. Macerate 50 to 100 gms. of dried 
fruit in a liter of water for several hours. Then stew them in the 
water. 
2. Pass through a coarse sieve. 
3. Boil. Filter. 
4. Tube and sterilize at 115° C. 
The liquid is slightly acid and is useful for cultivating molds. Other- 
wise make neutral. 
Barsiekow's Medium. Prepare and sterilize separately the two 
following solutions: 
A. Sodium chloride, NaCl 0.5 gm. 
Nutrose 1.0 gm. 
Water 75.0 gms. 
B. Water 25.0 gms. 
Litmus To give blue color 
Sugar (the one desired). 1.0 gm. 
Add sufficient litmus to give an amethyst color. After cooling 
mix and distribute in tubes. 
Bread. Soak some slices of white bread in distilled water and 
sterilize at 115° C. 
Crumble some dry bread and allow to dry. Grind when dry and 
put in Petri disher or Erlenmeyer flask and add water to moisten (about 
2| pts. of H2O to 1 pt. of bread by weight). 
Sterilize at 115° C. for twenty minutes. 60 
MEDIA AND THEIR PREPARATION 
Potato and Starch Media 
Potato Gelatin (Goadby). Add 100 gms. of gelatin to 1 liter of 
glycerol potato broth. Boil, adjust the reaction, filter, and sterilize. 
Glycerol Potato Broth. Wash, peel and grind 1000 gms. of potato. 
Soak this in a liter of water for twenty-four hours. After filtering 
add 3 to 5 per cent of glycerol and tube for sterilization. 
Potato Agar (McBeth, 1913). Pare, steam, and mash a quantity of 
potatoes. To 100 gms. of mashed potato add 800 c.c. of tap water and 
steam for one-half hour, filter through cotton. 
Potato solution 500 c.c. 
Agar 15 gms. 
Nutrient solution (same as for cellulose agar). 500 c.c. 
Starch Agar. Make 1 liter of ordinary peptone agar. Add to this 
a 2 per cent paste of potato starch. This should be added at as low 
a temperature as possible to the neutral agar. 
To use, pour plates with the sterile tubed starch agar and streak 
with the culture. After incubation for 5 days, pour over weak solution 
of Lugol's iodine. 
Starch Agar (Vedder, 1915). Prepare a liter of beef infusion in 
the usual way. Meat extract does not give as good results. To 
this add 1.5 per cent of agar. No peptone or salt should be added. 
Cook and clarify the medium and add 10 gms. of cornstarch to each 
liter. The starch is best rubbed up in a mortar in a little agar. One 
per cent of starch gives the best results. Tube and sterilize in the usual 
manner. This medium is especially valuable in the growth of gonococci. 
Elsner's Potato Medium. 1. Peel and grate 500 grams of potato. 
2. Grind up and soak in distilled water for twelve hours. 
3. Strain and allow to stand. Filter to clear. 
4. Make up to 1 liter and dissolve 15 per cent of gelatin. 
5. Adjust reaction to acid and sterilize. 
Potato Infusion. Wash and grind up a few potatoes. To each 
30 gms. of potato, add a liter of distilled water and allow to stand 
in the icebox over night. In the morning, filter and boil the filtrate. 
Filter again if necessary and tube for sterilization. If the infusion is too 
acid adjust the reaction as usual. 
Carrot Infusion. This medium may be prepared as above for 
potato infusion. 
Potato Mash. Clean and cut a large potato into slices. Boil until 
soft and grate or press through a ricer. Distribute this in layers in POTATO AND STARCH MEDIA 
61 
Petri dishes and sterilize at 120° C. for twenty minutes. The slices 
may be placed in the Petri dishes without mashing if desired. 
Starch Jelly (Smith, 1898). To 10 c.c. of Uschinsky's solution 
add 1 gm. of clean aseptic starch. Rub this up in the slanted fluid. 
Plug the tubes tightly and place in an inspissator or Arnold in the 
slanted condition. Heat for two hours on five successive days at 
85 to 93° C. If water is lost, this must be made up by the addition of 
distilled water. 
Potato Agar (Thomas, 1906). The potatoes are carefully washed, 
pared, sliced, then slowly heated for two hours in approximately two 
volumes of water. At the close of the heating the water is allowed 
to boil. The whole is then filtered through cloth, water being added 
to make up the loss of any evaporation. After filtering, 1 per cent 
shred agar is added to this fil rate. It may then be heated in an auto- 
clave for thirty minutes at 15 lbs. after which it may be tubed or fil- 
tered, if desired, before tubing. 
Cellulose Agar (McBeth, 1913). " Prepare 1 liter of a dilute 
ammonium-hydroxide solution by adding 3 parts water to 10 parts 
ammonia hydroxide, sp.gr. 0.99. Add a slight excess of copper car- 
bonate and shake vigorously, allow to stand overnight, and then siphon 
off the supernatant solution. Add 15 gms. of unwashed sheet filter 
paper and shake occasionally until the paper is dissolved. Dilute to 
10 liters and add slowly a one to five solution of hydrochloric acid, 
with vigorous shaking until the precipitation of the cellulose is com- 
plete. Dilute to 20 liters, allow the cellulose to settle, and decant the 
supernatant liquid. Wash by repeated changes of water, adding 
hydrochloric acid each time until the copper color disappears; then wash 
with water alone until the solution is free from chlorine. Allow it to 
settle several days and decant off as much of the clear solution as pos- 
sible. If the percentage of cellulose is still too low, a portion of the solu- 
tion is centrifugalized to bring the cellulose content up to 1 per cent." 
Cellulose solution 500 c.c. 
Agar 10 gms 
Nutrient solution, composed of: 
Potassium phosphate (dibasic) 1 gm. 
Magnesium sulphate 1 gm. 
Sodium chloride 1 gm. 
Ammonium sulphate 2 gms. 
500 c.c. 
Calcium carbonate 2 gms. 
Tap water 1000 c.c. 62 
MEDIA AND THEIR PREPARATION 
Starch Agar (McBeth, 1913). To 800 c.c. of boiling water add 10 
gms. of potato starch suspended in a little cold water. Concentrate 
by boiling to 500 c.c. This breaks up the starch grains and should give 
a nearly transparent starch solution. 
Starch solution 500 c.c. 
Agar 10 gms. 
Nutrient solution (same as for cellulose) 500 c.c. 
Synthetic Media 
Under the term synthetic the bacteriologist includes all media 
which may be prepared from pure chemical compounds. These may 
always be secured or brought to the same state of purity by the various 
methods well known to the chemist. The advantages of synthetic 
media are quite evident. 
1. They may be prepared in different lots which have a constant 
chemical composition. 
2. They may be prepared in different laboratories and render the 
data thus secured, more reliable. 
3. They lend themselves to the study of some one compound or 
element. For instance sulphur free media may be prepared to which 
certain sulphur compounds may be added. 
Parietti's Solution. 
Carbolic acid, CoHsOH 5 c.c. 
Hydrochloric acid, HC1 4 c.c. 
Water • 100 c.c. 
Drigalski and Conradi (1902) Medium. 
Dextrose (free broth) 2000 c.c. 
Nutrose 20 gms. 
Agar 40 gms. 
Boil, dissolve, neutralize to phenolphthalein, autoclave at 120° C. 
for five minutes. 
Mayer's Culture-Fluid (from Smith, 1905). 
Magnesium sulphate, MgSO4-7H2O 10 gms. 
Ammonium nitrate, NH4NO3 15 gms. 
Tribasic calcium phosphate, Ca3(PO4)2 0.01 gms. 
Monobasic potassium phosphate, KH2PO4.... 10 gms. 
Distilled water 1000 c.c. SYNTHETIC MEDIA 
63 
Dissolve cold and add sugar. Add NaCI (3 per cent) if it is to be 
used for luminous bacteria, and an excess of pure CaCOs if acid form- 
ing bacteria are to be grown. 
Prazmowski's Culture-Fluid. 
Dipotassium phosphate, K2HPO4 5 gms. 
Magnesium sulphate, MgSO^THoO 5 gms. 
Ammonium carbonate, (NH^COs 5 gms. 
Calcium chloride, CaCh 0.5 gms. 
Distilled water 1000 c.c. 
Naegeli's Medium (after Besson, 1913). 
Water 1000 gms. 
Ammonium tartrate 10 gms. 
Dipotassium phosphate, K2HPO4 1 gm. 
Magnesium sulphate, MgSO4 • 7H2O 0.2 gm. 
Calcium chloride, CaCh 0.12 gm. 
Cohn's Solution. 
Distilled water 1000 c.c. 
Dipotassium phosphate, K2HPO4 5 gms. 
Magnesium sulphate, MgSO4 • 7H2O 5 gms. 
Ammonium tartrate 10 gms. 
Potassium chloride, KC1 0.5 gm. 
Hydrolyzed Casein Medium (Cannon, 1916). Zipfel proposed the 
addition of tryptophane to a synthetic medium for the determination 
of indol formation. Cannon has realized the high cost of tryptophane 
and suggested the use of a hydrolyzed casein which is prepared as 
follows: Ten grams of casein are hydrolyzed by 200 c.c. of 10 per cent 
sulphuric acid. The mixture is kept on the water bath for twenty- 
four hours after which it is neutralized by saturated barium hydroxide. 
This will precipitate the sulphate. Evaporate the resulting solution 
until the amino acids crystallize. Half of the preparation is then dis- 
solved in 500 c.c. of Zipfel's synthetic medium, which has the following 
composition: 
As paragin 5.0 gms. 
Ammonium lactate 5.0 gms. 
Potassium acid phosphate 2.0 gms. 
Magnesium sulphate 0.2 gm. 
Distilled water 1000.0 gms. 64 
MEDIA AND THEIR PREPARATION 
Capaldi's Medium. 
Peptone 20 gms. 
Gelatin 10 gms. 
Agar 20 gms. 
Dextrose or mannit 10 gms. 
Sodium chloride, NaCl. 5 gms. 
Potassium chloride, KC1 5 gms. 
Distilled water 1000 c.c. 
Frankel's Solution. 
Sodium chloride, NaCl 5 gms. 
Monocalcium phosphate, Ca(H2PO4)2 2 gms. 
Ammonium lactate, CH3-CHOH-CONH2.. 6 gms. 
/CONH2 
Asparagin, C2H3(NH2)<f 4 gms. 
XCOOH 
Distilled water 1 liter 
Sodium hydroxide, NaOH 20 c.c. 
Sullivan's (1905) Medium. 
/CONH2 
Asparagin, C2H3(NH2)< 10 gms. 
\COOH 
Magnesium sulphate, MgSO4 2 gms. 
Dibasic potassium phosphate, K2HPO4 1 gm. 
This medium supports good pigment formation by Ps. pyocyaneus 
and Ps. fluoresceous liquefaciens. 
Van Delden's (1904) Solution. For SO4 reducing bacteria. 
Dibasic potassium phosphate, K2HPO4 0.5 gm. 
Sodium lactate, CH3-CHOH-COONa 5.0 gms. 
Magnesium sulphate MgSO4 1.0 gm. 
/CONH2 
Asparagin, C2H3(NH2)< 1.0 gm. 
\COOH 
Ferrous sulphate, FeSO4 Trace 
Tap water 1000 c.c. 
This medium is of special value for the determination of sulphate 
reduction. SYNTHETIC MEDIA 
65 
Dolt's Medium (1908). The following media were found to grow 
B. coli well in twenty-four hours. 
Medium I. 
/CONH2 
Asparagin, C2H3(NH2K 1 gm. 
XCOOH 
Dibasic sodium phosphate, Na2HPO4 0.2 gm. 
Distilled water 100 c.c. 
Medium II. 
Asparagin 1 gm. 
Dibasic ammonium phosphate (NH4)2HPO4. . 0.2 gm. 
Distilled water 100 c.c. 
The following solid media were prepared: 
Purified agar, 3 per cent solution 250 c.c. 
Asparagin 2.5 gms. 
Na2HPO4 0.5 gm. 
Distilled water 100 c.c. 
250 c.c. 
Nitrogen-free Medium for Bacteria (Smith, 1905). 
Triple-distilled water . 1000 c.c. 
Cane sugar 5 c.c. 
Monopotassium phosphate, KH2PO4 2 c.c. 
Magnesium sulphate, MgSCh • 7H2O 0.1 c.c. 
Sodium chloride, NaCl 0.5 c.c. 
All apparatus should be scrupuously clean and only chemicals of 
known purity .should be used. 
Dox's (1910) Solution for Fungi. Modified Czapek's Medium. 
(See Thom, 1910). 
Distilled water 3000 c.c. 
Magnesium sulphate, MgSO 1.5 gms. 
Dibasic potassium phosphate, K2HPO4 3.0 gms. 
Potassium chloride, KC1 1.5 gms. 
Ferrous sulphate, FeSO4 03 gm. 
Solidified media may be obtained by the addition of agar. 66 
MEDIA AND THEIR PREPARATION 
Czapek's Solution. Cultivation of the fungi. (From Smith, 1905.) 
Distilled water 1000 c.c. 
Magnesium sulphate, MgSCh • 7H2O 50 gm. 
Dipotassium phosphate, K2HPO4 1.00 gm. 
Potassium chloride, KC1 0.50 gm. 
Ferrous sulphate, FeSO4 0.01 gm. 
Sodium nitrate, NaNOs................... 2.00 gms. 
Cane sugar 30.00 gms. 
Pasteur's Medium (after Besson). 
Water 100 gms. 
Candied sugar 10 gms. 
Ammonium tartrate 0.1 gm. 
Ash of yeast 0.075 gm. 
Raulin's Solution for Molds. 
Distilled water. 1500 c.c. 
Cane sugar, C12H22O11 70 gms. 
Tartaric acid, (CHOH)2(COOH)2 4 gms. 
Ammonium nitrate, NH4NO3 4 gms. 
Ammonium phosphate, (NH4)2HPO4 0.6 gms. 
Magnesium carbonate, MgCOa 0.4 gm. 
Ammonium sulphate, (NH4)2SO4 0.25 gm. 
Zinc sulphate, ZnSO4 • 7H2O 0.07 gm. 
Iron sulphate, FeSO4-7H2O 0.07 gm. 
Potassium silicate, KSiO 0.07 gm. 
Lipman-Brown Medium (1910). 
Water 1000 c.c. 
Dextrose, CoH^Oo 10.00 gms. 
Dipotassium hydrogen phosphate, K2HPO4.. . 0.50 gm. 
Magnesium sulphate, MgSO4 • 7H2O 0.20 gms. 
Peptone 0.05 gm. 
Agar 20.00 gms. 
Jordan's Non-protein Medium. 
Redistilled water 1000 c.c. 
/CONH2 
Asparagin, C2H3(NH2)<; 2 gms 
\COOH 
Magnesium sulphate, MgSO4-7H2O 1 gm. 
Dipotassium hydrogen phosphate, K2HPO4.. . 1 gm. SYNTHETIC MEDIA 
67 
Beijerinck's Medium for Nitrate Reduction (after Fred). 
Dibasic potassium phosphate, K2HPO4 0.5 gm. 
Potassium nitrate, KNO3. 10.0 gms. 
Ethyl alcohol, C2H5OH 5.0 c.c. 
Tap water 1000 c.c. 
Winogradsky's Medium for Nitrite Formation (from Ferd., 1916). 
Ammonium sulphate, (NHA2SO4 1.0 gm. 
Dibasic potassium phosphate, (K2HPO4) 1.0 gm. 
Magnesium sulphate, MgSO4-7H2O 0.5 gm. 
Sodium chloride, NaCl. 2.0 gms. 
Ferrous sulphate, FeSCU-7H2O. 0.4 gm. 
Magnesium carbonate, MgCO3 in excess 5.0 gm. 
Distilled water 1000 c.c. 
Giltay and Ab er son's (1891) Solution. For Denitrifying Bacteria. 
Distilled water. 1000 c.c. 
Potassium nitrate, KNO3 2 gms. 
Magnesium sulphate, MgSO4 2 gms. 
Citric acid 5 gms. 
Dipotassium hydrogen phosphate, K2HPO4.. . 2 gms. 
Calcium chloride, CaCE 0.2 gm. 
Sodium carbonate, Na2COs 4.25 gms. 
/CONH2 
Asparagin, C2H3(NH2)< 1.00 gm. 
\COOH 
This medium should be placed in fermentation tubes before steril- 
ization. To prevent any decomposition, the potassium nitrate and 
asparagin should be dissolved together. The other salts should be 
dissolved in another portion of water and the whole brought up to a 
liter. 
Omelianski's and Winogradski's Medium. 
Ammonium sulphate, (NH)SO 1 gm. 
Potassium bi-phosphate, KH2PO4 1 gm. 
Magnesium sulphate, MgSO4 • 7H2O 0.5 gm. 
Sodium chloride, NaCl 2.0 gms. 
Ferrous sulphate, FeSCh-TthO 0.4 gm. 
Basic magnesium carbonate, 4MgCOs • Mg(0H)2 Excess 
Distilled water 1000 c.c. 68 
MEDIA AND THEIR PREPARATION 
MacKensie's Culture Fluid (from Smith, 1905). 
Acid ammonium tartrate 1.5 gms. 
Bipotassium phosphate, KH2PO4 2.5 gms. 
Potassium sulphate, K2SO4 1.5 gms. 
Sodium chloride, NaCl 0.5 gm. 
Glucose, 5.0 gms. 
Lactose, C12H22O11 ' 5.0 gms. 
Glycerol, CH2OHCHOHCH2OH 15.0 gms. 
Distilled water 1000 c.c. 
Alkaline to phenolphthalein. 
Proskauer and Beck's Culture Fluid (from Smith, 1905). 
Commercial ammonium carbonate (NH4)2CO3 3.5 gms. 
Potassium phosphate, K3PO4 1.5 gms. 
Magnesium sulphate, MgSO4 • 7H2O 2.5 gms. 
Glycerol, CH2OHCHOHCH2OH 15.0 gms. 
Distilled water . 1000 c.c. 
Fermi's Culture Fluid. 
Distilled water 1000 c.c. 
Magnesium sulphate, MgSCU • 7H2O 0.2 gm. 
Dipotassium hydrogen phosphate, HK2PO4.. . 1.0 gm. 
Ammonium phosphate, (NH4)2HP()4 10 gms. 
Glycerol, CH2OHCHOHCH2OH 45 gms. 
This may be added to agar in place of peptone broth or to silicate 
jelly in which case the volume of H2O must be reduced. (Smith.) 
Fraenkel and Voges' Solution (from Smith, 1905). 
Water 1000 c.c. 
Sodium chloride, NaCl 5 gms. 
Dipotassium phosphate, K2HPO4 2 gms. 
Ammonium lactate 6 gms. 
Sodium asparaginate 4 gms. 
Uschinsky's Protem Free Medium. 
Asparagin 3.4 gms. 
Ammonium lactate 10.0 gms. 
Sodium chloride, NaCl 5.0 gms. 
Magnesium sulphate, MgSO4-7H2O 0.2 gm. 
Calcium chloride, CaCl2 0.1 gm. 
Dipotassium phosphate, K2HPO4 1.0 gm. 
Distilled water 1000 c.c. BLOOD AND BLOOD SERUM MEDIA 
69 
When these substances are thoroughly dissolved, add 40 c.c. of 
glycerol. Tube and sterilize. 
Uschinsky's Medium (Smith's 1905 Modification). 
Distilled water 1000 c.c. 
Ammonium lactate 5 gms. 
Sodium asparaginate 2.5 gms. 
Sodium sulphate, NaSCU- lOIDO 2.5 gms. 
Sodium chloride, NaCl 2.5 gms. 
Dipotassium phosphate, K2HI O4 2.5 gms. 
Calcium chloride, CaCh 010 gm. 
Magnesium sulphate, MgSCh .010 gm. 
Recommended for use with starch jelly. 
Blood and Blood Serum Media 
Blood Serum. Beef blood should be collected at the abattoir in 
clean containers and placed in the refrigerator until a clot has formed. 
The clear serum should then be decanted or better siphoned off into a 
sterile flask. For the preparation of Loeffler's blood serum, 1 part of 
glucose broth is mixed with 3 parts of the serum. This is then dis- 
tributed into test tubes (or other containers) and placed in the inspis- 
sator in a slanted position. Small tin ointment boxes make good con- 
tainers when the blood serum is to be used for routine diphtheria exami- 
nations. After coagulation has been accomplished, the boiled serum 
should be sterilized in the autoclave at a temperature of about 112° C. 
If desired, intermittent heating in the Arnold may be used. 
Blood Agar (Besson). 1. Melt tubes of glycerol agar and cool to 
40° C. 
2. Add to each tube a small quantity (1 c.c.) of blood from a rabbit's 
artery. Mix without shaking the tubes and cool in sloping position. 
A solution of hemoglobin may be used instead of blood. 
Blood Agar for Streptococci (Becker, 1916). Standard agar used in 
water analysis is placed in quantities of 40 to 100 c.c. in flasks. These 
are heated until the agar is melted, and then cooled to 45° or 50° C. 
One cubic centimeter defibrinated human blood is added for each 6 c.c. 
of the agar base and the whole is thoroughly shaken. Approximately 
7 c.c. are used for each plate. Surface streaks are made and observa- 
tions made after twenty-four hours at 27° C. Isolated colonies only 
should be studied. 70 
MEDIA AND THEIR PREPARATION 
Serum Agar. 1. Dissolve 1.5 grams of agar in 100 c.c. distilled 
water. Filter, tube (5 c.c. in each tube) and sterilize. 
2. Cool to 40° C. To each tube add an equal volume of sterile serum. 
Mix gently by rolling the tube and cool in a slanting position. 
Alkaline Blood Agar (Dieudonne). Mix equal volumes of bovine 
blood and a normal solution of potassium hydroxide. Steam for one- 
half hour; mix three volumes of this alkalin blood with seven volumes 
of neutral (to litmus) meat broth agar and pour plates. Dry the plates 
at 60° C. for half a hour and keep at room temperature for at least 
twenty-four hours before using in order to free them from ammonia, 
the evolution of which would prevent the growth of Microspira cholerae. 
(Quoted from Goldberger, 1913.) 
Alkalin Hemoglobin Agar for Microspira Cholerae (Esch, 1910). 
Dissolve 5 gms. of horse hemoglobin (Merck) in 30 c.c. of a half normal 
sodium hydroxide solution. Steam for one-half hour. Mix 15 c.c. 
of this alkalin hemoglobin solution with 85 c.c. of neutral (to litmus) 
meat broth agar, pour plates and dry at room temperature with covers 
removed. The plates are ready for use as soon as dry-a matter of 
about a half a hour. (Quoted from Goldberger.) 
Milk Media 
Casein Agar (Ayres, 1913). Preparation of 1 liter. 
Casein solution: 
300 c.c. distilled water. 
10 gms. casein (Eimer & Amend C.P. casein prepared accord- 
ing to Hammarsten). 
7 c.c. normal sodium hydroxide. 
Agar solution: 
500 c.c. distilled water. 
10 gms. agar. 
After dissolving casein make up to 500 cubic centimeters. 
To 300 c.c. of water (distilled) add: 
10 gms. casein (Eimer & Amend C.P. casein prepared accord- 
ing to Hammarsten). 
7 c.c. normal sodium hydroxide. 
Dissolve casein by heating to boiling. It is desirable to let this 
solution stand for several hours to get a perfect solution. This is 
not necessary, however. Make up volume to 500 c:c. and bring the MILK MEDIA 
71 
reaction of the solution to between +0.1 and +0.2 Fuller's scale. 
Do not allow solution to become alkaline to phenolphthalein or over 
+ 0.2. If the casein is weighed accurately and the normal solution 
accurate the reaction will be about +0.2. 
The agar solution is prepared by dissolving 10 gms. agar in 500 c.c. 
of water. Both casein and agar solutions should be filtered, then mixed. 
Tube and sterilize in autoclave under pressure for twenty minutes; 
then cool the tubes quickly in cold water or ice water. The final 
reaction of the medium will be about +0.1 Fuller's scale. If the 
medium is alkaline the bacterial growth will be restricted. If the 
medium is more than +0.1 some of the casein may be precipitated 
during sterilization. The casein agar should be clear and almost 
colorless when poured in a Petri dish. Sometimes the casein will be 
slightly precipitated during sterilization or the cooling, but it is of no 
consequence since on pouring into plates the precipitate on account 
of its finely divided condition becomes invisible. 
Whey Agar. Add a few drops of acetic acid to boiling milk until 
the casein is precipitated. Neutralize, or bring to 1 per cent + . Dis- 
solve 1 per cent peptone, 2 per cent dextrose and 1.5 per cent agar. 
Filter, tube and sterilize. 
Plain Milk. This may be made from fresh skimmed milk. It 
may be used in many of the different laboratory apparatus. Very 
often it will be found convenient to make it from desiccated milk. 
Desiccated milk 100 gms. 
Distilled water 1000 gms. 
The milk powder should be added to about 200 c.c. of the distilled 
water and thoroughly mixed. Beating with an egg beater often serves 
this purpose. It should then be made up to a liter. Sterilize in the 
Arnold. 
Litmus Milk. Add sterile litmus or azolitmin to plain skimmed milk 
until the color is a distinct lavender. Tube in any of the laboratory 
apparatus and sterilize. 
Litmus Whey (Petruschky). Fresh milk is slightly warmed and 
treated with dilute hydrochloric acid to precipitate the casein. This 
is filtered off and dilute sodium carbonate added to the filtrate to 
neutrality. This is then steamed for two hours to precipitate any 
casein which is changed to acid albumin by the dilute hydrochloric 
acid. After filtration a clear neutral solution should be the result. 
This is colored with litmus, tubed and sterilized. After growth the 
amount of acid may be determined by standard reagents. 72 
MEDIA AND THEIR PREPARATION 
Rice Milk (Besson). 
Milk 150 gms. 
Peptone broth 50 gms. 
Powdered rice 100 gms. 
Distribute the mixture in Petri dishes 1-2 com. deep. Heat to 
115° C. for twenty minutes. The mixture solidifies and forms an 
opaque layer. 
Dialyzed Milk. Dialyzed milk may be prepared by the addition 
of .10 c.c. of hydrogen peroxide to 500 c.c. of milk. Two hours later 
hydrogen peroxide should be added again and after four to five hours 
the whole is dialyzed. The sugar content is i educed 50 per cent and 
the normal quantities of fat and proteins are preserved. The milk is 
diluted one-fifth. 
Milk Agar (Hastings, 1904). This medium is prepared by adding 
to ordinary nutrient agar, which has been melted and cooled to 50° C. 
10 per cent sterile skimmed milk. If the milk is added to the agar 
while hot, the casein will be precipitated in course flocculent masses. 
The medium is poured into sterile Petri dishes and after hardening is 
streaked with the cultures. After incubation, if proteolytic action 
is present, there will be a clear zone about the streak which will not 
change when treated with dilute acids. This distinguishes the acid 
formers from the proteolytic bacteria. 
BIBLIOGRAPHY 
Ayers, S. H. 1913. Casein Media Adapted to the Bacterial Examination of 
Milk. Ann. Rept. Bureau of Anim. Ind., 1913, 225-235. 
Banzhaf, E. J. and Hirschleifer, L. J. 1914. Studies in Preparation of 
Diphtheria Toxin Media (Analysis of Nutrient Materials) Collected Studies, 
Bureau of Laboratories, City of New York, 8, 223-225. 
Barlow, B. 1912. A Spoilage of Canned Corn Due to a Thermophilic 
Bacteria. Thesis for Master's Degree, LTniversity of Illinois, June, 1913. 
Becker, W. C. 1916. The Necessity of a Standard Blood Agar Plate for 
the Determination of Hemolysis by Streptococci. Jour. Inf. Dis., 19, 
754-759. 
Bendick, A. J. 1913. The Use of Calcium Carbonate in Solid Media for 
the Differentiation of Sugar Fermenting Bacteria, Especially of the Colon- 
typhoid Group. Trans. 15th Intern. Cong. Hygiene and Demography, 
Vol. II, 40. 
Berry Jane. 1914-15. Studies on Laboratory Media. Collected Studies, 
Bureau of Laboratories, City of New York, 8, 288-293. BIBLIOGRAPHY 
73 
Berthelot, A. 1917. Sur 1'emploi du Bouillon de Legumes comme milieu 
de Culture. Comp. Rend. soc. biol., 80, 131-132. 
Besson, A. 1913. Practical Bacteriology, Microbiology, and Serum Therapy. 
Translated by Hutchens, H. J. Longmans, Green & Co. 
Bigelow, W. D. and Cook, F. C. 1908. Meat Extracts and Similar Prepara- 
tions. U. S. Depart, of Agriculture, Bureau of Chemistry, Bulletin 114. 
Breed, R. S. 1915. Standard Method for Determining Nitrate Reduction. 
Science N. S., 41, 661. 
Buchanan, R. E. 1909. The Gum Produced by B. radicicola. Cent. 
Bakt. Abt., 2, 22, 392-396. 
Burling, H. A. and Levene, M. 1918. Concentration of Glucose and 
Lactose and Viability of Coli-like Bacteria. Amer. Jour. Pub. Health, 
8, 306-307. 
Cannon, P. R. 1916. A Rapid and Simple Indol Test. Jour, of Bact., 
1, 535-536. 
Cook, F. C. 1914. The Partition of Nitrogen of Plant, Yeast, and Meat 
Extracts. Jour. Amer. Chem. Soc., 36, 1551-1556. 
Conn, H. J. 1914. Culture Media for Use in the Plate Method of Counting 
Soil Bacteria. New York Agric. Exp. Sta., Bulletin 38. 
Conn, H. W. 1915. Standard Methods for Determining the Purity of Milk. 
Public Health Reports, 30, 3-48. Reprint 295. 
Dolt, M. L. 1908. Simple Synthetic Media for the Growth of B. coli and for 
its Isolation from Water. Jour. Inf. Diseases, 5, 616-626. 
Drigalski, V. and Conradi, H. 1902. Ueber ein Verfahren zum Nachweis 
der Typhusbacillen. Zeit. Hygiene, 39, 283. 
Emmett, A. D. and Gies, W. G. 1907. On the Chemical Relation between 
Collagen and Gelatin. Jour. Biol. Chem., 3, 33. 
Esch. 1910. Zum Bakteriologischen Cholera nachweise mittels der Blut- 
alkali-nahrboden. Deutsch, med. Wochenschr., 36, 559-561. 
Eyre, J. W. H. 1903. The Elements of Bacteriological Technique. W. B. 
Saunders & Co., Philadelphia. 
Fraenkel, C. 1894. Beitrage zur Kenntniss des Bakterienwachstums auf 
eiweissfreien Nahrlosungen. Hyg. Rundschau. 1894. 769. 
Fellers, C. F. 1916. The Analysis, Purification, and Some Chemical Prop- 
erties of Agar Agar. Jour. Ind. Eng. Chem., 8, 1128-1133. 
Fred, E. B. 1916. A Laboratory Manual of Soil Bacteriology. W. B. 
Saunders & Co., Philadelphia. 
Gage, S. H. and Adams, G. O. 1904. Studies on Media for the Quantita- 
tive Estimation of Bacteria in Water and Sewage. Jour. Inf. Diseases, 
1, 358. 
Giltay, E. and Aberson, J. H. 1891. Recherches sur un mode de Deni- 
trification et sur le Schizomycetes qui et Produit. Archives Neerlandaises 
des Sciences ex. et nat., 25, 341-361. 
Goadby, K. W. 1903. The Mycology of the Mouth. Longmans, Green & 
Co. 74 
MEDIA AND THEIR PREPARATION 
Gordon, M. II., Hine, T. G. M. and Flack, M. 1916. Cultural Require- 
ments of Meningococcus. Brit. Med. Jour., 1916, 678. 
Goldberger, J. 1913. Some New Cholera Selective Media. Bulletin 91, 
Hyg. Lab., Washington. 
Hastings, E. G. 1904. The Action of Various Classes of Bacteria on Casein 
as Shown by Milk Agar Plates. Cent. Bakt. Abt., 2, 12, 590-592. 
Hasseltine, H. E. 1917. The Bacteriological Examination of Water. Com- 
parative Studies of Media Used. Public Health Reports, 32, 1878-1887. 
Hesse, W. and Niedner. 1898. Die Methodik der Bakteriologischen Was- 
seruntersuchungen. Zeit. Hygiene, 29, 454-462. 
Hiss, P. H. 1897. On a Method of Identifying and Isolating Bacillus Typho- 
sus Based on a Study of Bacillus Typhosus and Members of the Colon 
Group in Semi-solid Culture Media. Jour. Exper. Med., 2, 701-710. 
Hiss, P. H. 1902. New and Simple Media for the Differentiation of the 
Colonies of Typhoid, Colon, and Allied Bacteria. Jour. Medical Research, 
8, 148. 
Houston, A. C. 1914. Eighth Annual Report Metropolitan Water Board, 
41. 
Kendall, A. I. and Day, A. 1911. The Rapid Isolation of Typhoid Para- 
typhoid and Dysentery Bacilli. Jour. Medical Research, 25, 95-99. 
Krumwiede, C., Pratt, C. S., and McWilliams, H. I. 1916. The Use of 
Brilliant Green for the Isolation of Typhoid and Paratyphoid Bacilli from 
Feces. Jour. Infectious Diseases, 18, 1-13. 
Levene, M. 1918. A Simplified Fuchsin-sulphite (Endo) Agar. Abstracts 
of Bacteriology, 2, 13. 
Levene, M. 1918. Presumptive Test for Bacillus Coli. Eng. Contr., 49, 
34. Jour. Inf. Dis., 23, 43-47. 
Lipman, J. G. and Brown, P. E. 1910. Media for the Quantitative Estima- 
tion of Soil Bacteria. Cent. Bakt. Abt., 2, 25, 447-454. 
MacConkey, A. 1900. Experiments on the Differentiation and Isolation 
from Mixtures of Bacillus Coli Communis and Bacillus Typhosus by the 
Use of Sugars and the Salts of Bile. The Thompson Yates Laboratory, 
Reports III, 41. 
McBeth, I. G. and Scales, F. M. 1913. The Destruction of Cellulose by 
Bacteria and Filamentious Fungi. U. S. Department of Agriculture, 
Bureau of Animal Industry, Bull. 266. 
Myer, E. M. 1917. The Use of a Three Per Cent Lactose Litmus Agar 
Plate for Demonstration of B. coli in Water Examination. Jour. Bact., 
2, 237-240. 
Mudge, C. S. 1917. The Effect of Sterilization upon Sugars in Culture 
Media. Jour. Bact., 2, 403-415. 
North, C. E. 1909. An Agar Gelatin Medium. Jour. Med. Research, 20, 
359-363. 
Petruschky. Quoted from Moir and Ritchie's Manual of Bacteriology. Mac- 
millan Company. 6th Edition. New York. BIBLIOGRAPHY 
75 
Redfield, H. W. 1912. A Study of Hydrogen Sulfid Production by Bacteria 
and its Significance in the Sanitary Examination of Water. Thesis, 
Cornell University, 1912. 
Rettger, L. F. 1906. Studies on Putrefaction. Jour. Biol. Chern., 2, 71-86. 
Russell, F. F. 1911. Isolation of B. typhosus from Feces and Urine. Jour. 
Med. Research, 25, 217. 
Savage, W. G. 1901. Neutral Red in the Routine Bacteriological Examina- 
tion of Water. Jour. Hyg., 1, 437. 
Smith, E. F. 1898. Potato as a Culture Medium. Proc. Am. Assoc. Adv. 
Science. 47, 411. Cent. Bakt. Abt., 2, 5, 102. 
Smith, E. F. 1905. Bacteria in Relation to Plant Diseases. Carnegie 
Institution of Washington. 
Sullivan, M. X. 1905. Synthetic Culture Media and the Biochemistry of 
Bacterial Pigments. Jour. Med. Research, 14, 109-160. 
Thom, C. A. 1906. Fungi in Cheese Ripening; Camembert and Roquefort. 
U. S. Department of Agriculture, Bureau of Animal Industry. Bulletin 82. 
Thom, C. A. 1910. Cultural Studies of Species Penicillium. U. S. Dept, 
of Agr., Bureau of Animal Industry. Bulletin 118. 
Teague, O. 1918. Stock Sodium Sulfite Solution for Endo Agar. Jour. 
Amer. Med. Assn., 70, 454. 
Teague, O. and Clurman, A. W. 1916. An Improved Brilliant-green Cul- 
ture Medium for the Isolation of Typhoid Bacilli from Stools. Jour. 
Inf. Diseases, 18, 647-652. 
Thiele, L. 1912. The Manufacture of Gelatin. Jour. Ind. Eng. Chern., 4, 
446-451. 
Uschinsky. 1893. Ueber eine eiweissfreie Nahrlosung fur pathogene Bakte- 
rien nebst einigen Bemerkungen liber Tetanusgift. Cent. Bakt. Abt., I, 
14, 316-319. 
Van Delden, A. 1904. Beitrag zur Kenntniss der Sulfatreduktion durch 
Bakterien. Cent. Bakt. Abt., II, 11, 88-119. 
X edder, E. B. 1915. Starch Agar as a Useful Culture Medium. Jour. 
Infectious Diseases, 16, 385-388. 
Whittaker, H. A. 1911. The Source, Manufacture and Composition of 
Commercial Agar-agar. Jour. American Public Health Assn., 1, 632-639. CHAPTER lib 
STAINING TECHNIQUE 
Since bacteria are such small organisms, they must be examined 
collectively and only in rare instances as individuals. Barber (1914) 
devised a method whereby single cells may be isolated, but this pro- 
cedure is better adapted to advanced work where very accurate results 
are desired. For ordinary work the bacteria may be grown either in 
liquid or solid media. From these media they may be put on slides 
according to instructions which are given later. After these slides 
or cover glasses have been examined they should be dropped into a 
disinfectant since Rettger (1913) has pointed out that the staining 
procedure may not kill the organism. 
Hanging Drop. This preparation enables the bacteriologist to 
observe bacteria under the microscope in the free condition and not 
fastened to the slide as in the staining methods. There are several 
adaptations of this method all of which lead to the same results, i.e., 
suspending the organisms in a drop of fluid in a well of some sort. 
Some of the more important of these are mentioned below. 
Koch's Concave Slide. This consists of a plain glass slide with a 
hollow well ground in the middle. The cover glass with the culture 
is placed over this. Such a slide is quite satisfactory for the exam- 
ination of bacteria, but may be too shallow for the study and continued 
observation of molds since the mycelium will soon fill the cell. For 
this purpose other slides of a similar nature will be found to give 
better results. 
Bottcher's Moist Chamber. This is essentially for the same pur- 
pose as the concave slide, but is constructed in such a way that a deeper 
well is secured. These may be made by fastening to an ordinary glass 
slide a glass or rubber ring about 16 mm. in diameter and 2 to 3 mm. 
high. These are inexpensive and may be easily glued to the slide. A 
preparation made with one of these may be securely sealed with vaseline 
and thereby prevent evaporation which is an important factor if con- 
tinued observations are to be made. Other forms of apparatus which 
may be used in this relation are shown in Fig. 30. 
76 STAINING TECHNIQUE 
77 
Hanging Block. This preparation allows the bacteria to develop 
but prevents their movement and thus holds them in one place for 
observation. In this way the relation of cell to cell may be studied. 
They may be prepared in the following manner. A sterile agar plate 
is poured and allowed to harden. From this by means of a sterile 
scalpel or spatula is cut a block of the agar which is picked up on the 
spatula. On its surface is rubbed a small amount of the culture which 
Fig. 30.-Types of Concave Slides and Moist Chambers for Making Hanging Drop 
and Hanging Block Mounts. 
is under study. The agar block is then put up on a sterile cover glass 
in such a way that the cells of the organism will be between agar block 
and the cover glass, cover slip. About the edge of the cover slip 
should be placed a small amount of vaseline to hold the slip in place 
and also to prevent evaporation if the mount is to be incubated. These 
preparations may be kept for some time if desired. Hill (1902) has 
discussed the application of this preparation to the observation of 
developing bacteria. 78 
STAINING TECHNIQUE 
Chinese Ink Preparations. The organisms are mounted in Chinese 
ink and are unstained. Therein lies the special advantage of such a 
preparation. Under the microscope the organisms appear as trans- 
parent spots in a dark field. Before using, the ink should be sterilized 
Fig. 31.-Types of Forceps for Use in Staining Microorganisms. 
A, Cornet Style; B, Two Types of Stewart Style; C, Type of Slide Forceps. 
C 
B 
/I 
because it may contain many bacteria. After the ink has dried 
examination may be made either with or without a cover glass. 
Dark Ground Illumination. By this method the bacteria are 
examined in the unstained condition. In order to be observable, the METHODS OF STAINING 
79 
objects must be of low refractive index. This method of observing 
bacteria has been especially valuable for the study of Treponema 
pallidum and flagella of ordinary bacteria. 
Theory and Methods of Staining 
The staining of bacteria should be regarded as a delicate procedure 
and one requiring much care. It should not be considered-although 
it probably is in many cases-as a saturation of the cell with the dye. 
Rather should it be regarded as a chemical reaction involving the 
same laws which govern chemical changes. It doubtless follows the 
law of mass action. 
Koch (1877) was one of the first to recognize the value of aniline 
dyes for staining. For convenience the aniline dyes have been divided 
into two classes-the basic and acid dyes. The basic aniline dyes 
contain amido groups while the acid dyes contain the hydroxyl groups. 
The basic dyes are usually used as the salts of hydrochloric acid while 
the acid dyes are secured as sodium or potassium salts. Unna (1888) 
has mentioned that these dyes are not always basic but may be salts 
neutral in character. They are called basic simply because the colored 
component is basic. The aniline dye does not separate into two parts, 
the basic or colored part attacking the cell protoplasm, but rather the 
entire molecule plays its part in the staining process. That the stain- 
ing ability is determined by the solvent condition of the dye is shown 
by the following observations. 
1. Pure alcoholic water-free solutions of the dyes do not stain. 
2. Pure absolute alcohol will not decolorize while alcohol diluted 
with water decolorizes quickly. 
3. The more perfect the solution of the dye in the solvent the 
lesser does its staining ability become. This helps to explain why 
absolute alcohol does not decolorize or why pure alcoholic solutions 
do not stain. 
One of the most important essentials of the staining procedure 
is clean apparatus. All grease and foreign matter must be removed 
from the slides and cover glasses according to the procedure outlined 
under cleaning apparatus in the first chapter. After the slides have 
been thoroughly cleaned they may be kept in a salt-mouth bottle in 
alcohol. The cover slips may be kept in a short wide-mouth bottle 
containing alcohol or ether. In this way both the slides and cover 
glasses will be free from grease provided they are thoroughly cleaned 
and rinsed in distilled water. 80 
STAINING TECHNIQUE 
Preparation of the Smear. The smear on the slide or cover glass 
should be even and not too thick. For making this, a clean cover 
glass is removed from the bottle and allowed to dry in the air or by 
flaming. By means of a sterile platinum loop place a small drop of 
sterile water on the slide and rub a little of the culture in it. It is 
best not to stir too much but by gentle tappings add enough of the 
growth to the drop of water. Spread the drop of water over the glass 
A 
B 
D 
c 
E 
Fig. 32.-Types of Staining Jars and Dishes. 
Coplin's; B, Moore's; C, Lellendahl's; D, Naples Jar; E, Series of Jars for Differential 
Staining. 
and allow to dry in the air or by gentle heating. After it has evap- 
orated fix by holding above the flame at a point which is not uncom- 
fortable for the hand. The procedure will give the smear. A good 
preparation should have the organisms evenly distributed over it 
and should not collect in droplets due to grease. The smear may 
now be subjected to any of the many staining processes. 
Staining Apparatus. While any method may be used to stain bac- 
teria certain pieces of apparatus are more convenient than others. 
When the organisms are mounted on a slide they may be covered STAINING SOLUTIONS 
81 
with the stain or immersed in it. For immersion, many different 
types of staining jars are available. When the staining procedure 
is carried out by the putting the dye on the bacteria on a slide or cover 
glass, it may be kept in ordinary dropping bottles. 
Stock Solutions of Dyes. These may be made up and kept on 
hand in glass-stoppered bottles. It is best to keep them in the dark. 
Such solutions may be made either in alcohol or water. 
Alcoholic Stains. Such stains are made by dissolving a little of 
the dye in alcohol. They are not much used in bacteriology and are 
usually diluted with distilled water. 
Aqueous Alcoholic Stains. These are prepared by adding to dis- 
tilled water a portion of the filtered stock solution of the dye in absolute 
alcohol. These are the stains which have the greatest use in the 
laboratory. From 1 to 5 c.c. of the saturated alcoholic solution of the 
dye are diluted with 100 c.c. of the water. 
Aqueous Stains. Pure aqueous solutions are weak stains and have 
limited use. They are prepared by adding to the distilled water 
enough of the dye to give a saturated solution. They have the great 
advantage that over staining with them is improbable. 
Mordants. They have the same purpose in staining that they have 
in dyeing. They are substances which unite with the aniline dye and 
with the cell protoplasm. They increase the rapidity of the staining 
procedure and render the color of the stained protoplasm more intense. 
The following substances are used as mordants: 
Iodine in LugoFs solution. 
Bromine in solution with potassium bromide. 
Tannin. 
Different acids. 
Alkalis. 
Gram Stain. Gram (1884) devised a differential staining procedure 
which has been of much service in bacteriology. The explanation of 
this stain is not definitely settled. Gram in his original work did not 
attempt to explain it. Unna (1887) put the explanation on a chemical 
basis. The Gram stain is an arbitrary procedure and a variation in 
the details of it are very important. It probably depends upon the 
fact that the pararosaniline (methyl violet, gentian violet) compounds 
form substances with iodine which are insoluble in alcohol. The iodine 
salts of rosaniline (fuchsin and methylene blue), however, are decomposed 
by alcohol. The Gram negative bacteria are stained only with the 
gentian violet. The alcohol washes out the iodine. The iodine in 82 
STAINING TECHNIQUE 
Gram positive bacteria forms a compound between the protoplasm 
of the cell and the dye which is insoluble in alcohol. In the Gram 
negative bacteria this compound is soluble in the alcohol and broken 
up. Kruse (1910) attempted to explain this stain by stating that the 
Gram positive bacteria are more resistant to autolysis, solution in 
strong alkali, etc., than the Gram negative bacteria. Eisenberg (1909) 
assumed that the differential Gram staining rested upon the presence 
of some special compound such as unsaturated fatty acids or phos- 
phatids in the cell membrane. These formed a compound with iodine 
which renders the cell wall impermeable to alcohol. 
Dreter, Kriegler and Walker (1911) in seeking an explanation for 
this stain proposed the presence of lipoidal substances in the Gram 
positive bacteria which bind the pararosaniline compounds. This, 
however, has little foundation according to our present knowledge. 
Brudny (1908) regarded differences in the permeability of the cell 
membrane as the important factor. With the Gram positive bac- 
teria the membrane is more easily penetrated by the iodine solution. 
The structure of these bacteria is probably " looser " and the stain 
gains an easy entrance. The Gram negative bacteria, however, are 
not penetrated and no deposit of the dye takes place on the interior 
of the cell. Benians (1912) places the explanation of this stain on a 
definite cell membrane. This prevents the removal of the dye by tlie 
alcohol while Hottinger (1916) explains it from the standpoint of col- 
loidal chemistry. He states that the relation of the bacteria to this 
stain depends upon the degree of dispersion of the nucleoproteins. In 
the Gram negative bacteria, the stained nucleoproteins are so widely 
dispersed that they are not seen. In the Gram positive bacteria the 
stained nucleoproteins form a coarse emulsoid. Gram positive bacteria 
are said to become Gram negative when the degree of dispersion is 
increased by the action of proteolytic enzymes. Gram positive bac- 
teria resist ferment action longer than Gram negative. Weinkopff 
(1911) in studying tryptic digestion of bacteria concluded that the 
difference between the Gram positive and Gram negative bacteria 
rested in the penetrability of cell protoplasm. Dreter et al. (1912) 
extracted a substance from Gram positive bacteria (staphylococci) 
which when applied to Gram negative B. coli made them Gram positive 
in many cases. When this ether extract was dried on a slide, the 
residue took the Gram stain. This investigator also treated B. coli 
with lecithin which made them Gram positive. That the Gram nega- 
tive bacteria are structurally different from the Gram positive bacteria 
seems to be indicated by the work of Larson, Hartzell and Diehl (1918). THEORY OF THE GRAM STAIN 
83 
These investigators stated that the Gram negative bacteria could be 
broken up by the sudden release of the pressure when the bacteria 
were in an atmosphere of carbon dioxide while the Gram positive 
bacteria resisted such treatment. The latter, although killed, under- 
went no morphological change. 
Procedure for the Gram Stain. Prepare a smear as outlined above. 
1. Flood with aniline gentian violet and heat gently for five minutes. 
2. Wash in water and immerse in Lugol's iodine solution for five 
minutes. 
3. Wash in water and place in strong alcohol until most of the dye 
is removed. It may be necessary to repeat this procedure from 2 
if any dye remains. 
4. Wash in water and examine or counterstain if desired. 
5. Counterstain with 10 per cent solution of eosin in water. 
There have been several modifications of the Gram method of 
staining all of which give good results in the hands of those who pro- 
posed them. 
Nicolles Modification of the Gram Stain. This is similar to the 
above except that carbol gentian violet is substituted for the aniline 
gentian violet in the regular Gram's procedure. Decolorization is 
also accomplished with acetone alcohol (1 part acetone and 2 parts of 
alcohol). 
GRAM POSITIVE BACTERIA 
Staphylococcus pyogenes aureus 
Staphylococcus pyogenes albus 
Staphylococcus pyogenes 
Pneumococcus 
Micrococcus tetragenus 
Bacillus Xerosis 
Bacillus hoffmanii 
Oidium albicans 
Bacillus tetanus 
Bacterium diphtheria) 
Bacillus orogenes capsulatus 
Bacillus lepra) 
Bacillus anthracis 
GRAM NEGATIVE BACTERIA 
Gonococcus 
Micrococcus catarrhalis 
Bacillus typhosus 
Bacillis coli communis 
Bacillus dysenteria) 
Micrococcus melitensis 
Pseudomonas pysocyaneus 
Bacillus pneumonia) Fried 
Bacillus proteus 
Bacillus mallei 
Bacillus influenza) 
Koch-Weeks bacillus 
Staining of Capsules 
Muir's Method. 
I. Mordant. 
Mercuric chloride (saturated aq. sol.) 2 c.c. 
Tannin (20 per cent aq. sol.) 2 c.c. 
Potassium alum (Sat. aq. sol.) 5 c.c. 84 
STAINING TECHNIQUE 
II. Stain. 
Carbol fuchsin. 
III. Counter stain. 
Methylene blue. 
The smear is mordanted two minutes, washed in alcohol and water and 
then stained two to three minutes with gentle heat. It is then washed 
with water and remordanted again for two or three minutes. Counter- 
stain with methylene blue. 
Welch's Method. Fix the smear in glacial acetic acid. After a 
few seconds pour off the acetic acid and flood with aniline gentian violet. 
Repeat until all acid is removed. Wash and examine. 
Huntoon's Method 
Preparaticn of Reagents. Solution 1. To be used as diluent. 
Three grams of nutrose are sifted into 100 c.c. of distilled water and 
heated to 100° C. in the Arnold sterilizer for an hour. Add 5 c.c. 
of a 2 per cent phenol solution to act as a preservative. Decant into 
a test tube and allow to settle. Employ the supernatant liquid as the 
diluent. (Since the supernatant liquid tends to become thinner by 
constant precipitation of the nutrose, the solution should occasionally 
be reboiled.) 
Solution 2. Fixing and staining solution. 
2 per cent aq. phenol solution 100 c.c. 
Concentrated lactic acid 0.25 to 0.5 c.c. 
1 per cent acetic acid solution Ic.c. 
Saturated fuchsin in alcohol 1 c.c. 
Carbol fuchsin, old solution 1 c.c. 
This staining solution must be kept tightly corked. 
Technique of Staining 
1. Employ the solution (No. 1) as a diluent emulsifying the bac- 
teria in 1 or 2 loopfuls and then spreading in as thin a film as possible 
with the loop. The use of the edge of a slide in spreading the film is 
not to be recommended. 
2. Allow to dry in the air. 
3. Cover the film with the fixative and the staining solution (No. 2) 
and allow to act for from 30 to 45 seconds. 
4. Wash quickly in water, dry and examine. STAINING OF FLAGELLA 
85 
Staining of Spores 
Neisser's Method. Flood the smear with hot aniline fuchsin for 
about an hour. Wash in water and decolorize with acid alcohol (1 part 
HC1 and 3 parts alcohol). Too long* decolorization will remove the 
stain from the spores also. Counterstain with methylene blue if 
desired. 
Chromic Acid Method. Place the smear in 1 to 20 aqueous chromic 
acid for five minutes. Wash in water and stain with carbol fuchsin 
for thirty minutes. Wash and examine. 
Staining of Flagella 
This has always been a difficult procedure for many bacteriologists. 
It may be due to lack of care in some of the preparatory procedures. 
Smith (1905) has mentioned some of the common errors. 
1. Oily or otherwise dirty cover glasses. 
2. Unsuitable cultures. 
3. Breaking off the flagella. 
4. Uneven or too copious distribution of the bacteria. 
5. Imperfect mordanting. 
6. Excessive mordanting. 
7. Understaining. 
8. Overstaining. 
9. Precipitates on the cover glass during some stage of the process. 
The greatest error probably lies in dirty slides and cover glasses. These 
should be cleaned in strong alkali in order to saponify any animal 
grease which may be on them. Otherwise, the smear will " roll " 
and not spread evenly. The culture which is stained must be young 
and vigorous. A small amount of this growth should be transferred 
to a test tube of sterile water and incubated at 37° C. After several 
hours, some of this suspension should be put on slides and allowed to 
dry by placing the slides in the 37° C. incubator. The smear should 
then be fixed and stained by any of the many processes. 
The author has secured the best results with Loeffler's method. 
A double boiler is prepared from two beakers. The inner is supported 
by pieces of cork. The staining solution should be put into the inner 
one and the temperature raised to about 65° C. or until the stain steams. 
The slides are immersed in this solution and stained as long as required. 86 
STAINING TECHNIQUE 
Loeffler's Mordant 
Tannic acid, 20 per cent aqueous 10 c.c. 
Ferrous sulphate, saturated aqueous 5 c.c. 
Basic fuchsin, saturated alcoholic 1 c.c. 
Mix and filter after a short time. 
Loeffler's Stain 
Basic fuchsin, saturated alcoholic 2.5 c.c. 
Carbolic acid 20.0 c.c. 
Van Ermengen's Flagella Stain. I. Van Ermengen's Mordant. 
Osmic acid 50 c.c. 
Tannic acid aqueous 10-25 per cent 100 
Four drops of glacial acetic acid should be added to the above. 
II. Silver Bath. A 0.25 to 0.5 per cent solution of silver nitrate 
in distilled water. 
III. Reducing Bath. 
Gallic acid 5 c.c. 
Tannin 3 c.c. 
Sodium acetate fused 10 c.c. 
Distilled water 350 c.c. 
The smear should be covered with the mordant for fifteen minutes 
at 50° C. The mordant is then washed off in distilled water and 
alcohol. The smear is then placed in a small amount of the silver 
bath and gently heated. Without washing, it is then put into the 
reducing bath and agitated until the solution begins to blacken. It 
is removed dried and mounted for examination. The apparatus used 
in this procedure must be absolutely free from animal grease. Kuntze 
(1902) has suggested some improvements in Van Ermengen's procedure 
for staining flagella, 
Acid Fast Staining 
This involves the formation of a permanent compound between the 
stain and the cell protoplasm-one which may not be removed by 
comparatively strong solutions of mineral acids. These bacteria DIFFERENTIAL STAINING OF BLOOD FILMS 
87 
require a strong stain with a mordant and once the cell is stained it is 
decolorized with great difficulty. Different reasons such as a thick 
membrane about the cell or peculiar structure of the cell contents, 
have been given to explain the acid fast staining procedure. Klebs 
(1896) has shown that the acid fast properties are lost if the tubercle 
bacillus is extracted with ether. Others (Bienstock, 1886) have stated 
that by increasing the fat content of the cell it may be made to 
take on acid fast properties. Miller (1916) grew tubercle bacilli in 
sperm oil and attributed the variations in the staining properties with 
carbol fuchsin to the production of free oleic acid in the interior of 
the rod. This acid was believed to be formed by the spores or round 
granules in the cell. Bulloch (1904) when studying the acid fast 
properties of bacteria isolated a wax and other substances in the nature 
of fats from acid fast bacteria. The other constituents of the cells 
were not acid fast. Baumgarten (1911) thought that unsaturated 
fatty acids were present in the acid fast bacteria which united with the 
dye. Benians (1912) claimed that the cell wall was the important 
factor in retaining the dye. He states " for the present the evidence 
points to the existence of a waxy layer enclosing a protoplasmic and 
fatty cell substance and conferring on the organism the property of 
resisting the penetration of acid and alcohol." Aronson (1910) claims 
that the peculiar staining properties of the tubercle bacillus are due 
to the fat content and not to any peculiar kind of protoplasm. This 
waxy substance may be extracted by trichlorethylen. 
Ziehl-Nielsen Method of Acid Fast Staining. Flood the smear 
with the stain and heat for ten minutes. Decolorize in 25 per cent 
mineral acid and wash. Counterstain if desired with eosin or Bismarck 
brown. The staining solution is prepared as follows: 
Basic fuchsin 1 part 
Absolute alcohol 10 parts 
Carbolic acid (1 to 20) 100 parts 
Wright's Method for the Differential Staining of Blood Finns and 
Malarial Parasites. The method of preparing the stain and applying 
it is outlined by Wright (1902) as follows: 
I. Preparation of the Staining Fluid. Make a 0.5 per cent solution 
of sodium bicarbonate in an Erlenmeyer flask and add to it 1.0 c.c. of 
methylene blue. Grubler's BX or Koch's or Erlich's rectified methy- 
lene blue may be used. The sodium bicarbonate should be entirely 
dissolved before the methylene blue is added. The mixture should be 88 
STAINING TECHNIQUE 
steamed in an Arnold steam sterilizer for one hour. The steaming 
of the alkalin solution of methylene blue effects certain changes in 
the methylene blue whereby a polychromatic property is given to it 
so that the compound with eosin, which is later to be formed with it, 
has not only the property of differentially staining the chromatine of 
the malarial parasite but also of differentiating and bringing out more 
sharply the nuclei and granules of the white blood corpuscles. 
When the steaming is completed the mixture is removed from the 
sterilizer and allowed to cool. When it is cold, without filtering, pour 
it into a large dish or flask and add to it shaking or stirring meanwhile 
a 1 to 1000 solution of eosin (Grubler, yellowish, soluble in water suffi- 
cient in quantity, until the mixture losing its blue color becomes pur- 
ple in color, and a scum with yellowish metallic luster form on the 
surface, while on close inspection a finely granular black precipitate 
appears in suspension. This will require about 500 c.c. of the eosin 
solution for 100 c.c. of the alkalin methylene blue solution. 
The precipitate is collected on a filter and allowed to dry thereon 
without washing. When thoroughly dry a saturated solution in pure 
methyl alcohol is made. Three-tenths of a gram of the dry substance 
will thoroughly saturate 100 c.c. of the methyl alcohol in a few minutes. 
The saturated alcoholic solution of the precipitate is next filtered 
and to the filtrate is then added 25 per cent of methyl alcohol; e.g., 
to 80 c.c. of the saturated alcoholic solution 20 c.c. of methyl alcohol 
are added. 
This somewhat diluted solution of the precipitate is the staining 
fluid. It is permanent and may be kept on hand. Care should be 
taken to prevent evaporation of the alcohol so that the fluid may not 
become too saturated. The object of the dilution is to prevent precipi- 
tations on the blood film in the process of staining. 
II. The Staining of the Blood Films. The films of blood which are 
spread thinly are allowed to dry in the air. As much of the staining 
fluid is poured upon the film as the cover glass or slide will readily 
hold without draining off. Allow the staining fluid to remain in con- 
tact with the film for one minute. Next, add to the staining fluid 
on the cover glass or slide, sufficient water drop by drop, until it becomes 
semi-translucent and a reddish tint becomes visible at its margin, 
while a metallic scum forms on its surface. The amount of water 
required will vary with the amount of staining fluid on the preparation. 
Generally 8 or 10 drops will be required if a seven-eighth square cover 
glass is used. The staining fluid thus diluted should be allowed to 
remain on the preparation for 2 or 3 minutes, during which time the DIFFERENTIAL STAINING OF BLOOD FILMS 
89 
real staining of the preparation takes place, and is then washed off 
with water. 
The differential staining of the preparation is accomplished by 
washing the preparation with distilled water. The washing is con- 
tinued until the better spread portions of the film appear yellowish 
or reddish in color. This method of differentiation of decolorization 
may require from one to three minutes according to the intensity of 
the staining and the tint sought in the red blood corpuscles. When 
the desired color is obtained in the red blood corpuscles the preparation 
is quickly dried between filter papers and mounted in balsam. It is 
important to stop the decolorization by drying the preparation as soon 
as the desired tint in the red blood corpuscles is obtained. A little 
experience will show the operator how far to carry the decolorization 
and the time required for to obtain the desired tint in the red blood 
corpuscles. 
Summary for the Method of Staining 
1. Make films of the blood, spread thinly and allow them to dry 
in the air. 
2. Cover the preparation with alcoholic solution of the dye for one 
minute. 
3. Add to the alcoholic solution of the dye on the preparation suffi- 
cient water, drop by drop, until the mixture becomes semi-translucent 
and a yellowish metallic scum forms on the surface. Allow the mixture 
to remain on the preparation for two or three minutes. 
4. Wash in distilled water until the film has a yellowish or pinkish 
tint in its thinner or better spread portions. 
5. Dry between filter paper and mount in balsam. 
III. Microscopical Appearance in Blood Films Stained by this 
Method. The red cells are orange or pink in color. Polychromeato- 
philia and punctate basophilia (the granular degeneration of Grawitz) 
are well brought out. The nucleated red cells have deep blue nuclei 
and the cytoplasm is usually of a bluish tint. The lymphocytes have a 
dark purplish-blue nucleus and a robin's egg blue cytoplasm in which a 
few dark blue or purplish granules are sometimes present. The poly- 
nuclear neutrophilic leucocytes have a dark blue or dark lilac-colored 
nucleus and the granules are usually of a reddish color. The eosino- 
philic leucocytes have blue or dark-colored lilac nuclei. The granules 
have the color of eosin, while the cytoplasm in which they are imbedded 
has a blue color. The large mononuclear leucocytes appear in at least 90 
STAINING TECHNIQUE 
two forms. Each form has a dark or lilac-colored nucleus. The 
cytoplasm of one form is pale blue and of the other form is blue with 
dark lilac or deep purple-colored granules, which are usually not so 
numerous as are the granules in the polynuclear neutrophilic leucocytes. 
The mast cells appear as cells of about the size of polynuclear leucocytes 
with purplish or dark-blue stained irregular-shaped nuclei and the 
cytoplasm sometimes bluish in which coarse spherical granules cf 
variable size are imbedded. These granules are of a dark-blue or purple 
color and may appear almost black. The myelocytes have a dark 
blue or dark lilac-colored nuclei and blue cytoplasm in which numerous 
dark lilac or reddish lilac-colored granules are imbedded. The blood 
plates are deeply stained and are a prominent feature of nearly all 
blood preparations. They appear as blue or purple rounded or oval 
bodies, usually of a diameter of one-third to one-half of that of a red 
blood corpuscle. They have ragged margins and present fine bluish 
or purplish dots or mottlings in their substance. In some instances 
the blood plate appears to be within the red blood corpuscle or partially 
within and partially without. The plates found within the red cells 
must not be mistaken for young malarial parasites. 
Preparation of Common Stains. Alkalin Methylene Blue (Loeffler's): 
Saturated alcholic methylene blue 30 c.c. 
Aqueous KOH solution (0.1 per cent) 100 c.c. 
Carbol Fuchsin (Ziehl): 
Saturated alcoholic fuchsin solution 10 c.c. 
Carbolic acid crystals 5 gms. 
' Distilled -water 100 c.c. 
Anilin Gentian Violet: 
Saturated alcoholic gentian violet 11 c.c. 
Absolute alcohol 10 c.c. 
Anilin water 100 c.c. 
The anilin water may be made by shaking 5-6 c.c. of anilin oil in 
distilled water and filtering. 
Lugol's Iodine Solution: 
Iodine 1 gm. 
Potassium iodide 2 gms. 
Distilled water 300 gms. DIFFERENTIAL STAINING OF BLOOD FILMS 
91 
Stabilized Gentian Violet (Stovall and Nicholls, 1916). To pre- 
vent the usual deterioration of gentian violet, the following stain is 
proposed: 
Aniline 28 c.c. 
Gentian violet 8 gms. 
95 per cent alcohol 100 c.c. 
Normal hydrochloric acid 5 c.c. 
Distilled water 1000 c.c. 
The gentian violet should be dissolved in the alcohol. The aniline and 
hydrochloric acid may be mixed and diluted to 900 c.c. Filter these 
solutions and add each other with a subsequent filtration. 
Another formula for gentian violet for use in the Gram stain has 
been given by Kilduffe (1909). This has been used in the author's 
laboratory and has given good results. 
Solvent 
5 c.c. commercial formalin. 
95 c.c. distilled water. 
Stain 
25 c.c. saturated alcoholic gentian violet. 
75 c.c. of the solvent. 
or 
16 c.c. saturated gentian violet. 
84 c.c. solvent. 
BIBLIOGRAPHY 
Aronson, H. 1910. In Regard to the Biology of the Tubercle Bacillus. 
Berlin, klin. Wochenschr., 47, 1917-1620. 
Barber, M. A. 1914. The Pipette Method in the Isolation of Single Micro- 
organisms and in the Inoculation of Substances into Living Cells. Philip- 
pine Jour. Science, 9B, 307-360. 
Baumgarten. 1911. Lehrbuch der pathogenen Mikroorganismen, p. 641. 
Original not seen. 
Bechtold. 1886. Fortschritt der Medizin, 1886, 4, 193. Original not seen. 
Bienstock, B. 1886. Zur Frage der sogenannten Syphilis Bacillen und der 
Tuberkelfarbung. Fortschr. der Med., 1886. 193-195. 
Brundny. 1908. Ueber die Beziehung der Farbbarkeit der Bakterien nach 
der Gram und ihrer Permeabilitat. Cent. Bakt. Abt., IL, 21, 62. 
Bulloch, W. and M'Leod, J. J. R. 1904. The Chemical Constitution of 
the Tubercle Bacillus. Jour. Hyg., 4, 1-10. 
Dreter, C., Kriegler, S. G., and Walker, E. W. 1911. The Effect of 
Certain Dyes on Bacteria. Jour. Path, and Bact., 15, 133-136. 92 
STAINING TECHNIQUE 
Dreter, C., Scott, S. A., and Walker, E. W. 1912. The Nature of Gram 
Staining. Jour. Path, and Bact., 16, 146. 
Eisenberg. 1909. II. Ueber das Ektoplasms und seine Veranderungen ini 
infizierten Tier. Cent. Bakt. Abt., 1, 49, 465-493. 
Gram, C. 1884. Ueber die isolierte Farbung der Schizomyceten in Schnitt 
und Trokenpraparate. Fort. der. Med., 2, 185-190. 
Hill, H. W. 1902. " Hanging Block " Preparations for the Microscopic Obser- 
vation of Developing Bacteria. Journal Medical Research, 7, 202-212. 
Hottinger, R. 1916. Contributions to the Theory of Gram's Stain from a 
Colloidal Chemical Standpoint. Cent. Bakt., 76, 367-383. 
Huntoon, F. M. 1917. A Simple Method for Staining the Capsules of 
Bacteria. Jour. Bact., 2, 241-244. 
Kilduffe, R. 1909. A New and Stable Solution of Gentian Violet for the 
Gram Stain. Jour. Amer. Med. Assn., 53, 2002. 
Klebs, E. 1896. Ueber heilende und immunisierende Substanzen aus 
Tuberkelbacillen. Cent. Bakt. Abt. 1., 20, 488-508. 
Koch. 1877. Cohn's Beitrage Biol. Pflanzen. 2, Original not seen. 
Kruse, 0. 1910. Beziehungen zwischen plasmolyse Verdaulichkeit, Los- 
lichkeit und Farbarkeit von Bakterien. Munchen, med. Wochenschr., 
1910, 685, No. 13. 
Kuntze. 1902. Einige Bemerkungen fiber die Farbung der Giesseln beson- 
ders uber das Verfahren von van Ermengen. Cent. Bakt. Orig., 32, 
555-560. 
Larson, W. P., Hartzell, T. B., and Diehl, H. S. 1918. The Effect of 
High Pressures on Bacteria. Journal of Infectious Diseases, 22, 271-279. 
Loeffler. 1889. Eine nene Methode zum Farben der Mikroorganismen, im 
besonderer ihrer Wimperhaare und Giesseln. Cent. Bakt., 6, 209-224. 
Miller, A. H. 1916. .On the Biochemistry of the Loss of Power of the 
Tubercle Bacillus to Stain with Carbol Fuchsin. Jour. Path, and Bact., 
21, 41-46. 
Rettger, L. F. 1913. Hygiene of the Bacteriological Laboratory. Amer. 
Jour. Pub. Health, 3, 697-700. 
Smith, I. F. 1905. Bacteria in Relation to Plant Diseases. Smithsonian 
Institution. Washington. 
Stovall, W. D., and Nicholls, M. S. 1916. Stabilized Gentian Violet. 
Jour. Amer. Med. Assn., 66, 1620-1631. 
Unna, P. G. 1881. Die Rosanilin und Pararosanilin. Eine bacteriologische 
Farbenstudie. Cent. Bakt. Abt., I, 2, 135-136. 
van Ermengen, E. 1894. Nouvelle methode de coloration des cils des 
bakteries. Reviewed in Cent. Bakt., 15, 969-970. 
Weinkopff, P. 1911. Tryptic Digestion of Gram Positive and Gram Nega- 
tive Bacteria. Zeit. Immunitat., 11, 1-17. Chemical Abstracts, 5, 3857. 
Wright, J. H. 1902. A Rapid Method for the Differential Staining of Blood 
Films and Malarial Parasites. Jour. Med. Research, 7, 139-143. 
Ziehl. 1882. Deutsch, med. Wochenschr. Original not seen. CHAPTER IV 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
The early investigators in bacteriology were impressed with the 
newness of the field and devoted more time to the isolation of new 
forms. At this time classification received less attention. They 
examined all sorts of substances and described many shapes and sizes. 
The condition for working also made it difficult to secure accurate 
data and this is quite essential in all systematic work. Pure cultures 
were difficult to secure and the microscope approached in no way the 
perfection of the modern instrument. Under such conditions it remained 
for them as for the pioneers in any field to reproduce the facts upon 
which the later workers could build. Consequently the arrangement 
of bacteria into groups or related masses has been left for the bacteri- 
ologists of a later day. 
One of the most important ideas which deviated attention from 
classification was the conception of pleomorphism. The early con- 
ception of this theory has been well stated by Fischer (1900). " The 
pleomorphists maintained that a coccus did not necessarily remain a 
coccus all its life long, but it could under certain conditions stretch 
itself and assume the shape of a bacillus, that this again could become 
curved and change into a vibrio, to return again later on to the coccus 
form that it commenced with. Words like Micrococcus, Bacillus, Vibrio, 
Spirillum, which we know now to have a definite taxonomic value, 
were in the eyes of the pleomorphists worthless designations of transient 
changes of shape." Such a conception of bacterial forms easily pre- 
vented or inhibited attempts at the arrangement of the described 
forms into a classification. This idea had its origin in the beginnings 
of biology and botany. In the early days of chemistry it was one of 
the earliest objects of the science to change copper into gold. 
In bacteriology this theory has constantly received some attention. 
Buchner revived it when he reported that he had changed Bacillus 
subtilis into the bacterium specific for anthrax. More recently Rosenow 
(1914) has reported that he has changed the streptococcus into the 
pneumococcus. He was able to repeat this several times. The serum 
93 94 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
reactions for the new strain possessed all of those of the true pneumo- 
coccus. In turn, he was able to change these serum reactions. This 
raises the question with regard to what characters may be depended 
upon for constancy. This work of Rosenow together with the recent 
reports on life cycles may make it necessary to introduce new methods 
of classification. In the study of the higher forms of life the term 
evolution form conveniently lends itself to abnormal shapes. 
Anton van Leewenhoek (1683) was probably one of the first to 
report different kinds of bacteria. From our present knowledge, it is 
doubtless true that he had a mixture of the various forms of micro- 
scopic life. At this time the methods of securing different dilutions 
were not perfected. In fact, no real attempt had been made up to 
this time to secure pure cultures. since their necessity for accurate 
bacteriological work had not been realized. Van Leewenhoek recog- 
nized curved and straight forms and presented drawings with his paper. 
This contribution may be considered as a classification only in the 
most general sense and then only to the extent that different shapes 
were observed. It is not certain that he saw many bacteria through his 
crude apparatus. 
Muller (1786) studied bacteria from a zoological standpoint and 
proposed a classification which never had very extended use. Probably 
he also observed protozoa with perhaps a few bacteria. 
Ehrenberg (1828) began his contributions to systematic bacteri- 
ology by founding the genus bacterium. A resume of his work is men- 
tioned by Smith. This genus he later divided into several parts. In 
1838 he published his work " Die Infusionsthierchen " and therein 
describes the genus bacterium as being " Die quergetheilten gehoren 
zu den Zitterheirchen (Vibrionen) die langsgehteilten zu den Stabtheir- 
chen (Bacillen)." Ehrenberg gave us several terms which have been 
retained up to the present time but to which different meanings are 
attached. 
Cohn (1872) published a classification which was used for some time. 
He divided the bacteria into the following four groups: 
I. Sphaerobacteria 
Species 1. Micrococcus. 
II. Microbacteria: 
Species 2. Bacterium. 
III. Desmobacteria: 
Species 3. Bacillus. 
Species 4. Vibrio. HISTORY OF CLASSIFICATION 
95 
IV. Spirobacteria: 
Species 5. Spirillum (Ehrenberg). 
Species 6. Spirochaete (Ehrenberg). 
Cohn's classification is not used at the present time. He did not 
make any provision for pleomorphism and thus immediately opposed 
himself to Klebs, Buchner and Lister. It was about this time that 
Buchner reported the changing of B. subtilis into B. anthracis. The 
publications of Cohn and Koch proved the constancy of this char- 
acteristic and struck a forceful blow at pleomorphism. The principal 
objectors to Cohn's classification were the medical men, but after the 
articles of Cohn and Koch they were more careful. 
Lister (1873) was one of the bitterest opponents of Cohn's system. 
He transferred a drop of sour milk in which he had observed cocci to 
broth and observed, after a short time, long chains. He used such 
data to support his views of pleomorphism. 
De Bary (1884) divided all bacteria into two classes, those forming 
endospores and those forming arthrospores. He used spore formation 
in a different sense than we now use it. Whenever he saw a cell which 
differed from the normal, he called it an arthrospore. His discussion 
on the formation and germination of spores is quite complete and 
interesting. 
Eisenberg (1891) for the first time attempted to use physiological 
characters. Bacteria were divided into pathogenic and non-patho- 
genic varieties. The pathogens were again divided with regard to their 
effect on man and animals. No attention was given to spore formation, 
or flagellation. 
Migula (1897) published a classification which has been generally 
used by bacteriologists. There are objections to it, but it is probably 
as good a working basis as any which is available. This is given as 
follows by Jordan (1910): 
Classification of Bacteria-(Migula) 
I. Cells globose in a free state, not elongating in 
any direction before division into one, two, 
or three planes 1. Coccacea?. 
II. Cells cylindrical, longer or shorter, and only 
dividing in one plane, and elongating to 
about twice the normal length before the 
division. 96 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
a. Cells straight, rod-shaped, without sheath, 
non-motile. or motile by means of 
flagella 2 Bacteriacea*. 
b. Cells crooked, without sheath 3. Spirillace®. 
c. Cells inclosed in a sheath 4. Chlamydobacteriace®. 
1. Coccacea* 
Cells without organs of motion: 
a. Division in one plane 1. Streptococcus. 
b. Division in two planes 2. Micrococcus. 
c. Division in three planes 3. Sarcina. 
Cells with organs of motion: 
a. Division in two planes 4. Pianococcus. 
b. Division in three planes 5. Planosarcina. 
2. Bacteriace® 
Cells without organs of motion 1. Bacterium. 
Cells with organs of motion (flagella): 
a. Flagella distributed over the whole body 2. Bacillus. 
6. Flagella polar 3. Pseudomonas. 
3. Spirillace® 
Cells rigid, not snake-like or flexuous: 
a. Cells without organs of motion 1. Spirosoma. 
b. Cells with organs of motion (flagella): 
1. Cells with one, very rarely two to three 
polar flagella 2. Microspira. 
2. Cells with polar flagella-tufts 3. Spirillum. 
Cells flexuous 4. Spiroch®ta. 
4. Chlamydobacteriace® (Higher Bacteria) 
Cell contents without granules of sulphur: 
a. Cell threads unbranched. 
I. Cell division always only in one plane.. . 1. Streptothrix. 
II. Cell division in three planes previous to 
the formation of gonidia: 
1. Cells surrounded by a very delicate, 
scarcely visible sheath (marine).. . 2. Phragmidiothrix. 
2. Sheath clearly visible (in fresh 
water) 3. Crenothrix. 
b. Cell threads branched 4. Cladothrix. 
Cell contents containing sulphur granules 5. Thiothrix. MIGULA'S CLASSIFICATION 
97 
Fischer (1897) proposed a classification and system of nomenclature 
in which the root of the generic name expressed the shape of the cell 
and the ending the arrangement of the flagella as follows: 
Baktron = rod 
Kloster = spindle 
Plectron = drum stick 
inium = monotrichous 
ilium = lopotrichous 
idium = peritrichous 
Using this system a peritrichous rod would be a " bactronidium." 
Fischer's system met with little approval and is not used by 
bacteriologists. 
Lehmann and Neumann (1899) published their classification which 
has had rather extended use in the science. It has been used to a great 
extent by the medical men. They proposed the following system: 
I. Coccace^e 
1. Streptococcus. Dividing in one plane. 
2. Sarcina. Dividing in three planes. 
3. Micrococcus. Irregular division, including all but clearly 
marked packets and chains. 
II. BACTERIACEiE 
1. Bacterium. With no endospores, rods usually 0.8-1.0 microns 
in diameter. 
2. Bacillus. With endospores, rods, may be more than 1.0 micron 
in diameter. 
III. Spirillace^e 
1. Vibrio. Short, rigid, slightly curved cells, with 1 to 2 polar 
flagella. 
2. Spirillum. Long rigid spiral cells, with lophotrichic flagella. 
3. Spirochaete. Long flexible spiral cells, flagella not observed; 
motility accomplished by undulating membrane. 
This is essentially a morphological classification and has many 
advantages. It was produced at about the same time that Migula 
proposed his system. 
Jensen (1909) made an attempt to group bacteria according to then- 
natural development. He made three divisions as follows: 
1. Those bacteria which do not require organic nitrogen or organic 
carbon. 98 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
2. Those bacteria which require organic carbon but are able to dis- 
pense with organic nitrogen. These forms utilize sugars, nitrogen, 
ammonia or nitrates. 
3. Those forms which require both organic nitrogen and carbon. 
Inorganic substances are of little use. 
Jensen attempted to devise a system which would show the rela- 
tionship of forms. Reasoning from the synthetic processes, he con- 
structed a family tree for bacteria. His methanomonas is proposed 
as the first bacterium on earth and he considered it the beginning of all 
life. The method of nomenclature in this system is cumbersome and 
so far his proposals have been of little more than theoretical interest. 
The present-day system and those which are developed in the 
future must rest upon the past work in this field. Fischer, in his book 
previously referred to, has stated that the very factor contributing 
to the progress of bacteriology, the number and variety of its students, 
has been a great hindrance. There are pathologists, chemists, brewers, 
botanists, all of whom are making species or varieties. Fischer does not 
claim that to any one class of investigators should be given the priv- 
ilege of systematizing the science, but that established principles 
should be followed. 
A student reviewing the systems which have been proposed for the 
classification of bacteria realizes that the problem is a difficult one. 
The number of systems which have been proposed supports this state- 
ment. Smith has said that no harm will come to anyone if all of these 
perplexing questions are not settled definitely within his own 
generation. 
The Society of American Bacteriologists has been wrestling with 
the classification of bacteria for some little time. The Committee 
(Winslow, 1917) has made an interesting report in which the more 
important classifications of the past have been discussed. This com- 
mittee has proposed the following eight families: 
Family 1. Nitrobacteriaccae. 
Family 2. Mycobacteriaceae. 
Family 3. Pseudomonadaceae. 
Family 4. Spirillaceae. 
Family 5. Coccaceae. 
Family 6. Bacteriaceae. 
Family 7. Lactobacillaceae. 
Family 8. Bacillaceae. 
The classification will probably demand much more study before a LIFE CYCLES OF BACTERIA 
99 
satisfactory one has been developed. Much more data may have to be 
secured with regard to the life histories of the bacteria. 
Bacteriological Groups. The arrangement of bacteria into physio- 
logical groups have been carried out merely for convenience. In 
some instances probably these groups are made up of very closely 
related organisms; in other cases a too insignificant characteristic 
has been taken as the standard. The first five groups of Fuller and 
Johnson's classification of water bacteria have been made on the basis 
of chromogenesis. Jordan also used this same characteristic when he 
systematized the bacteria which he isolated from the Illinois river. 
Rahn states, in Marshall's Microbiology, that this is a convenient and 
helpful method for handling the bacteria. As mentioned above many 
of the groups are arbitrary and often depend upon characteristics 
which are not fixed. Many of the groups need to be subjected to 
careful study. This will tell whether the groups approach natural 
units and, if they do, the relation and characters of the members. 
The groups of pathogenic bacteria and those which have sanitary 
significance have received careful study. 
Life Cycles of Bacteria. It has been the custom in bacteriology to re- 
gard bacteria as monomorphic. So firmly rooted has this idea become 
that few bacteriologists have given thought to the possibility of there 
being life cycles through which the bacteria pass. Involution form has 
been the term which has been applied to many of the forms which were 
regarded as 11 abnormal." In many cases these were explained as con- 
taminations and the culture discarded. That bacteria may possess more 
or less intricate life cycles has been proposed by different workers. 
When the data from these different sources are compared, one is im- 
pressed with the possibility that bacteriologists have been too hasty in 
excluding all of these abnormal forms. Lohnis (1916) has given this 
subject most intensive study. Some of the conclusions which this in- 
vestigator has reached are as follows: 
A comparative study of 42 strains of bacteria has shown that life 
cycles of these organisms are not less complicated than those of other 
microorganisms. As representatives of all groups of bacteria have been 
tested and all, without exception, behaved essentially in the same 
manner, in all probability analogous results may be expected with all 
species of bacteria. All bacteria live alternately in an organized and 
in an amorphous stage. The latter has been called the " symplastic " 
stage because at this time the living matter previously inclosed in 
the separate cell undergoes a thorough mixing, either by a complete 
disintegration of cell wall, as well as cell content, or by a " melting 100 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
together," of the content of many cells which leave their empty cell 
walls behind them. In the first case a readily stainable, in the latter 
case an unstainable " symplasm " is produced. Lohnis later states 
that all bacteria multiply not only by fission but by gonidia. These 
may become exospores, grow to full-sized cells, or enter the symplastic 
stage. Some of the gonidia are said to be filterable. Hort (1917) 
working independently in England studied B. tijphi and stated that 
simple binary fission was not the only method of reproduction. He 
had previously received similar evidence in a study of typhus fever. 
Rosenow (1917) in studying acute anterior poliomyelitis believed that 
he has observed a transmutation. All of these data from so many 
different sources point out the fact that bacteria may pass through 
different stages and that we are not justified in excluding all aberrant 
forms as contaminations. Should these data be further confirmed, 
the bacteriologist may find it necessary to alter his methods of analysis. 
The Descriptive Chart of the Society of American Bacteriologists. 
Bacteriologists, for some time, have been working out a system for 
classifying bacteria. The Descriptive Chart of the Society of American 
Bacteriologists represents a stage in the development of this effort. 
A history of the development of the card and the numerical system 
for recording the characters of bacteria has been prepared by Harding 
(1910), a very brief outline of which will be given here. 
Johnston (1895) first called attention to the possibility of using 
some such method as the Dewey decimal system for recording the 
important characteristics of bacteria. A committee which in its 
report suggested the beginnings of such a procedure was appointed. 
Conn adopted their suggestion and used such a system in his classifi- 
cation of dairy bacteria. A real attempt to use a group number such 
as our present one was made by Kendall (1903) at the Lawrence Experi- 
ment Station. This is reported by Gage and Phelps (1903). The 
genus classification of bacteria proposed by Migula was used as the 
basis for their report. 
The present chart has a group number depending on the determina- 
tion of ten characteristics. It has been recently described by Rahn 
and Harding (1916). This system of recording the characteristics of 
bacteria has not met with entire approval on the part of many of the 
workers. Ono of those who was instrumental in introducing this 
numerical system has said that it was a thing to be regretted. Whether 
it is or will be of any value may only be determined after it has been 
used to study certain groups. This, however, has been done by a very 
few bacteriologists. Harding (1910) reports the study of the Ps. Source Date of Isolation Name Group No 
Date of revivification Determined: When? By whom? Culture No 
DETAILED FEATURES 
Note.-Underscore required terms. Observe 
notes. 
Vegetative Cells, Medium used 
temp age days. 
Form, spheres, short rods, long rods, filaments, 
commas, short spirals, long spirals, spindled, 
cuneate, clavate, curved. 
Arrangement, single, pairs, chains, fours, cubi- 
cal packets. 
Limits of Size 
Size of Majority 
Ends, rounded, truncate, concave. 
Gelatin Colonies (continued). 
Elevation, flat, effuse, raised, convex, pulvinate, 
crateriform (liquefying). 
Edge, entire, undulate, lobate, erose, lacerate, 
fimbriate, filamentous, floccose, curled. 
Liquefaction, cup, saucer, spreading. 
Gelatin Stab. 
Growth, uniform, best at top, best at bottom. 
Line of puncture, filiform, beaded, papillate, 
villous, plumose, arborescent. 
Liquefaction, none, crateriform, napiform, in- 
fundibuliform, saccate, stratiform; begins 
in d, complete in d. 
Depth of liquefaction in tube of 10 mm. diam- 
eter, evenly inoculated at 20° C. for thirty 
days mm. 
Potato. 
Growth, scanty, moderate, abundant, transient, 
persistent. 
Form of growth, filiform, echinulate, beaded, 
spreading, plumose, arborescent, rhizoid. 
Elevation of growth flat effuse, raised, convex. 
Luster, glistening, dull, cretaceous. 
Topography, smooth, contoured, rugose, verru- 
cose. 
Chromogenesis Pigment in water 
insoluble, soluble; other solvents 
Odor, absent, decided, resembling 
Consistency, slimy, butyrous, viscid, membran- 
ous, coriaceous, brittle. 
Medium, grayed, browned, reddened, blued, 
greened. 
Potato Starch Jelly. 
Growth, scanty, copious, absent. 
Diastasic action, absent, feeble, profound. 
Medium stained 
Dextrine, present, absent. 
Temperature Relations. 
Optimum temperature for growth 20° C.; or 
37° C.; or °C. 
Maximum temperature for growth °C. 
Minimum temperature for growth °C. 
Cohn's Solution. 
Growth, copious, scanty, absent. 
Medium, fluorescent, nonfluorescent. 
Uschinsky's Solution. 
Growth, copious, scanty, absent. 
Fluid, viscid, not viscid. 
Loeffler's Blood Serum. 
Stroke, invisible, scanty, moderate, abundant. 
Form of growth, filiform, echinulate, beaded, 
spreading, plumose, arborescent, rhizoid. 
Elevation of growth, fat, effuse, raised, convex. 
Luster, glistening, dull, cretaceous. 
Topography, smooth, contoured, rugose, verru- 
cose. 
Chromogenesis 
Medium, grayed, browned, reddened, blued, 
greened. 
Liquefaction begins in d, complete in 
d. 
Milk. 
Clearing without coagulation. 
Coagulation, prompt, delayed, absent. 
Extrusion of whey begins in days. 
Coagulum, slowly peptonized, rapidly pepton- 
ized. 
Peptonization begins on .... d, complete on 
... .d. 
Reaction, Id... 2d... 4d... lOd... 20d... 
Consistency, slimy, viscid, unchanged. 
Medium, browned, reddened, blued, greened. 
Litmus Milk. 
Acid, alkaline, acid then alkaline, no change. 
Prompt reduction, no reduction, partial slow 
reduction. 
Indol Production, feeble, moderate, strong, 
absent. 
Hydrogen Sulphide, feeble, moderate, strong, 
absent. 
Ammonia Production, feeble, moderate, strong, 
absent, masked by adds. 
Nitrate in Nitrate Broth. Reduced, not 
reduced. 
Presence of nitrites Ammonia 
Presence of nitrates free nitrogen.... 
Nitrogen. Obtained from peptone, asparagin, 
glycocoll, urea, ammonia salts, nitrogen. 
Silicate Jelly (Fermi's Solution). 
Growth, copious, scanty, absent. 
Medium stained 
Best media for long-continued growth 
Quick test for differential purposes 
Endospores produced 100. 
Endospores not produced 200. 
Aerobic (Strict) 10. Facultative anaerobic 20. 
Anaerobic (Strict) 30. 
Gelatin liquefied 1. 
Gelatin not liquefied 2. 
Acid and gas .1. Acid without gas .2. 
No acid .3. No growth .4 with dextrose. 
Acid and gas .01. Acid without gas .02. 
No acid .03. No growth .04 with lactose. 
Acid and gas .001. Acid without gas .002. 
No acid .003. No growth .004 with sacchrose. 
Nitrates reduced with gas .0001. 
Nitrates reduced without gas .0002. 
Nitrates not reduced .0003. 
Fluorescent .00001. Violet .00002. Blue .00003. 
Green .00004. Yellow .00005. Orange .00006. 
Red .00007. Brown .00008. Pink .00009. 
Non-chromogenic .00000. 
Diastasic action on potato starch, strong .000001 
Feeble .000002 absent .000003. 
Acid and gas.0000001. Acid without gas.0000002 
No acid .0000003. No growth .0000004 with 
glycerine. 
BRIEF CHARACTERIZATION 
Diameter over Ip 
Chains, filaments 
Endospores 
Capsules 
Zoogloea, Pseudozoogloea 
Motile 
Involution forms 
Gram's Stain 
MORPHOLOGY | | CULTURAL FEATURES | BIOCHEMICAL FEATURES | DISTRIBUTION 
Committee on Revision of Chart Identification of Bacterial Spedes. 
Agar 
Hanging- 
Block 
Orientation (grouping) 
Chains (No. of elements) 
Orientation of Chains, parallel, 
irregular. 
Postfission movements, loop forming, folding, 
snapping, slipping. 
Sporangia, present, absent. Medium used 
temp age days. 
Form, elliptical, short rods, spindled, clavate, 
drumsticks. 
Limits of Size Size of Majority 
Broth | Agar I Gel. Plate | | Potato 
Cloudy, turbid 
Ring 
Pellicle 
Sediment 
Shining 
Dull _ 
Wrinkled 
Chromogenic 
Round 
Proteus-like 
Rhizoid 
Filamentous 
Curled 
Surface-growth 
Needle-growth 
Moderate, absent 
Abundant 
Discolored 
Starch destroyed 
Orientation (grouping) 
Chains (No. of elements) 
Orientation of Chains, parallel, 
irregular. 
Agar 
Hanging- 
Block 
Endospores, present, absent. 
Location of Endospores, central, polar. 
Form, spherical, elliptical, elongated. 
Limits of Size 
Size of Majority 
Wall, thick, thin. 
Sporangium wall, adherent, not adherent. 
Germination, equatorial, oblique, polar, bipolar, 
by stretching, by absorption of spore wall. 
Capsules, present on 
Zoogloea, Pseudozoogloea. 
Flagella, No Attachment, polar, bipolar 
lophotrichiate, peritrichiate. How Stained 
Involution Forms, on in.... days at 
°C. 
Staining Reactions. 
1 : 10 watery fuchsin, gentian violet, carbol 
fuchsin, Loeffler's alkaline methylene blue. 
Special Stains. 
Gram Glycogen 
Fat Acid fast 
Neisser 
Metachromatic granules, sporogenous gran- 
ules. 
Nutrient Broth. 
Surface growth, ring, pellicle, flocculent, mem- 
branous, none. 
Clouding, slight, moderate, strong, transient, 
persistent, none, fluid turbid. 
Odor, absent, dedded, resembling 
Sediment, compact, flocculent, granular, flaky, 
viscid on agitation, abundant, scant, none. 
Agar Stroke. 
Growth, invisible, scanty, moderate, abundant, 
none. 
Form of growth, filiform, echinulate, beaded, 
spreading, plumose, arborescent, rhizoid. 
Elevation of growth, flat, effuse, raised, convex. 
Luster, glistening, dull, cretaceous. 
Topography, smooth, contoured, rugose, verru- 
cose. 
Optical Characters, opaque, translucent, opales- 
cent, iridescent. 
Chromogenesis Photogenic, Fluorescent 
Odor, absent, dedded, resembling 
Consistency, slimy, butyrous, viscid, mem- 
branous, coriaceous, brittle. 
Medium, grayed, browned, reddened, blued, 
greened. 
Agar Colonies. 
Growth, slow, rapid, temperature 
Form, punctiform, drcular, irregular, ameboid, 
mycelioid, filamentous, rhizoid. 
Surface, smooth, rough, concentrially ringed, 
radiate, striate. 
Elevation, flat, effuse, raised, convex, pulvinate, 
umbonate. 
Edge, entire, undulate, lobate, erose, lacerate, 
fimbriate, floccose, curled. 
Internal structure, amorphous, finely-, coarsely- 
granular, grumose, filamentous, floccose 
curled. 
Gelatin Colonies. 
Growth, slow, rapid. 
Form, punctiform, drcular, irregular, ameboid, 
mycelioid, filamentous, rhizoid. 
Prepared by H. A. Harding 
H. J. Conn 
I. J. Kligler 
W. D. Frost 
M. J. Prucha 
1. Fermentation-tubes 
containing peptone- 
water or sugar-free 
bouillon. 
At °C. 
Gas production, in % 
Growth in closed arm 
Amt. of acid produced d. 
Amt. of acid produced d. 
Amt. of acid produced d. 
| Dextrose 
| Lactose 
| Saccharose 
| Glycerine 
Grows at 37° C. 
Grows in Cohn's Sol. 
Grows in Uschinsky's Sol. 
DESCRIPTIVE CHART 
SOCIETY OF AMERICAN BACTERIOLOGISTS 
Endorsed by the Sgdety for General Use at the Annual Meeting, Dec. 31, 1914 
Pathogenic to Animals. 
Insects, crustaceans, fishes, reptiles, birds, mice, 
rats, guinea pigs, rabbits, dogs, cats, sheep, 
goats, cattle, horses, monkeys, man 
Serum reactions, agglutinins, bactericidins, 
opsonins, precipitins, complement deviation 
Products of growth, soluble toxins, endotoxins. 
Pathogenic to Plants. 
Plants affected 
Products of growth 
Loss of Virulence on Culture Media, 
prompt, gradual, not observed in months. 
Acids Produced 
Alkalies Produced 
Alcohols Produced 
Ferments: peptase, tryptase, chymase, diastase, 
invertase, pectase, cytase, tyrosinase, oxidase, 
perooxidase, reductase, catalase, lipase, 
glucase, galactase, etc 
Crystals formed 
Toleration of Acids; Great, medium, slight. 
Acids tested 
Toleration of Alkalies: Great, medium, slight. 
Alkalies Tested 
Optimum reaction for growth in bouillon, 
stated in terms of Fuller's scale 
Killed readily by drying: resistant to drying. 
Per cent killed by freezing (salt and crushed ice 
or liquid air) .••••■. 
Sunlight: Exposure on ice in thinly sown agar 
plates: one-half plate covered (time fifteen 
minutes), sensitive, not sensitive. 
Per cent killed 
Vitality on culture media: brief, moderate, 
long. 
Thermal death-p int (ten minutes' exposure in 
nutrient broth when this is adapted to 
growth of organism) °C. 
Liquefaction : Milk 
Gelatin 
Blood-serum 
Casein 
Agar, mannan 
Acid curd 
Rennet curd 
Casein peptonized 
Indol 
Hydrogen sulphide 
Ammonia 
Nitrates reduced 
Fluorescent 
Luminous 
Animal pathogen, epizoon 
Plant pathogen, epiphyte 
Soil 
Mik 
Fresh water 
Salt water 
Sewage 
Iron bacterium 
Sulphur bacterium 
To face page 100. THE DESCRIPTIVE CHART 
101 
campestris group. For each strain the same group number was obtained 
which would indicated that it was well fitted to these bacteria. This 
author believes that it may not " carry the separation to a group 
synonymous with the ordinary conception of species." This statement 
seems to have been borne out by later investigations. Harding and 
Prucha (1908) used the Descriptive Chart in the study of bacteria 
from cheddar cheese. In their conclusions, they state that this method 
of recording the reaction of cultures is a marked advance in technique, 
and that shifts in the cheese flora may be thus traced more accurately. 
The sum and substance of their opinion of the Chart in its application 
to the study of cheese bacteria is that it is a valuable means to an end. 
Harding, Morse and Jones (1909) in their study of soft rot organisms 
used the group number. By examining their data it is apparent that 
the group number is a valuable aid. Harding reached essentially the 
same conclusion in 1910 when he studied the same group of bacteria. 
Conn .(1906) when classifying dairy bacteria drew no conclusions with 
regard to the value of the rudimentary group number which he used. 
More recently H. J. Conn (1915) reported the study of 130 strains of 
B. subtilis by means of the Descriptive Chart. In selecting these 
strains one-half of the determinations represented in the group number 
are fulfilled because they were implied in the definition of B. subtilis. 
He stated that different group numbers did not always represent dif- 
ferent species, and that better methods for making these ten deter- 
minations should be devised. This worker later used the Descriptive 
Chart in another investigation involving the study of about 1000 pure 
strains, isolated from soil (Conn, 1917). The Chart was found to be 
quite well adapted to the study of spore forms and was of value in the 
" preliminary study of other forms before learning what special tests 
were best adapted to them." The following tests were reported to 
give sufficiently consistent results to be of diagnostic value: shape of 
vegetative forms, size of vegetative forms, arrangement of flagella 
when present, presence of spores, size and shape of spores, growth in 
the absence of oxygen liquefaction of gelatin, nitrate reduction, chromo- 
genesis and form of growth in liquid and solid media. At times some 
of these gave inconsistent results. Edson and Carpenter (1912) made 
no statement in their study on sap bacteria about the value of the 
Descriptive Chart. Tanner (1918) used the Chart in the study of 
green fluorescent bacteria from water and found the Chart a very 
convenient method for carrying out such a study. 
Description of Microorganisms. The Descriptive Chart of the 
Society of American Bacteriologists has been given rather extended 102 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
FLAT 
TYPES OF SURFACE ELEVATION OF COLONIES 
RAISED 
CONVEX 
PULVINATE 
CAPITATE 
UMBILICATE 
UMBONATE 
ENTIRE 
UNDULATE 
REPAND 
LOBATE 
AURICULATE 
LACERATE 
Cl DATE 
EROSE 
TYPES 
OF MARGIN OF COLONIES 
FIMBRIATE 
AREOLATE 
GRUMOSE 
MORULOID 
CLOUDED 
GYRO5E 
MARMORATED 
RETICULATE 
TYPES OF INTERNAL STRUCTURE OF COLONIES 
Fig. 33.-Showing the Various Types of Elevation, Margins, and Internal Structure 
of Colonies. FORMS OF GROWTH 
103 
CRATERIFORM 
NAPIFORM 
INFUNOIBULI 
SACCATE 
STRATIFORM 
nPES OF LIQUEFACTION IN GELATIN STAB CULTURES 
FILIFORM 
ECHINULATE 
BEADED 
EFFUSE 
PLUMOSE 
ARBORESCENT 
RHIZOID 
FORMS OF GROWTH ON STREAK CULTURES 
Fxg, 34, 104 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
discussion in the previous paragraph. From this it will be seen that 
those who have really used the Chart regard it as an aid in the study 
of bacteria. Barring certain recognized limitations it is a convenient 
1 
f 
2 
5 
3 
4 
6 
7 
8 
9 
Structure of colonies: 1, conglomerate colony; 2, toruloid colony; 3, alveolate structure; 4, 
grumose in center; 5, moruloid; 6, clouded; 7, reticulate; 8, marmorated; 9, gyrose. 
(From Moore's Laboratory Directions for Beginners in Bacteriology, Ginn & Co., 1905.) 
1 
2 
3 
4 
5 
6 
Types of colonies: 1, cochleate; 2, amoeboid; 3, rhizoid; 4, mycelioid; 5, filamentous; 6, 
curled structure. 
(From Moore's Laboratory Directions for Beginners in Bacteriology, Ginn & Co., 1905.) 
Fig. 35.-Showing Characteristics of Colonies. These should not he regarded as 
absolutely constant. Other terms may be used which will more correctly 
describe a colony growth. 
method for recording the salient characteristics of bacteria. Many 
of those who criticize it most severely have never used it in any extended 
research. The student will find the Chart a convenient factor in the GLOSSARY OF TERMS 
105 
study of bacteria from foods. The following glossary of terms has 
been taken from the back of the 1912 Chart: 
Glossary of Terms 
Adherent, applied to sporangium wall, indicates that remnants of sporangium 
remain attached to endospore for some time. 
Aerobic, growing in the presence of free oxygen; strictly aerobic, growing only 
in the presence of free oxygen. 
Ameboid, assuming various shapes like an ameba. 
Amorphous, without visible differentiation in structure. 
Arborescent, a branched, tree-like growth. 
Anaerobic, growing in the absence of free oxygen; strictly anaerobic growing 
only in the absence of free oxygen; facultative anaerobic, growing both in 
presence and absence of free oxygen. 
Beaded, in slat or stroke culture, disjointed or semi-confluent colonies along the 
line of inoculation. 
Bipolar, at both poles or ends of the bacterial cell. 
Brief, a few days, a week. 
Brittle, growth, dry, friable under the platinum needle. 
Bullate, growth rising in convex prominences, like a blistered surface. 
Butyrous, growth of a butter-like consistence. 
Chains, four or more bacterial cells attached end to end. 
Chromogenesis, the production of color. 
Ciliate, having fine hair-like extensions, sometimes not visible to the naked 
eye, resembling cilia. 
Clavate, club shaped. 
Cloudy, said of fluid cultures which might indicate growth. 
Coagulation, the separation of casein from whey in milk. 
Contoured, an irregular, smoothly undulating surface, like that of a relief map. 
Convex, surface the segment of a sphere. 
Coprophyl, dung bacteria. 
Coriaceous, growth tough, leathery, not yielding to the platinum needle. 
Crateriform, a saucer shaped liquefaction of the medium. 
Crecateous, growth opaque and white, chalky. 
Cuneate, wedge-shaped. 
Curled, composed of parallel chains in wavy strands, as in anthrax colonies. 
Diastatic action, conversion of starch into simpler carbohydrates, such as dex- 
trins or sugars, by means of diatase. 
Echinulate, a growth along line of inoculation with toothed or pointed margins. 
Effuse, growth thin, veily, unusually spreading. 
Endospores, thick-walled spores formed within the bacterial cell; i.e., typical 
bacterial spores like those of B. anthracis or B. subtilis. 
Entire, with an even margin. 
Erose, border irregularly toothed. 106 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
Filaments, applied to morphology of bacteria, refers to thread-like forms, 
generally unsegmented; if .segmented, to be distinguished from chains 
(q.v.) by the absence of constrictions between the segments. 
Filamentous, growth composed of long, irregularly placed or interwoven threads. 
Filiform, in stroke or stab cultures, a uniform growth along line of inoculation. 
Fimbriate, border fringed with slender processes, larger than filaments. 
Floccose, growth composed of short curved chains, variously oriented. 
Flocculent, containing small adherent masses of bacteria of various shapes 
floating in the culture fluid. 
Fluorescent, having one color by transmitted light and another by reflected light. 
Grain's stain, a method of differential bleaching after gentian violet, methyl 
violet, etp. The + mark is to be given only when the bacteria are deep 
blue or remain blue after counter-staining with Bismarck brown. 
Grumose, clotted. 
Granular, composed of small granules. 
Infundibuliform, form of a funnel or inverted cone. 
Iridescent, exhibiting changing rainbow colors in reflected light. 
Lacerate, having the margin cut into irregular segments as if torn. 
Lobate, having the margin deeply undulate, producing lobes (see undulate). 
Long, many weeks or months. 
Luminous, glowing in the dark, phosphorescent. 
Maximum temperature, temperature above which growth does not take place. 
Medium, several weeks. 
Membranous, growth thin, coherent, like a membrane. 
Minimum temperature, temperature below which growth does not take place. 
Mycelioid, colonies having the radiately filamentous appearance of mold 
colonies. 
Napiform, liquefaction in form of a turnip. 
Nitrogen requirements, the necessary nitrogenous food. This is determined 
by adding to nitrogen-free media the nitrogen compound to be tested. 
Opalescent, resembling the color of an opal. 
Optimum temperature, temperature at which growth is most rapid. 
Papillate, growth beset with small nipple-like processes. 
Pellicle, bacterial growth forming either a continuous or an interrupted sheet 
over the culture fluid. 
Peptonization, rendering curdled milk soluble by the action of trypsin. 
Peritrichiate, covered with flagella over the entire surface. 
Persistent, lasting many weeks or months. 
Plumose, a fleecy or feathery growth. 
Polar, at the end or pole of the bacterial cell. 
Pseudozoogloea', clumps of bacteria, not dissolving readily in water, arising 
from imperfect separation, or more or less fusion of the components but 
not having the degree of compactness and gelatinization seen in zoogloea?. 
Pulvinate, decidedly convex, in the form of a cushion. 
Punctiform, very small, but visible to naked eye; under 1 min. in diameter. GLOSSARY OF TERMS 
107 
Radiate, showing ray-structure. 
Raised, growth thick, with abrupt or terraced edges. 
Reduction, removing oxygen from a chemical compound. Refers to the 
conversion of nitrate to nitrite, ammonia, or free nitrogen, and to the de- 
colorization of litmus. 
Rhizoid, growth of an irregular branched or root-like character, as in B. mycoides. 
Ring, growth at the upper margin of a liquid culture, adhering to the glass. 
Repand, wrinkled. 
Rapid, developing in twenty-four to forty-eight. 
Rugose, wrinkled. 
Saccate, liquefaction in form of an elongated sac, tubular, cylindrical. 
Scum, floating islands of bacteria, an interrupted pellicle or bacterial membrane. 
Slow, requiring five or six days for development. 
Short, applied to time, a few days, a week. 
Spindled, larger at the middle than at the ends. Applied to sporangia, refers 
to the forms frequently called clostridea. 
Sporangia, cells containing endospores. 
Spreading, growth extending much beyond the line of inoculation, i.e., several 
millimeters or more. 
Stratiform, liquefying to the walls of the tube at the top and then proceeding 
downwards horizontally. 
Thermal Death-point, the degree of heat required to kill young fluid cultures 
of an organism exposed for ten minutes (in thin-walled test tubes of a diam- 
eter not exceeding 20 mm.) in the thermal water-bath. The water must 
be kept agitated so that the temperature shall be uniform during the 
exposure. 
Transient, lasting a few days. 
Truncate, ends abrupt, square. 
Turbid, cloudy with flocculent particles, i.e., cloudy plus flocculence. 
Umbonate, having a button-like, raised center. 
Undulate, border wavy, with shallow sinuses. 
Verrucose, growth wart-like, with wart-like prominences. 
Vermiform-contoured, growth like a mass of worms, or intestinal coils. 
Villous, growth beset with hair-like extensions. 
Viscid, growth follows the needle when touched and withdrawn; sediment 
on shaking rises as a coherent swirl. 
Zoogloeae, firm gelatinous masses of bacteria, one of the most typical examples 
of. which is the streptococcus mesenterioides of sugar vats (leuconostoc 
mesenterioides), the bacterial chains being surrounded by an enormously 
thickened firm covering inside of which there may be one or many groups of 
the bacteria. 
Suggested Procedure for Studying Bacteria According to the 
Descriptive Chart of the Society of American Bacteriologists. The 
following procedures are suggested for use with the Descriptive Chart. 108 
CLASSI FIXATION AND DESCRIPTION OF BACTERIA 
One of Ilie factors which has inhibited a more extended use of the Chart 
has been the lack of standard methods to use with it. Several of the 
tests need more study. The Committee on the Chart for the Identi- 
fication of Bacterial Species of the Society of American Bacteriologists 
h is made a preliminary report on methods to be used with the Chart 
(Conn, II. J., 1918). Since their report represents the attempt on the 
part of an organized society to standardize technique, parts of their 
report will be reproduced here. 
Media. The importance of using carefully prepared media does 
not need emphasis. Greatest care should be used in adjusting the 
reaction to true neutrality by means of brom thymol blue. All media 
should be prepared after the Standard Methods of the American Public 
Health Association. 
Invigoration of Cultures. The Committee* has made the following 
recommendation with regard to this important factor: 
Provided a medium can be found upon which the organism to be studied 
grows vigorously, it should be invigorated before study, even though freshly 
isolated from its natural habitat. The procedure to be employed is as follows: 
Prepare duplicate subcultures in standard gluclose broth, and on standard agar 
slopes, placing cultures of each at 37° and 25°. On the basis of resulting growth 
the organism falls into one of the following series: 
Series I. Organisms which produce good growth (surface growth, dis- 
tinct turbidity, or heavy precipitate) in twenty-four hours at 37° in glucose 
broth. 
Series II. Organisms which do not produce good growth in twenty-four 
hours as above, but do in forty-eight hours at 25° in glucose broth. 
Series III. Organisms which do not grow well in glucose broth but do 
produce good growth on the surface of agar in twenty-four hours at 37°. 
Series IV. Organisms excluded from the above groups but which produce 
good growth on the surface of agar in forty-eight hours at 25° C. 
Record the series number on the chart at the proper place and proceed 
with the invigoration by inoculating into another tube of glucose broth for 
organisms of Series III and IV. Incubate this tube at the temperature, and 
for the time, called for in the series which it belongs; then transfer from this 
tube to a third tube and incubate as before. From this third culture make a 
gelatin or agar plate and incubate at the temperature previously used until 
colonies of sufficient size for isolation are obtained. 
Transfer from a typical colony to one or more agar slants and incubate 
one day at 37° or for two days at 25° according to the temperature relation of 
the organism studied. 
* The term " Committee " when used in this chapter will refer to the Committee 
on the Chart for Identification of Bacterial Species of the Society of American Bac- 
teriologists. PURE CULTURE METHODS 
109 
In case the organism does not produce growth on either of these media at 
either temperature, it should be invigorated with any medium and at any tem- 
perature known to be adapted to its growth. Under such circumstances, 
invigorate by the procedure just outlined but using the medium and tempera- 
ture found most favorable for the organism in question, recording on the chart 
the method of invigoration adapted. If no conditions are known under which 
the organism in question produces vigorous growth, it should be studied without 
preliminary cultivation as soon as possible after isolation from its natural 
habitat. Such an organism is not likely to give good growth on any ordinary 
media, and the results of the study called for by the chart will have little 
significance. 
Motility. Some care is necessary in interpreting the results of 
motility determinations. Positive results are satisfactory but negative 
results are convincing oniy after a number of attempts. The safest 
method to follow is to make flagella stains for if the organism possesses 
flagella, it is reasonable to assume that it is motile. The Committee 
states that even negative results do not absolutely prove that the 
organism is immotile. 
Vegetative Cells. Some bacteria go into the spore stage so quickly 
that in order to secure vegetative cells, as young a culture as possible 
should be used. For making the various determinations it is necessary 
to have a young and vigorous culture. This may be secured by passing 
it through a process of rejuvenation which should consist of continued 
transferring to a medium which is best adapted for growth. It may be 
necessary to determine the optimum temperature and incubate all 
cultures at this temperature. 
Endospores. These may usually be observed on the smears which 
have been stained with the ordinary alcoholic-aqueous stains. On 
account of a different type of protoplasm, possibly, they resist the 
staining process and appear on the smear as colorless bodies usually 
round or oval in shape. In some cells they will be observed in the 
ends or middle. In this case the cell has not decomposed to set them 
free. Special stains may be used for indicating the presence of spores, 
the procedure for which has been given in the Chapter on Staining. 
Very often these special methods are not attended with much success. 
Another method which is often reliable is to kill the vegetative 
cells by heating in broth culture for thirty minutes at 80° C. If after 
this procedure growth is secured by subculturing into other media, 
it is good indication that the culture was a spore former. 
Capsules. The presence of capsules may be indicated either by 
special staining procedures or the growth in the common media. The 110 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
special staining procedures have been described in the Chapter on 
Staining. Capsulated bacteria usually grow with a sticky, slimy growth 
which when touched with a needle, will pull out into a thread. In 
liquid media the sediment in the bottom of the culture tube will often 
show a slimy appearance. On the ordinary smears stained with aque- 
ous alcoholic solutions of the aniline dyes, the capsulated bacteria 
often show a halo about each cell. This seems to resist the staining 
procedure and may often be made more visible by slightly moving the 
fine adjustment of the microscope. 
Staining Reactions. The theory of staining and the preparation 
of smears has been fully described elsewhere. To make a complete 
study of the staining properties of an organism, all of the stains men- 
tioned on the Descriptive Chart should be used. The common special 
stains, such as the acid-fast, should also be tried. 
Oxygen Relations. The relation to oxygen may be determined 
from the closed arm of the fermentation tubes. Before using, however, 
it is advisable to reduce the dissolved oxygen to a minimum. This 
may be done either by a vacuum pump or boiling. 
The incubation of streak plates by means of Torrey's method will 
give good results. Also agar streaks of the organism may be incubated 
in a Novy jar. 
Nutrient Broth. After this medium has been inoculated from a 
young culture of the organism, it should be incubated at the optimum 
temperature for the organism. The characteristics of growth should 
be recorded on the Chart and the culture saved for other tests as here- 
after described. Before these observations are made the tube should 
be shaken as little as possible in order not to destroy any ring or pellicle 
formation. 
Agar Stroke. Streak the agar slant in a straight line from the 
bottom of the slant to the top. Care should be exercised not to cut 
through the surface of the slant. If the inoculum is deposited 
within the agar when it is intended for the surface anaerobic 
conditions may be established. With freshly prepared agar streaks, 
in the bottom of which is water of condensation, care must be 
used not to lay them flat on the desk since a spreading growth will 
usually result. 
Agar Colonies. Pour agar plates with a dilution which will allow 
few colonies on the plate. Ten to twenty are sufficient when cultural 
characteristics are to be determined. Spreading colonies should be 
avoided either by inverting the plates during incubation or the use of 
Hill's porous covers. PURE CULTURE METHODS 
111 
Gelatin Colonies. See the directions given for agar colonies. If 
the culture is a liquefier, observations should be made at frequent 
intervals before the colonies have spoiled the plate. 
Gelatin Stab. A straight needle should be used and the tube 
stabbed to the bottom. It should be incubated at 20° C. and frequent 
observations made to determine the amount of liquefaction. The 
amount of liquefaction may be determined by means of a millimeter 
rule. The Descriptive Chart adopted at the 1907 meeting of the 
Society of American Bacteriologists recommended that gelatin tubes 
should be held for six weeks to determine liquefaction. This is a test 
upon which some work must be carried out in order to secure better 
methods for determining gelatin liquefaction in the shortest time. 
There is little trouble or error involved in reporting positive results 
but the time is an important element with negative results. Some 
bacteria w'ill liquefy gelatin only after several months. This indicates 
that another method, by which to more sharply distinguish between 
the liquefiers and the non-liquefiers, is needed. 
The Committee has proposed to make the method of Rothberg 
(1917) provisional until it may be tried out. Rothberg gives the 
organism a preliminary cultivation in a 1 per cent gelatin solution at 
25° or 37°; then inoculate the surface of gelatin in a test tube and 
incubate for 15 days at 20° C. 
Potato. The inoculation of potato slants should be made in the 
same way as for agar slants. After the observations called for on the 
Descriptive Chart have been made, the potato may be tested for 
diastasic action according to the directions given under " Potato 
Starch Jelly." 
Potato Starch Jelly. Iodine should be used to determine the presence 
of diastasic action. Pour the contents of the culture tube into a beaker 
and dilute with distilled water. If the dilution is sufficient and if the 
organism possesses a diastase, the various colors of the starch decom- 
position products with iodine will be obtained. Some of these are as 
follows: 
Starch blue color with iodine 
Soluble starch blue color with iodine 
Erythordextrin red color with iodine 
Achroodextrin no color with iodine 
Maltose no color with iodine 
Dextrose no color with iodine 112 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
To secure success with this method both the iodine and starch solutions 
must be very dilute. The same method has been applied to potato 
slants. In this procedure the slants are thoroughly mashed and after 
dilution with water are treated as above. 
Allen (11)18) has described another convenient method which has 
had rather extended use. This involves the use of a 0.2 per cent thymol 
starch agar which is poured into a sterile Petri dish and allowed to 
harden. Streaks are made on this and after incubation the dish may be 
flooded with iodine solution. A clear halo about the growth indicates 
diastasic action on starch. In order to satisfy the requirements of the 
Descriptive Chart Allen has stated that a clear zone of more than 2 mm. 
width should be regarded as strong while a feeble action is denoted by 
a width of less than 2 mm. This differentiation called for on Descrip- 
tive Chart is rather ambiguous since to distinguish between feeble 
and strong diastasic action is probably unnecessary. 
Edson and Carpenter (1912) to determine the presence or absence 
of diastasic action added a 2 per cent thymol starch paste to a 10-day- 
old broth culture. After incubation for about eight hours, the culture 
was tested for reducing sugars by means of Fehling's solution. 
Temperature Relations. The determination of this characteristic 
has been usually limited to 37° C. and 20° C. Other temperatures may 
be used as desired. The organism should be inoculated into a medium 
in which it will grow well. This culture should then be incubated at 
different temperatures. The possibility of other factors inhibiting 
growth should be carefully guarded against. It may be necessary to 
study this characteristic before starting the other culture work in 
order to determine the optimum temperature. 
Cohn's and Uschinsky's Media. After inoculation they should 
be incubated at the optimum temperature. The observations which 
should be made are mentioned on the Chart. These media may also 
be used as a base to which other compounds, such as fats, proteins, etc:, 
may be added. 
Loeffler's Blood Serum. Treat in the same way as described for 
" Agar Stroke." Make the necessary records on the Chart. 
Milk. Freshly skimmed milk should be sterilized either in test 
tubes or Erlenmeyer flasks. The various observations should be 
recorded on the Chart. The proteolysis of casein may be determined 
either by the appearance of the tube or by means of the Hastings (1904) 
milk agar plate. For preparing this milk agar plate, sterile skimmed 
milk is added to melted plain agar which has been cooled to 50° C. 
and poured into a sterile Petri dish to harden. The strain which is STARCH AGAR PLATES 
113 
Fig. 36.-Showing Strong, Feeble and Weak Diastasic Action by Bacteria. (After 
Allen, 1918). 114 
CLASSIFIGATION AND DESCRIPTION OF BACTERIA 
being studied should be streaked across the surface of the agar. After 
incubation a clear zone will be visible, which, if due to true proteolysis, 
will not be rendered opaque if treated with weak acid. If a clear zone is 
present after incubation, it may be due to either proteolysis or the 
acid formed in the metabolism of the organism. To prove that proteo- 
lytic enzymes produced it, flood the plate with dilute acetic acid and 
if the clear zone remains, the presence of a protease is indicated. 
The Descriptive Chart calls for the determination of the reaction 
on certain days. In the past, this has been determined by titrating 
5 c.c. of the culture with N/20 NaOH or HC1. That this method is 
not accurate has been shown by Clark and Lubs. In order to take 
advantage of their work the Committee has advised the following 
procedure: 
Acid production in milk can be detected by adding brom cresol purple to 
the culture and comparing with the color obtained by adding the same propor- 
tionate quantity of the indicator to sterile milk. (Brom thymol blue does not 
give satisfactory results in milk.) Four degrees of acidity that can be recog- 
nized in milk are listed in Table II. They correspond closely to those listed in 
Table I, differing only on that brom cresol purple is used instead of brom thymol 
blue to show "neutrality" and that the curdling point (Ph = 4.7) is used to 
separate between " moderate " and " strong " acidity instead of the less 
definite point of maximum red to methyl red. The same method of expression 
used in recording acidity in clear media should be used in recording that of 
milk. 
Table VI 
DEGREES OF ACIDITY EASILY RECOGNIZED IN MILK 
(After Conn el al., 1918) 
Acidity. 
Indicator Reaction, etc. 
Approximate 
Value. 
Neutral 
Same color with brom cresol purple * as sterile milk; 
i.e., blue to gray green. 
6.2-6.8 
Weak 
Color with brom cresol purple lighter than in sterile 
milk; i.e., gray green to greenish yellow. 
5.2-6.0 
Moderate 
Yellow with brom cresol purple. Not curdled 
4.7-5.0 
Strong 
Curdled. Blue or green to brom phenol blue 
3.2-4.6 
Very strong.. . . 
Yellow to brom phenol blue 
Under 3.0 
* Use a 0.04 per cent solution. PURE CULTURE METHODS 
115 
Table VII 
SHOWING DEGREES OF ACIDITY EASILY RECOGNIZED IN CLEAR 
MEDIA 
(After Conn et al., 1918) 
Acidity. 
Indicator Reactions. 
Approximate 
Ph Value. 
Neutral 
Blue or green to brom thymol blue.* 
Over 6.2 
Weak 
Yellow to brom thymol blue 
Purple to brom cresol purple.* 
5.2-6.0 
Moderate 
Yellow to brom cresol purple 
Orange to methyl red.f 
Maximum red to methyl red. 
4.6-5.0 
Strong 
Blue or green to brom phenol blue.* 
3.2-4.4 
Very strong.. . . 
Yellow to bromphenol blue 
Under 3.0 
* Use a 0.04 per cent alcoholic solution, 
t Use a 0.02 per cent alcoholic solution. 
Litmus Milk. Growth in this medium is not much different from 
that in plain milk. If the tube turns to a white with a pink layer 
at the surface which is in contact with air, the presence of a reductase 
for litmus is indicated. Clark and Lubs (1917) have stated that brom 
cresol purple may be used as a substitute for litmus in milk. The 
committee regards this as not always to be recommended since this 
dye does not show the reduction phenomena. 
Indol Production. Indol is formed from tryptophane and is pro- 
duced in media which contains proteins or their split products con- 
taining this amino acid. Under routine conditions the production of 
indol may be determined in Dunham's medium or in the plain broth 
culture. Zipfel (1912) has shown that trytophane in an inorganic 
medium will show indol formation in twenty-four hours. Since tryp- 
tophane is so expensive Cannon (1916) has prepared a hydrolyzed 
casein medium with which it is claimed that good indol formation is 
secured. This is prepared according to directions which are given in 
the Chapter on Media. The following tests for indol may be used: 
Nitroso-Indol Nitrate Test: Add 0.5 c.c. of concentrated sulphuric 
acid and 0.5 c.c. of dilute sodium nitrite to the culture. If a red color 
is obtained the presence of indol is indicated. 
Ehrlich's Test: Add 0.5 c.c. of paradimethylamidobenzaldehyde 
to the culture and if'indol is present, a deep crimson color will be 
formed. 116 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
Hydrogen Sulphide. Hydrogen may be formed from many of the 
sulphur-containing compounds. When formed from proteins it probably 
comes from cystine or some other sulphur linkage. It may be detected 
by suspending a strip of bibulous paper saturated with lead acetate to 
which a little glycerol has been added. This method has been found 
to yield satisfactory results by the author. 
Ammonia Production. Dilute the culture with ammonia-free water 
and add 1 c.c. of Nessler's reagent. The presence of a yellowish-green 
color indicates the formation of ammonia. 
Nitrate in Nitrate Broth. Ammonia, see above. Nitrate. The 
amount of nitrate decomposed by an organism may be determined 
quantitatively by the aluminum-reduction method which has been 
outlined in the Chapter on Water Analysis. For qualitative results 
the presence of nitrites or ammonia in a medium which was free from 
these substances before inoculation may be taken as sufficient evidence 
of nitrate reduction. 
Test for Nitrites. The same method may be used for the qualita- 
tive estimation of the presence of nitrites that is used in the quantitative 
determination. The Griess method has had much application and 
depends on the formation of azobenzolnaphthylamin whenever naph- 
thylamin and sulphanilic acid are present in an acid solution of nitrites. 
Mason (1912) gives the reaction as follows: 
N=N 
0 I 
NH2-CgH4-HSO3 + HNO2 = CGH4 II I 4- 2H2O 
0 
- N-N H II - N N 
9 1 h/V^h 
CgH4 + , 
II u \/ 
o nh2 h hso3 nh2 h 
This is a very delicate test and is regarded by a few bacteriologists 
as too delicate for bacteriological work. If a control tube is made 
accurate results should be secured by its use. 
To determine nitrites, dilute 2 or 3 c.c. of the culture with ammonia- 
free water and add | c.c. each of sulphanilic acid and naphthylamin 
hydrochloride. The presence of a red color when viewed the long way 
of the Nessler tube indicates the presence of nitrites. PURE CULTURE METHODS 
117 
Smith (1905) regards the iodine starch test as most satisfactory for 
microbiologists. He gives the procedure.as follows: " Twenty-five c.c. 
of distilled water are added to | gm. (more or less) of pure potato 
starch and the fluid boiled. One cubic centimeter of this starch water 
and 1 c.c. of freshly prepared potassium iodide water (1:250) are now 
put into the culture fluid, to which is then added a few drops of strong 
sulphuric acid water (2:1). If any appreciable quantity of nitrite is 
present the culture immediately becomes blue black from the libera- 
tion of free iodine which acts upon the starch. Old potassium iodide 
should never be used without first testing carefully as it usually con- 
tains some free iodine." Blank determination should be made. 
Silicate Jelly. Make the required observations and record on the 
Chart. 
Fermentation Reactions. The various compounds upon which it is 
desired to study the action of microorganisms, are added to plain broth. 
Great care should be used in the sterilization of these media since 
Mudge (1917) and Hasseltine (1917) have shown that polysaccharides 
are easily hydrolyzed to monosaccharides. The latter of these investi- 
gators found that serious errors were introduced in the study of B. 
proteus, for instance, on common carbohydrates. For investigations 
requiring accurate data other methods of sterilization than moist heat 
should be used. The broth may be sterilized by filtration after the 
carbohydrates have been added, or the carbohydrate solutions may be 
sterilized by filtration and added to the broth by means of a sterile 
pipette just before the tubes are inoculated. In determinating the 
reaction of the fermentation tubes, it has been the custom to titrate 
5 c.c. of the medium with N/20 NaOH or HC1 with phenolphthalein 
as the indicator. The work of Clark and Lubs has shown that this 
method does not allow accurate results. They have advised the deter- 
mination of H-ion concentration, but such a procedure is not adapted 
to routine work on account of the time consumed. In place of this, 
these workers have prepared a series of indicators with a sufficient 
range to meet the requirements of bacteriological media. These 
should be used where it is possible. They are given in the appendix. 
Pathogenicity to Animals. The suspected organism should be fed 
to different laboratory animals and also injected into different tissues. 
The animal should be kept under observation for a short period before 
the experiment begins. After injection, it should be constantly watched 
and weighed. 
The Committee recommends the use of these indicators and makes 
the following statement: 118 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
In careful research work the exact shade of the indicator should be compared 
with that obtained in standard " buffer " solutions, and the results recorded 
in terms of Ph. In laboratories where these solutions cannot be obtained, it is 
better to record the results as simply + or - according to the reaction of the 
culture to litmus, than to use the titration method. Under such conditions it 
is possible, however, to obtain a rough idea of the hydrogen-ion concentration 
by the use of Clark and Lubs' series of indicators without making accurate 
determinations of Ph. Four different degrees of acidity can be easily distin- 
guished by this simple method in sugar broth with initial reaction of neutrality. 
The indicator reactions for these different degrees of acidity are listed in table 
II together with the approximate range of Ph to which each corresponds. In 
the absence of accurate determinations, these degrees of acidity may be recorded 
by the indefinite terms, " weak," " moderate," " strong," and " very strong," 
or by the symbols +, ++, + ++, and + + + + . 
If this procedure is used in order to secure data, it is possible that 
a more satisfactory method may be developed whereby the actual 
acidities of the culture tubes may be defined. 
Pathogenicity to Plants. Apply a broth culture of the organism 
to that part of the plant for which it is supposed to be specific. 
Loss of Virulence on Culture Media. The loss of any characteristic 
may be determined by comparing it before and after prolonged culti- 
vation on artificial media. Attempts should also be made to bring it 
back after it has been demonstrated to have been lost. 
Ferments. The presence of enzymes may be determined by the 
decompositions which the organisms accomplish. In searching for the 
presence of any special enzyme, a substrate containing the special 
compound should be used. Frankel's medium or any other such 
medium may be used as a base. 
Toleration to Acids or Alkalis. This characteristic may be deter- 
mined by adding different amounts of normal acid to alkali to plain 
broth before inoculating with the organism. Quite often a large 
amount of acid or alkali may precipitate the proteins which are in 
the media. This precipitation must not be confused with growth. 
Thermal Death-point. The Committee on Identification of Species 
of the Society of American Bacteriologists has recommended the expo- 
sure of the organism in nutrient broth for ten minutes. The medium 
should be prepared according to standard methods and the time very 
carefully limited to ten minutes. BIBLIOGRAPHY 
119 
BIBLIOGRAPHY 
Allen, P. W. 1918. A Simple Method for the Classification of Bacteria as 
to Diastase Production. Jour. Bact., 3, 15-17. 
Cannon, P. R. 1916. A Rapid and Simple Indol Test. Journal of 
Bacteriology, 1, 535-536. 
Chester. 1901. Manual of Determinative Bacteriology. New York. 1901. 
Clark, W. M. and Lubs, H. A. 1917. A Substitute for Litmus for Use in 
Milk Cultures. Jour. Agr. Research, 10, 105-111. 
Cohn, F. 1872. Untersuchungen uber Bakterien. Cohn's Beitrage zur Biol, 
der Pflanzen. Bd. I. Hefte, 2, 127-224. 
Conn, H. W. 1906. Classification of Dairy Bacteria. Ann. Rept. Storrs 
Agr. Exp. Sta., 107. 
Conn, H. J. 1915. A Study of B. Subtilis by Means of the Classification 
Card. Science N. S., 41, 618. 
Conn, H. J. 1917. Soil Flora Studies. Part I. Jour. Bact., 2, 35-45. 
DeBary. 1888. Comparative Morphology Biology of the Fungi, Mycetozoa, 
and Bacteria. 
Edson, H. A. and Carpenter, C. W. 1912. Microorganisms in Maple Sap. 
Vermont Agr. Exp. Sta., Bulletin 167. 
Edson, H. A. and Carpenter, C. W. 1912. The Green Fluorescent Bacteria 
Occurring in Maple Sap. Vermont Agr. Exp. Sta., Bulletin 167, 521-599. 
Eisenberg. 1891. Bakteriologische Diagnostik III. 
Ellis, H. 1909. Outlines of Bacteriology. Longmans, Green & Co., New- 
York. 
Fisher. 1900. The Structure and Functions of Bacteria. Oxford. 
Flugge. 1886. Die Mikroorganismen. II Auf., 1886. 
Gage, S. M., and Phelps, E. B. 1903. On the Classification and Identifica- 
tion of Bacteria with a Description of the Card System in Use at the Law- 
rence Experiment Station with Record of Species. Proceedings, American 
Public Health Association, 28, 494-505. 
Harding, H. A. 1910. The Constancy of Certain Physiological Characters 
in the Classification of Bacteria. Tech. Bull. 13., New York Agr. Exp. 
Sta., Geneva. 
Harding, H. A., Morse, W. J. and Jones, L. R. 1909. The Bacterial Soft 
Rot of Certain Vegetables. Tech. Bull., N. Y. Agr. Exp. Sta., Geneva. 
Harding, H. A. and PrucAa, M. J. 1908. The Bacterial Flora of Cheddar 
Cheese. Tech. Bull., N. Y. Agr. Exp. Sta., Geneva. 
Hasseltine, H. E. 1917. The Bacteriological Examination of Water. 
Reprint No. 43 from Public Health Reports, Nov. 9, 1917. 
Hastings, E. G. 1904. The Action of Various Classes of Bacteria on Casein 
as Shown by Milk Agar Plates. Cent. Bakt. Abt. II, 12, 590-592. 
Hort, E. C. 1917. Morphological Studies in the Life-Histories of Bacteria. 
Proc. Royal Soc. B., Vol. 89, 468-480. 120 
CLASSIFICATION AND DESCRIPTION OF BACTERIA 
Jensen, 0. 1909. Die Hauptlinien des natiirlichen Bakterien systems. Cent. 
Bakt. II, Abt., 22, 305. 
Jensen, 0. The Main Lines of the Natural Bacteria System and the Bacteri- 
ological Nomenclature. VII Intern. Cong. Applied Chern., Part IV, 
176-181. 
Johnston, W. 1894. On the Grouping of Water Bacteria. Amer. Pub. 
Health Assn. Proc., 20, 445-449. 
Jordan, E. O. 1912. General Bacteriology. 107-109. 
Kendall, A. I. 1903. Reported by Gage and Phelps. On the Classification 
and Identification of Bacteria with a Description of the Card System in 
Use at the Lawrence Experiment Station for Records of Species. Amer. 
Pub. Health Assn. Proc., 28, 494-505. 
Lehmann and Heumann. 1901. Atlas and Principles of Bacteriology. 
Philadelphia. 
Lister, J. 1872. On the Germ Theory of Fermentation and Other Fermenta- 
tive Changes. Nature, July 10, and 17, 1872. 
Lister, J. 1873. A Further Contribution to the Natural History of Bacteria 
and the Germ Theory of Fermentation Changes. Quarterly Jour. Micro- 
scop. Soc., 380. 
Lohnis, F. 1916. Life Cycles of Bacteria. Jour. Agr. Research, 6, 675-702. 
Mason, W. P. 1912. Examination of Water. New York, John Wiley & 
Sons. 
Migula, Die Bakterien. 
Mudge, C. S. 1917. The Effect of Sterilization upon Sugars in Cultures 
Media. Jour. Bact. 2, 403-415. 
Rahn, O. 1911. Marshall's Microbiology. P. Blakiston's Son & Co., 
Philadelphia. 
Rahn, O. and Harding, H. A. 1916. Die Bemiihumgen zur einheitlichen 
Beschreibung der Bakterien im Amerika. Cent. Bakt. Abt. II, 42, 385-393. 
Rosenow, E. C. 1914. Transmutation within the Streptococcus-pneumo- 
coccus Group. Jour. Inf. Diseases, 14, 1. 
Rosenow, E. C. and Towne, E. B. 1917. Bacteriological Observations in 
Experimental Poliomyelitis. Jour. Med. Research, 36. • 
Rothberg, W. 1917. Observations on Some Methods for the Study of 
Gelatin Liquefaction. Paper read before the Society of American Bacteri- 
ologists, December, 1917. 
Smith, I. F. 1905. Bacteria in Relation to Plant Diseases. 
Tanner, F. W. 1918. A Study of the Green Fluorescent Bacteria from 
Water. Jour. Bact., 3, 63-101. 
Winslow, C. E. A. 1917. The Families and Genera of Bacteria. Jour. 
Bact., 2, 505-566. 
Zipfel. Cent. Bakt. Orig. Abt., I, 64, 65. 
Zopf. 1884, Die Bakterien I. Aufl., 1885. CHAPTER V 
STERILIZATION AND DISINFECTION 
Bacteria do not exist in nature as pure cultures. Since there are so 
many varieties, the bacteriologist must use media and apparatus which 
are free from not only bacteria but all forms of life. This is accom- 
plished by sterilization, which is to be distinguished from disinfection- 
the removal of pathogenic bacteria only. The methods for sterilization 
may be classified as follows: 
I. Dry heat: 
a. Flaming and incineration. 
b. Hot air oven. 
II. Moist heat: * 
a. Inspissator. 
b. Streaming steam in the Arnold. 
c. High pressure steam in the autoclave. 
d. Boiling. 
III. Filtration: 
a. Through liquids. 
1. Sodium hydroxide, sulphuric acid, etc. 
b. Through solids. 
1. Sand, cotton, glass wool, porcelain, etc. 
IV. Light: 
a. Sunlight. 
b. Ultraviolet. 
Dry Heat 
Death by drying may be the result of two processes. The cell 
protoplasm may decompose or oxidation may take place. This has been 
studied by Paul (1909) and his co-workers who found, in general, that 
the rate of death in dry heat is proportional to the oxygen concentration. 
This would seem to indicate that death in dry heat was an oxidation 
process, although other reactions may enter. With regard to moist 
121 122 
STERILIZATION AND DISINFECTION 
bacteria, the oxidation processes would be limited by the solubility 
of the oxygen in water. According to Winkler (1889) this is 10.14 
parts per million at 15° C. and 760 mm. 
Incineration. As a means of sterilization, this method needs no 
discussion. It is plainly a process of oxidation and very efficient. 
Small incinerators may be purchased which will handle practically all 
ordinary material. 
Flaming. This simple method is a serviceable one for the bac- 
teriologist. Platinum wires may be sterilized by this method. Watch 
glasses, slides, etc., may be completely sterilized if a little care is used 
during the flaming process. The Bunsen burner or alcohol lamp may 
furnish the heat. 
Hot-air Oven. The apparatus for this method is constructed much 
like the ordinary baking oven. Quite often an attempt is made to 
insulate it either by means of a double wall or some special material. 
The temperature is usually maintained by means of a gas flame or 
electricity. Some hot-air sterilizers are so well insulated that after 
the temperature has been raised to 180° C., this temperature is main- 
tained for a long time even though the source of heat is removed. The 
hot-air oven is used for sterilizing all dry glassware. This should 
be put into the sterilizer when it is cool and gradually raised to the 
sterilization temperature. A temperature of 180° C. for from one hour 
to one hour and a half will suffice for all ordinary apparatus. This 
method has definite limitations and should not be applied to the sterili- 
zation of media, thick glassware, etc. 
Moist Heat 
The theory of sterilization by moist heat has been studied by 
Chick (1910). Here three essentially different processes take place: 
1: Direct effect of heat on bacterial protein. 
2. Effect of water possibly hydrolytic on these proteins at high 
temperature. 
3. Desiccation of bacteria. 
It was pointed out by Miss Chick that an analogy exists between 
the disinfection of bacteria by hot water and the " heat coagulation " 
of proteins. This may help to account for the difficulty with which 
spores are disinfected by hot water when compared with the vegetative 
cells. The protein in the spores may be more resistant to hydrolysis 
than that in vegetative cells. Both changes follow the monomolecular 
law and both are greatly increased by the presence of minute amounts STERILIZATION BY MOIST HEAT 
123 
of acid. Chick (1910) states, " The striking similarity between the 
effects of temperature (dry) on the one hand and hot water on the other 
indicate that disinfection by the latter is due to the action of water 
(coagulation and alteration) upon some one protein which is essential 
for life of the bacterium and that the reaction is conditioned by the 
chemical action of water upon its constituent proteins." The follow- 
Fig. 37.-Hot-air Sterilizer, Lautenschlager Type. 
ing facts with regard to disinfection by moist heat were established 
by Chick: 
1. Disinfection proceeds according to the logarithmic law the rate 
of disinfection being proportional, at any moment, to the concentration 
of surviving bacteria. 
2. The presence of minute quantities of acid or alkali too small to 
produce any direct disinfectant action increases the reaction greatly. 
The acid, however, gave the greatest increase. This presents close 
analogy to the " heat coagulation " of proteins. 124 
STERILIZATION AND DISINFECTION 
Rubner (1913) has reviewed the* application of steam to sterili- 
zation. He points out that vegetative cells contain much more water 
than spores and thus succumb more quickly to the action of heat. 
Spores contain hygroscopic water which soon evaporates leaving dry 
bacterial protein. This will resist heating for some time. Steam at 
100° C. is an important chemical agent since hydrogen sulphide, 
ammonia and carbon dioxide are liberated from keratin, casein, and 
dried bacteria when they are subjected to its influence. Rubner found 
the saturation of the steam to be about as important as the temperature. 
Steam at 100° C. with a saturation of 80 per cent requires five times 
as long to kill bacteria as does saturated steam and steam with a 
saturation of 70 per cent requires twenty-two times as long as saturated 
steam. If steam at 100° C. is superheated, it is altered in two ways. 
The temperature is raised which makes it a more powerful disinfectant 
and the saturation is lowered which makes it a weaker disinfectant. 
In superheated steam at 110° C. made from steam at 100° C. spores 
lived twice as long and at 127° C. ten times as long. This suggests the 
correlation between the temperature and saturation which are very 
important factors in sterilization. According to the laws of disin- 
fection, time is the other factor and it cannot be separated from the 
first two. 
High-pressure Steam. This method has extensive use in bacteri- 
ology. The apparatus is called an autoclave or dressing sterilizer. 
Several types are used, the most convenient of which are connected 
to a steam main from a power plant. Where this is impossible, it may 
be necessary to generate the steam under the sterilizer. This usually 
requires more time since it takes some time to get up the pressure. 
Often it is necessary to keep water in the bottom of the sterilizer in 
order to prevent the superheating of the steam. Under practical 
conditions this is usually not necessary since the steam will be saturated. 
The following table will show the relation between the usual pressures 
and temperatures required for sterilization in the autoclave: 
Gage Pressure. 
Temperature, 
Centigrade. 
Gage Pressure. 
Temperature, 
Centigrade. 
0 
100 
15 
121.5 
5 
109 
20 
126 
10 
115.5 
40 
141 
• 
In the presence of water, Chick has shown that the temperature 
is an important factor in sterilization. The temperature coefficient HIGH PRESSURE STEAM 
125 
was determined for B. typhosus and was reported to be 1.635 per 1° C. 
or 136 per 10° C. Here again the killing of bacteria in the presence 
of water was found to be analogous to the 11 heat coagulation " of their 
constituent proteins. 
Fig. 38.-Autoclave or Dressing Sterilizer, Kny-Schereer Type. 
The autoclave may be used for sterilizing many pieces of apparatus 
and many media. It has the advantage over other methods of taking 
less time. It has been recently shown that less hydrolysis of poly- 
saccharides is secured in the autoclave than in the Arnold. This is 
contrary to what was believed, for it is stated in many places that 126 
STERILIZATION AND DISINFECTION 
the Arnold steam sterilizer too should be used on such substances as 
sugars to prevent hydrolysis. 
Boiling. Practically no special apparatus is required by this 
method and any that is demanded may be quickly secured. Probably 
fifteen minutes is sufficient,-for vegetative cells by spores are too resistant 
to be quickly killed. Surgical instruments may be sterilized by boiling. 
The water should be boiled for ten minutes before they are put in in 
order to prevent the possibility of rusting. 
Streaming Steam. The apparatus which is used in this method is 
much like the ordinary steamer which is available in the kitchen. 
Fig. 39.-Arnold Steam Sterilizer. (Boston Board of Health Pattern.) 
In bacteriology the apparatus is known as the Arnold steam sterilizer. 
It was devised by Tyndall, and improved by Koch and Arnold. It 
consists of essentially a copper box with a false bottom through which 
the steam rises to escape at the top. There is no superheating in the 
Arnold as with the autoclave and it requires less attention. It may be 
used in two ways-continuous or intermittently. 
With the continuous method the material to be sterilized is heated 
for from thirty minutes to a hour and a half. This method has the 
disadvantage that such prolonged heating may cause changes in the 
materials. Di-, tri-, and poly-saccharides may be hydrolyzed proteins STERILIZATION BY FILTRATION 
127 
caogulated, etc. Such has been found to be the case as mentioned 
elsewhere. 
With the intermittent method the material is heated for twenty to 
thirty minutes on three successive days. On the first day the vegetative 
cells are killed. The second day's heating will destroy those vegetative 
cells which have come from spores which passed through the first day's 
heating. On the third day the process is simply checked up. 
Fig. 40.-Bath for Sterilization at Low Temperatures (Inspissator). 
Inspissator. Some media which are used by bacteriologists must 
be sterilized at low temperatures. For this purpose a serum coagulator 
or inspissator is used. The media is heated at from 57° to 60° C. 
for different lengths of time. Blood serum may be heated for periods 
of one hour or for a longer period at one time. 
Sterilization by Filtration 
Filtration is an efficient method of sterilization which has certain 
distinct advantages over other methods. It leaves the filtrate sterile 128 
STERILIZATION AND DISINFECTION 
and unchanged. Body ext racts and sera may be thus treated to render 
them sterile. 
Filtration through Liquids. The action is entirely mechanical 
unless some strong liquid such as sodium hydroxide or sulphuric acid 
are used. In these cases the bacterial protein is decomposed by the 
chemical. Rettger applied this method to the bacterial examination 
of air. 
Filtration through Solids. The same principle is involved here 
that is involved in the use of the cotton plug in test tubes and culture 
flasks. Different substances have been used but most attention has 
Fig. 41.-Diatomaceous Filters for the Sterilization of Bacterial Extracts and Body 
Fluids. 
been given to cotton, glass, wool and sand. Sand is used in the standard 
method for the bacterial examination of air and also in the purification 
of water. Stone or porcelain filters are the best substances for labora- 
tory use. They may be secured in many sizes and are easily sterilized 
and cleaned. 
Dialysis. In this medium the semi-permeable membrane is used 
as the filtering medium. Collodion sacs have had quite a little use 
in bacterial work. 
Light 
Although light does exert an antiseptic action on bacteria and 
may even totally destroy them after a long period, it is scarcely used STERILIZATION BY LIGHT 
129 
by the bacteriologist to sterilize media or apparatus. The work on the 
germicidal action of light is extensive and those wishing bibliographies 
on the subject should consult the papers of Dieudonne (1894), Ward 
(1895) and Weinzirl (1914). 
Ward (1895) using anthrax bacilli and spores found that exposures 
of one-half to one hour were necessary for inhibition of growth and 
that one and a half hours were necessary for sterilization. 
Dieudonne (1894) worked with pathogenic bacteria. With B. 
fluorescens puditus and B. prodigiosus he found that one and a half 
hours were required in March, July and August and nearly twice as 
long in November. 
Kruse found that an atmosphere of oxygen shortened the length 
of life by one-half over that of hydrogen when bacteria were exposed 
to sunlight. 
Weinzirl (1914) devised a new technique which involved exposure 
of the bacteria on paper directly to the sunlight. He stated that non- 
spore-forming bacteria were killed in from two to ten minutes. Such 
conditions are rarely secured in nature where the organisms are often 
accompanied with organic matter. For spores from two to eight 
hours were necessary. 
The various colors of the spectrum exert a different action on bac- 
teria. The intensity of this action increases as one goes from the red 
toward the violet. Apparent destruction is noticed only after the 
yellow is passed. From the yellow through the violet and ultra- 
violet, the germicidal action is at its maximum. In fact it is now well 
established that the shorter the wave length the greater is the bacteri- 
cidal action. Newcomer (1917) has reported experiments which indi- 
cate that X-ray fluorescence is very bactericidal. X-rays alone exerted 
a partial bactericidal effect on suspensions of typhoid bacilli. Kempster 
(1917) exposed tubercle bacilli to X-rays and secured a reduction in 
their multiplication. Inoculations of tubercle bacilli, which have been 
exposed to X-rays, into guinea pigs caused no tubercular lesions to 
develop. He stated that the phagocytes were able to overcome the 
bacteria if their multiplication was reduced. 
Ultra Violet Light. This light on account of its short wave length 
possesses decided bactericidal effects. Ward (1892) in studying the 
effect of arc spectra on bacteria found that the infected plates were 
sterilized whenever exposed to the violet or ultra violet rays. He 
successively treated Thames River water by submitting it the effects 
of ultra violet rays. Houghton and Davis (1913) give the following 
conclusions to their work: 130 
STERILIZATION AND DISINFECTION 
A. The ultra viole t rays produced by the Cooper Hewitt mercury 
arc have a strong 
bactericidal power. 
B. Certain varieties 
of bacteria in aqueous 
suspension, including 
spore-forming organ- 
isms, are killed by 
exposure to the rays. 
Molds, however, are 
only partially de- 
stroyed by the ultra 
violet light. 
C. The action seems 
to be photo-mechani- 
cal and is in all proba- 
bility due to absorp- 
tion of ultra violet 
rays by bacterial pro- 
toplasm. 
D. Proteins and 
other bodies of high 
molecular weight in- 
terfere with the action 
of the rays. Turbidity, 
both organic and in- 
organic, has a similar 
action. Color within 
certain limits seems 
to have no action. 
Ultra violet light 
was first used for the 
sterilization of milk 
and since this time its 
use has been extended 
to other materials. 
Von Recklinghausen 
studied the action of 
ultra violet light on 
bacteria in water. 
Spores are said to be 
Fig. 42.-Seconds Necessary to Kill Different Types of Microorganisms at 200 mm. from a Quartz Lamp Burning at 66 Volts 
and 3.5 Amperes. (After von Recklinghausen, 1914.) 
Seconds 
Staphylococcus aureus 
Microspira cholera* 
Bacillus typhosus 
B. dysenteriae Shiga 
B. dysenteriae Dopter 
B. coli 
Bacillus anthrax 
Pneumobacillus Friedlander _ 
Sarcina alba 
B. aerogenes capsulatus 
Bacillus tetanus 
Bacillus megatherium.. _ 
B. phleole 
Bacillus subtilis 
Sarcina lutea 
Paramecia <■ 
Yeast DISINFECTION 
131 
1.5 and five times as resistant as vegetative cells. The resistivity 
of some of the common bacteria is shown in Fig. 42. This author 
does not believe that hydrogen peroxide is formed during the 
action of these rays. Burge has demonstrated that these radiations 
exert a strong coagulation of the protein and probably the same 
change is secured on bacterial proteins. He declares (Burge, 1917) 
that the theory of the destruction of the intracellular enzymes to 
explain death, is not true. He killed some bacteria by ultra violet 
radiation and after grinding found that gelatin was liquefied. In this 
case the intracellular enzymes were not destroyed when the cells had 
been killed. This same opinion was advanced by Oker-Blom (1913). 
He argues that the rays act directly on living protoplasm and do not 
act through ozone, hydrogen peroxide or nitrous acid. In this con- 
nection the greater resistance of the fluorescent bacteria to ultra violet 
rays found by Burge and Neill (1915) is interesting. They found the 
fluorescent bacteria to be much more resistant than the non-fluorescent. 
Harris and Hoyt (1917) state that visible light rays are not as a rule 
absorbed by protoplasm. Ultra violet rays are generally toxic and they 
attempted to determine what constituent of the protoplasm was 
responsible for absorption of these rays. While their experiments 
were carried out with protozoa, the results are probably applicable to 
bacteria. The paramecia were suspended in different solutions such as 
urea, cane sugar, alanin, leucine, and gelatin. They state the sus- 
ceptibility of protoplasm to ultra violet light is conditioned by the 
absorption of the toxic rays by amino acrd radicles in the proteins. 
Newcomer (1917) reported that the typhoid bacilli are about 1/200 
as sensitive to ultra violet radiations of wave lengths 2100-2800 Ang- 
strom units as a photographic plate. This decreases to almost zero 
at 2970 units. Browning and Russ (1917) working the Staphylococcus 
pyogenes aureus, B. coll and B. typhi reported that ultra violet radia- 
tions with wave lengths between 3800 and 2960 A possessed no marked 
bactericidal action. Wave lengths between 2960 and 2100 A were 
markedly bactericidal. 
Disinfection 
The distinction between sterilization and disinfection is quite 
artificial in many cases. In both processes the death of bacteria is 
concerned. Disinfection is generally regarded as the removal of patho- 
genic bacteria from materials while sterilization is applied to those 
processes which remove all kinds of life. Obviously all methods of 132 
STERILIZATION AND DISINFECTION 
sterilization are disinfection processes. Even with this close relation 
of the process there are certain procedures and reagents which are 
used more as disinfectants than as sterilizing agents. Many of these 
could not be applied to media and some apparatus since some of the 
disinfectant remains after the treatment. 
Disinfection is a chemical process and therefore follows chemical 
laws. Chick (1908) has given the best work on this subject and much of 
her data form the basis upon which our knowledge with regard to 
disinfection rests. The chemical reaction in this case would take place 
between the disinfectant and the bacterium protoplasm. The disin- 
fection process follows the well-known law of physical chemistry-the 
monomolecular law. The same equation may be used provided legi- 
timate substitutions are made in it to make it fit disinfection. 
at 
or 
1 , ci 
- log -= A 
t2~t\ & C2 
This may be changed to 
rtrlog- = K. 
t2~ti n2 
In this equation ci and C2 have been substituted for ni and W2 the 
numbers of surviving bacteria. For such a discussion, we may define 
the monomolecular law as one governing a reaction between two sub- 
stances where the change in concentration of one may be measured. 
This law seems to be followed in all cases of disinfection when any 
agent is used to destroy bacteria. It is apparent that the rate of death 
is always proportional to the number of cells which are living at any 
given time. This simply means that where there are larger amounts 
of reacting substances, more reaction is secured. Consequently a 
greater drop is secured in the curve during the first units of time. 
Some objections may be raised against regarding disinfection in this 
light. The bacterial cell must be regarded as a molecule. It is quite 
different since it possesses " life " and is surrounded by a membrane. 
It is also necessary to neglect the changfi in concentration of the dis- 
infectant. At present the disinfectant is usually added in such excess 
that, any change which might occur is neglected. 
Different opinions exist with regard to how the disinfection process 
should be explained. For disinfection purposes, a bacterial culture is 
regarded as made up of a uniform population. Hewlett (1909) and INORGANIC DISINFECTANTS 
133 
Reichel (1909) explain the time element in disinfection by assuming 
that the different cells in a bacterial suspension possess different 
resistances-the weakest or less resistant cells dying first. If such 
were the case it is reasonable that most of the cells would possess the 
same resistance and consequently would succumb at about the same 
time and that at each end of the process we would be dealing with the 
weakest (less resistant) and the strongest (most resistant) cells. Such 
a unimodal curve is usually secured where the element of chance or 
the law of probability enter. This same monomolecular law is followed 
among human beings where similar individuals and a multitude of 
causes of death obtain. The disinfectant may act as a catalyzer since 
the process demands the presence of water. Where water is absent 
disinfection is inhibited. 
Factors which Influence the Value and Use of Disinfectants. The 
ideal disinfectant does not exist. If one could be found, it would con- 
form to the following characteristics: 
1. Low cost. 
2. Non-toxicity to man and animals. 
3. Solubility in ordinary reagents in sufficient amounts to destroy 
bacteria. 
4. Minimum time for destruction of bacteria. 
5. Great penetrating power. 
6. Should be a stable compound under ordinary conditions. 
7. Should not react with cloth dyes or wood finishes. 
Inorganic Compounds 
Lusini (1912) has pointed out with regard to the cations that 
although the disinfecting property is not a function of the atomic 
weight the heavy metals exert the greatest action. With a few excep- 
tions, the disinfecting action increases with the chemical affinity of 
the combined elements. The alkali group is less powerful than the 
alkalin earth group. The last mentioned group does not fit in with the 
generalizations above since its disinfecting power is in inverse ratio 
to the atomic weight. Among the other elements of the periodic 
system iron has a lower activity corresponding to its low atomic weight; 
tin has a higher and lead still higher activity. This has been somewhat 
confirmed by other investigations. Bitter (1912) found that the fol- 
lowing metals exerted an antagonistic action toward bacteria drying 
on them. The intensity of action is in the following order: Copper, 
brass, silver, gold, platinuin, lead, cast iron, steel, aluminum, nickel. 134 
STERILIZATION AND DISINFECTION 
zinc and tin. The action was found to be the same for both polished 
and corroded metal. Such data are interesting when it is remembered 
that some of these elements are used for the manufacture of toilet 
articles and mouth pieces for public drinking fountains. Under these 
conditions a disinfecting action could hardly be expected. Bacteria 
will exhibit great development in cultures containing the heavy metals 
in large pieces. 
Halogens. The halogens have rather important positions as dis- 
infectants. Chlorine has had much use as an internal disinfectant in 
diphtheria, scarlet fever, etc. It is contained in water in a concen- 
tration of about 0.4 per cent.* The action of chlorine is generally 
admitted to be an oxidation reaction. Recently it has replaced bleach- 
ing powder in the treatment of public water supplies. It is distributed 
in the liquid form and has many advantages over calcium hypochlorite. 
Bromins. This is used as a saturated aqueous solution containing 
about 3 per cent of the element. When used internally the strength 
of the solution must be greatly reduced to contain about 0.3 per cent 
It is a strong oxidizing agent and readily attacks metals. This places 
some limitations on its use as a disinfectant. 
Iodine. A 5 per cent solution with 10 per cent of potassium iodide 
is a reliable disinfectant. It is available as a tincture which is an 
alcoholic solution of the element. It is soluble in alcohol to the extent 
of about 1 part in 10. Free iodine exerts a powerful toxic action on 
bacteria since it unites directly with the bacterial protoplasm. Iodo- 
form is an iodine compound (CHI3) used in disinfection. It is applied 
directly to wounds and unites with the organic matter. Free iodine 
results and iodoform owes its action to the iodine which is thus given 
off. Often it is made into an ointment by mixing with some carrier. 
Other forms of iodine which may be used as antiseptics are: 
lodoformogen-iodoform albuminate. 
lodo-Hemol Merck-Iodized hemol. 
Iodol- (C4 H4NH) iodocarbamid. 
lodolnuth-Bismuth iodine compound. 
Mahon and White (1915) report that iodine in alcohol is about four 
times as powerful as a disinfectant as phenol. Their work was done 
on naked organisms with B. typhi as the test organism. Eugling (1912) 
in studying the germicidal action of iodoform could not detect a reduc- 
ing action of bacteria resulting in the liberation of free iodine. 
Silver Salts. Silver precipitates proteins from solution as insoluble 
salts. It probably acts in the same way on bacterial protoplasm. SILVER SALTS AS DISINFECTANTS 
135 
Biasiotti (1910) reported that colloidal silver electrically prepared 
would kill Staphylococcus pyogenes aureus, B. typhi, and Bacterium 
diphtheria? in a few hours. The silver is probably united to the proto- 
plasm in some way because Gram positive bacteria are made Gram 
negative. Simpson and Hewlett (1914) experimented with colloidal 
silver in the form of "collosol " on the typhoid bacillus and found it 
an active bactericide. The 11 collasols " are said to be non-poisonous 
but slow of action and very expensive. 
Silver is combined with several substances which have wide appli- 
cation in certain phases of the disinfection problem. Argyrol is the 
silver salt of vitellin and is used in solutions of from 1 to 4 per thousand. 
Protargol is a compound of silver with protein. It contains 8 per cent 
of silver nitrate. Albargin is a silver salt of galactose. It may be used 
on mucous membranes, which explains its use in the venereal diseases. 
It contains about 10 to 15 per cent of silver. Often it is applied as 
10 to 20 per cent solutions in 20 per cent glycerol. Argentamin, a 10 
per cent solution of silver nitrate in 10 per cent ethylene-diamin, has 
much use in gonorrheal infections. It is usually diluted to about 1 
part in 300. Argentose is a silver compound of nucleoprotein and is 
used like silver nitrate. Argonin, a silver caseinate, is used in a 3 per 
cent solution for treatment of gonorrhea and ophthalmia neonatorum. 
Silver Nitrate, AgNOa. This is a common silver disinfectant. It 
is not as powerful as mercuric chloride but does not precipitate proteins 
as does mercuric chloride. 
Silver Citrate, AgaCcHsOr. This is used as a wash for wounds and 
for gonorrhea in dilutions of about 0.25 per cent. Great precautions 
must be used to obtain fresh solutions. It is also known as itrol. 
Silver Lactate. This compound of silver is used as a mild disin- 
fectant. It has about the same applications as has silver citrate. 
Bismuth Compounds. Bismuth subiodide, bismuth subsalicylate, 
bismuth nitrate are all used as weak disinfectants. They are used in 
solutions of 1 to 1000 to 1 to 4000 and are probably not very reliable. 
Calcium Compounds. Chloride of Lime. This is also known 
under the names of bleach, hypo and bleaching powder. It has attained 
an important position in sanitation probably for two reasons; it pos- 
sesses great bactericidal properties and may be secured for a low price. 
Thus it satisfies two of the characteristics of a good disinfectant. In 
the dry condition chloride of lime is calcium oxychloride which, in 
the presence of water takes on another atom of oxygen. 
2CaOCl2 = Ca(OCl)2 136 
STERILIZATION AND DISINFECTION 
Chloride of lime is soluble according to Hooker (1913) in about ^0 
times its own weight of water. It leaves an insoluble residue com- 
posed mostly of calcium hydroxide. It is prepared from Ca(OH)2 as 
follows: 
/OH HC1 /Cl 
Ca^ 4- = Ca^ 4- 2H2O. 
XOH H0C1 XOC1 
The H0C1 is made by treating water with chlorine. Some care 
should be used to secure clear solutions for use in disinfection. This is 
especially true in water treatment. The potential part of this com- 
pound is very soluble in water while the part with no sterilizing 
properties quickly settles out. Hooker (1913) gives the following rules 
for the use of chloride of lime: 
First, do not mix too stiff a paste, otherwise a gelatinizing action takes place 
and greater difficulty in settling out is encountered. Never mix a paste with 
less than | gal. of water for 1 lb. of chloride of lime. Second, it is not necessary 
nor desirable to grind nor break up the lumps too thoroughly; the available 
chlorine nearly all dissolves readily and too much agitation is detrimental to 
prompt settling. 
The above precautions should be borne in mind in any application 
of chloride of lime to sanitation. 
The strength of chloride of lime is measured in terms of " available 
chlorine." This is an unfortunate term since it implies that the dis- 
infecting properties of the compound rest in the chlorine. The work 
in the past indicates that it is an oxidation and not a chlorination. 
The oxygen is formed in the following manner: 
Ca(OCl)2+H2CO3 = CaC03+2HC10., 
2HC1O = 2HC1+O2 
It has been suggested by some sanitarians that the term " available 
chlorine " be substituted by the term " potential oxygen." Lager (1916) 
has made some interesting statements in regard to the action of calcium 
hypochlorite. He states that the disinfecting action does not depend 
upon the available chlorine nor the length of time the chlorine acts. 
He states that the time for killing depends upon the resistance of the 
organism. 
The United States Pharmacopoeia specifies that chloride of lime shall 
contain 35 per cent of available chlorine. As it is purchased in small 
cans on the open market it probably falls far short of this and the use CALCIUM HYPOCHLORITE 
137 
of such a product allows a false sense of security. Hooker, in the book 
mentioned above, presents a table which contains much useful infor- 
mation. It appears here as Table VIII and is based on 30 per cent 
bleach. 
Table VIII 
SHOWING STRENGTH OF SOLUTIONS OF CALCIUM HYPOCHLORITE 
(After Hooker, 1913) 
Pounds 
Bleach per 
1,000,000 
. Gallons 
Water. 
Parts Bleach 
per 1,000,000 
Parts Water. 
Parts Chlorine 
per 1,000,000 
Parts Water. 
Grains Bleach 
per Gallon 
Water. 
Grains Avail- 
able Chlorine 
per Gallon 
Water. 
Drops Bleach 
Solution, 2 Per 
Cent or | Lb. 
in Gallon Used 
per Gallon 
Water. 
2 
.24 
.08 
.104 
.005 
.25 
4 
.48 
.16 
.028 
.009 
.50 
6 
.72 
.24 
.042 
.014 
.75 
8 
.96 
.32 
.056 
.019 
1.00 
10 
1.20 
.40 
.070 
.023 
1.25 
12 
1.44 
.48 
.084 
.028 
1.50 
14 
1.68 
.56 
.098 
.033 
.1.75 
16 
1.92 
.64 
.112 
.037 
2.00 
18 
2.16 
.72 
.126 
.042 
2.25 
20 
2.40 
.80 
.140 
.047 
2.50 
22 
2.64 
.88 
.154 
.051 
2.75 
24 
2.88 
.96 
.168 
.056 
3.00 
26 
3.12 
1.04 
.182 
.061 
3.25 
28 
3.36 
1.12 
.196 
.065 
3.50 
30 
3.60 
1.20 
.210 
.070 
3.75 
The objections to chloride of lime are important. It takes the 
color out and exerts great reactivity with organic matter. When it is 
used in the presence of large amounts of organic matter, a sufficient 
amount should be used to satisfy the requirements of it and leave 
enough left over for destroying the bacteria. This is difficult to 
determine. 
Labarraque's Solution. This is a solution of chloride of lime con- 
taining 2.4 per cent of available chlorine. 
Dakin-Carrel Solution. This solution has been found to be of great 
value in the treatment of wounds. Different methods may be used 
in the preparation of this solution. Below are two formulae: 
Formula No. 1. Dissolve 140 gms. of sodium carbonate in 10 liters 
of distilled water and add 200 gms. of calcium hypochlorite. Shake 
this solution thoroughly and allow the sediment to settle. After sedi- 138 
STERILIZATION AND DISINFECTION 
* 
mentation has taken place decant the clear liquid and add 40 gms. of 
boric acid. The solution is then ready to use. 
Formula No. 2. Dissolve 100 gms. of sodium carbonate, 80 gms. of 
calcium hypochlorite in 10 liters of water. This should give a solution 
of about | per cent of hypochlorite. 
In the use of this solution the wound should be kept constantly 
moist. Doctor Carrel applies the solution every hour or two hours, 
the amount used generally depending upon the character of the wound. 
Generally 5 to 10 c.c. were applied every two hours. The use of this 
solution is being extended to other infections such as puerperal sepsis, etc. 
The absolute germicidal power of the chemical was studied by 
Nissen (1890). He used pure cultures and secured the following 
results which are quoted from Hooker: 
Organisms Killed. 
Minutes. 
By Dilutions of 
Chloride of Lime. 
Bacillus typhosus 
1 
1-800 
Bacillus typhosus 
10 
1-1600 
Bacillus cholera; 
1 
1-800 
Bacillus cholera; 
10 
1-600 
Bacillus anthracis 
1 
1-1000 
Staph, pyogenes aureus 
5 
1-800 
Streptococcus erysipelatus 
1 
1-200 
Anthrax spores 
15 
1-20 
Anthrax spores 
20 
1-100 
Hooker has collected an excellent resume of the literature up to 1913. 
Calcium Oxide, CaO. This is known as quick lime and is caustic 
in the presence of organic matter. It may be used in two forms as 
slaked lime and milk of lime. Slaked lime is prepared by adding a pint 
or pound of water in two pounds of lime. The reaction proceeds as 
follows: 
CaO+H2O = Ca(OH)2. 
This will react with carbon dioxide to give calcium carbonate as 
follows: 
Ca(OH) 2+C02 = CaCO3+H20. 
Milk of lime is a thick mixture containing 4 to 6 parts of slaked lime. 
This milk of lime reacts with carbon dioxide to form the carbonate as 
does the slaked lime. Therefore these mixtures should be used as 
soon after preparation as possible. COPPER SALTS AS DISINFECTANTS 
139 
Chloramine. Toluene parasulfondichloramin is formed when nascent 
chlorine formed in aqueous solutions of hypochlorite is brought in 
contact with suppurating wounds. Dakin (1916, 1917) has proposed 
the name dichloramine-T for this compound. He has shown that 
most of the substances containing a NCI group are strongly germicidal. 
The presence of more than one such groups does not seem to increase 
the germicidal properties to any marked degree. It may be used in 
stronger concentrations than with some of the other disinfectants. It 
is claimed that its use is as simple as the use of tincture of iodine. 
Zinc Chloride. This compound had quite a reputation as a disin- 
fectant at one time, but recent knowledge has caused the compound 
to fall into disuse. McClintic (1905) found that B. coli was not killed 
in one hour's exposure to a 5 per cent solution and that it required ten 
minutes for a 25 per . ent solution to kill the same organism. The 
spores of B. subtilis and B. anthracis were found to resist 100 per cent 
and 50 per cent solutions for 30 and 40 days respectively. 
Copper Salts. These have been used in the treatment of public 
water supplies but not generally in ordinary disinfection. Kellerman 
and Beckwith (1906) have given a resume of the literature up to the 
time when their paper was published. Moore and Kellerman (1905) 
reviewed the application of copper sulphate to water sterilization and 
the removal of algae. About 10 lbs. per million gallons of water were 
sufficient. Its toxic properties are said to be due to the copper salts 
which are formed with proteins. Embrey (1912) reported that copper 
sulphate would destroy algae in a dilution of 1 to 3 million. DeWitt and 
Sherman (1916) studied the effects of both copper sulphate and cupric 
chloride on bacteria. Their results showed that copper was not a 
reliable disinfectant. They state that 2.5 parts of cupric chloride 
and 4 parts of copper sulphate per million would kill B. coli and 
B. typhi. 
Ferrous Sulphate, FeSO4. This has little germicidal value unless 
in solutions of over 2 per cent. When it is present above 5 per cent, 
it will restrain development of putrefactive changes. McLaughlin 
(1903) secured no disinfection in applying it in saturated solution to 
feces. 
Boric Acid, H3BO3. Boric acid is a mild disinfectant and is used 
in 5 to 100 per cent solution. It is of especial value for use on mucous 
membranes such as those of the eye and the nose. Ochsner (1916) 
stated that boric acid does not destroy pathogenic bacteria but does 
diminish their virulence. Wet dressings of boric acid were much more 
effective on the pyogenic cocci than on other infections. 140 
STERILIZATION AND DISINFECTION 
Carbon. Renon (1914) produced colloidal carbon electrolytically 
so that the size of the particles was 4 to G/x/z- Ue studied the antiseptic 
properties by measuring the acidification of milk. The antiseptic 
action increased with the amount of carbon. 
Organic Compounds 
The infinite number of possibilities for securing compounds de- 
structive to bacterial life renders a discussion of the organic compounds, 
in this connection, rather difficult. Kligler (1918) has made a study 
of the action of aniline and some of its derivatives and of the triphenyl- 
methane dyes on bacteria. He stated from this investigation that 
the action on bacteria might be a function of the benzine nucleus and 
in the case of the dyes a function of the quinoid structure of the nucleus. 
Increasing the number of alkyl radicals increased the antiseptic proper- 
ties. " The antiseptic power is enhanced to a greater extent by an 
ethyl than by a methyl group and the second alkyl produces a pro- 
portionately greater increase than the first. It appears that the relative 
position of the introduced group may be a factor in determining the 
relative improvement in the effectiveness of the compound." An 
interesting fact is mentioned by Kligler that the Gram positive bacteria 
are more susceptible to the aniline dyes than are the Gram negative 
bacteria. 
Alcohol. Ethyl alcohol is usually used. It precipitates proteins 
unaltered and if they are allowed to remain in contact with the alcohol 
they are changed. The precipitation is probably due to dehydration. 
The greatest disinfective power is secured in strengths of between 50 
and 70 per cent. These solutions contain sufficient water for the 
coagulation of the protein. Beyer (1912) found that concentrations 
of alcohol under 60 per cent and over 80 per cent were practically 
worthless as disinfectants. Absolute alcohol, on account of its drying 
action exhibited a drying and preserving action. Frey (1912) found that 
treatment with various concentrations of alcohol influenced the swelling 
and solubility of protein in water. Alcohol diluted to 10 per cent 
dissolved a little of the protein; above 20 per cent of alcohol solution 
of the protein stops and swelling decreases; above 90 per cent the 
protein dissolves. The greatest action of alcohol on protein is at 60 to 
70 per cent while the greatest disinfecting power is 70 per cent. Frey 
explains the use of alcohol as a disinfectant in the irreversibility of the 
precipitation of protein after treatment in alcohol. 141 
COAL TAR DISINFECTANTS 
Phenol and Cresol: 
ch3 
/\0H 
CH3 
\/0H 
CH3 
on 
^OH 
m-cresol 
p-cresol 
phenol 
o-cresol 
The action of these substances as disinfectants has been studied 
by Cooper (1912), who found that the entrance of the methyl and nitro 
groups increased the bactericidal and protein penetrating powers of 
phenol while the entrance of the hydroxyl group decreased these 
properties. Solutions of phenol in alcohol and in fat had.no bactericidal 
action on spores and did not penetrate solutions of gelatin. The 
precipitating action of phenol is increased by adding acid. Cooper 
points out that the selective action of phenol on different bacteria 
is connected with the different susceptibilities of proteins to its pre- 
cipitating action. The absorption of phenol by bacteria is only the 
beginning of disinfection. No chemical union follows between the 
phenol and the bacterial protoplasm but it is associated with de-emul- 
sification of the collodial suspension as shown by the precipitation of 
proteins when a certain phenol concentration is reached. 
The superiority of m-cresol over phenol as a bactericide is due to 
the fact that cresol precipitates proteins in a lower concentration than 
phenol. Steenhauer (1916) showed that m-cresol is the strongest 
and p-cresol the next strongest disinfectant. The cresol solutions 
secured on the market are mixtures of the o-, m-, and p-cresols. The 
introduction of the methyl group has decreased the solubility. On 
account of this the cresols are often suspended in or mixed with some 
carrier. Both hard and soft soaps, for instance may be used. Lysol, 
a highly bactericidal substance, is prepared by saponifying linseed oil 
with commercial cresol in alcoholic solution. This is readily soluble 
in water and is used in from 2 to 5 per cent solutions. Weaker solu- 
tions must be used for disinfection of mucous membranes as the lining 
of the mouth and vagina. Steenhauer reported that m-cresol is the 
strongest bactericide with p-cresol next. He recommended the use 
of 5 per cent solutions. 
Thymol. This compound is probably of little value as a disin- 
fectant. Schmidt (1910) stated that thymol does not affect certain 
putrefactive bacteria and is not entirely efficient as an antiseptic in 142 
STERILIZATION AND DISINFECTION 
enzymological work. It is used in many pharmaceutical preparations 
but probably does no harm. 
CH3 
^OH 
CH(CH3)2 
Formaldehyde. This is a powerful disinfectant sold in 35 per cent 
solutions under the name of formalin. Formaldehyde changes to para- 
formaldehyde by polymerization when kept at room temperatures. It 
unites easily with many substances especially the organic compounds. 
For instance when added to gelatin it forms a compound which is 
insoluble in many of the common reagents. Fermi attempted to secure 
a gelatin medium in this way which would not melt at room tem- 
perature. It may act in the same way on bacterial protein. It is 
important to have plenty of moisture present when using formaldehyde 
as a disinfectant. Koch (1901) reported that a 0.05 per cent solution 
of this compound would kill growing yeast while a 0.005 per cent solu- 
tion would not. Since carbon dioxide was formed after the cell had 
been killed, the zymase was not destroyed. 
Glycerol. Rosenau has made an interesting study of this com- 
pound in its relation to dinsinfection. He found that a 50 per cent 
solution of glycerol would restrain all bacterial growth and that lower 
precentages would allow better growth. Bacteria would not grow 
in media containing 32 per cent of glycerol but molds grew in stronger 
solutions of 40 and 49 per cent. Glycerol was found to have a distinct 
but slight germicidal action and probably acted by virtue of its abstract- 
ing water from the bacterial cell. Spores were not killed and anthrax 
spores lived 200 days in strong glycerol solutions. The compound 
may be used by many bacteria as a food. It is then probably oxidized 
to glyceric aldehyde and glyceric acid. Glycerol may be used, however, 
as a carrier for many other disinfectants such as phenol, cresol, etc. 
Goodrich (1917), however, has shown that glycerol more or less com- 
pletely destroys the antiseptic power of thymol, phenol, boric acid, and 
mercuric chloride in aqueous solution. Ruediger (1914) found a 
distinct but weak bactericidal action of. glycerol which was stronger 
at 30° than at 15° C. The action is limited by the diluent. Glycerol 
diluted with normal salt solution killed bacteria more quickly than 
when diluted with bouillon. In dilutions of 50 per cent anthrax STANDARDIZATION OF DISINFECTANTS 
143 
organisms were not killed in fifteen days. A selective action was 
noticed. 
Quinone. Bactericidal activity of quinone has been found to be 
due to its chemical action on certain constituent proteins of the bacterial 
cell. According to Cooper quinone is a stronger bactericide than 
phenol, quinol, or acetone, on account of its reactivity towards proteins 
in much lower concentrations. 
Dyes as Disinfectants. Recently it has been observed that dye- 
stuffs exert a distinct retarding action on bacteria. Churchman (1912) 
made one of the most important contributions to this question. He 
reported that gentian violet in dilutions of 1 to 100,000 when added to 
media would inhibit the development of certain bacteria. The Gram 
negative bacteria grew fairly well on the gentian violet media while 
generally the Gram positive would not. The subject has been greatly 
enriched by much recent work especially that of Krumwiede and his 
co-workers. Some of these researches are mentioned in other places. 
Table IX 
SHOWING ANTISEPTIC VALUES OF SOME COMMON SUBSTANCES 
(After Park and Williams) 
Allim 1 : 222 
Aluminum acetate 1 : 6000 
Boric acid 1:9 
Calcium chloride 1 : 143 
Calcium hypochlorite 1 : 25 
Carbolic acid 1 : 1000 
Chloral hydrate 1 : 333 
Cupric sulphate 1 : 107 
Ferrous sulphate 1 : 2000 
Formaldehyde 1 : 10,000 
Hydrogen peroxide 1 : 20,000 
Mercuric chloride 1 : 14,300 
Mercuric iodide 1 : 40,000 
Potassium bromide 1 : 10 
Potassium iodide. 1 : 10 
Potassium permanganate. . 1 : 300 
Pure formaldehyde 1 : 25,000 
Quinine sulphate 1 : 800 
Silver nitrate 1 : 12,500 
Sodium borate 1:14 
Sodium chloride 1:6 
Zinc sulphate 1 : 20 
Standardization of Disinfectants 
It is essential to know as far as possible just how toxic to the 
bacterial cell certain chemicals are. This knowledge is important in 
the purchase of large amounts of disinfectants and in their application 
to disinfection problems. That the chemical analysis would not 
furnish this information is apparent. Furthermore, many of the 
disinfecting compounds are of such complicated nature that a chemical 
analysis would be impossible. In view of these facts, many investi- 
gators have attempted to devise a purely bacteriological method since 144 
STERILIZATION AND DISINFECTION 
in disinfection it is the aim to destroy bacteria and the reaction must 
take place between the bacterial cell and the disinfectant. 
The bactericidal activity of chemicals has been studied by many 
investigators. Robert Koch in the early days of the science gave 
some attention to the question. He devised a method wherein the 
bacteria were dried on silk threads. These were then suspended in 
known dilutions of the disinfectant. After the process had been com- 
pleted these threads were then transferred to media in order to deter- 
mine whether the bacteria had been killed. He used Bacillus anthracis 
for the test organism. Sternberg mixed 5 c.c. of a young culture 
with equal quantities of the germicide. At regular intervals, loops of 
this mixture were removed and put in broth. Kronig and Paul (1897) 
dipped garnets into a culture of Bacillus anthracis containing spores 
and allowed them to dry. These garnets were then put into the dis- 
infecting solutions and a few taken out at intervals. They were 
shaken in a definite amount of water and plates made from this. In 
this way they determined the rate of death. They realized that the 
value of any disinfectant varied with the conditions under which 
it acted. 
Upon such data as these our present methods have been devised. 
These represent the present stage in the effort to secure satisfactory 
methods with which to measure strength of disinfectants. Walters 
(1917) has pointed out that disinfectants possess a certain specificity 
for certain bacteria. He further argues that the strength of a disin- 
fectant may only be stated in terms of the organism on which it is to 
be used in actual practice. 
Rideal-Walker Method. Anderson and McClintic (1912) have 
summed up this method as follows: 
Briefly stated, the carbolic coefficient in the Rideal-Walker method 
is arrived at by dividing the figure indicating the degree of dilution 
of the disinfectant that kills an organism in a given time by that 
expressing the degree of dilution of the carbolic acid that kills the 
same organism in the same time under exactly similar conditions. 
Leaving out details, the determination of the Rideal-Walker coefficient 
is substantially as follows: 
Certain standard conditions are considered essential to the proper 
performance of the test. Phenol solutions of known strength are 
used; cultures are grown in a standard medium, transplants being 
made every twenty-four hours; the loops used for all inoculations are 
of a standard size (about 4 mm. in diameter). Usually four dilutions 
of suitable strengths of the disinfectant to be used are made. Phenol RIDEAL-WALKER METHOD 
145 
controls of a suitable strength are also prepared. Five c.c. of each 
of these dilutions are placed in sterile test tubes, to which are added 
at intervals of one-half minute a twenty-four-hour broth culture of 
B. typhosus in the proportion of 1 drop of culture to each cubic centi- 
meter of disinfectant used (according to Partridge, 1 drop of culture 
equals about 0.1 c.c.). 
At the end of two and a half minutes a loopful of each of the mix- 
tures is inoculated into a test tube containing 5 c.c. of standard broth, 
an interval of half a minute being thus allowed between taking the 
samples from the different dilutions. This is repeated at five, seven and 
a half, ten, twelve and a half, and fifteen minutes. The broth tubes, 
after being incubated at 37° C. for forty-eight hours, are examined for 
growth. 
The results of the examination are then noted, and if suitable 
comparative strengths of the disinfectant and carbolic acid have been 
selected the carbolic acid coefficient is determined as above stated. 
The following table (Table 10) illustrates the manner of determining 
the carbolic acid coefficient of a disinfectant according to the Rideal- 
Walker method: 
Table X 
Name, " A." 
Temperature of medication, 20° C. 
Culture used, B. typhosus, twenty-four-hour, extract broth, filtered. 
Proportion of culture and disinfectant, 0.1 c.c.+5 c.c. 
Sample. 
Dilution. 
Time Culture Exposed to Action 
of Disinfectant for Minutes. 
Phenol Coeffi- 
cient. 
21 
J 5 
71 
10 
121 
15 
Phenol 
1 : 90 
1 : 100 
+ 
+ 
1 + 
+ 
- 
- 
- 
100)550 _ 
5.5' 
coefficient. 
Disinfectant " A " 
1 : 500 
+ 
+ 
- 
- 
- 
- 
• 
1 : 550 
1 : 600 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
- 
- 
This method has had to be greatly modified. There are certain 
things taken for granted which may materially alter the results. It is 
assumed that the rate of death is proportional to the concentration 
of the disinfectant. Bacteria are regarded as molecules in their reaction 
with the disinfectant. Chick has recommended thirty minutes as the 
standard time in place of fifteen minutes. She pointed out the error 146 
STERILIZATION AND DISINFECTION 
in the method by working with anthrax spores. The Rideal-Walker 
method does not consider the presence of organic matter. 
Chick and Martin's Modification of the Rideal-Walker Method. 
These authors have pointed out that an arbitrary time must be selected 
during which the disinfectant shall be allowed to act. The Rideal- 
Walker method is modified with this in view. The procedure is as 
follows: 
Without Organic Matter 
Everything used in the experiment, tubes, pipettes, etc., being 
previously sterilized, a series of tubes containing 5 c.c. of disinfectant 
in different concentrations are placed in a water bath at 20° C. When 
the tubes have taken the temperature of the bath, they are one after 
another inoculated with five drops of a twenty-four hour culture of B. 
typhosus from a standard pipette, the time being registered by a 
chronograph. Exactly one minute is allowed to pass between each 
inoculation. When thirty minutes have elapsed since the first tube 
was inoculated, samples in duplicate arc taken from it with a platinum 
loop, and sown in 10 c.c. glucose broth containing litmus. One min- 
ute later the second tube is sampled and so on. These test cultures 
are incubated at 37° C. and always kept four days under observation. 
Supposing the value of the disinfectant to be tested is totally 
unknown, the first series of observations must be scattered over a 
wide range, e.g., concentrations from 1 in 10 to 1 in 10,000. Having 
ascertained that the concentration necessary to kill in thirty minutes 
is between, say, 10 in 1000 and 1 in 1000 the second series is arranged 
to narrow it down to between, say, 4 and 5 per 1000, and a third series 
may determine the necessary concentration as between 4.2 and 4.5 
per 1000. At this last trial a series of tubes, containing various 
strengths of pure phenol, are simultaneously tested. 
With Organic Matter 
In order to determine the effect of the presence of organic matter, 
Chick and Martin use dried, pulverized feces as follows: 
The feces used were' dried first in a water bath and subsequently 
at 105° C., ground to a fine powder, and passed through a fine sieve 
with a mesh of 130 to the inch. Quantities of 0.15 germs were weighed 
out and placed in test tubes. To each test tube 2.5 c.c. distilled water 
was added and the tube sterilized in the autoclave (ten minutes at LANCET METHOD 
147 
120° C.) The tubes were covered with india-rubber caps and kept in 
jars with greased lids to prevent evaporation. 
At the time of the experiment different amounts of a suitable 
dilution of the disinfectant were added to each tube together with 
enough distilled water to make the total volume up to 5 c.c. The 
tubes then contained different concentrations of the disinfectant in 
question in the presence of 3 per cent feces. The tubes were inoculated 
and sampled in exactly the same way as when the test was made in 
distilled water without organic matter. 
Lancet Method. This is a modification of the Rideal-Walker 
method. The following method was advised: 
Bacillus coli communis from a twenty-four hour broth culture 
which had been incubated at 37° C. was used as the test organism. 
The disinfectants were diluted with distilled water and placed in special 
containers 2| by | ins. Platinum spoons were advised instead of loops 
for the seeding. By this technique more fluid could be carried than 
on any ordinary loop. These spoons carried about 0.08 c.c. of water. 
MacConkey's lactose bile broth was advised for the secondary culture 
tubes. Samples were to be removed every two and a half minutes 
from each container up to fifteen minutes. After this the time interval 
was to be fifteen, twenty, twenty-five and thirty minutes. All tests 
were carried out between 62° and 67° F. The carbolic acid coefficient 
was deduced as follows: " The figure representing the percentage 
strength of the weakest lethal dilution of the carbolic acid control was 
divided by the figure representing the percentage strength of the 
weakest lethal dilution of the disinfectant being tested. This was done 
both at the two-and-a-half-minute line and at a thirty-minute line and a 
mean of the resulting figures was taken as the carbolic acid coefficient." 
Determinations of the phenol coefficient have not yielded consistent 
results in the hands of the various investigators. This may not be due 
to the method itself, although it is not perfect in its present technique, 
but to the media used in the various laboratories. Phelps has pointed 
out some of the limitations and Supfle and Dlugeler, Norton, and 
have emphasized the necessity of great care in the preparation of the 
media. The H-ion concentration appears to be very important. De- 
spite these objections, the phenol coefficient is probably the best method 
available at present, for the comparison of the disinfecting properties 
of the coal-tar products. 148 
STERILIZATION AND DISINFECTION 
Hygienic Laboratory Phenol Coefficient 
A. WITHOUT ORGANIC MATTER 
Having discussed the necessity for a satisfactory method of stand- 
ardizing disinfectants and the factors involved in the examination 
of disinfectants, we present below the method we* have devised. 
When this method is used for the standardization of disinfectants 
we recommend that it be referred to as the " Hygienic Laboratory 
phenol coefficient." 
We prefer to use the word " phenol " instead of " carbolic acid " 
when speaking of the coefficient, especially since certain dealers adver- 
tise for sale carbolic acids which vary greatly in the proportion of 
phenol present. 
Media. Standard extract broth is used, both for the culture to be 
tested and for the subcultures made after exposure to the disinfectant. 
The broth is made from Liebig's extract of beef and is in exact accord- 
ance with the standard methods adopted by the American Public Health 
Association for water analysis. Ten c.c. of the broth are put into each 
test tube. This amount of broth has been found sufficient to avoid any 
antiseptic action of the disinfectant carried over. It is important that 
the reaction of the media is just +1.5. 
Organism. For the test organism a twenty-four-hour-old broth 
culture in extract broth of the B. typhosus (Hopkins) is used. Before 
beginning a test the culture should be carried over every twenty-four 
hours on at least three successive days. For carrying over the culture 
one loopful of a 4 mm. platinum loop is used. 
Before being added to the disinfectant the culture is well shaken, 
filtered through sterile filter paper, and placed in the water bath in 
order that it may reach a temperature of 20° C. before being added to 
the disinfectant. 
Temperature. A standard temperature of 20° C. has been adopted 
for all experiments. This temperature is obtained by the use of a 
specially devised water bath. The cultures and dilutions of the dis- 
infectant are brought to this temperature before the beginning of the 
test. 
Proportion of Culture to Disinfectant. One-tenth c.c. of the culture 
is used, added to 5 c.c. of the disinfectant dilution. The amount of 
culture is measured with a pipette graduated in tenths of a cubic cen- 
timeter. 
* This method is quoted from Anderson and McClintic's original publication. HYGIENIC LABORATORY METHOD 
149 
Inoculation Loops. For making the transfer of the culture after 
exposure to the disinfectant a platinum loop 4 mm. in diameter of 23 
United States standard gauge wire is used. We have found it is of 
advantage to have at least 4, and preferably 6, loops. In order to save 
time in flaming the following method was devised: 
A block about 3 ins. wide, 10 ins. high, and 12 ins. long, containing 
4 or 6 grooves, spaced 2 ins. apart, is used. Into each of the grooves 
the platinum loop is laid so that the end of the loops extend about 5 ins. 
beyond the side of the block. The first step in the operation is to ster- 
ilize each loop by flaming with a fantail Bunsen burner before beginning 
the experiment. 
When ready to begin the operation the loop farthest from the operator 
is taken in the right hand and the inoculation made. It is then replaced 
in the groove with the right hand and the Bunsen burner (fantail) 
placed under it with the left hand. The next loop is then used replaced 
in its groove, and the Bunsen burner placed under it with the left hand, 
the first loop having been heated to redness while the second loop was in 
use. This procedure is then continued until all the inoculations have 
been made. The time required in making the inoculations and in 
replacing the loop is short, it being found that fifteen seconds is ample. 
Incubation. The subcultures are incubated forty-eight hours at 
37° C., and the results then read off and tabulated. 
Dilutions. Capacity pipettes for the original dilutions are invariably 
used. For the phenol controls a standard dilution of pure phenol 
(" Merck's Silver Label ") is made and standardized by the U. S. P. 
method (Koppeschaar) to contain exactly 5 per cent of pure phenol by 
weight. From this stock solution the higher dilutions are made fresh 
each day for that day's test. 
For the dilutions of the disinfectant a 5 per cent solution is made 
by adding 5 c.c. of the disinfectant to 95 c.c. of sterile distilled water. 
A standardized 5 c.c. capacity pipette is used for this, and after filling 
the pipette all excess of the disinfectant on the outside of the pipette is 
wiped off with sterile gauze. The contents of the pipette are then 
delivered into a cylinder containing 95 c.c. of sterile distilled water and 
the pipette washed out as clean as possible by aspiration and blowing 
out the contents of the pipette into the cylinder. The contents of the 
cylinder are then thoroughly shaken and the dilutions up to 1 : 500 
made from it, using delivery pipettes for measuring. For those dis- 
infectants which do not readily form a 5 per cent solution we make a 
1 per cent stock solution, and from this make the dilutions greater than 
1 : 100 in accordance with the second table of dilutions. If greater 150 
STERILIZATION AND DISINFECTION 
dilutions than 1 : 500 are to be made a 1 per cent solution is made 
from the 5 per cent solution and the higher dilutions made from 
this. 
We have adopted the following scale for making dilutions: 
For dilutions up to 1 : 70, increase or decrease by a difference of 5 
(i.e., 5 parts of water). 
From 1 : 70 to 1 : 60 by a difference of 10. 
From 1 : 160 to 1 : 200 by a difference of 20. 
From 1 : 200 to 1 : 400 by a difference of 25. 
From 1 : 400 to 1 : 900 by a difference of 50. 
From 1 : 900 to 1 : 1800 by a difference of 100. 
From 1 : 1800 to 1 : 3200 by a difference of 200. 
And so on if higher dilutions are necessary. 
It is important that the cylinders used for making the dilutions be 
correctly graduated, as we have found disregard of this factor an im- 
portant source of error. It is preferable to use standardized cylinders 
and pipettes, and we recommend that they be used whenever possible. 
They, of course, should be perfectly clean. For making the dilutions 
in accordance with the above scheme we have found the following tables 
of much service: 
Seeding Tubes. The seeding tubes are glass test tubes 1 in. in 
diameter and about 3 ins. long, with round bottoms. In order to 
measure the disinfectant into them they are placed in a suitable wooden 
stand to receive them. We found it convenient to use a wooden block 
containing 6 rows of 15 holes each for the disinfectant to be tested and 
a separate stand for the phenol controls. The tubes are placed in the 
stand and each marked with the strength of dilution it is to 
contain. 
Starting with the lowest dilution (i.e., the strongest), the cylinder 
is shaken, then 5 c.c. are measured into the tubes marked to receive 
that strength, using a 5-c.c. delivery pipette. In order to economize 
glassware, the same pipette is used for measuring out the next dilution, 
first blowing out as much of the remaining liquid as possible, then 
drawing a pipetteful of the next dilution to be used and discarding that; 
then filling the pipette a second time which is emptied into the seeding 
tube. 
The measuring out being completed, the tubqs are placed in the 
water bath and allowed to stand a few minutes in order that the dis- 
infectant solution may reach the standard temperature. We have HYGIENIC LABORATORY METHOD 
151 
not found it necessary to use cotton plugs in the seeding tubes. They 
are sterilized in paper-lined wire baskets, with the closed end of the 
tubes up. 
Table XI 
(FOR DILUTIONS)-STOCK 5 PER CENT SOLUTION 
[5 c.c. Disinfectant+95 c.c. Distilled Water = Solution A] 
1 
c.c. 
of 
A. 
: 20 = 20 
+ 
c.c. 
dist. 
water 
0 
or 
c.c. 
of 
A. 
10 - 
c.c. 
dist. 
water 
0 
c.c. 
of 
A. 
or 4 
+ 
c.c. 
dist 
water. 
0 
1 
: 25 = 20 
+ 
5 
or 
10 + 
21 
or 4 
+ 
1 
1 
: 30 = 20 
+ 
10 
or 
10 + 
5 
or 4 
+ 
2 
1 
: 35 = 20 
+ 
15 
or 
10 + 
71 
or 4 
+ 
3 
1 : 
: 40 = 20 
+ 
20 or 
10 + 
10 
or 4 
+ 
4 
1 : 
: 45 = 20 
+ 
25 or 
10 + 
12j 
or 4 
+ 
5 
1 : 
: 50 = 20 
+ 
30 
or 
10 + 
15 
or 4 
+ 
6 
1 
: 55 = 20 
+ 
35 
or 
10 + 
171 
or 4 
+ 
7 
1 
: 60 = 20 
+ 
40 
or 
io + 
20 
or 4 
+ 
8 
1 
: 65 = 20 
+ 
45 
or 
10 + 
221 
or 4 
+ 
9 
1 
: 70 = 20 
+ 
50 
or 
10 + 
25 
or 4 
+ 
10 
1 
: 70 = 20 
+ 
50 or 
10 + 
25 
or 4 
+ 
10 
1 
: 80 = 20 
+ 
60 
or 
10 + 
30 
or 4 
+ 
12 
1 
: 90 = 20 
+ 
70 or 
10 + 
35 
or 4 
+ 
14 
1 
: 100 = 20 
+ 
80 or 10 + 
40 
or 4 
+ 
16 
1 
: 110 = 20 
+ 
90 or 10 4- 
45 
or 4 
+ 
18 
1 
: 120 = 20 
+ 
100 
or 
10 + 
50 
or 4 
+ 
20 
1 
: 139 = 20 
+ 
110 or 10 + 
55 
or 4 
+ 
22 
1 
: 140 = 20 
+ 
120 
or 
10 + 
60 
or 4 
+ 
24 
1 
: 150 = 20 
+ 
130 
or 
10 + 
65 
or 4 
+ 
26 ■ 
1 
: 160 = 20 
+ 
140 
or 
10 + 
70 
or 4 
+ 
28 
1 
: 160 = 20 
+ 
140 
or 
10 + 
70 
or 4 
+ 
28 
1 
: 180 = 20 
+ 
160 
or 
10 + 
80 
or 4 
+ 
32 
1 
: 200 = 20 
+ 
180 
or 
10 + 
90 
or 4 
+ 
36 
1 
: 200 = 20 
+ 
180 
or 
4 + 
36 
or 2 
+ 
18 
1 
: 225 = 20 
+ 
205 
or 
4 + 
41 
or 2 
+ 
20J 
1 
: 250 = 20 
+ 
230 
or 
4 + 
46 
or 2 
- 
23 
1 
: 275 = 20 
+ 
255 
or 
4 + 
51 
or 2 
+ 
251 
1 
: 300 = 20 
+ 
280 
or 
4 + 
56 
or 2 
+ 
28 
1 
: 325 = 20 
+ 
305 
or 
4 + 
61 
or 2 
+ 
301 
1 
: 350 = 20 
+ 
330 
or 
4 + 
66 
or 2 
+ 
33 
1 
: 375 = 20 
+ 
355 
or 
4 + 
71 
or 2 
+ 
351 
1 
: 400 = 20 
+ 
380 
or 
4 + 
76 
or 2 
+ 
38 
1 
: 450 = 20 
+ 
430 
or 
4 + 
86 
or 2 
+ 
43 
1 
: 500 = 20 
+ 
480 
or 
4 + 
96 
or 2 
+ 
48 
• 152 
STERILIZATION AND DISINFECTION 
Table XII 
(FOR DILUTIONS)-STOCK 1 PER CENT SOLUTION 
[1 c.c. Disinfectant+99 c.c. Distilled Water = Solution B] 
1 : 
1 : 
1 : 
1 : 
1 : 
1 
1 
c.c. 
of 
B. 
: 100 = 100 
: 110 = 100 
: 120 = 100 
: 130 = 100 
: 140 = 100 
: 150 = 100 
: 160 = 100 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
c.c. 
dist. 
water. 
0 or 
10 or 
20 or 
30 or 
40 or 
50 or 
60 or 
c.c. 
of 
B. 
10 
10 
10 
10 
10 
10 
10 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
c.c. 
dist. 
water 
0 
1 
2 
3 
4 
5 
6 
c.c. 
of 
B. 
c.c. 
dist. 
water. 
1 
: 160 = 
100 
+ 
60 or 
10 
+ 
6 
1 
: 180 = 
100 
+ 
80 or 
10 
+ 
8 
1 
: 200 = 
100 
+ 
100 or 
10 
+ 
10 
1 
: 200 = 
100 
+ 
100 or 
10 
+ 
10 
or 4 
+ 
4 
1 
: 225 = 
100 
+ 
125 or 
10 
+ 
12$ 
or 4 
+ 
5 
1 
: 250 = 
100 
+ 
150 or 
10 
+ 
15 
or 4 
+ 
6 
1 
: 275 = 
100 
+ 
175 or 
10 
+ 
17| 
or 4 
+ 
7 
1 
: 300 = 
100 
+ 
200 or 
10 
+ 
20 
or 4 
+ 
8 
1 
: 325 = 
100 
+ 
225 or 
10 
+ 
22| 
or 4 
+ 
9 
1 
: 350 = 
100 
+ 
250 or 
10 
+ 
25 
or 4 
+ 
10 
1 
: 375 = 
100 
+ 
275 or 
10 
+ 
27| 
or 4 
+ 
11 
1 
: 400 = 
100 
+ 
300 or 
10 
+ 
30 
or 4 
+ 
12 
1 
: 400 = 
10 
+ 
30 or 
4 
+ 
12 
or 2 
+ 
6 
1 
: 450 = 
10 
+ 
35 or 
4 
+ 
14 
or 2 
+ 
7 
1 
: 500 = 
10 
+ 
40 or 
4 
+ 
16 
or 2 
+ 
8 
1 
: 550 = 
10 
+ 
45 or 
4 
+ 
18 
or 2 
+ 
9 
1 
: 600 = 
10 
+ 
50 or 
4 
+ 
20 
or 2 
+ 
10 
1 
: 650 = 
10 
+ 
55 or 
4 
+ 
22 
or 2 
+ 
11 
1 
: 700 = 
10 
+ 
60 or 
4 
+ 
24 
or 2 
+ 
12 
1 
: 750 = 
10 
+ 
65 or 
4 
+ 
26 
or 2 
+ 
13 
1 
: 800 = 
10 
+ 
70 or 
4 
+ 
28 
or 2 
+ 
14 
1 
: 850 = 
10 
+ 
75 or 
4 
+ 
30 
or 2 
+ 
15 
1 : 
: 900 = 
10 
+ 
80 or 
4 
+ 
32 
or 2 
+ 
16 
1 : 
: 900 = 
5 
+ 
40 or 
4 
+ 
32 
or 2 
+ 
16 
1 : 
: 1000 = 
5 
+ 
45 or 
4 
+ 
36 
or 2 
+ 
18 
1 : 
: 1100 = 
5 
+ 
50 or 
4 
+ 
40 
or 2 
+ 
20 
1 : 
: 1200 = 
5 
+ 
55 or 
4 
+ 
44 
or 2 
+ 
22 
1 : 
: 1300 = 
5 
+ 
60 or 
4 
+ 
48 
or 2 
+ 
24 
1 : 
: 1400 = 
5 
+ 
65 or 
4 
+ 
52 
or 2 
+ 
26 
1 : 
: 1500 = 
5 
+ 
70 or 
4 
+ 
56 
or 2 
+ 
28 
1 : 
: 1600 = 
5 
+ 
75 or 
4 
+ 
60 
or 2 
+ 
30 
1 : 
: 1700 = 
5 
+ 
80 or 
4 
+ 
64 
or 2 
+ 
32 
1 : 
« 
: 1800 = 
5 
+ 
85 or 
4 
+ 
68 
or 2 
+ 
34 HYGIENIC LABORATORY METHOD 
153 
Table XII-Continued 
(FOR DILUTIONS)-STOCK 1 PER CENT SOLUTION 
[1 c.c. Disinfectant+99 cc. Distilled Water = Solution B] 
c.c. 
of 
B. 
cc. 
dist. 
water. 
c.c. 
of 
B. 
c.c. 
dist. 
wrater. 
c.c. 
of 
B. 
c.c. 
dist. 
water. 
1 
: 1800 = 
5 
+ 
85 or 
4 
+ 
68 
or 2 
+ 
34 
1 
: 2000 = 
5 
+ 
95 or 
4 
+ 
76 
or 2 
+ 
38 
1 
: 2200 = 
5 
+ 
105 or 
4 
+ 
84 
or 2 
+ 
42 
1 
: 2400 = 
5 
+ 
115 or 
4 
+ 
92 
or 2 
+ 
46 
1 
: 2600 = 
5 
+ 
125 or 
4 
+ 
100 
or 2 
+ 
50 
1 
: 2800 = 
5 
+ 
135 or 
4 
+ 
108 
or 2 
+ 
54 
1 
: 3000 = 
5 
+ 
145 or 
4 
+ 
116 
or 2 
+ 
58 
1 
: 3200 = 
5 
+ 
155 or 
4 
+ 
124 
or 2 
+ 
62 
Subculture Tube Racks. Wooden racks, with 5 rows of 14 holes 
each, are used for holding the subculture tubes, and as plants are made 
from each mixture of culture and disinfectant every 2| minutes up 
to fifteen minutes, 6 tubes are required for each dilution. Thus, in 
each rack we have 10 rows of 6 tubes each with 2 empty cross rows of 
Fig. 43.-Block for Subculture Tubes. 
holes remaining, which are utilized by placing over in the next row each 
tube as it is planted. This makes it easy to keep run of the tubes that 
are planted. It is well also always to plant from the seeding tube in a 
certain hole in the water bath into a certain row of tubes in the rack. 
This, after a little practice, will help to avoid errors in planting. 
Method of Conducting the Test. If there are in one experiment 
more than 10 dilutions of the disinfectant, including the phenol controls, 
the stronger solutions of the disinfectant and phenol are tested first, 
as it will not be necessary to plant them after seven and a half minutes. 
The weaker solutions are then immediately done and are then planted 
every two and a half minutes for fifteen minutes. 
For keeping the time a stop watch can be used, but an ordinary 
watch will serve the same purpose by simply starting on the two-and- 
one-half- or five-minute periods. 154 
STERILIZATION AND DISINFECTION 
When everything is in readiness the culture is added to the dis- 
infectant solutions with a sterile pipette in quantities of a tenth of 
a cubic centimeter to each dilution. 
To add the culture, the seeding tube containing the disinfectant 
is removed from the water bath with the left hand and slanted at 
an angle of about 45°, and with the right hand the end of the pipette 
containing the culture is introduced and lightly touched against the 
side of the tube where the liquid has run away on account of the 
slanting. At the proper time the culture is allowed to run into the 
disinfectant solution, the pipette removed, the tube straightened up, 
gently shaken three times, and 
replaced in the water bath. The 
other tubes are done the same way 
in succession, and it will be found 
that fifteen seconds is ample time for 
each tube. By adding the culture 
to the disinfectant with a pipette 
touched against the side of the 
seeding tube, accurate measure- 
ments can be made and each tube 
receive exactly the same amount 
of " seeding," which is not the case 
when the culture is added by the 
" drop." 
If ten tubes are to be inocu- 
lated, only a few seconds will remain 
after inoculating the last tube before 
a plant from the first tube will have 
to be made. 
The mixing tubes are not removed or disturbed in making the 
planting except to insert the loop or spoon into them, touch the botton, 
withdraw, and then make the plant in broth. Every effort is made to 
insert and withdraw the loops or spoons in a uniform manner. The 
loops and spoons are bent to an angle of about 45° where they are 
jointed to the shank, and therefore are always filled with the mixture 
when withdrawn from the seeding tubes. After making the plants 
the loops or spoons are flamed as already described. 
After an experiment is finished the date and any necessary details 
can be marked on one of the broth tubes and the rack placed in the 
incubator at 37° C. for forty-eight hours. At the end of this time 
the results are recorded on a chart specially devised for the purpose. 
Fig. 44.-Cross-section of Water 
Bath Showing Seeding Tubes in 
Place. HYGIENIC LABORATORY METHOD 
155 
Determining the Coefficient. After a large number of experiments 
we have concluded that the method employed by the Lancet commission, 
with certain modifications, is the best one for determining the coeffi- 
cient, i.e., the mean between the strength and time coefficients. 
In performing the test, plants are made every two and a half minutes 
up to and including fifteen minutes. To determine the coefficient, the 
figure representing the degree of dilution of the weakest strength of the 
disinfectant that kills within two and a half minutes is divided by 
the figure representing the degree of dilution of the weakest strength of 
Fig. 45.-Device for Flaming Inoculating Loops. 
the phenol control that kills within the same time. The same is done for 
the weakest strength that kills in fifteen minutes. The mean of the 
two is the coefficient. The method of determining the coefficient will 
be seen in Table XIII. 
B. THE DETERMINATION OF THE COEFFICIENT WITH THE ADDITION OF 
ORGANIC MATTER 
It may be well to briefly discuss here the meaning or significance 
of the term " phenol coefficient," particularly when it is determined 
in the presence of organic matter. In general terms the coefficient 156 
STERILIZATION AND DISINFECTION 
of a disinfectant may, for practical purposes, be defined as the figure 
that represents the ratio of the germicidal power of the disinfectant 
to the germicidal power of carbolic acid, both having been tested under 
the same conditions. 
Table XIII 
Name " A." 
Temperature of medication, 20° C. 
Culture used, B. typhosus, 24-hour, extract, broth, filtered. 
Proportion of culture and disinfectant, 0.1 c.c.+5 c. c. 
Time Culture Exposed to Action 
of Disinfectant for Minutes. 
Sample. 
Dilution. 
Coefficient. 
21 
5 
71 
10 
121 
15 
Phenol 
1 : 80 
375 650 
1 : 90 
4- 
- 
- 
- 
80 + 10 _ 
1 : 100 
+ 
4- 
4- 
- 
- 
- 
2 
1 : 110 
+ 
4- 
+ 
4- 
4" 
4.694-5.91 
Disinfectant " A " 
1 : 350 
- 
- 
2 
1 : 375 
- 
- 
- 
5.30 
1 : 400 
4- 
- 
- 
- 
1 : 425 
4- 
+ 
- 
- 
- 
- 
1 : 450 
+ 
4- 
- 
- 
- 
- 
1 : 500 
+ 
+ 
- 
- 
- 
- 
1 : 550 
+ 
4- 
+ 
- 
- 
- 
- 
1 : 600 
+ 
+ 
4- 
4- 
- 
- 
1 : 650 
4- 
4- 
4- 
4- 
4- 
- 
1 : 700 
+ 
+ 
+ 
4- 
+ 
4- 
• 
1 : 750 
+ 
4- 
4- 
+ 
+ 
4- 
Although the germicidal power of carbolic acid is taken as the unit 
of comparison, it is influenced to a certain extent by the conditions, par- 
ticularly the addition of organic matter, or, in other words, it is not 
a constant unit. This has to be borne in mind when making a com- 
parison of the relative values of the phenol coefficients of a disin- 
fectant determined with and without the addition of organic matter, 
respectively. It will readily be seen, for instance, that if the germi- 
cidal powers of a disinfectant and of carbolic acid were proportion- 
ately reduced by the addition of organic matter the coefficient of the 
disinfectant would remain unchanged regardless of whether or not 
organic matter was used. However, the germicidal power of car- 
bolic acid, like the other pure phenols, is, as compared with most 
other disinfectants, only slightly affected by the addition of organic 157 
HYGIENIC LABORATORY METHOD 
matter, and therefore serves as a fairly accurate means of estimat- 
ing, in the presence of organic matter, the germicidal values of dis- 
infectants in general. 
The method of determining the coefficient of disinfectants with 
the addition of organic matter is identical in many respects with the 
method in which no organic matter is used, the former differing from the 
latter principally in the strength of the disinfectant dilutions that 
have to be prepared and in the prepa- 
ration and addition of the organic 
matter to the disinfectant dilutions 
when performing the test. As the 
method in which no organic matter 
is added has already been given in 
detail, further description here will 
consist only of the differences between 
the two methods. 
Dilutions. In making the dilutions 
of a disinfectant for determining its 
coefficient in the presence of organic 
matter, allowance must be made for the 
further dilution of the disinfectant when 
the volume of organic matter is added 
thereto. For instance, if 1 c.c. of 
organic matter is added to 5 c.c. of a 
1 per cent dilution of a disinfectant the 
percentage strength of the disin- 
fectant is proportionately reduced, it 
then being about 0.83 per cent. 
The volume of organic matter that 
can be added to the disinfectant 
dilution is, of course, variable, as 
is also the volume of disinfectant 
dilution to which it can be added. Consequently, we have decided 
rather arbitrarily to use the organic matter by adding 1 c.c. of it to 
4 c.c. of the disinfectant dilution contained in a seeding tube. It 
will be seen that the strength of the disinfectant is reduced 20 per 
cent by the addition of the organic matter and that the dilutions of 
the disinfectant must be made accordingly. For example, if it is 
desired to test a 1 per cent strength of a disinfectant it is necessary 
to prepare a strength of 1.25 per cent, 4 c.c. of which becomes a 1 per 
cent strength when 1 c.c. of the organic matter is added to it. 
Fig. 46-Water Bath Showing Po- 
sition of Holes for Seeding Tubes 
and Thermometer in Place. 158 
STERILIZATION AND DISINFECTION 
We also tried adding 2.5 c.c. of the organic matter to 2.5 c.c. of 
the disinfectant dilution, but found it rather difficult and cumber- 
some to do, particularly when the experiment has to be performed 
with a number of different strengths of the disinfectant. When using 
the proportions as just stated, the dilutions of the disinfectant are 
made double the strengths it is desired to test, thus allowing for the 
further dilution when an equal volume of organic matter is added 
thereto. 
In using the proportions of 1 c.c. of organic matter to 4 c.c. of the 
disinfectant dilution we have found the following tables (XIV and XV) 
of service in preparing the dilutions of the disinfectant: 
It will be seen that by preparing the dilutions of the disinfectant 
according to the strengths shown in the second column of Table XIV, 
and then adding 1 c.c. of organic matter to 4 c.c. of the dilutions, the 
final dilutions of the disinfectant become as represented in the first 
column of the table given above. 
Preparation and Use of the Peptone-gelatine Organic Matter. 
As already stated, we prefer to use a mixture of peptone and gelatine 
dissolved in distilled water. It is prepared from Witte's peptone 
(siccum) and " Best French Gold Label " gelatine. The stock prepara- 
tion is made to contain 10 per cent peptone and 5 per cent gelatine. 
Proportionate amounts of peptone and gelatine, respectively, are 
weighed out and liquefied separately in small quantities of water by 
means of heat. They are then mixed in a graduate and sufficient 
water added thereto to make a mixture containing 10 per cent of pep- 
tone and 5 per cent of gelatine. It is then placed in bottles of appro- 
priate size and sterilized on three successive days. When the mixture 
has become cold it will be observed that some of the peptone settles to 
the bottom of the bottles as a flocculent deposit. Consequently, the 
bottle should be shaken before using it. The stock preparation con- 
taining 5 per cent gelatin becomes semi-solid if it is kept in the cold room 
at a temperature of 16° C. However, by warming it until it becomes 
perfectly liquefied and then not allowing it to go below the temperature 
of 20° C. we found that it remains liquid and is easily measured in the 
pipette. 
By adding 1 c.c. of the stock preparation of 10 per cent peptone 
and 5 per cent gelatin to 4 c.c. of the disinfectant dilutions a result- 
ing mixture is obtained containing 2 per cent of peptone and 1 per cent 
of gelatin, or a total of 3 per cent organic matter. 
The Method of Conducting the Test. Four c.c. of each disinfectant 
dilution, including the phenol controls, are accurately measured into HYGIENIC LABORATORY METHOD 
159 
Table XIV 
STOCK 5 PER CENT SOLUTION 
[5 c.c. Disinfectant+95 c.c. Distilled Water = Solution A] 
Strength to 
be tested 
Strength to 
be made. 
c.c. of 
A. 
c.c. dist. 
water. 
c.c. of 
A, 
c.c. dist. 
water. 
1 
: 30 
1 
: 24 
= 
20 
+ 
4 
or 
10 
+ 
2 
1 
: 35 
1 
: 28 
= 
20 
+ 
8 
or 
10 
+ 
4 
1 
: 40 
1 
: 32 
= 
20 
+ 
12 
or 
10 
+ 
6 
1 
: 45 
1 
: 36 
= 
20 
+ 
16 
or 
10 
+ 
8 
1 
: 50 
1 
: 40 
= 
20 
+ 
20 
or 
10 
+ 
10 
1 
: 55 
1 
: 44 
= 
20 
+ 
24 
or 
10 
+ 
12 
1 
: 60 
1 
: 48 
= 
20 
+ 
28 
or 
10 
+ 
14 
1 
: 65 
1 
: 52 
SB 
20 
+ 
32 
or 
10 
+ 
16 
1 
: 70 
1 
: 56 
a® 
20 
+ 
36 
or 
10 
+ 
18 
1 
: 70 
1 
: 56 
= 
10 
+ 
18 
or 
5 
+ 
9 
1 
: 80 
1 
: 54 
= 
10 
+ 
22 
or 
5 
+ 
11 
1 
: 90 
1 
: 72 
= 
10 
+ 
26 
or 
5 
+ 
13 
1 
: 100 
1 
: 80 
= 
10 
+ 
30 
or 
5 
+ 
15 
1 
: 110 
1 
: 88 
SB 
10 
+ 
34 
or 
5 
+ 
17 
1 
: 120 
1 
: 96 
= 
10 
+ 
38 
or 
5 
+ 
19 
1 : 
: 130 
1 : 
: 104 
sa 
10 
+ 
42 
or 
5 
+ 
21 
1 : 
: 140 
1 : 
: 112 
= 
10 
+ 
46 
or 
5 
+ 
23 
1 : 
: 150 
1 : 
: 120 
= 
10 
= 
50 
or 
5 
+ 
25 
1 : 
: 160 
1 : 
: 128 
* 
10 
+ 
54 
or 
5 
+ 
27 
1 
: 160 
1 
: 128 
= 
10 
+ 
54 
or 
5 
+ 
27 
1 
: 180 
1 : 
: 144 
= 
10 
+ 
62 
or 
5 
+ 
31 
1 
: 200 
1 : 
: 169 
10 
+ 
70 
or 
5 
+ 
35 
1 
: 200 
1 
: 160 
= 
5 
+ 
35 
or 
2 
+ 
14 
1 
: 225 
1 
: 180 
= 
5 
+ 
40 
or 
2 
+ 
16 
1 
: 250 
1 
: 200 
= 
5 
+ 
45 
or 
2 
+ 
18 
1 
: 275 
1 
: 220 
= 
5 
+ 
50 
or 
2 
+ 
20 
1 
: 300 
1 
: 240 
= 
5 
+ 
55 
or 
2 
+ 
22 
1 
: 325 
1 
: 260 
= 
5 
+ 
60 
or 
2 
+ 
24 
1 
: 350 
1 
: 280 
= 
5 
+ 
65 
or 
2 
+ 
26 
1 
: 375 
1 
: 300 
= 
5 
+ 
70 
or 
2 
+ 
28 
1 
: 400 
1 
: 320 
= 
5 
+ 
75 
or 
2 
+ 
30 
1 
: 400 
1 
: 320 
= 
5 
+ 
75 
or 
2 
+ 
30 
1 
: 450 
1 
: 360 
= 
5 
+ 
80 
or 
2 
+ 
32 
1 
: 500 
1 
: 400 
= 
5 
+ 
85 
or 
2 
+ 
34 
1 
: 500 
1 
: 440 
= 
5 
+ 
90 
or 
2 
+ 
36 
1 
: 600 
1 
: 480 
= 
5 
+ 
95 
or 
2 
+ 
38 
1 
: 650 
1 
: 520 
= 
5 
+ 
100 
or 
2 
+ 
40 160 
STERILIZATION AND DISINFECTION 
Table XV 
STOCK 1 PER CENT SOLUTION 
[1 c.c. Disinfectant+99 c.c. Distilled Water = Solution B] 
Strength to 
Strength to 
c.c. of 
c.e. dist. 
be tested 
be made. 
B. 
water. 
1 : 500 
1 : 400 
5 
+ 
15 
1 : 550 
1 : 440 
5 
+ 
17 
1 : 600 
1 : 480 
5 
+ 
19 
1 : 650 
1 : 520 
5 
+ 
21 
1 : 700 
1 : 560 
5 
+ 
23 
1 : 750 
1 : 600 
5 
+ 
25 
1 : 800 
1 : 640 
5 
+ 
27 
1 : 850 
1 : 680 
5 
+ 
29 
1 : 900 
1 : 720 
5 
+ 
31 
1 : 900 
1 : 720 
.5 
+ 
31 
1 : 1000 
1 : 800 
5 
+ 
35 
1 : 1100 
1 : 880 
5 
+ 
39 
1 : 1200 
1 : 960 
5 
+ 
43 
1 : 1300 
1 : 1040 
5 
+ 
47 
1 : 1400 
1 : 1120 
5 
+ 
51 
1 : 1500 
1 : 1200 
5 
+ 
55 
1 : 1600 
1 : 1280 
5 
+ 
59 
1 : 1700 
1 : 1360 
5 
+ 
63 
1 : 1800 
1 : 1440 
5 
+ 
67 
the seeding tubes and placed in the water bath. For reasons that will 
be obvious later, it is difficult to handle more than nine dilutions at one 
time in making a test. The broth culture of B. typhosus is filtered and 
placed in the water bath. The desired quantity of stock peptone- 
gelatine mixture is measured into a large test tube or flask and also 
placed in the water bath. It is necessary to have a slight excess of the 
organic matter, so that there will be no trouble in getting the 10 c.c. 
pipette full when adding the organic matter and culture to the disin- 
fectant. Thus, for nine tubes we usually measure out 15 c.c. of the 
organic matter. When the disinfectant dilutions, the typhoid culture, 
and the organic matter have reached the temperature of the water bath 
(20° C.) 1.5 c.c. of the typhoid culture is added to the 15 c.c. of organic 
matter and thoroughly mixed by means of a 10-c.c. pipette. With the 
same pipette the seeding tubes containing the disinfectant dilutions 
then have added to them, successively every fifteen seconds, 1.1. cc. of 
the mixture of the organic matter and typhoid culture. The technique 
of shaking, planting, etc., is then as has already been described. 
From the above it will be seen that each seeding tube, as the test 
is conducted, contains 4 c.c. of the disinfectant dilution plus 1 c.c. HYGIENIC LABORATORY METHOD 
161 
of organic matter, plus 0.1 c.c. of typhoid culture, and that the quan- 
tity of organic matter represented is 3 per cent. 
In computing the strengths of the disinfectant and the percentage 
of organic matter present in the seeding tubes when the test is made, 
we have disregarded the slight change resulting from the addition of the 
0.1 c.c. of typhoid culture to each tube. By so doing the experiment 
is very much simplified, as it would be very difficult to prepare the 
dilutions of the disinfectant on the basis of allowing for so slight a 
change as is caused by the addition of 0.1 c.c. of liquid. 
Many of the details of conducting the experiment and the method 
of determining the coefficient will be seen in Table No. 16: 
Table XVI 
Name, "A." 
Temperature of medication, 20° C. 
Culture used, B. typhosus, twenty-four-hour, extract broth, filtered. 
Proportion of culture, organic matter and disinfectant, 0.1 c.c.-|-l c.c.+4 c.c. 
Percentage of organic matter, 2 per cent peptone and 1 per cent gelatin. 
Total percentage of organic matter, 3 per cent. 
Sample. 
Dilution. 
Time Culture Exposed to Action 
Phenol coeffi- 
cient. 
of 
Disinfectant for 
Minutes. 
21 
5 
71 
10 
121 
15 
Phenol 
1 
80 
180 375 
1 
90 
+ 
+ 
- 
- 
- 
- 
"80 + 90" 
1 
100 
+ 
+ 
+ 
+ 
+ 
+ 
2 
1 
110 
+ 
+ 
4- 
+ 
+ 
4- 
2.25+4.17 
Disinfectant " A " 
1 
180 
- 
2 
1 
200 
+ 
- 
- 
- 
- 
1 
225 
+ 
- 
- 
- 
- 
- 
1 
250 
+ 
- 
- 
- 
- 
- 
1 
300 
+ 
+ 
- 
- 
- 
- 
1 
325 
+ 
+ 
- 
- 
- 
- 
1 
350 
+ 
+ 
+ 
- 
- 
- 
1 
375 
+ 
4- 
4- 
+ 
- 
- 
1 
400 
+ 
+ 
4- 
+ 
4- 
4- 
1 
450 
+ 
+ 
+ 
+ 
+ 
4- 
To Determine the Cost of a Disinfectant per Unit of Disinfecting 
Efficiency as Compared with Pure Phenol. It is manifestly cheaper to 
purchase a disinfectant for 60 cents a gallon than it is to purchase one 
for 30 cents a gallon, provided the former has four times the efficiency of 
the latter. The true cost of a disinfectant can only be determined by 162 
STERILIZATION AND DISINFECTION 
taking into consideration the phenol coefficient and the cost of the disin- 
fectant per gallon. 
The cost of a disinfectant per 100 units of efficiency as compared 
with pure phenol is obtained by first dividing the cost per gallon of 
the disinfectant by the cost per gallon of pure phenol; the efficiency 
ratio of course is obtained by dividing the coefficient of the disinfectant 
by the coefficient of phenol, but as the coefficient is always 1 the effi- 
ciency ratio is represented by the phenol coefficient of the disinfectant. 
The cost ratio divided by the efficiency ratio (the coefficient of the dis- 
infectant) gives the cost of the disinfectant per unit of efficiency as com- 
pared with the cost per unit of efficiency of pure phenol = 1. By mul- 
tiplying by 100 the relative cost per 100 units is obtained. Thus, 
Cost of disinfectant per gallon, . .Coefficient of disinfectant 
Cost of phenol per gallon ( a Coefficient of phenol( = l) 
( = efficiency ratio) = the cost of the disinfectant per unit of efficiency 
as compared with phenol =1. And by multiplying by 100 the cost 
per 100 units is obtained. 
For instance, the cost of disinfectant " Car " is $0.30 per gallon, and 
it has a coefficient of 2.12; the cost of phenol is $2.67 and has a coeffi- 
cient of 1. Then. 
.20 2.12 
2.67^ -='°52 
Therefore, the comparative cost per unit of efficiency of " Car " and 
phenol, respectively, is as 0.052 is to 1; or, by multiplying by 100, 
the relative cost per 100 units-5.2 to 100-is obtained. 
The following table illustrates the value of the determination of the 
cost per 100 units of efficiency of a disinfectant as compared with the 
cost of 100 units of pure phenol: 
Table XVII 
Disinfectant. 
Price per 
Gallon. 
Cost, Ratio. 
Efficiency 
Ratio, which 
is the Phenol 
Coefficient. 
Relative cost 
per 100 units 
of Efficiency 
as Compared 
with Pure 
Phenol = 100 
SO.30 
0.112 
2.12 
5.2 
Chi 
1.00 
.374 
4.44 
8.4 
Phi 
.37 
.138 
1.40 
9.9 
.44 
.164 
1.13 
14.5 
.41 
.153 
.44 
34.8 
Zod 
.40 
.150 
.25 
60.0 
Pure phenol 
2.67 
1.00 
1.00 
100.0 DETERMINATION OF PENETRATING POWER 
163 
It will be seen that the disinfectant Chi has a higher coefficient than 
any of the others in the table, but its higher cost per gallon results in 
its being placed second in cost per 100 units of efficiency. 
When bids are solicited for supplying disinfectants they should be 
required to be made so as to show the cost per 100 units of efficiency 
of the disinfectant as compared with the cost of 100 units of efficiency 
of pure phenol. In that way only can a contract for supplying disin- 
fectants be awarded in the combined interests of economy and effi- 
ciency. 
It may be proper to state here that in the specifications for bids for 
supplies for the fiscal year beginning July 1, 1912, prepared by the 
General Supply Committee, is the following specification for bids on 
disinfectants: 
Item 4136 (d), noncorrosive, cresol, phenol, or analogous compound; must 
be uniform material at 43° F. and 'dilute with water and form a practically 
perfect emulsion or solution and have a phenol coefficient according to present 
method of Hygienic Laboratory of not less than 2; 1 gal. sample required. 
Note.-The phenol coefficient of disinfectants on which bids are made must be 
stated. 
The General Supply Committee prepares specifications on which 
bids are solicited for supplies for all the departments in Washington. 
McClintic (1912) determined the phenol coefficient of some common 
disinfectants. The results are indicated in Table XVIII. 
Kendall and Edward's Method for Determining Penetrating Power. 
The authors have attempted to broaden the standardization of dis- 
infectants by determining the relative penetrating power of these agents. 
The important features of the Rideal-Walker method are retained. 
(1) Prepare a twenty-four-hour culture of B. Coli in plain broth 
of standard composition and reaction; (2) add 10 c.c. of this standard 
culture to 1 liter, of agar (1.5 per cent agar) and mix thoroughly; (3) 
pour the infected agar into sterile tubes of convenient length (1 meter) 
and of exactly 1.5 cm. diameter and allow to harden, after closing the 
ends with sterile rubber stoppers; (4) allow to harden at 20°. C.; (5) 
prepare dilutions of the desired disinfectants and a standard 5 per cent 
carbolic acid solution-the latter is the standard to which the other 
disinfectants are referred; (6) place the disinfectants so prepared in 
sterile beakers, allowing 50 c.c. for each agar cylinder; (7) remove 
stoppers from the infected agar tubes, and permit the contents to run 
out slowly as a long cylinder; with a sterile knife cut off portions by 
transverse cuts of 2 cm. length, and allow these smaller cylinders to 164 
STERILIZATION AND DISINFECTION 
Table XVIII 
SHOWING THE PHENOL COEFFICIENTS OF SOME COMMON 
DISINFECTANTS 
(McClintic, 1912) 
Name of Disinfectant. 
With Organic 
Matter. 
Without Organic 
Matter. 
Bacterol 
1 34 
1 58 
Benetol 
0.92 
1 23 
Cabot's silpho-naphthol 
2 33 
3 87 
Carbolene 
0.65 
1 36 
Carbolozone 
0 48 
1 48 
Car-sul 
1.75 
2 (X) 
Chloro-naphtholeum 
3 21 
6 06 
Cremoline 
0.69 
1 26 
Creo-carboline 
2.26 
4 03 
Creolin-Pearson 
2.52 
3 25 
Cresoleum 
1.75 
2.90 
Crude carbolic acid 
2.63 
2 75 
Dusenberry's liquid creoleum 
0 40 
1 00 
Germol 
1.79 
2.12 
Hycol ... 
9.37 
12 30 
Kreosota 
0 65 
1 26 
Kreotas 
0.30 
1 10 
Kreso 
2.32 
3.92 
Kresolig 
1 48 
2 13 
Liquor cresolis compositus (U. S. P.) 
1.87 
3.00 
Lysol 
1 57 
2 12 
Phenoco disinfecting fluid 
9.86 
5 00 
Saponified cresol 
0.57 
1.03 
Tarola 
1.93 
3.12 
Trikresol 
2.50 
2.62 
Zonol 
1.57 
2.37 
Creola disinfectant 
.52 
fall directly into the disinfectant solutions, one cylinder for each time 
interval selected; (8) note the temperature of the solutions (they should 
be kept at exactly 20° C. during the experiment); (9) note the time at 
which the cylinders were dropped into the disinfectant solutions; 
(10) at the end of stated intervals (usually at hourly intervals for pre- 
liminary tests) remove one cylinder from each solution with one of the 
sterile holders mentioned above, wash it thoroughly with sterile water, 
and remove a core from the center with a sterile piece of quill tubing 
(3-mm. bore); (11) place these cores in lactose fermentation tubes, 
after they are properly labeled, and incubate at 37° C. for several days, A. P. H. A. PHENOL COEFFICIENT 
165 
making daily observations; (12) compare the killing times of the 
various solutions tested with that obtained from carbolic acid, and 
determine the carbolic coefficient of germicidal and penetrating powers 
combined. 
In tests with some common disinfectants and a seventy-two-hour 
incubation, there was no growth after one hour's exposure to 4 per cent 
formalin; no growth after three hours' exposure to 1 per cent formalin, or 
1 per cent corrosive sublimate solution; and no growth after five hours' 
exposure to 5 per cent carbolic acid, 0.25 per cent formalin, and 10 per 
cent chloride of lime. Even after five hours' exposure, however, there 
was growth with 1 per cent carbolic acid, 0.1 per cent corrosive subli- 
mate, 4 per cent chloride of lime, 2 per cent hyco, 1 per cent cresol, and 
2 per cent sulphonaphthol. 
Table XIX 
SHOWING COMBINED GERMICIDAL AND PENETRATING POWERS 
OF SOME COMMON DISINFECTANTS 
(After Kendall and Edwards.) 
Disinfectants. 
Dilution, 
Per Cent. 
Time of Exposure 
1 Hour. 
3 Hours 
5 Hours. 
Carbolic acid 
5 
+ 
+ 
Carbolic acid 
1 
-1- 
+ 
4- 
Formalin 
4 
Formalin 
1 
+ 
Formalin 
0.25 
+ 
4- 
Mercuric chloride 
0.1 
+ 
4- 
Mercuric chloride. 
1.0 
+ 
Chloride of lime 
10.0 
4- 
4- 
Chloride of lime 
4.0 
4- 
4- 
4- 
Hvco 
2.0 
+ 
4- 
4- 
Cresol 
1.0 
+ 
4- 
4- 
Sulphonaphthol 
2.0 
+ 
+ 
+ 
The American Public Health Association Standard Phenol Coeffi- 
cient. This association appointed a committee to report on the stand- 
ardization of disinfectants and the material given below is taken from 
that report as published in the American Journal of Public Health for 
July, 1918. 
Apparatus: Subculture Tubes. A test-tube rack of eight rows of 
ten holes each may be used for the subcultures. If the test be of 166 
STERILIZATION AND DISINFECTION 
twenty dilutions, eighty tubes are numbered serially and placed in the 
rack which is placed conveniently at the worker's left hand. 
Bunsen Burner. A Bunsen burner with a fantail top is led from 
the left and back of the bench with a little spare tubing. 
Loop Rack. This is made of four pieces of |-in. wood, each 4 ins. 
by 9 ins. joined in a frame so that the top is about 10 ins. high. The 
top is provided with about six grooves for the loops. The latter are 
placed in their grooves and the rack is placed in front of the worker's 
right shoulder, close to the front of the bench and almost at right angles 
to the front edge but with a slight slant to the right and rear. The 
loops point to the left and the fantail burner is so adjusted as to prop- 
erly envelop the loop which is over it. The loops are used in succession 
and, after each one is returned to its place, the burner is moved under it 
thus providing at all times for a sterile and cool loop. 
Loops. The loops used for transferring the culture after exposure 
to the disinfectant are made as follows: A close cylindrical spiral is 
made by winding nichrome wire No. 23 B. & S., as tightly as possible 
about a piece of steel or other hard wire having a diameter of .072 in. 
(B. & S., No. 13). Wind about five full turns, bend the remainder of 
the wire sharply at a right angle to the wound portion and parallel to 
the axis of the cylinder. Remove from the core and cut off the lower 
end to leave exactly four complete turns. When completed the suc- 
cessive turns of the spiral must touch one another continuously. 
Seeding Tubes. The characters of the seeding tubes to be used 
depends entirely upon local conditions. In places in which there is 
any doubt as to air contamination cotton plugged tubes are to be pre- 
ferred. However, under conditions as exist in most laboratories the 
open seeding tubes recommended in the present standard methods are 
most convenient and quite satisfactory. 
Seeding Tube Holder. Cut down an oblong wire test-tube basket 
to about 2.5 ins. in depth and to the top fit a piece of |-in. wood through 
which three rows of seven holes each have been bored, the holes being 
of proper size to accommodate the tubes loosely. An ordinary tripod 
is used to support this rack in the water bath. The legs of the tripod 
are cut so that the tops of the seeding tubes are a little above the sur- 
face of the water. 
Inoculator. The inoculations of the diluted disinfectant with the 
culture are made with an apparatus described by Rosenau (Hyg. Lab. 
Bull. 21, p. 60). A capillary pipette graduated in tenths c.c. is fitted 
with a rubber bulb and clamped to a ring stand. The bulb is actuated 
by a second spring clamp the jaws of which engage it. This apparatus A. P. H. A. PHENOL COEFFICIENT 
167 
is placed at the right of the worker so that he can push the loop rack 
away from him and draw the inoculator toward him and when through 
inoculating reverse the action. 
Water Bath. The water bath consists of a wooden box about 18 ins. 
square and deep, containing a three- or four-gallon pail packed around 
with sawdust which is lightly oiled so as not to become dusty. The 
bath is raised from the floor about 6 ins. by cleats and castors, and when 
not in use is rolled under the bench. During the test it is on the floor 
at the right of the worker. 
Timepiece. A watch or small clock of fair accuracy, provided 
with a second hand, is so mounted as to be conveniently and com- 
fortably read by the worker from his place at the bench. 
Culture Media, Broth. Ten grams of peptone (Witte's), 3 gms. of 
extract of meat (Liebig's), 5 gms. of sodium chloride, C. P. Distilled 
water 1000 c.c. Boil for fifteen minutes, make up to weight, filter tube, 
sterilize at 10 lbs. pressure for twenty minutes. No adjustment what- 
ever of the acidity should be attempted. Determine and record the 
Ph value. This should be between 6 and 7. 
Agar. To 1000 c.c. of the standard broth add 15 gms. of best grade 
agar, boil thirty minutes, make up to weight, filter and sterilize at 1 lb. 
for twenty minutes. Do not use egg albumen to clarify. 
Organism. For the standard test organism in determining the 
coefficient against the typhoid organism a twenty-four-hour-old broth 
culture of B. typhosus (Hopkins) is used. Before beginning a test the 
culture is carried over in broth at 37° every twenty-four hours for at 
least five successive days, and the last culture well shaken and filtered 
through sterile filter paper. For carrying over the culture one standard 
loop is used. It is important that the transfers be made as nearly as 
possible as exactly on the twenty-four-hour interval, although a varia- 
tion of not more than two hours is allowable. The stock culture of B. 
typhosus (Hopkins) is kept on agar slants. These cultures are incubated 
at 37° C. for twenty-four hours and then placed in the refrigerator 
until ready for use. In no case should they be kept more than 
one month. 
Phenol. While pure phenol crystals are undoubtedly the best stand- 
ard which we have at present, they are far fiom being entirely satis- 
factory. Commercial phenol crystals are often contaminated with 
cresols and other phenol homologues. It is important that nothing 
but the highest grade of pure white synthetic phenol crystals be used 
of a solidifying point not less than 40° C. as determined by the method 
of Weiss and Downs. 168 
STERILIZATION AND DISINFECTION 
Phenol crystals themselves as well as solutions change in germicidal 
strength upon standing, particularly when exposed to direct sunlight 
and to high temperature. Phenol changes more readily when melted 
or in the presence of moisture than when in crystalline form. It is 
important, therefore, that the crystals of phenol be kept in a tightly 
stoppered bottle, in a cool, dry place and exposed to the light and mois- 
ture as little as possible. Phenol which has been exposed to the air and 
allowed to absorb enough water to alter its melting-point or which has 
become at all red should be discarded for the purpose of this test. The 
5 per cent stock solution is prepared as follows: Melt the phenol in the 
original container and pour out approximately 50 gms. into a closed 
weighing bottle; weigh and dissolve in the proper amount of water to 
give a 5 per cent solution. Do not attempt any further standardization. 
This stock solution is kept in the refrigerator in ambered-colored bottles 
of not more than 500-c.c. capacity, and for a period of not longer than 
three months. 
Dilutions, Distilled Water. Sterile distilled water is recommended 
for all dilutions. Attention is directed to the danger of metallic copper 
in the distilled water prepared from the common type *of laboratory 
still in which the condensation surface is of tin-coated copper or brass. 
If the tin-plate is worn away sufficient copper may be dissolved to 
materially influence the coefficient. 
Five Per Cent Stock Solution. Accurately graduated capacity 
pipettes are to be used in preparing the stock dilution of disinfectant. 
Five c.c. of disinfectant are measured into a flask containing 95 c.c. of 
sterile distilled water the interior of the pipette being rinsed several 
times with the contents of the flask. From this solution or emulsion, 
and from the stock 5 per cent phenol solution the required dilutions are 
prepared. 
Initial Dilution from 5 Per Cent Dilution. Take 50 c,c. of the 5 
per cent stock solution, place in a 100-c.c. graduated flask and make 
up to the mark with sterile distilled water. Mix by pouring. This 
gives a dilution of 1 : 40. With 50 c.c. of this dilution repeat the process 
to obtain a dilution of 1 : 80 and so on by successive dilutions until the 
following initial dilutions or such as these as are necessary for the test, 
are prepared: 
1 : 20 
1 : 40 
1 : 80 
1 : 160 
1 : 320 
1 : 640 
Final Dilutions from Initial Dilutions. With sterile delivery pipettes 
measure the quantities of initial dilutions and sterile distilled water A. P. H. A. PHENOL COEFFICIENT 
169 
required in accordance with the following table to obtain the desired 
final dilutions: 
Table XX 
Initial 
Dilution 
+Water (c.c) 
Initial Dilution. 
1 : 20 
1 : 40 
1 : 80 
1 : 160 
1 : 320 
1 : 64 
Final Dilution. 
4+4 
4+5 
4+6 
4+7 
4+8 
4+9 
4 + 10 
1 : 40 
1 : 45 
1 : 50 
1 : 55 
1 : 60 
• 1 : 65 
1 : 70 
1 : 80 
1 : 90 
1 : 100 
1 : 110 
1 : 120 
1 : 130 
1 : 140 
1 : 160 
1 : 180 
1 : 200 
1 : 220 
1 : 240 
1 : 260 
1 : 280 
1 : 320 
1 : 360 
1 : 400 
1 : 440 
1 : 480 
1 : 520 
1 : 560 
1 : 640 
1 : 720 
1 : 800 
1 : 880 
1 : 960 
1 : 1040 
1 : 1120 
1 : 1280 
1 : 1440 
1 : 1600 
1 : 1760 
1 : 1920 
1 : 2080 
1 : 2240 
Temperature. The standard temperature at which the organism is 
exposed to the action of the disinfectant is 20° C., but a variation of 
not more than 0.5° on either side of this figure is allowable. The tem- 
perature should be maintained by the use of a water bath, the design 
of which may be left to the individual operator. It is important, how- 
ever, that the bath be so arranged that the water will rise to a height 
greater than that of the dilution of disinfectant contained in these seed- 
ing tubes. The cultures and dilutions of disinfectants should be brought 
to a temperature of 20° C. before starting the test. In very warm 
weather, it will be found that there is a material increase in the tempera- 
ture of the water bath during the twenty-minute period. If, however, 
the bath is brought at the start to a temperature of 19.5° C. it will not 
generally exceed 20.5° C. during the test. For the determination of the 
temperature coefficient a temperature of 30° C. is also employed. 
All subcultures are incubated at 37° C. for forty-eight hours. The 
temperature of the incubator should be maintained between 36° and 38°. 
Technique. In the test of a new preparation the coefficient of which 
is unknown it will be necessary to make a set of range-finding tests. 
For this purpose use the series 1 : 40, 1 : 80, etc., at the head of the col- 
umns in the table of final dilutions and select for the actual test with 
phenol a series of final dilutions ranging from the highest dilution which 
killed in five minutes to the lowest dilution which failed to kill in twenty 
minutes. If some approximate idea of the coefficient is available, or if 
the preliminary test indicates a too extensive range of dilutions for the 170 
STERILIZATION AND DISINFECTION 
final test, a closer range may be obtained by restricting the test to fewer 
columns and using the middle row of dilutions together with the top row. 
The seeding tubes having been properly sterilized are brought to 
the bench and placed in the seeding racks to the number required. 
Five c.c. of each of the dilutions of phenol and disinfectant to be tested 
are placed in order in these seeding tubes, which are appropriately num- 
bered, a separate 5-c.c. delivery tube being used for each dilution. The 
tube containing the filtered culture is next placed in one of the holes of 
the seeding-tube holder which is then placed in the water bath and 
allowed to stand there for sufficient time to bring the contents to tem- 
perature. The inoculator is then filled with culture and, at the beginning 
of an even five-minute period the first seeding tube is inoculated with 
0.1 c.c. of culture and the succeeding tubes are then inoculated at 
appropriate intervals. If the manipulator is sufficiently skilled to make 
inoculations and transfers at fifteen-second intervals the total number 
of dilutions including the phenol dilutions that can be carried through 
a test simultaneously is 20. Five of these are necessary for the phenol, 
1 : 80 to 1 : 120, inclusive, leaving a maximum of 15 for the disinfectant. 
This will permit the employment of two of the columns of the table of 
final dilutions, if necessary. In practice, however, it will seldom be 
found necessary to extend the range over more than one complete col- 
umn or eight dilutions which with five phenol dilutions gives thirteen 
in all. This will permit the making of inoculations and transfers at 
twenty-second intervals, with one minute leeway at the end of each 
round, a safer and more comfortable schedule. For the beginner thirty- 
second intervals are recommended which permit the carrying of ten 
dilutions simultaneously. Whatever the interval decided upon it is 
quite desirable that a plan of the test showing the dilutions to be used, 
intervals between inoculations, and times of making subcultures be 
carefully laid out on paper so that there shall be no confusion in these 
matters during the actual test. 
When the tubes have been inoculated, the inoculator is pushed away 
diagonally and the loop holder is drawn up to the edge of the bench and 
just before the expiration of the time interval, subculture tube No. 1 
is taken in the hand, the plug withdrawn, and a loop dipped perpen- 
dicularly into the first seeding tube. At the expiration of the time 
interval, the loop is withdrawn, and the transfer consummated. The 
loop is returned to its groove on the loop holder, the burner moved in 
place under it and the subculture tube given a rapid whirling movement 
to mix its contents and replaced. Another tube and another loop are 
then taken in hand for the next transfer. At the completion of the test COAGULATION COEFFICIENT 
171 
the subculture tubes are placed in the incubator and forty-eight hours 
later they are removed, read and recorded. 
Determining the Coefficient. The concentrations of the highest dilu- 
tions which gave negative results in five, ten, fifteen and twenty minutes, 
respectively, are divided by the corresponding concentrations of phenol 
to obtain a series of four coefficients. The arithmetical mean of the four 
is taken as the " Mean Phenol Coefficient against Typhoid 20° C., 
five to twenty minutes, A. P. H. A." It is recommended that a final 
report be based upon the results of not less than three separate tests. 
The coefficient will be known for brevity as the " A. P. H. A. Standard 
Phenol Coefficient," but for any other organism temperature, time 
ranges that may be employed the word " Standard " will be omitted 
and the full expression given. The determination of these special 
coefficient for special uses is recommended. 
Determination of Coagulation Coefficient. (After Schneider, 1912). 
Certain chemicals which are used as disinfectants precipitate proteins 
and may be used up in that way. Mercuric chloride, for instance, is 
such a disinfectant and does not do its maximum work under such con- 
ditions. Many of the disinfectants do not coagulate albumin in the 
dilutions which are used for disinfecting. There seems to be no relation 
between the " phenol " coefficient and the " coagulating coefficient " 
but it is often desirable to know just how active a certain substance is 
in the precipitation of proteins. For this purpose Schneider (1912) 
devised the following procedure; 
Albumen Test Solution 
Based upon the results of the preliminary experiments a 1 per cent 
aqueous (distilled water) solution of pure dried egg albumen is recom- 
mended as the substance upon which the different strength solutions 
of the various disinfectants is to act. 
The following methods for making the albumen solution are sub- 
mitted for consideration, hoping that other investigators may try them 
out comparatively: 
(a) Gravimetric Method A. Place 2 gms. of pure powdered egg 
albumen in 100 c.c. of boiled distilled water, shake and set aside for 
six to twelve hours, shaking frequently. Filter through a fared filter 
paper which has been dried (at 100° C.) to constant weight. Filtering 
is slow, requiring perhaps one hour's time. When the last drop has 
filtered through, dry the filter paper with the unfiltered albumen residue 
upon it to constant weight and weigh. Deduct from this weight the 172 
STERILIZATION AND DISINFECTION 
weight of the albumin residue. From 1 gm. of egg albumen dried to 
constant weight determine the percentage of moisture. From the data 
thus obtained it is easy to determine the amount of boiled distilled 
water which must be added to the filtrate (100 c.c.) to make 1 per cent 
dried albumen solution. 
We will suppose that the dried filter paper to be used in filtering the 
albumen solution weighs 1.570 gms. and this same paper with the 
undissolved albumen residue (also dried at 100° C. to constant weight) 
weighs 1.965 gms., then the weight of the undissolved dried albumen 
residue equals 0.395 gm. We will suppose that 1 gm. of albumen loses 
0.126 gm. on drying, or 12.6 per cent moisture. 0.395 gm. raised to its 
normal air moisture (0.395 gm.+ 12.6 per cent of 0.395 gm. = 0.444 gm.) 
and subtracted from 2.00 gms. leaves 1.556 gms., the amount of albumen 
that passed through the filter paper. 12.6 per cent of 1.556 gms. = 0.196 
gm. and 1.446 gms. less 0.196 gm. = 1.360 gms. which represents the 
amount of albumen, dried to constant weight, that passed into solution. 
Therefore to make a 1 per cent solution it is necessary to add enough 
boiled distilled water to the filtrate to make 1/100, in this case add water 
up to the 136 c.c. mark. We now have a 1 per cent solution sufficiently 
accurate for all practical purposes. 
This albumen test solution is now ready for use but it must be kept 
in mind that it is readily attacked by microbes. Howrever, if carefully 
prepared with pure albumen, boiled distilled water, in sterile vessels, 
and put on ice or in a cool place, it will keep for perhaps four days. 
Any quantity of albumen solution may be made, it merely being 
advised not to prepare more than may be required for the tests contem- 
plated. 
(6) Gravimetric Method B. In a dried and tarred platinum dish 
place 5 c.c. of the albumen filtrate (2 gms. in 100 c.c. of boiled distilled 
water), evaporate over water bath and dry to constant weight, and from 
this determine the percentage of albumen in the solution and the 
amount of water that must be added to the albumen filtrate to make 
1 per cent. 
(c) Nitrogen Determination. By means of the Kjeldahl apparatus 
determine the percentage of nitrogen representing the albumen in 
solution. 
Having prepared the egg albumen solution the next step is to make 
the phenol control solution, the primary stock solutions of the disin- 
fectants to be tested for coagulating powers, and, from these, the 
secondary or sub-stock solutions, from which the final test dilutions 
are made as the experiment progresses. COAGULATION COEFFICIENT 
173 
The standard of comparison is the opacity produced in 5 c.c. of the 
1 per cent egg albumen solution when 5 c.c. of 5 per cent phenol solu- 
tion is added (in a standard test tube of about 15 c.c. capacity). This 
phenol tube is placed against a black background. In making a test, 
varying dilutions of the disinfectant are added to the egg albumen 
solution until the opacity produced is the same as that in the phenol 
tube. In each test 5 c.c. of the dilution is added to 5 c.c. of the egg 
albumen in a standard test tube and the two tubes compared, placed 
against the black background. The following tentative procedure for 
making dilutions is suggested: 
Dilutions of Disinfectants to be Used 
The phenol control solution (5 per cent) is made as for the Anderson- 
McClintic method of standardizing disinfectants, using only pure 
phenol crystals. 
Of the disinfectants to be tested 10 per cent and 1 per cent primary 
stock solutions are made; 10 per cent solutions of liquid disinfectants 
as alcohol, formalin, and acids, and 1 per cent solutions of the salts of 
heavy metals and of soluble substances generally. From these primary 
stock solutions the following secondary dilutions or sub-stock solutions 
are made, always in those amounts which will serve the purpose, that is, 
in amounts for perhaps ten sub-dilutions for each and every disinfectant 
to be tested: 
1/10 (of liquids only) 
1/100 
1/1000 
1/10,000 
1/100,000. 
Method of Testing 
(a) Phenol Standard. Pour 5 c.c. of the egg albumen solution in a 
standard test tube, using a 5-c.c. pipette having a free outflow. Add 
to this 5 c.c. of the phenol stock solution (5 per cent). Set the tube 
in the standard test rack (with black background made of cardboard 
covered with black tissue paper). The degree of opacity developed is 
to serve as the standard of comparison. 
(6) Preliminary Testing. The albumen coagulating power of the 
disinfectant being unknown, much time and labor can be saved by 
testing with the four or five sub-stock solutions, adding 5 c.c. to 5 c.c. of 174 
STERILIZATION AND DISINFECTION 
the egg albumen test solution, in order to find that dilution of the disin- 
fectant which fails to show any opacity. We will suppose that the 
1/1000 sub-stock solution shows very marked opacity or precipitation, 
then the 1/10,000 solution might be tried, which may also show quite 
marked opacity, then the 1/100,000 may be tried. If this gives nega- 
tive results then we know that the phenol standard lies between 
1/10,000 and 1/100,000, with the probabilities that it is nearer 1/10,000. 
Table XXI 
PLAN FOR TEN DILUTIONS AT THIRTY-SECOND INTERVALS 
Seeding 
Tube 
Dilution. 
Inoculate. 
5-Min. Test. 
10-Min.Test. 
15-Min.Test. 
20-Min.Test. 
Min. 
Sec. 
Min. 
Sec. 
Min. 
Sec. 
Min. 
Sec. 
Min. 
Sec. 
1 
1 : 80 
00 
00 
5 
00 
10 
00 
15 
00 
20 
00 
2 
1 : 90 
00 
30 
5 
30 
10 
30 
15 
30 
20 
30 
3 
1 : 100 
1 
00 
6 
00 
11 
00 
16 
00 
21 
00 
4 
1 : 110 
1 
30 
6 
30 
11 
30 
16 
30 
21 
30 
5 
1 : 120 
2 
00 
7 
00 
12 
00 
17 
00 
22 
00 
6 
1 : 360 ' 
2 
30 
7 
30 
12 
30 
17 
30 
22 
30 
7 
1 : 400 
3 
00 
8 
00 
13 
00 
18 
00 
23 
00 
8 
1 : 440 
3 
30 
8 
30 
13 
30 
18 
30 
23 
30 
9 
1 : 480 
4 
00 
9 
00 
14 
00 
19 
00 
24 
00 
10 
1 : 520 
4 
30 
9 
30 
14 
30 
19 
30 
24 
30 
(c) Concluding Testing. Going back to the 1/10,000 dilution make 
ten sub-dilutions, increasing the dilutions by a difference of 1000 by 
simply adding the required parts of distilled water, using small quan- 
tities, thus: 
10 parts of 1/10,000+1 part water = 1/11,000. 
10 parts of 1/10,000+2 parts water = 1/12,000. 
10 parts of 1/10,000+3 parts water = 1/13,000, etc. 
Any other quantity proportions may be used, however, as 10, 15 
20, etc., parts of the sub-stock solution with the required parts of dis- 
tilled water. If the 1/1000 sub-stock solution is to be used then the 
dilutions should be increased by 100 as follows: 
10+1 = 1/1100 
10+2=1/1200 
10+3 = 1/1300 
10+4=1/1400; HALE TOXICITY COEFFICIENT 
175 
or any other equal proportion of stock solution and distilled water may 
be used, as 5+0.5, 100+10, or 1000+100, etc. 
If the highest stock dilution (1/100,000) is to be used, then the 
increase should be by 10,000, thus: 
10+1 = 1/110,000 
10+2=1/120,000 
10+3 = 1/130,000, etc. 
Determination of the Phenol Coefficient 
Having determined that dilution which gives the same coagulation 
opacity as the 5 per cent carbolic acid, it is a very simple matter to 
determine the phenol albumen coagulating coefficient by simply dividing 
the strength of the dilution of the disinfectant tested by the phenol 
dilution (1/20). 
We conclude by giving the coagulating coefficients of a few disin- 
fectants: 
Name of Disinfectant. 
Reaction 
Limit. 
Phenol 
Coefficient. 
Phenol 
1/20 
1.00 
Copper sulphate 
1/15,000 
750.00 
Mercuric chloride 
1/10,000 
500.00 
Silver nitrate 
1/9,500 
475.00 
Alcohol 
1/3 
0.15 
The Hale Toxicity Coefficient of Disinfectants. Hale has attempted 
in this procedure to establish the toxicity of different substances and to 
give them a definite numerical relation to other toxic agents. He 
further proposed that this toxicity coefficient be established for groups 
of substances having, in general, a similar pharmacological action. 
The following procedure was proposed for determining the toxicity 
coefficient of phenol-like compounds: 
The animal upon which the substance in question is to be tested 
shall be the white mouse of not less than 15 nor more than 30 gms. 
weight. The dose is to be calculated per gram of body weight and 
should, when diluted, equal between 0.03 and 0.04 c.c. per gram weight; 
that is, 0.06 to 0.-08 c.c. for a 20-gm. mouse. The diluent is to be dis- 176 
STERILIZATION AND DISINFECTION 
tilled water and primary dilutions are to be made of such strength that 
the dose is easily measured with a 1-c.c. pipette graduated in hun- 
dredths. This is most easily accomplished by the use of the substance 
in greater concentration than that required to kill in the above volume 
dose. After the required dose of the diluted disinfectant has been 
estimated it is measured into a suitable dish and is then diluted further 
to the required volume by adding water in sufficient quantity. A series 
of mice are then to be injected with varying amounts of the substance 
until the least fatal dose is determined, the mice being kept under 
observation for a period of twenty-four hours unless death results in a 
shorter time. 
Mice of the same lot are similarly injected with pure phenol prop- 
erly diluted to make the measurement of the dose easy and then further 
diluted in a small dish to equal a volume dose of 0.03 to 0.04 c.c. per gram 
of body weight and the least fatal dose determined as above. The dose 
thus obtained is considered unity and the least fatal dose of the sub- 
stance in question is estimated in per cent of this, as is illustrated in the 
table following: 
Mouse 
Weight. 
Dose per Gram 
of Body 
Weight. 
Results. 
Time. 
Hrs. Min. 
Disinfectant A 
21.13 
0.0012 
Survived 
20.64 
0.0016 
Survived 
18.32 
0.0018 
Lethal 
10 30 
• 
19.05 
0.0020 
Lethal 
2 15 
Phenol . . 
18 46 
0 00035 
Survived 
20.10 
0.00040 
Survived 
19.23 
0.00045 
Lethal 
1 15 
18.90 
0.00050 
Lethal 
0 25 
The least fatal dose of disinfectant A was estimated to be 0.0018; 
that of phenol 0.00045. The phenol toxicity coefficient of disinfectant A 
therefore is, according to the proportion 4.5 : 18:: X : 100, 25 per cent. 
The following are some toxicity coefficients secured by Hale in the 
application of his procedure to some commercial disinfectants. For 
the sake of comparison, the " phenol coefficients " as determined by 
McClintic have been included: BIBLIOGRAPHY ON DISINFECTION 
177 
T oxicity 
Coefficient. 
Phenol Coefficient. 
Without Or- 
ganic Matter. 
With Or- 
ganic Matter. 
Bacteriol 
45 
1.58 
1.34 
Benetal 
33 
1.23 
0.92 
Creola 
12.8 
Creolin-Pearson 
18.0 
3.25 
2.52 
Cresoleum 
11 
2 90 
1 75 
Ger mol 
16 
2.12 
1.79 
Hycol 
32 
12 30 
9 37 
Kreotas 
5.6 
1.10 
0 30 
Kreso 
22.5 
3.92 
2.32 
Lysol 
2 12 
1 57 
Phenol sodique 
4.5 
Trikresol 
90 
2.62 
2.50 
BIBLIOGRAPHY 
Anderson, J. F. and McClintic, T. B. 1912. I. Method of Standardizing 
Disinfectants with or without Organic Matter. Bull. 82, Hygienic Labora- 
tory U. S. Pub. Health Service, Wash. 
Bazzoni, C. B. 1914. The Destruction of Bacteria through Light. Amer. 
Jour. Pub. Health, 4, 975-992. 
Beyer, A. 1912. Alcohol as a Disinfectant. Zeit. Hyg., 70, 225. 
Biassioti, A. 1910. The Action of Colloidal Metals on Pathogenic Bacteria. 
Cent. Bakt. Abt. L, Ref. 45, 680. 
Bitter, L. 1912. The Destruction of Bacteria by the Heavy Metals and 
Building Materials. Zeit. Hyg., 69, 483-512. 
Browning, C. H. and Russ, S. 1917. Germicidal Action of the Ultra-violet 
Radiation and its Correlation with Selective Absorption. Proc. Roy. Soc., 
London, (B) 90, 33-38. 
Burge, W. E. 1917. The Action of Ultra-xiolet Light in Killing Living Cells 
such as Bacteria. American Journal Physiology, 43, 429-432. 
Burge, W. E. and Neill, A. 1915. The Comparative Rate at which Fluores- 
cent and Non-fluorescent Bacteria are Killed on Exposure to Ultra-violet 
Light. Amer. Jour. Physiology, 38, 399-403. 
Chick, H. 1908. An Investigation of the Laws of Disinfection. Jour. 
Hygiene, 8, 92. 
Chick, H. 1910. The Process of Disinfection by Chemical Agencies and 
Hot Water. Journal of Hygiene, 10, 237. 
Chick, H. 1912. The Factors Influencing the Velocity of Disinfection. 
Eighth Intern. Cong. Appl. Chern., 26, 167-197. 178 
STERILIZATION AND DISINFECTION 
Chick, II. and Martin, C. F. 1908. The Principles Involved in the Stand- 
ardization of Disinfectants and the Influence of Organic Matter on the 
Germicidal Value. Journal Hygiene, 8, 654. 
Chick, H. and Martin, C. J. 1910. On the Heat Coagulation of Proteins. 
Journal of Physiology, 40, 404. 
Chick, H. and Martin, C. F. 1908. A Comparison of the Power of a Germi- 
cide Emulsified or Dissolved with an Interpretation of their Superiority 
of the Emulsified Form. Journal of Hygiene, 8, 698. 
Churchman, J. W. 1912. The Selective Bactericidal Action of Gentian 
Violet. Journal Experimental Medicine, 16, 221-247. 
Cooper, E. A. 1912. Bactericidal Action of Cresols and Allied Substances 
and the Best Means of Employing Them. Brit. Med. Journal, 1912, I. 
1234-1240. 
Cooper, E. A. 1912. Relations of Phenols and their Derivatives to Proteins. 
A Contribution to Our Knowledge of the Mechanism of Disinfection. 
Biochem Jour., 6, 362-387; 7 (1913), 175-96. 
Dakin, H. D., Cohen, J. B., Daufresne, M., and Kenyon, J. 1916. Pro- 
ceedings Royal Society, London, Series B, 89, 232-251. 
Dakin, H. D., Cohen, J. B., and Kenyon, J. 1916. Chloramin, Its Prepara- 
tion, Properties, and Use. Brit. Med. Journal No. 2874, 160-162. 
Dakin, H. D., Lee, W. E., Sweet, J. E., and Hendrix, B. M. 1917. A 
Report of the Use of Dichloramin-T (Toluene-parasulphondichloramin) in 
the Treatment of Infected Wounds. Jour Amer. Med. Assn., 69, 27-31. 
DeWitt, L. M. and Sherman, H. 1916. The Bactericidal and Fungicidal 
Action of Copper. Journal Infectious Diseases, 18, 368-382. 
Dieudonne, A. 1894. Beitriige zur Beurtheilung der Einwirkung des Lichtes 
auf Bakterien. Art. a. d. kais. Gesundheitsamte, 9, 405. 
Duyser, C. A. and Lewis, W. K. 1914. A New Method for Determining the 
Value of Disinfectants. Chern. Eng., 19, 113; Jour. Ind. Eng. Chern., 6, 
198-200. 
Eijkman, C. 1908. Die Ueberlebungskurve bei Abtodtung von Bakterien 
durch Hitze. Biochem. Jour., 11, 12. 
Embrey, G. 1912. The Use of Copper Sulfate in Purifying Water Supplies. 
Canadian Engineer, 23, 245-6. 
Eugling, M. 1912. The Disinfective Action of Iodoform and Novoidin. 
Cent. Bakt. Abt., I, 60, 397-416. 
Findlay, L. and Martin, W. B. M. 1915. The Effect of Daylight and Dry- 
ing on the Human and Bovine Type of Tubercle Bacilli. Brit. Med. J., 
110-111. 
Frey, E. 1912. Why is Seventy Per Cent Alcohol so Strongly Bactericidal. 
Dent. Med. Wochenschr., 38, 1633-5. Chern. Absts., 6: 1912, 3843-4. 
Goodrich, H. P. 1917. Glycerol and Antiseptics. British Med. Jour., 1917, 
647-648. 
Gossl and Herzog. Theory of Disinfection. II. Disinfectants that Dissolve 
Lipooid. Zeit. Physiol. Chern., 88 (1), 103-8. BIBLIOGRAPHY ON DISINFECTION 
179 
Hamilton, H. C. 1917. Facts and Fallacies in Disinfection. Am. J. Pub. 
Health, 7, 282-95. 
Hamilton, H. C. and Ohno, T. 1913. Standardization of Disinfectants. 
Am. J. of Pub. Health, 3, 583. 
Hamilton, H. C. and Ohno, T. 1914. Bacteriological Standardization of 
Disinfectants; Amer..Jour. Pub. Health, 4, 486. 
Harris, F. J. and Hoyt, H. S. Possible Origin of the Toxicity of Ultra Violet 
Light. Science, 46, 318-20. 
Herzog and Betzel. 1911. Zur Theorie der Desinfection. Zeit. Physiol. 
Chemie, 67 (1910), 303, 74, 221. 
Hewlett. 1909. Milroy Lectures on Disinfection and Disinfectants. Lancet, 
March 13, 20-27. 
Hewlett and Hall. 1911. The Influence of Culture Media on the Germina- 
tion of Anthrax Spores with Special Reference to Disinfection Experiments. 
Jour. Hyg., 11, 473. 
Hooker, A. H. 1913. Chloride of Lime in Sanitation. John Wiley & Sons, 
New York. 
Houghton, E., Maud, Davis L. 1913. A Study of the Germicidal Action of 
the Ultra Violet Rays. Amer. Jour. Pub. Health, 4, 224-240. 
Kendall, A. I. and Edwards, M. R. 1911. A Method for Determining the 
Germicidal Value and Penetrating Power of Liquid Disinfectants. Jour. 
Infectious Diseases, 8, 250-57. 
Kempster, C. 1917. The Effect of X-Rays upon Diseases of Bacterial 
Origin. Lancet, 1917, II, 423-424. 
Kingzett, C. T. and Woodcock, R. C. The Bacterial Testing of Disinfectants: 
A Practical Criticism. Analyst, 38, 190-200. 
Kingzett, C. T. and Woodcock, R. C. 1910. Bacteriological Testing of 
Certain Disinfectants and the Results as Effected by Varying Conditions. 
Pharm. Jour., 85, 157-8. 
Kligler, I. J. 1918. A Study of the Antiseptic Properties of Certain Organic 
Compounds. Journal of Experimental Medicine, 27, 463-478. 
Koch. 1901. The Physiological Action of Formaldehyde. Amer. Jour. 
Physiology, 6, 325-329. 
Kronig and Paul. 1897. Die chemischen Grundlagen der Lehre von der 
Giftwirkung und Disinfection. Zeit. f. Hyg., 25, 1. 
Lager, H. 1916. Disinfection of Water with Calcium Hypochlorite. Zeit. 
Hyg., 81, 296-310. Chern. Abts., 12 (1918), 394. 
Lusini. 1912. Disinfecting Action of Cations According to the Law of the 
Periodic System. R. Acad, fisicrit. March 20, 1910. Chem. Abts., 6, 
1912. 240. 
Mahon,' T. and White, J. S. 1915. Germicidal Value of Iodine. J. Soc. 
Chem. Ind, 34 (1915), 244. Chem. and Druggists, 1915, 144. 
McClintic, T. B. 1905. Chloride of Zinc as a Deodorant, Antiseptic and 
Germicide. Bull. No. 22, Hyg. Lab. U. S. Pub. Health Service, Wash- 
ington. 180 
STERILIZATION AND DISINFECTION 
McLaughlin, A. J. 1903. Inefficiency of Ferrous Sulfate as an Antiseptic 
and Germicide. Bull. 15, Hyg. Lab. U. S. Pub. Health Service, Wash. 
Moore, G. T. and Kellerman, K. F. 1905. Copper as a Disinfectant and 
Algicide in Water Supplies. U. S. Dept. Agr. Bureau of Plant Industry 
Bull., 76. 
Madsen and Nyman. 1907. Zur Theorie der Disinfection. Zeit. Hyg., 57, 
388. 
Newcomer, H. S. 1917. Bactericidal Fluorescence Excited by X-Rays. 
Jour. Exper. Med., 26, 657-668. 
Newcomer, H. S. 1917. The Abiotic Action of Ultra Violet Light. Jour. 
Exper. Med., 26, 841-848. 
Nissen, F. 1890. Ueber die Desinficirende Eigenschaft des Chlorkalks. 
Zeit. Hyg., 8, 62-77. 
Norton, J. F. The Coefficients of Germicides. Jour. Ind. Eng. Chern., 8, 
197. 
Norton, J. F. and Hsu, P. H. 1916. Physical Chemistry of Disinfection. 
Jour. Inf. Diseases, 18, 180-195. 
Ochsner, E. H. 1916. The Biochemistry of Topical Applications with Spe- 
cial Reference to the Use of Boric Acid in Septic Infections. Chemical Ab- 
stracts, 12 (1918), 191. 
Oker-Blom, M. 1913. The Nature of the Action of Ultra Violet Rays on 
Bacteria. Zeit. Hyg., 74, 242-247. 
Paul Birstein and Reuss. 1910. Beitrage zur Kinetik der Giftwirkung von 
Geloeteten Stoffen. I. Einfluss der Konzentration. II. Einfluss der Neu- 
tralsalze. Einfluss und der Temperature. Biochem. Zeit., 29, 202 and 249. 
Paul Birstein and Reuss. 1910. Beitrag zur Kinetik des Absterben tro- 
kener Bakterien in Sauerstoff verschiedener Konzentration und ver- 
schiedenen Teinperaturen. Biochem. Zeit., 25, 367. 
Phelps, E. B. 1911. On the Application of Certain Laws of Physical Chem- 
istry in the Standardization of Disinfectants. Jour. Inf. Dis., 8, 27. 
Phelps, E. B. 1913. The Limitations of the Phenol Coefficient in the Stand- 
ardization of Disinfectants. Amer. Jour. Pub. Health, 3, 53-57. 
Reichel, H. 1909. Zur Theorie der Disinfection. Biochem Zeit., 22, 149; 
177 and 201. 
Renob, L. (et oZ). 1914. Antiseptic Role of Insoluble Substances. Comp. 
Rend. Soc. Biol., 76, 64-66. 
Reudiger, E. H. 1914. Germicidal Power of Glycerol on Various Micro- 
organisms under Various Conditions. Philippine Journal of Science., 
9, B, 564-477. 
Rideal, S. 1912. Suggested International Tests for Disinfectants. Eighth 
Intern. Cong. Applied Chemistry, 26, 261. 
Rideal, S. and Walker, A. 1903. The Standardization of Disinfectants. 
Jour. Roy. San. Institute, 24, 424. 
Rideal, {el al). 1915. A Note on Testing Disinfectants. Amer. Jour. Pub. 
Health, 5, 1175-1176. BIBLIOGRAPHY ON DISINFECTION 
181 
Robertson, T. B. 1914. Disinfection. Jour. Hyg., 14, 143-148. 
Robertson, T. B. 1914. On the Conditions under which Discontinuous 
Events may be Employed as a Measure of Continuous Processes with 
Special Reference to the Killing of Bacteria by Disinfectants. Journal of 
Hygiene, 14, 143-148. 
Rosenau, M. J. The Antiseptic and Germicidal Properties of Glycerine. Bull., 
16, Hyg. Lab. U. S. Pub. Health Service, Washington. 
Rubner, M. 1913. Modern Steam Sterilization. Jour. Am. Med. Assoc., 
60, 1344. 
Schmidt, E. W. 1910. Bactericidal Value of Thymol. Zeit. Physiol. Chern., 
67, 412-432. 
Schneider, F. 1912. Albumin Coagulation Coefficient for Disinfectants. 
Pacific Pharmacist, 5, 335-339. 
Simpson, W. J. and Hewlett, R. T. 1914. Germicidal Action of Colloidal 
Silver. Lancet, Dec. 12,1914, 1359. Chemical Absts., 8 (1915), 640, 641. 
Sommerville, D. 1913. Antiseptics and Germicides. Jour. Roy. Soc. 
Arts, 61, 927-937; 945-952. 
Steenhauer, A. J. 1916. The Disinfecting Power of o-, m-, and para-Cresol 
and Basic Mercuric Cyanide. Pharm. Weekbladt, 53, 680-685. 
Straus. 1917. Disinfecting Properties of Alcohol. Schweiz, apoth. Ztg., 55, 
36. 
Supfle, K. and Deugeler, A. 1916. Culture Media for Use after Disin- 
fection Experiments. Arch. Hyg., 85, 189-197. 
Von Recklinghausen, M. 1914. Purification of Water by the Ultra Violet 
Rays. Jour. Amer. Water Works Assn., 1, 565-588. 
Walker, A. 1916. The Standardization of Disinfectants. New York Med. 
Journal, 1916, 500-505. 
Walters, A. L. 1917. The Specificity of Disinfectants and its Bearing on 
their Standardization. Amer. Jour. Pub. Health, 7, 1030-1036. 
Ward, H. M. 1895. The Action of Light on Bacteria. Phil. Trans. Roy. 
Soc. 1895, 961. 
Watson, H. E. 1908. A Note on the Variation of the Rate of Disinfection 
with Change in Concentration of the Disinfectant. Jour. Hygiene, 8, 536. 
Weinzirl, J. 1914. The Germicidal Action of Sunlight upon Bacterial 
Spores, Amer. Jour. Pub. Health, 4, 969-974. 
Winkler, L. W. 1888. Die Bestimmung des im Wasser gelosten Sauerstoffs. 
Berichte, 21, 2, 2843-2854. 
Wright, J. H. 1917. The Importance of Uniform Culture Media on the 
Bacteriological Examination of Disinfectants. Jour. Bact., 2, 315-346. CHAPTER VI 
PROTEINS AND CARBOHYDRATES 
Classification of Proteins. Committees for the American Society of 
Biological Chemists and the American Physiological Society have made 
the following recommendations concerning the proteins: 
First. The word proteid should be abandoned. 
Second. The word protein should designate that group of sub- 
stances which consists, so far as at present is known, essentially of com- 
binations of the a-amino acids and their derivatives, e.g., a-amino acetic 
acid or glycocoll; a-amino propionic acid or alanin; phenyl-a-amino 
propionic acid or. phenylalanin; guanidin-amino valerianic acid or 
arginin, etc., and are; therefore, essentially polypeptids. 
Third. That the following terms be used to designate the various 
groups of proteins: 
I. Simple Proteins. Protein substances which yield only a-amino 
acids or their derivatives on hydrolysis. 
Although no means are at present available whereby the chemical 
individuality of any protein can be established, a number of simple 
proteins have been isolated from animal and vegetable tissues which 
have been so well characterized by Constance of ultimate composition 
and uniformity of physical properties that they may be treated as 
chemical individuals until further knowledge makes it possible to char- 
acterize them more definitely. 
The various groups of simple proteins may be designated as follows: 
(a) Albumins. Simple proteins soluble in pure water and coagulable 
by heat. 
(b) Globulins. Simple proteins insoluble in pure water but soluble 
in neutral solutions of salts of strong bases with strong acids. 
(c) Glutelins. Simple proteins insoluble in all neutral solvents but 
readily soluble in very dilute acids and alkalies. 
(d) Alcohol Soluble Proteins. Simple proteins soluble in relatively 
strong alcohol (70 to 80 per cent) but insoluble in water, absolute 
alcohol, and other neutral solvents. 
(e) Albuminoids. Simple proteins which possess essentially the 
182 CLASSIFICATION OF PROTEINS 
183 
same chemical structure as the other proteins, but are characterized by 
a great insolubility in all neutral solvents. 
(f) Histones. Soluble in water and insoluble in very dilute ammonia 
and, in the absence of ammonium salts, insoluble even in an excess of 
ammonia; yield precipitates with solutions of other proteins and a 
coagulum on heating, which is easily soluble in very dilute acids. On 
hydrolysis they yield a large number of amino acids, among which 
the basic ones predominate. 
(^) Protamins. Simpler polypeptids than the proteins including the 
preceding groups. They are soluble in water uncoagulable by heat, 
have the property of precipitating aqueous solutions of other proteins, 
possess strong basic properties and form stable salts with strong mineral 
acids, among which the basic amino acids predominate. 
II Conjugated Proteins. Substances which contain the protein 
molecule with nucleic acid. 
(a) Nucleoproteins. Compounds of one or more protein molecules 
with nucleic acid. 
(6) Glycoproteins. Compounds of the protein molecule with a sub- 
stance or substances containing a carbohydrate group other than a 
nucleic acid. 
(c) Phosphoproteins. Compounds of the protein molecule with 
some, as yet undefined, phosphorus containing substance other than a 
nucleic acid or lecithin. 
(d) Hemoglobins. Compounds of the protein molecule with hema- 
tin or some similar substance. 
(e) Lecitho-proteins. Compounds of the protein molecule with 
lecithins (lecithans, phosphatids). 
III. Derived Proteins. 
1. Primary Protein Derivatives. Derivatives of the protein mole- 
cule apparently formed through hydrolytic changes which involve only 
slight alterations of the protein molecule. 
(a) Proteins. Insoluble products which apparently result from the 
incipient action of water, very dilute acids or enzymes. 
(6) Metaproteins. Products of the further action of acids and alka- 
lies whereby the molecule is so far altered as to form products soluble 
in very weak acids and alkalies but insoluble in neutral fluids. 
This group will include the familiar " acid proteins " and " alkali 
proteins " not the salts of proteins with acids. 
(c) Coagulated Proteins. Insoluble products which result from (1) 
the action of heat on their solutions, (2) the action of alcohols on the 
protein. 184 
PROTEINS AND CARBOHYDRATES 
2. Secondary Protein Derivatives. Products of the further hydrolytic 
cleavage of the protein molecule. 
(a) Proteoses. Soluble in water, uncoagulable by heat and pre- 
cipitated by saturating their solutions with ammonium sulphate or zinc 
sulphate. 
(b) Peptones. Soluble in water, uncoagulable by heat, but not 
precipitated by saturating their solutions with ammonium sulphate. 
(c) Peptids. Definitely characterized combinations of two or more 
amino acids, the carboxyl group of one being united with the amino 
group of the other, with the elimination of a molecule of water. 
Structure of Proteins. Proteins are combinations of amino acids 
the unions being between the carboxyl group of one acid and the amino 
group of the other. The following will serve to illustrate the linkage. 
H H 
H !OH H H-C-H H H-C-H 
H-C-C-O >N-C-C< =H-C-C-N-C-0=0 
I HZ | \H | || | | \H 
H IT NH20 H H n 
Glycine 
Alanin 
Glycyl-alanin 
The free amino and carboxyl groups on the glycyl-alanin molecule 
are free to react in the same way. This yields infinite possibilities with 
regard to the size of the protein molecule. Fischer has done much 
toward synthesizing proteins from these smaller units the amino acids. 
The amino acids which have been isolated from proteins may be 
classified as follows: 
I. Monoamino Monobasic Acids. 
Glycocoll, a-amino acetic acid. 
H 
I //® 
H-C-C-OH 
/ nh2 
Alanin, a-amino propionic acid. 
H ' H 
I I z° 
H-C-C-C-OH 
H NH2 STRUCTURE OF AMINO ACIDS 
185 
Phenylalanin, a-amino, 0-phenyl propionic acid. 
II H 
/\ I I Z° 
z y-c-c-C-oh 
H H nh2 
Serine, a-amino, /3-hydroxy propionic acid. 
H H 
I I z° 
H-C-C-C-OH 
OH NH2 
Tyrosine, a-amino, 0-para hydroxy phenyl propionic acid. 
H H 
z \- C-C-C-OH 
H NH2 
OH 
Cystine, a-diamino, /3-dithiolactylic acid. 
S S 
I I 
H-C-H H-C-H 
H-C-NH2 H-C-NH2 
I I 
COOH COOH 
Leucine, isobutyl a-amino acetic acid. 
CH3x 
>CH-CH2-CH-COOH 
CH3z I 
NH2 
Isoleucine ethyl methyl, a-amino propionic acid. 
CH3 /CH-CH-COOH 
CH3-ch2/ 
nh2 
Valine, a-amino, isovalerianic acid. 
ch3 nh2 
I I Z° 
H-C C-C^-OH 
I I 
ch3 h 186 
PROTEINS AND CARBOHYDRATES 
II. Dicarboxylic acids. 
Aspartic acids, a-amino succinic acid. 
II 
I ^0 
NH2-C-C^-OH 
I 
H-C-C^-OH 
I 
H 
Glutamic acid, a-amino, n-glutaric acid. 
NH2 
I . 
H-C-C-OH 
H-C-H 
I z° 
H-C-C-OH 
H 
III. Diamino acids. 
Lysine, a-e-diamino caproic acids. 
NH2 H H H nh2 
I I I I I ZO 
H-C C-C-C-C C-OH 
H H H H H 
Arginine, d-guanidine, amino valerianic acid. 
H H H NH, 
Illi 
N-C-C-C-C-C^-OH 
H H H H 
C=NH 
nh2 
IV. Heterocyclic amino acids. 
Tryptophane, /3-indole, a-amino propionic acid. 
H H 
/ \ I I z° 
< >--C-C-C-C-OH 
|| H NH2 
Vy% 
N 11 
H 187 
ELEMENTS IN ORGANIC COMPOUNDS 
Histidine, a-amino imidazol propionic acid. 
H NH2 
I । 
H-C=C-C-C-C-OH 
H-H H 
C 
H 
Proline, a-pyrrolidine carboxylic acid. 
H2C CH2 
I I /Hz° 
H2C^/O-C-OH 
N 
I 
H 
That amino acids are utilized by bacteria has been pointed out in 
other places. The commercial peptones which are used in the prepara- 
tion of media are, among other things, mixtures of amino acids. The 
changes induced by bacteria on certain amino acids in the intestinal 
tract are discussed in that chapter. Raistrick (1917) has shown that 
the bacteria of the coli-typhi group are able to change histidine into 
urocanic acid (iminazolylacrylic acid). Most of the other amino acids 
may be changed by bacterial enzymes to smaller molecules. Bacterial 
changes induced in other amino acids have been discussed elsewhere in 
this book. 
Qualitative Analysis for the Elements in Organic Compounds. The 
inorganic elements may be detected by heating some of the sample on a 
piece of platinum foil or porcelain. During this ignition the organic 
elements are driven off. The organic elements are determined by 
ignition with sodium. It often happens that an element is present in 
both organic and inorganic forms. 
Inorganic Elements. The substance is ignited on a piece of platinum 
foil or porcelain until all of the organic portion is destroyed. If the 
sample chars during this procedure, the presence of an organic substance 
is shown. If an inorganic substance is shown in the charring it may 
further be determined by the procedures of qualitative analysis. 
Organic Elements. Place a small piece of sodium in an ignition 
tube about 4 ins. long. Incline the tube in a slanting position and 188 
PROTEINS AND CARBOHYDRATES 
heat the closed end very gently at first and later heat the bottom to 
a dull red. As soon as the sodium has begun to volatilize and condense 
on the sides of the tube, add a few drops of the sample or a few pieces of 
a solid, being careful to keep off the sides of the tube. As soon as the 
tube has cooled any excess of sodium may be removed by adding a little 
alcohol or if the sodium excess is not large the tube may be plunged into 
warm water. This solution must be filtered free of free carbon. The 
filtrate may then be tested for the halogens, nitrogen and sulphur. 
Hydrogen and oxygen are assumed to be present. 
Test for Organic Sulphur. Treat about 1 c.c. of the solution secured 
from the sodium ignition with lead acetate solution. If sulphur is 
present a black precipitate will be formed according to the following 
equation. The solution must be strongly alkaline. 
Na2S+Pb(C2H3O2)2 = PbS+2NaC2H3O2. 
Sodium nitro-prusside may also be used to test for organic sulphur. 
Add a few drops to about 1 c.c. of the filtrate secured from the sodium 
ignition and if a violet color results, the presence of sulphur is indicated. 
Test for Organic Nitrogen. Add to about 3 c.c. of the filtrate four 
drops of NaOH solution, four drops of FeSO4 solution and one drop of 
FeCl3. Add a little concentrated HC1 to dissolve the precipitated 
ferrous and ferric hydroxides and boil. If N is present in the sample a 
precipitate of Prussian blue will come down. It may be necessary to 
heat the tube gently and leave it for a time. The following reactions 
take place: 
6NaCN+FeSO4 = Na4Fe(CN)6+Na2SO4, 
FeCl3+NaOH = Fe(OH)3+3NaCl. 
4Fe(OH)3+3Na4Fe(CN)6+12HCl = Fe4(Fe(CN)6)3+ 12H?O+ 12NaCl. 
Test for Sulphur and Nitrogen when Present Together. Acidify 
about 1 c.c. of the solution with HC1 and add three drops of ferric chloride 
solution. A dark red indicates the presence of sulphur and nitrogen. 
3NaSCN+FeCl3 = Fe (SCN) 3+3NaCl. 
To be certain of results the above tests for sulphur and nitrogen should 
be made along with this test. 
Test for Organic Halogens: Group Test. This is carried out by 
removing H2S, and cyanide. To do this the solution is acidified with 
dilute nitric acid and boiling. The addition of silver nitrate at this ELEMENTS IN ORGANIC COMPOUNDS 
189 
point will precipitate the halids of silver which have the characteristic 
appearance. 
Tests for Bromine and Iodine. Acidify a few cubic centimeters of 
the sample and boil to remove HoS and the cyanids. Add a few drops 
of carbon bisulphide and a little chlorine water. If iodine is present, 
it is taken up by the heavier bisulphide which settle to the bottom 
of the tube with a distinct violet color. 
Nai+H3SO4 = HI+NaHSO4. 
2HI+Cl3 = H4O+l2. 
Qualitative Tests for Elements in Proteins. Carbon. Heat the 
protein on a piece of platinum foil and if charring takes place the pres- 
ence of carbon is indicated. 
Hydrogen. Test for water in the top of the test tube. 
Nitrogen. Heat with soda lime and test for ammonia by smell and 
moist litmus paper. 
Sulphur. Heat with concentrated potassium hydroxide which will 
set free potassium sulphide. Cool and add lead acetate solution, which 
will precipitate black lead sulphide. After adding the lead acetate solu- 
tion acidify, boil and suspend lead acetate paper above the liquid. 
Detection of Proteins. (1) The coagulation of proteins by heat 
especially in slightly acid solutions may be used in their detection. 
This precipitate does not disappear on continued addition of acid. 
(2) Alcohol in excess will form a precipitate which is soluble in water 
at first; if the precipitate is left in contact with the alcohol, it is changed. 
The proteins come down unchanged but are probably dehydrated when 
left in contact with the alcohol. The fixing of tissue in histological work 
rests upon this fact. (3) Heller's Ring Test rests upon the fact that 
strong mineral acids will coagulate proteins. A small amount of con- 
centrated nitric acid is put into a test tube and some of the test solution 
is floated upon it. If proteins are present a whitish layer will be formed 
at the point of contact between the two liquids. 
Action of Bacteria on Proteins. The hydrolysis of proteins by 
bacteria has been studied by different investigators with the production 
of data which is not in agreement. Bainbridge (1911) found that bac- 
teria could not decompose 11 purified native proteins." This was later 
verified by Sperry and Rettger (1915) who worked with purified serum 
albumin, egg albumin and edestin and the common proteolytic bacteria. 
They reported that when all other forms of nitrogen were removed, 190 
PROTEINS AND CARBOHYDRATES 
bacteria could not start the decomposition of the proteins. Rettger, 
Berman and Sturges (1916) have continued this work and reported that 
proteoses and peptones follow essentially the same law of resistance to 
bacterial action as do the native proteins. They found that if there 
was sufficient nitrogen in other combinations to allow growth of the 
bacteria in order that proteoses could be secreted, the proteins were 
hydrolyzed. When no growth took place due to the lack of available 
nitrogen, no hydrolysis occurred. The bacteria used in the above 
experiment might be regarded as " starved " and thus not in a normal 
condition. Robinson and Tartar (1917) when studying this question 
made no attempts to use purified proteins: blood fibrin, egg albumin, 
casein; gliadin and peptone were split by bacteria to ammonia. The 
chief source of ammonia seemed to be the monoamino and diamino 
nitrogen. The work of these investigators simulates more closely the 
conditions in our common media and in nature. 
Action of Bacteria on Polypeptids. Sasaki (1912) repoited the 
B. coli split glycyl-Z-tyrosin and glycylglycine into their component 
acids and from this they conclude that bacteria probably plays a role in 
digestion. The same author in two other papers reports investigations 
wherein liquefying and non-liquefying bacteria were used. With non- 
liquefying types such as typhi, dysentery, etc., glycyl-Z-tyrosine and 
glycylglycine were decomposed to their component acids. Sasaki 
regards this change as due to an ereptase-like enzyme, excluding pep- 
tase because synthetic polypeptids were used. The various liquefying 
bacteria which he used were also able to split the polypeptids. In 1914 
Sasaki killed B. coli with toluene and, after action on polypeptids, 
demonstrated that they were split into their amino acids. Somewhat 
similar results have been reported by Mito (1918). He tested the 
enzymes of B. coli communis and Staph, aureus on di-leucylyblycine. 
Asymetric cleavages were secured. The copper salt of Z-leucine was 
isolated. The mother liquor exhibited an optical activity compatible 
with d-leucylglycine. Sasaki (1917) grew B. coli communis and B. 
proteus vulgaris in tyrosine containing media of two kinds. One of these 
media contained lactose to allow acid formation while the other con- 
tained a mixture of phosphates to maintain neutrality. In the lactose 
media both bacteria formed p-hydroxyphenyl-ethy lamin. In the lac- 
tose free media no amine could be isolated but rather large amounts of 
d-p-hydroxyphenyllactic acid were secured. 
Bacterial Action on Other Bodies Related to Proteins. There exist 
a class of compounds which stand between the carbohydrates and pro- 
teins-glucosamin, REACTIONS OF PROTEINS 
191 
OH OH OH OH H 
H C C C C C C=O 
H H H H NH2 H 
This compound possesses an amino group in place of a hydroxyl 
group and this amino group, to a certain extent, connects it with the 
proteins although it retains many of its carbohydrate properties. In 
light of the controversy concerning the action of bacteria on proteins 
their action on this compound is interesting. 
Meyer, in attempting to explain the action of bacteria on d-glucosa- 
min, studied the action on acetyl derivatives. He believes that the NH2 
group is probably split off after which a further degradation takes place. 
Color Reactions of Proteins. By means of these much information 
may be secured concerning the amino-acids in a protein. 
Biuret Reaction. The protein solution is warmed and a little strong 
sodium hydroxide and dilute copper sulphate is added. Care should 
be exercised to use dilute copper sulphate two or three drops in a test 
tube of water. A violet color is obtained. The color is due to the 
formation of biuret, 
z 
C-N-H 
I 
N-H £ 
I /H 
C-N-H 
The test is given by those substances which have two CONH2 
groups. 
Xantho-proteic Reaction. The protein is treated with concentrated 
nitric acid. The solution or protein will turn yellow. The presence 
of a CcHo group is necessary. This reacts with the HNO3 to yield 
nitro derivatives of benzine. Nitro-benzine is formed. 
Millon's Reaction. The protein or protein solution is heated with 
Millon's reagent. The protein is turned red or, if a solution is used a 
reddish precipitate is formed. The reaction is given by those proteins 
which possess a hydroxy-phenyl group. Since tyrosine is the only 
amino acid having this group which has been isolated from proteins, 
the presence of this amino acid is indicated by a positive test with 
Millon's reagent. Care must be exercised in the use of Millon's reagent 
since it may be decomposed by some of the inorganic salts which precip- 
itate mercury. 192 
PROTEINS AND CARBOHYDRATES 
Liebermann's Reaction. Boil the protein solution with about 
4 c.c. of concentrated hydrochloric acid. A violet-lavender color 
will result if tryptophane is present. 
Bromine Reaction for Tryptophane. Uncombined tryptophane 
when treated with bromine water will give a violet color. 
Test for Tyrosine. Add a few drops of formol solution to concen- 
trated sulphuric acid. On warming with tyrosine, a brown red color 
is obtained, which, on addition of acetic acid becomes green. Neither 
proteins nor peptones give the reaction. (Nasse's modification of 
Demge's test. Quoted from Smith, 1905.) 
Schmidt Test for Tyrosine. Dissolve by boiling in water and add 
a solution of mercuric nitrate. The red reaction is sharper if a little 
fuming nitric acid diluted in water is added. Try also the violet reaction 
with neutral iron chloride. (Quoted from Smith, 1905.) 
Precipitation Reactions. The precipitation reactions of proteins 
are important in removing them from solution and also for detecting 
them in solution. Most of the metals will precipitate them. Mercury 
will throw down a heavy white precipitate of mercury proteinate. Cop- 
per, when added to a protein solution as the sulphate, will throw down a 
bluish precipitate. Iron and lead also give heavy precipitates, the one 
with iron being dissolved by an excess of that compound. Picric acid, 
trichloracetic acid, phosphotungstic acid, tannic acid and bromine 
also form precipitates in protein solutions. 
Carbohydrates 
The carbohydrates may be classified in different ways. The fol- 
lowing is a convenient method: 
I. Monosaccharides 
A. Pentoses, C5H10O5 
1. Xylose 
2. Arabinose 
B. Hexoses, CoHiaOe 
1. Dextrose 
2. Levulose 
3. Galactose 
II. Disaccharides 
1. Sucrose 
2. Maltose 
3. Lactose CLASSIFICATION OF CARBOHYDRATES 
193 
III. Trisaccharides 
1. Raffinose 
IV. Polysaccharides 
A. Starches 
1. Starch 
2. Glycogen 
3. Inulin 
B. Celluloses 
1. Cellulose 
2. Hemicellulose 
a. Pentosans 
Gum arabic 
b. Hexosans 
Galactans 
Agar-agar 
Action of Bacteria on Carbohydrates. This class of substances 
serves bacteria for both energy and building purposes. The action of 
microorganisms on carbohydrates is a subject too large for extensive 
treatment here.* The enzymes which will attack carbohydrates are 
widely distributed among microorganisms. Foods containing car- 
bohydrates are especially susceptible to attack by bacteria unless the 
sugar is too concentrated. If there is too little moisture present the 
organisms are unable to carry on their activities. 
The changes induced in sugar by bacteria may be discussed in dif- 
ferent ways. It is quite natural to discuss them on a physiological basis. 
Lactic Acid Fermentation. This is brought about by bacteria by 
first splitting the lactose into two molecules of a monosaccharide, each 
of which is further decomposed to lactic acid according to the following 
equation: 
C12H22O11+HOH = 2C6H12O6, 
CeH^Oe = C3H6O3. 
Since the lactic acid which is thus formed possesses so much latent 
energy, it may be further decomposed to other compounds such as 
butyric acid. 
The kind of lactic acid formed has been the subject of many investiga- 
tions. Four lactic acids are known, three of which have the same formula, 
* Those wishing an exhaustive treatment of the subject are referred to Kruse's 
Allgemeine Mikrobiologie, or Lafar's Handbuch der technischen Mycologie. 194 
PROTEINS AND CARBOHYDRATES 
CH3CH2OHCOOH. The other has the formula CH2OHCH2COOH. 
The last-mentioned lactic acid since it does not possess an asymetric 
carbon atom is not optically active. The ethylidene lactic acids possess 
asymetric carbon atoms and are therefore optically active. 
Thomas (1916) has shown that the lactic acid produced by Matzoon 
in synthetic culture media is inactive but that it may be resolved into 
two active components. According to Heinemann (1907) milk which 
sours naturally at room temperature contains chiefly d-acid; at 37° C. 
chiefly r-acid with Z-acid in excess if allowed to stand a few days. He 
also found that the kind of organism present determined the kind of 
lactic acid which was formed. The temperature was also believed to 
have an effect. 
Alcoholic Fermentation. These are those changes in sugar and 
decomposition products of sugar which result in the formation of large 
amounts of alcohol. Historically it has been known for a very long 
time and was not thoroughly analyzed until Pasteur proved that it was a 
biological phenomenon. The chemistry of alcoholic fermentation is 
now fairly well understood and there is much data available m the 
literature to review at this time. Kruse believes that dextrose breaks 
up in a series of reactions while Harden maintains that this takes place 
in one reaction. Kruse's reactions will add up to equal that of Harden's: 
3CgHi2O6 = 2C2H5OH+2CO2; 
2CgHi2Og = 3CH3COOH; 
6CgH12Og = 2C3HgO3; 
CgH i2O6+6H2O = 6CO2+6H2O; 
12CgHi2Og+H2O = 12C3H6O3+6C2H5OH+6 CH3-COOH 
+ I2CO2+12H2O. 
Ethyl Alcohol. Iodoform Reaction. Add about an equal amount 
of iodine solution to the sample and treat with sodium hydroxide until 
decolorized. Warm the mixture very carefully and try to detect the 
odor of iodoform. A yellow precipitate of iodoform may result. This 
test is not specific as it is given by acetone, aldehyde, acetic ester, and 
other similar substances. 
Acetaldehyde Reaction. Add a few drops of potassium dichromate 
and dilute sulphuric acid to the sample and heat. An odor of aldehyde 
will be observed if ethyl alcohol is present. REACTIONS OF CARBOHYDRATES 
195 
Ethyl Acetate Reaction. Add a little sodium acetate and concen- 
trated sulphuric acid to the sample and heat. The fruity odor of ethyl 
acetate will be detected if alcohol was present in the sample. Quanti- 
tative methods for this may be obtained from chemical texts. 
Acetone. Iodoform Reaction. This is the same test as is used to 
detect ethyl alcohol except that it is carried out in the cold. Add to 
the same a few drops of sodium hydroxide and very slowly iodine- 
potassium-iodide solution until the solution is colored yellow. If acetone 
is present iodoform will settle out. 
Nitroprusside Test. Add a few drops of a freshly prepared solution 
of sodium nitroprusside and about 1 c.c. of sodium hydroxide. A red 
color is produced which will fade to a yellow on standing. 
Molisch Reaction. Add a few drops of Molisch reagent to the 
sugar solution in a test tube. Incline the tube and pour a few cubic 
centimeters of concentrated H2SO4 down the side of the test tube. A 
lavender color will be formed if sugars are present. If the H2SO4 is put 
into the test tube first, and the sugar-Molisch reagent mixture run in 
on top, a ring test may be observed. 
Seliwanoff's Reaction for Ketoses. Add a small amount of the 
sugar or unknown solution to a few cubic centimeters of Seliwanoff's 
reagent and heat to boiling. A reddish coloration indicates the presence 
of ketose sugars. 
Fehling's Test. This depends upon the action of reducing sugars. 
This action is generally attributed to the presence of an aldehyde or 
ketone group. Some regard other parts of the sugar molecule as in- 
volved in this reduction. 
Two solutions are used in the test. These are kept apart until used. 
If they are mixed and allowed to stand, the copper will be slowly reduced 
by the tartrate. The solutions have the following composition: 
Solution I. 
Copper sulphate 34.65 gms. 
Distilled water 500.00 c.c. 
Solution II. 
Potassium hydroxide 125.00 gms. 
Sodium potassium tartrate 173.00 gms. 
Distilled water 500.00 c.c. 
In testing for reducing sugars, mix equal parts of these two solutions 
and heat to boiling. This will determine whether the solutions are good. 
If any copper is precipitated by this heating, the solutions must be dis- 196 
PROTEINS AND CARBOHYDRATES 
carded. If the solutions are satisfactory, the sugar solution should be 
added very slowly to the hot Fehling's solution with boiling after each 
addition. The presence of reducing sugars will be indicated by a yellow- 
brown precipitate. 
Benedict's Method for Reducing Sugars. This is a modification of 
Fehling's test for reducing sugars. Instead of using the strong NaOH, 
Benedict has substituted Na2C0s. Sodium citrate is used to keep the 
Cu(0H)2 in solution. The method for conducting the test is much like 
that for Fehling's test. The solution should be boiled before using and 
allowed to cool. If reducing sugar is present the solution will turn first 
a green and later a yellow color. Benedict's test is more delicate than 
the original Fehling's method. The solutions are prepared as follows: 
Solution I. 
Copper sulphate 34.65 gms. 
Distilled water 500.00 c.c. 
Solution II. 
Anhydrous sodium carbonate... . 100.00 gms. 
Rochelle salt 173.00 gms. 
Distilled water 500.00 c.c. 
Nylander's Test for Reducing Sugars. Heat a few cubic centimeters 
of the solution under examination with £ c.c. of Nylander's reagent. A 
black color is formed if a reducing sugar is formed which is due to a 
precipitation of the bismuth in the reagent. 
Phenyl-Hydrazine Reaction. With phenyl hydrazine osazones are 
formed by action with certain sugars. These are yellow crystalline 
substances which are characterized by a definite form typical for each 
different sugar. The test is carried out as follows: Put about 5 c.c. of 
the sugar solution into a small beaker or test tube and add about 1.5 c.c. 
of phenyl-hydrazine acetate solution. Heat on a water bath for three- 
quarters of an hour. Cool and examine the crystals under the micro- 
scope. 
Quantitative Determination of Dextrose. (Allihn's Method.) 
Reagents. 
(a) Copper sulphate solution: 
Copper sulphate CuSO4 • 5H20 34.639 gms. 
Distilled water 500.000 c.c. 
(6) Alkalin tartrate solution: 
Rochelle salts 173 gms. 
Potassium hydroxide 125 gms. 
Distilled water 500 c.c. DETERMINATION OF DEXTROSE 
197 
Procedure. Place 30 c.c. of the copper sulphate solution, 30 ?.c. of 
the alkaline tartrate solution and 60 c.c. of water in a beaker and heat to 
boiling. Add 25 c.c. of the solution of the material to be examined, 
which must be so prepared as not to contain more than 0.250 gm. of 
dextrose, and boil for two minutes. Filter immediately through asbes- 
tos without diluting. Wash the precipitate with hot water. Dry, 
ignite and weigh as cupric oxide. To calculate the amount of copper 
and its equivalent in dextrose consult Allihn's table in any chemical 
hand-book. 
Benedict's Method for Determination of Dextrose. Twenty-five 
c.c. of Benedict's special reagent (see Appendix) are measured into a 
porcelain evaporating dish, 25-30 cm. in diameter, 10-12 gms. of 
crystalline sodium carbonate, and a small amount of pumice, are added. 
The solution is boiled vigorously over a free flame and the sugar solution 
is run in until a white precipitate is formed and the blue color is dimin- 
ished. xXfter this the sugar solution is run in a few drops at a time until 
the blue color has entirely disappeared. Water may be added to replace 
that driven off by evaporation. Twenty-five c.c. of the reagent =0.05 
gm. glucose or .053 gm. levulose. 
Fats 
• The fats or lipins are esters of glycerol and certain organic acids 
which have come to be known as fatty acids. When three molecules of 
palmitic acid unite with glycerol, the fat plamitin is secured, as follows: 
HO HO 
I % I z 
H-C-OH HO-C-C15H31 H-C-O-C-C15H31 
O I O 
I % I Z 
H-C-OH HO-C-C15H31 H-C-O-C-C15H3i+3H2O 
O I O 
I \ I z 
H-C-OH HO-C-C15H31 H-C-O-C-C15H31 
IT Jr 
Glycerol 
Palmitic acid 
Palmitin 
Water 
When fats are broken up either by bacterial enzyme or other action, 
they are resolved into glycerol and fatty acids. This process is known 
as saponification. 
Action of Bacteria on Fats. There is much evidence that fats are 
split to fatty acids and glycerol by bacteria, although many text books 198 
PROTEINS AND CARBOHYDRATES 
state that lipase is not widely distributed in the bacterial world or, 
at least, this subject is little or almost not at all treated. Rancidity in 
butter has been attributed by some to the formation of butyric acid 
(Duclaux, 1887). Others maintain that the fat in butter is not attacked, 
Reinmann (1900) tried the effect of pure cultures on sterile butter and 
found that most bacteria did not attack fat. In cheese Laxa (1902) 
demonstrated that Ps. fluorescens decomposed fat quite extensively. 
Two other peptonizing bacteria and a yeast were also found which de- 
composed fat. Rahn (1905) stated that Penicillium glaucum and a few 
bacteria possessed strong fat-splitting properties. Kendall and Simonds 
(1914) found that sterile filtrates of plain and dextrose broth cultures of 
B. typhi liberated acid from ethyl butyrate. This was also demon- 
strated in connection with acid-fast bacteria. 
Determination of the Iodine Absorption Number (Hanus Method.) 
The unsaturated fatty acids will react with the halogens. The amount 
of iodine differs according to the fat, and consequently this iodine 
absorption number is of some value for identification of fats. It 
(Iodine number) may be defined as the number of grams of iodine 
absorbed by 100 gms. of fat. 
Reagents. Hanus Iodine Solution. Dissolve 13.2 gms. of iodine 
in 1000 c.c. of glacial acetic acid (99.5 per cent) showing no reduction 
with bichromate and sulphuric acid. Add 3 c.c. of bromine to the cold 
solution. 
Starch Paste. Boil a gram of starch in 200 c.c. of distilled water for 
ten minutes and cool. 
Sodium Thiosulphate. Standardize a N/10 solution. 
Procedure. Weigh about 0.5 gm. of fat into a 250-c.c. glass-stoppered 
bottle and dissolve by adding 10 c.c. of chloroform. After solution add 
30 c.c. of the iodine monobromide solution. Place the bottle in the 
dark and allow to stand for thirty minutes. The time factor is impor- 
tant. The excess of iodine should be at least as much as is absorbed. 
One or two blanks should be made under identical conditions. If the 
first addition of iodine is used up, another addition should be made. 
Add 10 c.c. of potassium iodide solution and shake after which add 100 
c.c. of distilled water washing any iodine on the stopper of the bottle 
and sides back into the bottle. Titrate the iodine with the N/10 
sodium thiosulphate to a yellow color. Then add a few drops of the 
starch solution and continue the addition of sodium thiosulphate until 
the blue color has disappeared. Toward the end of the reaction stopper 
the bottle and shake violently so that any iodine remaining in the 
chloroform may be taken up by the potassium iodide solution. The DETERMINATIONS ON FATS 
199 
number of cubic centimeters of iodine used in the blank minus the num- 
ber used for the sample gives the thiosulphate value of the sample. 
Saponification Number or Koettstorfer Number. Since the amount 
of alkali which will react with a fat is dependent upon the glycerides in it, 
the saponification number is valuable for identifying fats and oils. The 
saponification is the number of milligrams of sodium hydroxide neces- 
sary to saponify 1 gm. fat. 
Reagents. Sodium Hydroxide. Use a N/10 solution each cubic 
centimeter of which contains 0.0040 gm. NaOH and neutralizes 0.0088 
gm. of butyric acid. 
Alcoholic Potash Solution. Dissolve 40 gms. of pure potassium 
hydroxide in 1000 c.c. 95 per cent alcohol. 
Acid Solution. N/2 HC1. 
Indicator. Phenolphthalein. 
Procedure. Conduct the saponification in wide-mouth Erlenmeyer 
flasks of 250 c.c. capacity. Place about 5 gms. of the fat in a tarred 
flask and weigh. Add exactly 2 cubic centimeters of the alcoholic 
potash solution, connect with a reflux condenser and boil for thirty 
minutes or until the fat is completely saponified. Cool and titrate with 
the N/2 hydrochloric acid. The Koettstorfer number is determined 
as follows: Subtract the number of cubic centimeters of hydrochloric 
acid used to neutralize the excess of alkali after saponification from the 
number of cubic centimeters necessary to neutralize the 50 c.c. of alkali 
added; multiply the result by 28.06 and divide by the number of grams 
of fat used. Conduct two blanks using the same pipettes and con- 
ditions. (Official and Provisional Methods, 1912. A. O. A. C.) 
Determination of Volatile Fatty Acids (Reichert-Meissl Method). 
This determination must be carried out under standard conditions. 
Under these, the Reichert-Meissl number is the number of cubic cen- 
timeters of N/10 sodium hydroxide required by the soluble fatty acids 
distilled from 5 gms. of fat. 
Procedure. Into two clean Erlenmeyer flasks weigh 5 gms. of 
fhe fat. Add 10 c.c. of 95 per cent alcohol and 2 c.c. of sodium hydrox- 
ide (100 gms. in 100 c.c. FRO). Attach to a reflux condenser and heat 
on fhe steam bath until saponification is complete. After the sapon- 
ification, in case alcohol was used, remove this by dipping the flasks in a 
steam bath up to their necks. When the alcohol is nearly gone frothing 
may occur. Dissolve the soap by adding 135 c.c. of boiled water and 
warming on the water bath until a clear solution results. After the 
solution has cooled to about 60° C., free the fatty acids by adding 8 c.c. 
of dilute sulphuric acid (200 c.c. in 1000 c.c. of water). Connect to a 200 
PROTEINS AND CARBOHYDRATES 
condenser and heat slowly on a water bath until the fatty acids form a 
transparent layer on the surface. Cool to room temperature and add a 
few pieces of pumice stone and distill at a rate that will allow about 
110 c.c. to be driven over in thirty minutes. The pumice should be 
prepared by heating small pieces to a white heat and dropping them into 
distilled water. They should be kept under distilled water to keep 
them free from air. Mix the distillate, filter and titrate 100 c.c. with 
standard N/10 sodium hydroxide solution using phenolphthalein. 
To obtain the Reichert-Meissl number, increase the cubic centimeters of 
N/10 sodium hydroxide used by 100 c.c. of filtrate, by one-tenth, divide 
by the weight of fat taken, and multiply by 5. 
BIBLIOGRAPHY 
Bainbridge, F. A. 1911. The Action of Certain Bacteria on Proteins. Jour- 
nal of Hygiene, 11, 341. 
Duclaux, E. 1887. Le Lait Etudes Chimique et Biologiques, Paris. 
Heinemann, P. G. Kinds of Lactic Acid Produced by Lactic Acid Bacteria. 
Jour. Biological Chemistry, 2, 612. 
Kendall, A. I. and Simonds, J. P. 1914. The Esterase Activity of Plain and 
Dextrose Broth Cultures of the Typhoid Bacillus. Jour. Inf. Diseases, 15, 
354-356. 
Laxa, O. Ueber die Spaltung des Butterfetts durch Mikroorganismen. Arch. 
Hyg.,41, 119. 
Meyer, K. 1914. Bacterial Decomposition of d-Glu cosamine. Bichem. 
Zeit., 58, 415-416. 
Meyer, K. 1914. The Behavior of Some Bacteria Towards d-Glucosamine. 
Biochem. Zeit., 57, 297-299. 
Mito, I. L. 1918. The Asymetric Decomposition of Racemic Polypeptids by 
Dead Bacteria. Chem. Abstracts, 12, 816. 
Raistrick, H. 1917. A New Type of Chemical Change Produced by Bac- 
teria. The conversion of histidine into urocanic acid by the bacteria of 
the coli-typhosus group. Biochem. Jour., 11, 71-77. 
Reinmann, R. 1900. Investigations on the Causes of Rancidity in Butter. 
Cent. Bakt. Abt., II, 6, 31. 
Rettger, L. F., Berman, N. and Sturges, W. T. 1916. The LHilization of 
Protein and Non-protein Nitrogen. Jour. Bact., 1, 15. 
Robinson, R. H. and Tartar, H. V. 1917. The Decomposition of Protein 
Substances through the Action of Bacteria. Jour. Biol. Chem., 30,135-144. 
Sasaki, T. 1912. Cleavage, of Polypeptids by Bacteria I. Bacterium Coli 
Commune. Biochem. Zeit., 41. 174-179. 
Sasaki, T. 1913. The Decomposition of Certain Polypeptids by Bacteria. 
III. Investigations with Liquefying Bacteria. Biochem. Zeit., 47,472-481. BIBLIOGRAPHY ON FAI'S 
201 
Sasaki, T. 1913. The Decomposition of Certain Polypeptids by Bacteria. 
II. Investigations with Non-liquefying Bacteria. Biochem. Zeit., 47, 
462-471. 
Sasaki, T. 1916. Bacterial Decomposition of Polypeptids. Physiol. Ab- 
stracts, 1917, 2, 15. 
Sasaki, T. 1917. The Influence of Conditions of Bacterial Cleavage of Pro- 
teins on the Cleavage Products. Jour. Biol. Chem., 32, 527-532. 
Sasaki, T., and Otsuka, 1. 1917. The Stereochemistry of the Bacterial 
Decomposition of Albumin. Jour. Biol. Chem., 32, 533-8. 
Sperry, J. A., and Rettger, L. F. 1915. The Behavior of Certain Bacteria 
toward Animal and Vegetable Proteins. Jour. Biol. Chem., 20, 445. 
Taylor, J. W., and Hall, I. W. 1912. Action of Saliva, Tissue Fluids, 
Bacteria and Bacterial Extracts upon Polypeptids. Jour. Path, and Bact., 
17, 121-123. 
Thomas, S. J. 1916. A Study of Stereoisomerism of Fermentation Lactic 
Acid. Jour. Ind. Eng. Chem., 8, 821-823. 
Tsudji, M. 1918. The Asymetric Decomposition of Racemic Tyrosine by 
Bacillus Proteus Vulgaris and B. Subtilis (also a Biological Preparation of 
d-tyrosine). Chem. Abstracts, 12, 816. CHAPTER VII 
YEASTS AND MOLDS 
The molds may be responsible for many changes in foods. Their 
action, however, should be considered from two standpoints since they 
bring about desirable and undesirable changes. A type of the former 
in the manufacture of Camembert and Roquefort cheese while the 
spoilage of strawberries and citrus fruits may be offered as an example 
of the latter. Tas Yu, the Chinese drink, is also said to be fermented 
in part by molds. 
Fungi. These are plants which possess no chlorophyl and conse- 
quently are unable to utilize the energy in sunlight. All of it must come 
from analytic processes. They do not construct their own starch but 
use that which has been built up by other organisms. For convenience, 
fungi have been divided into the following main groups. To the food 
microbiologist, these may not be so important but may be helpful in 
the identification of unknown fungi. 
Ascomycetes. This class produces ascospores or spores in an ascus. 
The yeasts since they possess this characteristic are put into this group. 
The aspergillaceae are ascomycetes. Some of these form perithecia 
which contain asci. 
Basidiomycetes. The spore-bearing bodies are termed basidia and 
possess a typical morphology. The conidia are produced from this on 
the sterigmata which may be either branched or unbranched. 
Phycomycetes. In this group are placed the algae. None of the 
fungi belonging to this group are commonly met with in food micro- 
biology. 
Fungi Imperfecti. Into this group are placed many fungi which 
do not come under any of the above groups. The fungi imperfecti 
are characterized by no definite fruiting bodies. Oidium lactis is the 
best example and in this fungus each mycelial thread breaks up to 
form oidia which may develop into a new plant.. 
Structure of Molds. The body of the mold is made up of a cotton- 
like structure called the mycelium. Each individual thread is called a 
mycelial thread or hypha. The hyphae are of two kinds-fertile and 
202 FRUCTIFICATION IN MOLDS 
203 
vegetative. Fertile hypha) are those which bear the fruiting body and 
produce the spores. The vegetative hyphae secure the nutriment and 
get rid of the excess moisture. 
The structure of the hyphae differs among the molds. Some have 
cross walls or septa. In other molds these have not been observed. 
The presence or absence of these septa is an important factor in the 
Fig. 47.-Rhizopus nigricans Ehrenberg. (After Brefeld.) 
(a) is the extremity of a stolon, which has developed into the appressorium (h). This latter 
is the starting point of the sporangiophores (t) four of which are shown with the sporangia (s) 
unbroken, whilst the columella (e) is all that remains of the fifth. Magn. 30. 
identification of molds. The mucoraceae do not possess these cross 
walls ordinarily. 
Fructification. This takes place on the fertile hyphae which bear 
the fruiting bodies. Enormous numbers df spores are formed each of 
which may develop into a typical mold plant. The spores are of two 
kinds, sexual and asexual. 
The sexual spores are formed by the union of two cells. The result- 
ing cell then possesses the characteristics which are algebraic sums of 204 
YEASTS AND MOLDS 
the characteristics inherent in the parent, cells. When a sexual spore is 
formed on a non-septate hyphae, it is termed a zygospore; if on a sep- 
tate hyphae it is an ascospore. Zygospores are formed by the union of 
two terminal cells on adjacent hyphae. The ascospore is formed by the 
Fig. 48.-Mucor Mucedo. Formation of the Zygospores. (After Brefeld.) 
1, two hyphae in terminal contact; 2, articulation into gamete a and suspensor b; 3, fusion of 
the gametes a; the membrane thickens; 4, ripe zygospore b supported by the suspensors aa; 
5, germination of the zygospore to a sporangium stem. Mag. about 60. 
union of adjacent cells. In this mass are formed the asci which hold the 
spores. Most of the spores formed by molds are asexual being formed 
mostly at the ends of the fertile hyphae from typical fruiting bodies. 
Classification. The molds are thread fungi and it is often difficult to 
draw the line between them and some other classes of fungi. Oidium RHIZOPUS-MUCOR-OIDIUM 
205 
lactis (oospora lactis) is often classified with yeasts and has then been 
given the name Mycoderma lactis. The molds are set apart from bac- 
teria and yeasts in that they are multicellular. The following varieties 
may be met with in a microbiological examination of foods. 
Rhizopus. The most common member of this group is Rhizopus 
nigricans. It is the common black bread mold and may be responsible 
for other food spoilage. Stevens (1917) has shown that this fungus 
causes the rot of strawberries during shipment. This work verifies 
Fig. 49.-Oidium Lactis. (After Thom.) 
a, b, dichotomous branching of growing hyphae; c, d, g, simple chains of oidia breaking through 
substratum at dotted line s-y, dotted portions submerged; e, f, chains of oidia from a branching 
outgrowth of a submerged cell; h, branching chain of oidia; k, I, m, n, o, p, s, types of germination 
of oidia under varying conditions; t, diagram of a portion of a colony showing habit of Oidium 
lactis as seen in culture media. 
some done by other investigators on the same subject. Schneider- 
Orelli (1911) have shown that R. nigricans is important in bringing about 
the decay of over-ripe pears during storage. 
Mucor. The mucors have the same structure as Rhizopus molds. 
They are separated, however, by the fact that the sporangiophores 
spring from the stolons singly. 
Oidium. The sour milk mold Oidium ladis is a common variety 
of this genus. The most of this mold is below the surface of the medium. 206 
YEASTS AND MOLDS 
That part which is not submerged resembles a layer of velvet on the 
surface of the medium. 
Penicillium. Penicillium molds are important in the manufacture 
of certain cheeses. Thorn (1906, 1905) has given much study to this 
question. Dox (1910) has reported an extended study of the intracellu- 
lar enzymes in Penicillium camemberti. The following enzymes were 
demonstrated to be present: erepsin, nuclease, amidase, lipase, emulsin, 
Fig. 50.-Roquefort Penicillium (P. roqueforti). (After Thom, 1906.) 
a, part of conidiophore and of bas of fructification, highly magnified showing the production of 
basidia on the sides as well as at the apex of the basidiophore; b, c, other types of branching; 
d, young conidiophore just branching; e, f, basidia and the formation of conidia, highly magnified; 
g, h, j, diagrams of types of fructification as seen under low power ( X80); k. I, m, n, germination of 
coonidia and new conidia produced directly on the first hyphse. 
amylase, inulase, raffinase, sucrase, maltase, and lactase. The presence 
of these enzymes seems to indicate an ability to utilize various food 
products. Penicillia are widely distributed in nature and many of the 
molds which contaminate foods belong to this genus. Thorn (1910) 
has reported cultural studies on the species of penicillium. He has 
worked out a key which is of much assistance in studying molds and their 
relation to food spoilage. KEY TO PENCILLIA 
207 
KEY TO PENICILLIUM MOLDS 
(After Thom, 1910) 
4. Species fruiting typically by coremia (vertical and definite). 
a. Coremia long (3-15 mm.). 
1. Conidial masses strictly terminal, olive green, 
fragrant P. claviforme 
2. Upper third of coremia fertile, conidia green.. P. duclauxii 
aa. Coremia small. 
1. Coremia definite, densely crowded, colony 
orange below P. granulatum 
2. Coremifonn characters indicated incultures by 
clustering of conidiophores, definite coremia 
only in old cultures becoming large and def- 
inite on apples P. expansum 
AA. Species not (or rarely) producing coremia in culture. 
B. Species constantly producing sclerotia or ascigerous 
masses. 
b. Producing asciginous masses, yellow or red- 
dish P. luteum 
bb. Sclerotia appearing as white masses in old 
cultures P. italicum 
bbb. Sclerotia reddish or pink, globose or ellip- 
tical, 500 microns or less in diameter. 
c. Conidial fructification in column. 
1. Column dense long, sclerotia partially 
buried in substrate P. No. 30 
2. Column formed of loose chains, scle- 
rotia numerous, exposed P. No. 29 
cc. Conidial fructification of divergent chains: 
1. Rapid liquefier, spores globose. 2-5 
microns P. No. 31 
2. Slow liquefier, spores elliptical, 3.5-4 
by 2.5-3 microns P. No. 32 
BB. Sclerotia not(or rarely) produced under special conditions. 
C. Rapid liquefiers (abundant liquid in 5 to 12 days). 
D. With definite strong ammoniacal odor: 
1. Yellowish brown-avellaneous spores rough.. . P. brevicaule 
2. White or cream spores rough P. brevicaule 
3. White or cream, spores smooth P. brevicaule, ver. 
glabrum 208 
YEASTS AND MOLDS 
DD. Without ammoniacal odor. 
E. With yellow coloration of liquefied gelatin (not of 
mycelium. in reverse). 
1. Colonies small, conidiophores 100-150 mi- 
crons in length P. citrinum 
2. Colonies broadly spreading conidiophores 
250-300 microns P. chrysogenum 
EE. Without yellow color in liquefied medium (or slight traces 
only). 
e. Colonies white or pink or salmon P. roseum 
ee. Colonies some* shade of green. 
f. Colonies floccose, margin spreading by stolons.... P. stoloniferum 
ff. Colonies velvety surface growth of fruiting hyphae 
only. 
g. Conidiophores very short (100-200 microns). 
1 P. No. 12 
2 P. No. 37 
gg. Conidiophores longer (200-400 microns): 
1. Conidiophores variously branched, reverse 
always colorless P. No. 24 
• 2. Conidiophores each with a verticle of branches- 
each branch wearing a columnar fructification 
-reverse and medium darkening in sugar 
media P. atramentosum 
CC. Liquefaction of gelatin none or slower than 10-12 days, 
or only partial. 
h. Colonies yellowish brown spores elliptical P. divaricatum 
hh. Colonies white to lilac, liquefier, 14-16 days P. lilacinum 
hhh. Colonies floccose or creamy white: 
1. Conidiophores long, typical penicillate 
branching P.camembertivar. 
rogeri 
2. Conidial chains borne upon short branches 
of floccose hyph® P. No. 33 
GG. Colonies some shade of green. 
H. Surface with hyph® definitely in ropes or trailing, 
bearing numerous conidiophores, as short 
branches distinctly traceable to their origin in 
such hyph®. 
i. Colonies usually red below and reddening the 
substratum. 
1. Fruiting areas dark green P. funiculosum 
2. Fruiting areas mixed yellow and green.P. pinophilum KEY TO PENC1LLIA 
209 
, ii. Colonies not producing red color. 
1. Colonies gray rarely greenish, very loose 
floccose P. intricatum 
2. Colonies green, conidial chains in simple 
compact column P. No. 48 
3. Colonies gray to green, hyphee scattered 
creeping P. decumbens 
HH. Surface hypha1 not in well-defined ropes, nor trailing. 
j. Surface hyphae woven floccose, course of hyphae 
not traceable. 
1. Gray green long conidiophores, no odor. ... P. camemberti 
2. Gray green, shorter conidiophores, strong 
odor P. biforme 
jj. Surface growth at margin simple conidiophores, in 
older parts both hyphae and conidiophores. 
1. Gray greenish, branching of conidiophores 
rather loose, odor none or slight P. No. 22 
2. Green, conidial fructifications rather com- 
pact, odor definite "moldy" P. commune 
N. B. There is probably a number of races in 
this group. 
jjj. Fruiting surface velvety-of simple conidiophores 
or conidiophores borne so close to surface of 
substratum as to appear simple: 
k. Conidial mass a dense column of conidial chains. 
1. Column from a single verticil of basidia... .P. spinulosum 
2. Column from a verticil of branchlets with 
verticillate cells and chains P. rubrum 
kk. Elements of conidial fructification not in column. 
kk. Elements of fructification not in a column. 
I. Conidia smooth. 
1. Green, broadly speaking, ripe conidia glo- 
bose, 4-5 microns P. roquefort 
2. Green, less spreading, conidia elliptical, 
medium commonly purpled P. purpurogenum 
3. Gray or olive green, conidia 5-7 by 3-5 
microns P. digitatum 
U. Conidia delicate rugulose P. rugulosum 210 
YEASTS AND MOLDS 
SPECIES OF PENICILLIA DETERMINABLE FROM THE SUBSTRATA 
(After Thom, 1910) 
(In these species the substratum establishes a presumption of identity.) 
Cheese (Camembert and Brie): 
1. Floccose, white unchangeable, no odor P. camemberti 
var. rogeri 
2. Floccose, white to gray green, no odor P. camemberti 
3. Powdery, yellowish white, spores smooth, ammoniacal 
odor P. brevicaule var. 
Glabr. 
4. Powdery, yellowish white, spores, tuberculate, ammoni- 
acal odor P. brevicaule var. 
album 
5. Forming yellowish brown areas, spores rough, ammoni- 
acal odor P. brevicaule 
Cheese Roquefort: 
1. Green streaks inside the cheese P. roqueforti 
Citrus fruits: 
1. Colonies of mold blue green P. italicum 
2. Colonies of mold olive green P. digitatum-oli- 
vaceum 
Pomaceous fruits: 
1. Blue green colonies finally producing coremia P. expansum 
Polyporacese (Boleti, Polypori, etc.): 
1. Colonies green (yellowish green) spreading by stolons.. .P. stoloniferum 
Wood (pine): 
Producing orange to red stains in pine wood P. piniphilum 
Aspergillus. This is a common variety of mold and is responsible 
for many decomposition processes in nature. The spores are borne 
from a club-shaped fruiting body called the basidium. 
SUMMARY OF THE ASPERGILLUS SPECIES GROUPED ACCORDING 
TO COLOR OF THE CONIDIAL VEGETATION, THE CHAR- 
ACTER OF THE STERIGMATA AND THE EXISTENCE OF 
ASCOSPORES 
(After Lafar, 1910) 
1. Green (gray, bluish green or yellow green), viz.: 
(a) With simple sterigmata. A. glaucus, Link, with ascospores (naked 
peritheca); A. clavatus, Desmazieres; A. fumigatus, Fesenius 
ascospores (cased perithecia); A. oryz.e (Ahlburg) Cohn; A. 
varians, Wehmer; A. minimus, Wehiner; A. flaws, Link; with 
sterile sclerotia; A. giganteus, Wehmer; A. coesiellus, Saito; 
A. Tokelau, Wehmer; A. penicillopsis (Hennings) Raciborski. KEY TO PENCILLIA 
211 
(6) With branches sterigmata: A. nidulans, Eidam, ascospores (en- 
sheathed sclerotia); A. pseudodavatus, Puriewitsch, ascospores 
(naked perithecia); A. variabilis, Gasperini; A. ver si color, 
Vuillemin. 
Fig. 51.-Aspergillus Oryzae. (After Wehmer.) 
1-2, conidiophores with clavate and almost spherical globule; 2, in optical section; 3-5, develop- 
ment of a small conidiophore, distension of the hypha, protrusion of sterigmata and incipient for- 
mation of conidia; la, optical section of tough stem; 6, sterigmata; 7, conidal herbage, slightly 
magnified; 8, conidia. Approximate magnification of 1-5, 75; of 6, 400; of 8, 900. 
2. White, viz.: 
(a) With branched sterigmata (associated with simple sterigmata in the 
case of A. candidus, I.; A. candidus, I.; Wehmer, A. albus, Wil- 
helm. 
(&) With simple sterigmata: A. candidus (Link) Saccardo. 212 
YEASTS AND MOLDS 
3. Blackish brown, viz.: 
(a) With branched sterigmata. A. niger (Cramer), van Tieghm, with 
sterile sclerotia; A. phoenicis, Fat, and Delacr.; A. strychni, 
Lindau; A. pulverulenta, MacAlpine; A. atropzirpureus, Zimmer- 
mann; A. vioUiceo-fuscus, Gasperini. 
(6) With simple sterigmata. A. luchuensis, Inui; A. calyptratus Oude- 
mans. 
4. Brownish yellow, yellow brown and reddish, viz.: 
(a) With simple sterigmata: A. ostianus, Wehmer, A. wentii Wehmer; 
A. perniciosus, Inui; A. giganteo-sulfureus, Saito; A. citrisporus, 
von Hbhnel. 
(6) With branched sterigmata (occasionally associated with simple ones). 
A. sulfureus, Fresenius; A. ochraceus, Wilhelm (with sterile scle- 
rotia); A. rehmii, Zukal (with ensheathed perithecia); A. spurius, 
Schroter; A. elegans, Gasperini; A. auricomus, Gueguen (with 
sterile sclerotia). 
Examination of Molds. If molds are present in the foods or material 
under examination, they will appear on the agar plates after incubation 
although it may take a longer incubation period than for the bacteria. 
In the examination of foods or other substances where the entire flora 
is desired for study, dextrose agar should be used since this will permit 
the development of bacteria, yeasts and molds. These colonies should 
then be transferred to dextrose agar slants and, in this way, preserved 
for further study and identification. 
Total Organism. The structure and makeup of the total organism 
may be secured from a water mount on a plain slide or a hanging drop 
preparation. The water mount is made by carefully dispersing some 
of the mold mycelium in a drop of water on a slide. A cover glass is 
then dropped on the preparation after which it is ready for examination. 
Such a water mount will yield satisfactory results with objectives with 
the greater working distances. With the oil-immersion objective, it 
may be unsatisfactory on account of the very short working distance. 
From this preparation, the shape and makeup of the entire mold should 
be determined. 
Structure of Hyphae and Fruiting Body. The examination of this 
part of the fungus will require higher magnifications. Hanging-drop 
preparations of the hyphae should be prepared for examination with the 
oil-immersion objective. From this, the presence or absence of septa 
and other facts about the internal structure should be determined. 
The fruiting body should be carefully gone over in the same manner. 
The structure of the fruiting head, production of the spores, and other CLASSIFICATION OF YEASTS 
213 
data should be carefully worked out. All of this is necessary when 
comparing the new fungus to the described varieties. Illustrations 
of the more common varieties have been included in this chapter. 
Yeasts 
The yeasts are fungi which cause many changes in food. Many of 
them are desirable changes while some are undesirable. For cen- 
turies they have played an important part in the domestic life of peoples 
and are the bases for great industries. Foods which have a high con- 
tent of carbohydrates are especially susceptible to the attack of yeasts. 
Fig. 52.-Types of Cells in Common Yeasts. 
A, Saccharomyces cerevisirs; B, Saccharomyces ellipsoideus; C, Mycoderma rini. (Adapted from 
Guilliermond's Les Levures.) 
True and Pseudo Yeasts. True yeasts are budding fungi which 
produce endospores. The false yeasts resemble the true yeasts very 
closely but differ in the fact that no endospores are formed. These have 
some of the properties of the molds. The torute (Turpin), mycodermae 
(Persoona) and cryptococcus (Kutzing-Vuillemin) and representatives 
of the false yeasts. 
Classification of Yeasts. The classification of the yeasts is rather 
confused. This is in part due to the different points from which the 
subject may be viewed. The fermentologist is concerned with the 
physiological changes which are brought about by yeasts and does not 
give much attention to nomenclature. His cultures may be designated 214 
YEASTS AND MOLDS 
by such " pseudoscientific " names as " beer yeast," " wine yeast," etc., 
or they may be even numbered. The taxonomist, however, must be 
concerned with keeping this group of fungi classified and so far as pos- 
sible properly named. Many investigators have classified the budding 
fungi. Anderson (1917) has reviewed the various investigations and a 
student will find a good summary with discussion of the various classi- 
fications in his paper. 
Hansen has done most work on the classification of the yeasts. 
His classification which appeared in 1904 is now accepted as the basis 
for classification of the true yeasts. He made two families which 
are divided into nine genera. Not all of these have been accepted by 
microbiologists. Guillierimond (1912) does not recognize the schizo- 
saccharomycetes which Hansen separated from the saccharomycetes 
by the fact that they reproduced by fission. He divides the saccharo- 
mycetes into five groups. Hansen's classification is as follows: 
KEY TO GENERA OF TRUE YEASTS 
(Hansen after Buchanan) 
Family I. Vegetative reproduction by budding Saccharomycetacese 
A. Celis do not form a surface membrane at 
once on sugar media, i.e., do not 
grow exclusively at the top of the 
medium. 
1. Spores having a single membrane. 
a. Cells fusing in pairs before 
spore formation Zygosaccharomycete 
b. Cells not fusing in pairs before 
spore formation. 
(1) Spores germinate by or- 
dinary budding Saccharomyses 
(2) Spores germinate by 
means of promycel- 
ium Saccharomycodes 
2. Spores having two membranes. . . . Saccharoinycopsis 
B. Cells forming a surface membrane at 
once by sugar media. 
1. Spores spherical, hemispherical or 
irregular Pichia 
2. Spores lemon shaped with pointed 
ends Willia 
Family II. Vegetative reproduction by fission Schizosaccharomycetes KEY TO BUDDING FUNGI 
215 
FALSE YEASTS 
(After Guilliermond) 
1. Cells generally spherical, often forming a 
pellicle but only after fermentation; 
pellicle always viscous without inter- 
vention of air Torula 
2. Cells generally elongated. Pellicle ap- 
pears at the beginning of develop- 
ment with intervention of air Mycoderma 
3. Yeasts without ascospores, parasitic to 
animals Cryptococcus 
Anderson (1917), after a careful study of the field, has proposed the 
following key, in which the budding phase is predominant. He has 
retained many of the names proposed by Hansen: 
KEY TO GENERA OF BUDDING FUNGI 
(Anderson, 1917) 
I. Ascospores known. 
Vegetative cells single or attached in irregular col- 
onies, mycelium not developed, ascospores 
formed within isolated cells (Saccharomycetacese) * 
Spores globose or void: 
Spores on germination forming typical yeast 
cells. 
Ascus formation preceded by the conjugation 
of gametes 1. Zygosaccharomyces 
Ascus formation not preceded by the conju- 
gation of gametes: 
Spore membrane single 2. Saccharomyces 
Spore membrane double 3. Sacchromycopsis 
Spores on germination forming a poorly de- 
veloped promycelium 4. Saccharomycodes 
Spores pileioform or limoniform, costate.... 5. Willia 
Spores hemispheric, angular or irregular in 
form on germination forming an ex- 
tended promycelium 6. Pichia 
Vegetative cells produced predominately by 
budding but forming a mycelium under 
some conditions asci terminal or inter- 
calary, differentiated from the mycelium.7. Endomyces 
* The genus Sehizosaccharomyces, which does not bud, and the relatively unim- 
portant genera, Monospora and Nematospora, are not included in this key. 216 
YEASTS AND MOLDS 
II. Ascospores not known, i.e., Fungi Imperfecti. 
Heavy dry pellicle formed on liquid media 8. Mycoderma 
No distinct pellicle formed: 
Vegetative cells forming a septate mycelium 
under exceptional conditions but predom- 
inately budding 9. Parasaccharomyces 
Vegetative cells formed only by budding. 
Cells apiculate, limoniform 10. Pseudosaccharomyces 
Cells frequently elongated into narrow non- 
septate hyphal threads 11. Pseudomonilia 
Cells typically yeast-like 12. Cryptococcus 
Compressed Yeast. The greater part of the procedure in the 
manufacture of compressed yeast is concerned with the preparation of 
the medium or " mash." This is commonly prepared from a mixture 
of grains, rye, corn and barley being commonly used. After thorough 
cleaning, the barley is malted in the usual manner. The malt is later 
put into a kiln to stop germination. The mash is then prepared by 
mixing the ground grains with the malt and maintaining at a constant 
temperature of 60° until all of the starch has been changed to soluble 
sugars. The mash is then soured for two reasons. It renders the nitro- 
genous matter available to the yeasts and also prevents the changes 
induced by the putrefactive bacteria. The souring process is continued 
until about 1.5 per cent of acid has been produced. The mash is then 
passed through filter presses which gives a clear filtrate called the wort. 
This supplies the foods for the yeasts. It is inoculated either by a pure 
culture or a portion of the previous day's run. An optimum tempera- 
ture is maintained during which there is a vigorous multiplication of 
the yeast cells. The cells are carefully filtered out and pressed into 
cakes with or without starch. The steps in the preparation of com- 
pressed yeast are graphically shown in Fig. 53. 
Yeast Foods. The use of these substances in bread making has 
been investigated by Kohman (1916) and his co-workers. The inves- 
tigation apparently had its origin in the effect of different natural 
waters on the yeast fermentation in bread as noticed by a large baking 
company. Kohman showed that by the use of small quantities of 
ammonium and calcium salts and potassium bromide brought about 
a saving of from 50 to 65 per cent in the usual amount of yeast. Other 
distinct advantages connected with the use of these nutriments are also 
noted. Hoffman (1917) in discussing the same subject has stated: 
"Since there is so much evidence in favor of all the ammonium chloride 
being changed to protein by the yeast, we can calculate how much-protein we DIAGRAM OF YEAST MANUFACTURE 
217 
Corn 
Barley 
Malt 
Rye 
Water 
Sprouts 
Cleaner 
Cleaner 
Cleaner 
Filter 
Cleaner 
I Mill I 
Mill 
[ Mill | 
Corn 
Cooker 
Mash Tub 
[Mal tj 
|Rye| 
pVaten 
Small 
Lactic Acid. 
Mash 
Souring 
Tank 
Filter Tub 
Culture 
of 
Pure 
Yeast 
Cooler 
Fermenter 
where 
yeast 
grows 
Yeast 
Seperators 
Seperated 
Yeast 
Cooler 
Receiving 
Tank 
Filter 
Press 
Mixing Machine 
Shipping 
• Boxes 
Refrigerator 
Cars to Agencies 
Fig. 53.-Diagram of Yeast Manufacture. (Fleischmann Co.) 218 
YEASTS AND MOLDS 
gain in the form of yeast protein, from adding 0.5 lb. of ammonium chloride in a 
dough batch containing 1000 lbs. of flour and how much protein we lose by 
deducting one-half the quantity of yeast (5 lbs. would be an average amount) 
regularly used; 0.5 lb. ammonium chloride contains 0.131 lb. nitrogen; this 
times 6.25 gives 0.82 lb. protein gained by converting the salt into albuminous 
matter. Compressed bakers' yeast contains 27 per cent dry matter; 5 lbs. of 
yeast then contains 1.35 lbs. of dry matter, 50 per cent, or 0.68 lb. of which is 
albuminous in character. We have not only as much protein produced by the 
yeast from the ammonium salt but actually more than that lost by deducting 
half the yeast in the dough batch. The economic value of a process which can 
utilize such simple substances as sugar and ammonium salts and build there- 
from complex but useful food proteins cannot be overestimated." 
Table XXII is taken from his work and shows clearly the effect of 
ammonium chloride on gas formation in bread. 
Table XXII 
SHOWING THE INCREASE IN GAS PRODUCTION IN DOUGH USING 
AMMONIUM CHLORIDE 
(Hoffman) 
Fermentation 
Hours. 
Control Dough, 
c.c. 
Difference. 
Dough Contain- 
ing 0.2 Gram 
NHC1 per 100 
Grams Flour. 
Difference. 
0 
110 
0 
110 c.c. 
0 
0.5 
180 
80 
180 
80 
1.0 
285 
105 
280 
100 
1.5 
390 
105 
390 
110 
2.0 
470 
80 
500 
110 
2.5 
570 
100 
640 
140 
3.0 
665 
95 
785 
145 
3.5 
790 
125 
955 
170 
4.0 
880 
90 
1105 
150 
4.5 
980 
100 
1260 
155 
5.0 
1080 
100 
1400 
140 
5.5 
1160 
80 
1520 
120 
6.0 
1230 
70 
1625 
105 
Total eas in six hours 
1120 c.c. 
1515 3 
Per cent increase due to ammonium chloride. 
35.3 
Methods for Study of Yeasts. The study of yeasts demands a pure 
culture. The securing of this pure culture is not often an easy per- 
formance. Two methods have been used and are available to micro- 
biologists at the present time. IDENTIFICATION OF YEASTS 
219 
Physiological Method. In this method an environment is created 
which is favorable to the desired fungus and unfavorable to the others. 
In applying this method to the yeasts, an acid medium may be used. 
Bacteria, unless they are acidophiles, are unable to grow in a 2 per cent 
tartaric acid solution. Pasteur and Hansen grew yeasts in such a 
medium to free them from bacteria. This method was accompanied 
with some uncertainty and was replaced by the dilution methods. 
Dilution Methods. These have been described before. They con- 
sist of diluting or breaking up the yeast's masses in a definite amount of 
sterile water or physiological salt solution. From these diluted mix- 
tures definite portions are taken for plating on a solid medium. After 
incubation and growth, cultures should be picked from the colonies and 
transferred to dextrose agar slants. The yeast is then ready for a cul- 
tural and morphological examination. 
Moist Chamber Preparations. These are essentially the hanging 
drop mounts which have been described before. A small portion of 
the pure culture of yeast should be gently emulsified in a drop of dex- 
trose broth or wort. This should be transferred to a clean cover glass 
and suspended over a depression in a concave slide or ring slide. The 
edges of the cover glass should be carefully lined with vaseline in order to 
make a tight seal and allow no evaporation during incubation. Such a 
mount will permit observations on a few cells to allow determine bud- 
ding and cell shape. 
Scheme for Identifying a Yeast. Whether the fungus is really a 
yeast or borderline mold should be determined first of all. The following 
points may be of assistance: 
I. Spore or non-spore former 
A. Spore former 
1. Shape, morphology 
2. Scum or membrane 
3. Top or bottom yeast 
4. Per cent of alcohol formed 
B. Non-spore former 
1. Non-scum former 
a. Color 
b. Shape, morphology 
2. Scum former 
a. Color 
b. Morphology 220 
YEASTS AND MOLDS 
Microscopic Examination of Compressed Yeasts. Prepare a thin 
mixture of the yeast in distilled water. To this mixture or a small por- 
tion of it add a little of a 0.1 per cent solution of methylene blue. Mount 
a drop of this under a cover glass on a slide and enumerate the dead and 
living bacteria. The dead cells will stain but the living cells will not. 
Test for Adulteration with Beer Yeast. Practically all of the beer 
yeasts ferment raffinose while the bread yeasts generally do not. One 
gm. of the yeast is mixed with about 10 c.c. of a 1 per cent solution of 
raffinose broth and placed into a fermentation tube. Incubate this 
tube for a!bout twenty-four hours at 30° C. A blank tube without 
raffinose should be prepared in order to test for gas produced from 
stored glycogen. If the difference between the two tubes is decided 
(about 50 per cent) adulteration with beer yeast is probable. A greater 
difference renders the test more conclusive. 
Efficiency of Bread Yeasts. This may be determined in different 
ways. A representative sample of the yeast should be obtained and 
divided into three parts. Store one part at 
20°, 30°, and 40° C. At regular intervals test 
each part for dead cells, gas formation, and 
dough-rising power. Test once or twice for 
adulteration with beer yeast and with myco- 
derma. 
Dough-rising Power of Bread Yeast. Mix 
100 gms. of flour, 2 gms. of yeast and 60 c.c. 
of distilled water by first making a thin batter 
of the yeast, water and a little of the flour. 
Then add the rest of the flour and knead for 
five minutes. About 3 gms. of flour should 
be kept with which to clean the glass rod. 
The dough should be formed into a cylinder 
and dropped into a warmed graduate cyl- 
inder which has been greased or powdered 
with flour. Press the dough down into the 
cylinder and place at 30° C. The volume is 
read every thirty minutes until the maximum is reached. Compute 
the maximum volume in terms of the original. Plot a curve which 
will show the activity of the yeast. 
Determination of the Fermenting Power of Yeasts, Meissl Method. 
Prepare a saccharose phosphate mixture as follows: 400 gms. of sac- 
charose, 25 gms. ammonium phosphate, 25 gms. potassium phosphate. 
Mix this thoroughly and weigh 4.5 gms. into an Erlenmeyer flask which 
Fig. 54.-Culture Flask 
for Fermentation Tests. 
Fitted with Alwood 
( 19 0 8) Ventilation 
Tube. EXAMINATION OF YEASTS 
221 
carries a fermentation valve. Add 
50 c.c. of distilled water and 1 gm. 
of the yeast to be tested and incu- 
bate at 30° C. 
Different types of valves are 
available. Alwood (1908) used a 
convenient type for studying the 
fermenting capacity of pure strains 
of yeasts. The valves are so con- 
structed that the activities of the 
yeast may be observed and the gases 
from the fermentation may escape. 
The moisture is retained by some 
absorbent such as calcium chloride 
or sulphuric acid. The apparatus 
should be weighed before and after 
the run. The Alwood valve is 
shown in Figs. 54 and 55. 
Determination of the Ferment- 
ing Power of Yeasts, Kusserow's 
Method. Put 400 c.c. of 10 per cent 
saccharose solution and 10 gms. of 
the yeast under examination into the 
fermentation flask of the Kusserow 
apparatus. Warm the flask and solution to 30° C. before starting. 
After the apparatus has stood for an hour at 30° C. it is connected and 
the amount of gas formed in the next half hour is 
measured; it should be 250 c.c. The amount of gas 
for the first and second half hours is usually about 
50 and 150 c.c., respectively. Five gms. of yeast 
in 400 c.c. of 10 per cent saccharose solution in a 
flask fitted with a fermentation valve will cause a 
loss of about 6.5 gms. of carbon dioxide in twenty- 
four hours if distilled water is used; with tap water 
the loss should approximate 8 to 12 gms. 
Determination of Mycoderma in Bread Yeast. 
Press the yeast with a sterile spatula into the bot- 
tom of a small Petri dish and incubate at 37° C. 
for two days. If the yeast contains a mycoderma, 
pure white characteristic colonics will appear on 
the yellowish background of the bread yeast. 
Fig. 55.-Detail of Ventilation Tubt 
for Culture Flask. (Alwood, 1908.) 
Fig. 56.-Types of 
Ventilation Valves 
which may be Used 
with Erlenmeyer 
Flasks for Testing 
the Fermenting 
Power of Fungi. 222 
YEASTS AND MOLDS 
Bread Making 
The flour is one of the first things to which the baker pays special 
attention. Miner (1917), in describing the manufacture of flour, divides 
it into three general types: 
I. Low grade 
II. Clear flour 
III. Patent flour 
The baker demands a uniform flour and to secure it he may resort to 
blending. Most of them use a baker's " patent " flour which may be 
superior to the domestic " patent." This is carefully sifted and the 
other ingredients added such as salt, sugar, lard, malt extract, milk, 
water, yeasts, etc. In the straight dough method all of these are mixed 
and allowed to ferment for about five hours in a long trough. The 
temperature of this room is maintained at 27° C. with a relative humidity 
of 75 per cent. From the fermenting room the dough is taken to a 
divider where it is cut into any desired size. It may be worked a little, 
allowed to rise and molded for baking. In the larger bakeries this may 
be entirely a machine process. 
Yeast in Bread Making. Bread is one of the oldest foods in the 
preparation of which fermentative changes are involved. In the early 
days before commercial yeasts were available, the housewife had to 
reply on spontaneous fermentations. The organisms which induced 
these chance fermentations got into the sponge either from the air or 
along with one of the ingredients. Quite often, a portion of one batch 
was taken out and saved over to inoculate the next batch of bread. 
To-day the baker does not have to rely on liquid brewers' yeast nor the 
housewife on the uncertain fermentations of wild yeasts. The pro- 
duction of compressed and dried yeasts caused a distinct advance to be 
made in the production of leavened foods. A mixture of yeast and flour 
will ferment, but the addition* of other substances such as potato water, 
malt water, will greatly accelerate the action of the yeasts. All of the 
constituents of the flour which are given above are concerned in bread 
making. The carbohydrates are precursors of the carbon dioxide and 
in the baked loaf they impart a rich brown color to the crust. Accord- 
ing to Wardall (1910) the flavor of the bread is not dependent on the 
strain of yeast which is used as a leaven. She used thirty-three pure 
strains of yeasts and could observe no difference in the flavor of bread 
leavened by them. She regarded the time of incubation as too short 
for the development of any special flavor. SELF-RISING BREAD 
223 
Self-rising Bread. This has been known for generations under the 
name of " salt-rising " bread. As suggested by the name, the fermenta- 
tion is a spontaneous one. Hunt and Wessling (1917) give the following 
instructions for making: 
1 cup milk 
2 tablespoons cornmeal, white 
1 teaspoon salt 
1 tablespoon sugar 
Butter (if used) 1 tablespoon 
Flour. " Scald the milk. Allow it to cool until it is lukewarm; 
then add salt, sugar and cornmeal. Place in a fruit can or heavy crock 
or pitcher and surround by water at about 120° F. Allow the mixture 
to stand six or seven hours or until it shows signs of fermentation. If it 
has been fermented sufficiently, the gas can be heard as it escapes." 
As shown in the above paragraph, the fermentation is a chance one. 
It is, then, almost an uncontrolled process. The agent inducing this 
fermentation has received much study. Kohman (1912) has given a 
review of the literature and some of the data and references mentioned 
here have been taken from his interesting paper. There seems to be two 
sides to the question of the organism which is concerned. One side 
argues that the fermentation is induced by wild yeasts which get in 
from the air. In support of this are the investigations of Mitchell 
(1908), Bailey (1914), and others. That it is a bacterial change is sup- 
ported by the work of Heinemann and Hefferan (1909) and Kohman 
(1912). The middle ground that both yeasts and bacteria are con- 
cerned is taken by Lehman (1894), Peters (1889) and perhaps Harrison 
(1902). Lehman isolated an organism which he called Bacillus levans. 
Heinemann and Hefferan isolated a bacillus from corn meal which had 
many of the characteristics of Bacillus bulgaricus. They proved that 
the organism came from the corn meal and believed that, in self-rising 
bread, the high amount of acid formed by this bacillus (1.65 per cent) 
from the milk, united with the inorganic salts to form carbon dioxide. 
The extensive work of Kohman (1912) indicated that bacteria were the 
important agents in the fermentation. He believes that members of 
the colon group are concerned. The gases produced by the salt-rising 
bacteria consisted of two-thirds hydrogen and about one-third carbon 
dioxide. These bacteria aerate the bread by decomposing some of the 
constituents possibly the carbohydrates. The organisms are probably 
of the type B. levans (Lehman). Woodward (1911) believed that the 
fermentation in salt-rising bread was due to the presence of organisms 
which are introduced with the corn meal. The organism which was 
isolated was not a yeast but belonged to another group. It was advised 224 
YEASTS AND MOLDS 
that the milk be sterilized before the bat (er was mixed since this allowed 
more uniform results and decreased the possibilities of the bad flavors 
which are so often inherent in this type of bread. 
Examination of Bread. The bacterial examination of bread is 
important for two reasons: first, the possibility of bread spreading dis- 
ease bacteria; and secondly, the relation of bacteria to undesirable 
fermentations and changes which may take place. 
Bread is generally regarded as an absolutely safe article in the diet 
since it is heated before eaten. Roussel (1908) stated that the interior 
of the loaf reached 101 103° C.; the crust reached 125-140.5° C. 
Such temperatures were regarded as sufficient to kill the vegetative cells 
but not the spores except they be present in the crust. Tubercle bacilli 
in the dough were reported to have retained their vitality. From these 
data Roussel states that mechanical mixers should be used in order to 
prevent the spread of disease. Auche (1909, 1910) introduced turbercle 
bacilli into dough before it was made into loaves. The loaves weighed 
up to 2 kg. The bacilli were killed in the baking process but the author 
regards it q,s a possibility that bacteria may pass through the baking 
process. In the latter paper cited, this same author added such bac- 
teria as the following to bread dough before baking: B. typhi, B. para- 
typhi, B. dysenteries (Shiga), B. dysenteries (Flexner), B. coli, Strept. 
pyogenes, Staph, aureus and a variety of proteus. Cultures made from 
the loaves after baking were sterile. Auche states that possibly more 
resistant bacteria would pass through the baking process. He states 
that the greatest care must be exercised to prevent contamination after 
the loaves have left the oven. Fenyvessy and Denies (1911) found that 
if the temperature of bread reached 94-104° C. during the baking process, 
the pathogenic bacteria, if accidentally present, would be destroyed. 
Gaujoux (1911), discusses some studies of this question. He found the 
Koch's bacillus (Bacillus tuberculosis) did not survive the temperature 
of baking bread but assumed that it might pass through the temperatures 
employed for pastries. Howell (1912) examined bread collected from 
various districts in Chicago. In general it was found that bread made 
under clean conditions had a lower bacteria content and fewer strepto- 
cocci. Jacobs, LeClerc and Mason (1914) studied the effect of wrapping. 
Wrapped bread had fewer bacteria on the surface. From the above 
discussion it would seem that bread may carry infection. The evidence 
is not altogether convincing, however, that it does. Hansen (1913) has 
reported a typhoid fever epidemic which may have been caused by bread. 
In this case the bread may have been contaminated after baking. 
Slimy or ropy bread has received quite a little attention from the 225 
EXAMINATION OF BREAD 
bacteriologists. Russell (1903) examined a sample of slimy bread and 
isolated B. mesentericus vulgatus. Temperature studies indicated that 
the organisms could pass through the baking temperature. Watkins 
(1906) has studied the question and advised that the flour be subjected 
to examination when this infection appears. Kornauth (1912) also 
advised an examination of the flour for the organism. Williams (1912) 
found a capsulated organism in a case of slimy bread. Kayser (1911) 
found that the bacteria causing this infection were introduced with the 
yeast. He stated that 1 to 2 liters of vinegar added to each 100 kg. of 
the flour would prevent the infection. Kayser and Delavel (1912) 
isolated bacteria which were related to the mesentericus group. When 
a bakery once becomes infected with members of this group, it is disin- 
fected with great difficulty. Kayser suggests the use of acidulated 
water for washing all of the apparatus and even states that some appa- 
ratus may have to be discarded. 
Wright (1916), when studying musty bread, found an Aspergillus 
and Rhizopus nigricans. Further experimental work indicated that the 
R. nigricans produced a mustiness in bread while Aspergillus produced 
a sourness. It was believed that the mustiness resulted from proteolytic 
enzymes from the R. nigricans. 
Watkins' Method for Detecting in the Flour the Organisms which Cause Ropy 
Bread. This author (Watkins, 1906) finds the following procedure a delicate one 
for determining whether a flour contains those bacteria which cause ropy bread. 
The test is rapid and the author states that there is little possibility of a sound 
failing to pass the test because they do not yield appearances of ropiness in the 
time proposed as a limit. Ten large test tubes (6 in. by 1 in.), are thoroughly boiled 
in water for one hour, washed and drained. When drained sterilize in the oven 
at 232° C. for three hours. The tubes should be thoroughly sterilized. Cool 
and then place into each tube a finger of bread 3 in. by 1 in., cut from the center 
of the same two-day old loaf. The average weight of each loaf should be 5 gms. 
Moisten with distilled water and plug the tubes. Sterilize by boiling in water on 
three successive days. To test the flour, 2 gms. are well mixed with water and placed 
in boiling water for thirty minutes. To the series of ten tubes add successively 
from 1 to 7 c.c. of the flour mixture, leaving three tubes to serve as checks. Number 
the tubes in rotation. Incubate at 28° C. and at the end of twenty-four hours 
examine for ropiness. 
Kornauth's Method for Detecting Organisms in Flour which Cause Ropy 
Bread. This is a modification of Kuhl's method and is carried out as follows: 280 
gms. of flour, 140 c.c. of salt water, and 0.6 gm. of yeast are made into a loaf. 
After baking, the loaf should be stored at 25° C. for forty-eight hours. If the 
organisms causing ropy bread were present in the flour, the loaf will have a char- 
acteristic odor. Several loaves should be made at the same time with sound flour 
as a check. 226 
YEASTS AND MOLDS 
BIBLIOGRAPHY 
Alwood, W. B. 1908. The Fermenting Power of Some Pure Yeasts and Some 
Associated Fungi. U. S. Dept. Agriculture, Bureau of Chern., Bull. 111. 
Anderson, H. W. 1917. Yeast-like Fungi of the Human Intestinal Tract. 
Jour. Infect. Dis., 21, 341-386. 
Auche, B. 1910. Bacteria in Bread. Semaine Med., 35, Brit. Med. Jour., 
1910, No. 2569, 771-772. 
Auche, B. 1909. The Destruction of Tubercle Bacilli in Bread by Cooking. 
Comp. Rend. Soc. Biol., 66, 800-802. 
Bailey, E. H. S. 1914. Sanitary and Applied Chemistry. The Macmillan Co., 
New York. 
Balland, M. 1894. Sur la temperature interiure du pain sortant du four. 
Comp. Rend. Acad. Sci., 117, 519-521. 
Cadwell, H. V. 1917. Laboratory Control in Yeast Making. Amer. Food 
Jour., 12, 151. 
Fenyvessy, B. von, Denies, L. 1911. Is Baked Bread Sterile? Zeit. Hyg., 
69, 223-224. 
Gaujoux, E. 1911. Concerning Bread and Pastry. Rev. Hyg. et Pol. Sanit., 
33, 1176-1180. 
Gould, R. G. 1917. The Manufacture of Commercial Yeast. American 
Food Journal, 12, 143-148. 
Gould, R. G. The Manufacture of Bread. Amer. Food Jour., 12, 195-201. 
Hansen, P. 1913. A Study of Typhoid Fever in Rockford, Illinois, in the 
Late Summer and Fall of 1913. Illinois State Water Survey Bull., 11, 
384-430. 
Harrison, F. C. 1902. Yeast and its Household Use. Ontario Ag. Bull., 
118, 17 pp. 
Heinemann, P. G., and Hefferan, M. 1909. Note on a Lactic Acid-forming 
Bacillus Closely Resembling B. bulgaricus Isolated from Corn Meal. 
Science, 29, 1011. 
Hoffman, C. H. 1917. The Utilization of Ammonium Chloride by Yeast. 
Jour. Ind. Eng. Chem., 9, 148-153. 
Howell, K. 1912. The Bacterial Examination of Bread. Amer. Jour. Pub. 
Health, 2, 321-324. 
Jacobs, B. R., LeClerc, J. A., and Mason, Maud L. 1914. A report on a 
Chemical and Bacteriological Study of Wrapped Bread. Amer. Jour. Pub. 
Health, 4, 721-732. 
Kayser, E. 1912. Bacterial Changes in Bread. Chem. Abstracts, 6, 1067. 
Kayser, E., and Delavel, H. 1912. A Study of Stringy Bread. Comp. 
Rend., 153, 576-578. Chem. Abstracts, 6, 515. 
Kohman, H. 1912. Salt Rising Bread and Somp Comparisons with Bread 
Made from Yeasts. Jour. Eng. Ind. Chem., 4, 20-30; 100-106. 
Kohman, H. A., et al. 1916. On the Use of Certain Yeast Nutriments in 
Bread Making. Jour. Ind. Eng. Chem., 8, 781-789. BIBLIOGRAPHY 
227 
Kohman, H. A. 1917. Feeding the Yeast in Bread Making. American Food 
Journal, 12, 35-38. 
Kornauth, K. 1912. Ropy Bread. Chem. Abstracts, 7, 847. 
Lehman, K. B. 1894. Ueber die Sauerteiggarung und Beziehungen des 
Bacillus levans zum Bacillus coli communis. Cent. Bakt., 15, 350-354. 
Marchland, H. 1909. The Cooking of Bread. Meun. Franc., 25, 208-10. 
Exp. Sta. Rec., 22, 64. 
Miner, A. 1917. The Manufacture of Flour. Amer. Food Jour., 12, 27-31. 
Mitchell, M. 1908. Course in Cereal Foods and their Preparation for Mov- 
able Schools of Agriculture. U. S. Dept. Agric. Office Exp. Stations. Bull., 
200. 
Peters, W. L. 1889. Die Organismen des Sauerteigs und Ihrer Bedeutung 
fur Brotgahrung. Botan. Zeit., 47, 405, 420 and 435. 
Roussel, J. 1907. The Survival of Pathogenic Bacteria in Bread after 
Baking. Rev. Intend. Mil., 20, 122-123. Chem. Absts., 2, 1168. 
Russell, H. L. 1903. Sticky and Slimy Bread and its Causes. Ann. Rept. 
Wis. Ag. Exp. Sta., 565. 
Schnoeder-Orelli, O. 1911. Investigations on the Growth and Spread 
of Decay Fungi in Storage Fruit. Landw. Jahrb. Schweiz., 25, 225-226. 
Cent. Bakt. Abt. II., 32, 161-169. 
Stevens, N. 1917. Rhizopus Rot of Strawberries in Transit. U. S. Dept. 
Agriculture, Bull., 531. 
Thom, C. 1905. Some Investigations from the Study of Dairy Fungi. Jour- 
nal of Mycology, 2, 117-124. 
Thom, C. 1906. Fungi in Cheese Ripening. U. S. Dept. Agriculture, Bureau 
of Animal Industry Bull., 82. 
Thom, C. 1910. Cultural Studies of Species Penicillium. U. S. Dept. Agri- 
culture, Bureau of Animal Industry Bull., 118. 
Wardall, Ruth A. 1910. The Relation of Yeast to Flavor in Bread. Jour. 
Home Economics, 1910, 75-91. 
Watkins, E. J. 1906. Ropiness in Flour and Bread and its Detection and 
Prevention. Jour. Soc. Chem. Ind., 25, 350-357. 
Williams, Anna W. 1912. A Study of Ropy Bread. Biochem. Bull., 1, 
529-534. 
Woodward, W. 1911. The Leavening Agent in Salt Rising Bread. Jour. 
Home Economics, 3, 100-101. 
Wright, A. M. 1916. A Cause of Mustiness in Bread. Jour. Soc. Chem. 
Ind., 35, 1045-1046. 4 CHAPTER VIII 
INTESTINAL BACTERIA 
The flora of the mouth is dependent upon many factors and is, 
therefore, continually changing. Excluding the various infections of 
the mouth which will not be considered here, the bacteria in the mouth 
are not especially significant. There are, however, several features 
which, compared with other body fluids, are worthy of mention. The 
mouth often contains a large number of bacteria and many kinds. 
Germicidal Action of Saliva. The work which is available on this 
subject is neither conclusive nor extensive. Huggenschmidt (1896) 
studied the various factors which contribute to the protection of the 
oral cavity and from such factors as bactericidal action of the sputum, 
mechanical action of the saliva, action of the mucous membrane and 
bacterial antagonism concluded that the mechanical action of the 
sputum was the most important factor. Sanarelli (1891) filtered saliva 
through a Chamberland filter and tested the bactericidal action of the 
filtrate. He found that saliva did possess a germicidal action quite 
similar to that exhibited by other body fluids, such as blood, aqueous 
humor, etc. The speed of the reaction depended upon the initial num- 
ber of bacteria introduced into the saliva. If a small number of cells 
(three or four hundred) were introduced, all were killed in a short time 
but when a few thousand cells were introduced, there was usually a 
decrease followed by an indefinite increase. With Bacterium diphtheria 
and the pneumococcus no germicidal action was noticed. They were 
found alive after twenty-eight and forty days. The cause of the ger- 
micidal action was not determined. Gordon (1916) carried out his 
experiments on this subject in culture media and found a germicidal 
action against the meningococcus, which he attributed to the antagon- 
istic effect of other bacteria in the saliva. These were supposed to be 
mixed streptococci. 
Effects of Tobacco Smoke on Bacteria in the Mouth. This is a 
subject of more popular interest and has been of much use to those who 
wish to justify or decry the habit of smoking. Koerner (1896) found 
a decided reduction of bacteria in the mouth due to tobacco smoke. 
228 GERMICIDAL ACTION OF GASTRIC JUICE 
229 
From the facts secured, this author states that strong smokers (con- 
suming more than one dozen cigars a day) seldom have dental caries. 
Dunnon (1902) found no bactericidal action on B. tetanus, B. typhi or 
Leptothrix bucchalis and staphylococcus. Bacillus tuberculosis was 
inhibited. The action was not due to the nicotine content but to 
products of combustion. Arnold (1907) compared the bactericidal 
activity of hay and tobacco smoke. Rideal (1903) reported a slight 
bactericidal action of tobacco smoke. Ellis (1909) has stated that any 
beneficial effects resulting from the use of tobacco, is easily overbalanced 
by the habit of spitting so prevalent among smokers. 
The Stomach 
The bacteriological aspects of the stomach have been concerned 
mostly with its diseases. The bacteria of the stomach gain entrance 
with the food and drink. The germicidal action of the saliva may 
probably be neglected as a factor influencing the number of bacteria 
passing through the mouth. 
Germicidal Action of Gastric Juice. The gastric juice is the normal 
secretion of the stomach. It contains between 2 to 3 per cent solids 
and about 0.2 per cent of free hydrochloric acid, which is the most 
powerful of any acid combination in the stomach. A condition of hyper- 
acidity exists when a decidedly greater amount of hydrochloric acid is 
present than normally, and hyperacidity when the amount of acid is 
below normal. During conditions of hypoacidity, fermentation may be 
active with the formation of lactic and butyric acids. Some hold that 
gastric ulcer and gastric catarrh may be due to a reduced acidity. 
That the gastric juice is germicidal was noticed by Spallanzani in 
1734. When he moistened meat with gastric juice, it did not putrefy. 
Meat which was not so treated but which was moistened with other 
substances, such as water, developed a putrid odor very quickly. He 
opened a snake eighteen days after it had swallowed a lizard and while 
the lizard was partly digested, it had not decomposed. Other experi- 
ments by this early scientist demonstrated that putrefaction could be 
stopped by gastric juice. 
Koch (1884) fed cholera vibrios to dogs and after a few hours found 
that they had been destroyed. He stated that cholera vibrios were 
destroyed in the stomach under normal conditions and that if they 
passed through, it was due to some abnormal condition. Falk (1883) 
secured no action on B. tuberculosis. Kurloff and Wagner (1889) 
found a selective action of the gastric juice. Cholera vibrio, B. typhi 230 
INTESTINAL BACTERIA 
and Ps. pyocyaneus were destroyed but B. tuberculosis, B. anthrax, and 
B. tetanus (spores) were not affected. Strauss and Wurtz (1889) showed 
that anthrax bacilli were killed in fifteen minutes and that the destructive 
factor in the gastric juice was the hydrochloric acid. The same con- 
clusion was reached by Kabrhel (1890). Hamburger (1890) first called 
attention to the fact that some of the acid was bound and that the free 
acid had a stronger germicidal effect. This was proven by testing the 
action of a solution of HC1 with one of the same strength containing 
peptone. Kianowsky (1891) showed a direct relation between the 
number of bacteria in stomachs and the amount of acid. With patients 
possessing a faulty acid secretion many bacteria were found. Cadeac 
and Bournay (1893) found that Ps. pyocyaneus was destroyed after 
six hours and that B. anthrax and B. tuberculosis were not killed. Pet- 
tenkofer (1893) reported some startling work which was carried out in 
1893. This was done soon after the great epidemic of cholera in Ham- 
burg and Altoona. He proposed the X, Y, Z theory for cholera where 
X = bacterium; 
Y = host or soil; 
Z = environment. 
He stated that X without Y and Z would not cause cholera. So 
thoroughly did these workers believe in this theory that Pettenkofer and 
Emmerich drank pure cultures of the cholera vibrio. Each mixed 1 c.c. 
of a broth culture with 100 c.c. of 1 per cent sodium bicarbonate solution 
and drank it. Pettenkofer did not change his usual manner of living 
and, after having a diarrhoea for four days, came back to normal. 
Emmerich's case ran a longer time. In this case the sodium bicarbonate 
neutralized the HC1 of the gastric juice. The bacteria thus passed 
through the stomach and produced the usual results. 
Stern (1908) does not regard the gastric juice as such a barrier 
against bacterial invasion as some of the former workers. He regards 
persons with hyperacidity as fortunate from this standpoint, but in 
no way absolutely protected. Bile was found to decrease the bacteri- 
cidal action of the gastric juice. When proteins were present, cholera 
vibrios were found to live a long time. Pepsin was found to strengthen 
the action of the acid. Hansen (1912) infected food with B. coli and 
secured only a slight decrease of bacteria in the stomach. Schultz- 
Schultzenstein (1908) found the gastric juice to be more germicidal 
than hydrochloric acid solution of the same strength. Gregersen (1916) 
reported some of the more recent work on this subject. He studied 
the action of the acid in the juice and whether any other factors were GERMICIDAL ACTION OF GASTRIC JUICE 
231 
important. Tables XXIII and XXIV are taken from his paper and 
are quite significant. 
Table XXIII 
STOMACH CONTENT % HOUR AFTER EWALD'S TEST BREAKFAST. 
TEMPERATURE 37° C. STAPHYLOCOCCUS PYOGENES AUREUS 
(After Gregersen, 1916) 
Diagnosis. 
G 
K 
Al 
Ph 
Pepsin. 
Time of Destruction. 
1 
Cancer ventriculi.... 
-30 
-14 
2 
6 
0 
No destruction 
2 
Neurasthenia 
-24 
-10 
4 
10 
0 
No destruction 
3 
Alcoholismus chron. . 
-20 
-12 
0 
1 
0 
No destruction 
4 
Cancer ventriculi.. . . 
-18 
- 6 
1 
4 
0 
No destruction 
5 
Alcoholismus chron. . 
-16 
-10 
1 
5 
0 
No destruction 
6 
Vicus ventriculi 
-14 
0 
24 
28 
4 
No destruction 
7 
Cancer ventriculi.. . . 
- 3 
6 
14 
20 
3.5 
90 minutes 
8 
Arthroctis chron 
- 3 
10 
17 
22 
3 
90 minutes 
9 
Anaemia 
0 
6 
10 
12 
4.5 
90 minutes 
10 
Neurasthenia 
0 
12 
24 
32 
4 
38 minutes 
11 
Ulcus ventriculi 
0 
23 
35 
47 
5 
38 minutes 
12 
Ulcus ventriculi 
0 
8 
12 
20 
6 
45 minutes 
13 
Cirrhosis hepatis.... 
0 
14 
34 
44 
5 
45 minutes 
14 
Neurasthenia 
3 
10 
16 
24 
5 
9 minutes 
15 
Colitis 
5 
14 
20 
24 
4.5 
6 minutes 
16 
Neurasthenia 
5 
18 
22 
30 
5 
6 minutes 
17 
Ulcus ventriculi 
5 
14 
20 
24 
6 
8 minutes 
18 
Colitis 
6 
12 
16 
22 
4 
5 minutes 
19 
Abstipatic 
8 
22 
29 
34 
6 
6 minutes 
20 
Colitis 
10 
22 
30 
38 
6 
5 minutes 
21 
Ulcus ventriculi 
13 
34 
50 
60 
6 
3 minutes 
22 
Colitis 
20 
28 
30 
34 
4.5 
1.5 minutes 
23 
Ulcus ventriculi 
20 
40 
54 
60 
5.5 
2 minutes 
24 
Ulcus ventriculi 
26 
48 
58 
66 
5.5 
1.5 minutes 
25 
Ulcus ventriculi 
32 
42 
50 
56 
6 
1 minute 
26 
Ulcus ventriculi 
46 
58 
66 
74 
6 
. 5 minute 
27 
Ulcus ventriculi 
50 
64 
70 
78 
6.5 
. 5 minute 
The letters at the top of the columns refer to the reaction as deter- 
mined by the following indicators: 
G = Gunzberg's reagent. Indicates free hydrochloric acid. 
K = Congo red. The blue color indicates free hydrochloric acid. 
Other mineral acids may also be indicated by this reagent. 
Al = Sodium alizarin sulphonate, which indicates all available except 
that which is combined. 
Ph = Phenolphthalein, which indicates total acidity. 232 
INTESTINAL BACTERIA 
It will be noticed from the table that, without free acid, the stomach 
may not be depended upon to kill bacteria. As the amount of free 
hydrochloric acid increases, however, the time required for the destruc- 
tion of bacteria is reduced. The same facta were obtained experi- 
mentally by this author and are reported in Table No. XXIV. 
Table XXIV 
STOMACH CONTENTS WITH OR WITHOUT HC1 OR NaOH. STAPH. 
PYOGENES AUREUS. TEMPERATURE 37° C. 
(After Gregersen, 1916)) 
G 
K 
Al 
ph 
Pep- 
sin. 
Destruction 
Time. 
No. 4. Without addition 
-18 
-6 
1 
4 
0 
No destruction 
After addition of HC1 
-12 
0 
7 
11 
No destruction 
After addition of HC1 
0 
0 
19 
23 
45 minutes 
After addition of HC1 
5 
17 
24 
27 
- . 
6 minutes 
After addition of HC1 
10 
22 
29 
32 
3 minutes 
No. 11. After addition of NaOH. . . 
-23 
0 
12 
24 
5 
No destruction 
Without addition 
0 
23 
35 
47 
38 minutes 
After addition of HC1 
5 
28 
40 
52 
6 minutes 
After addition of HC1 
10 
33 
45 
57 
3 minutes 
After addition of HC1 
20 
43 
55 
67 
1.5 minutes 
After addition of HC1 
40 
63 
75 
87 
1 minute 
No. 23. After addition of NaOH. . . 
-20 
0 
14 
20 
No destruction 
After addition of NaOH . . . 
0 
20 
34 
40 
45 minutes 
After addition of NaOH.. . 
5 
25 
39 
45 
8 minutes 
After addition of NaOH . . . 
10 
30 
44 
50 
3 minutes 
Without addition 
20 
40 
54 
60 
5.5 
1.5 minutes 
No. 22. After addition of NaOH. . . 
- 8 
0 
2 
6 
No destruction 
After addition of NaOH . . . 
5 
13 
15 
19 
9 minutes 
After addition of NaOH. . . 
10 
18 
20 
24 
4 minutes 
Without addition 
20 
28 
30 
34 
4.5 
1.5 minutes 
No. 27. After addition of NaOH. . . 
10 
24 
30 
38 
6 
2 minutes 
Without addition 
50 
64 
70 
6.5 
.5 minute 
The bacterial content of the stomach as shown above is quite depend- 
ent upon the acidity of the stomach. In order to standardize these 
determinations, the acidity of the stomach is usually examined after 
a test meal. The Ewald test breakfast is much used, which consists 
of two pieces of toast and 400 c.c. of water. The stomach is pumped 
out at the end of about one hour. ACIDITY IN STOMACH 
233 
The contents should be carefully filtered and the following deter- 
minations made. 
Total Acidity. Filter 10 c.c. of the gastric contents and add about 
four drops of phenolphthalein. Titrate to a faint pink color with 
N/10 NaOH. Calculate the number of cubic centimeters of N/10 
NaOH required for 100 c.c. of stomach contents. This is often termed 
degrees. Some prefer to express the results in terms of per cent hydro- 
chloric acid. (1 c.c. of N/10 HC1 = .00365 gm. HC1.) 
Free Acidity. This gives both the organic and inorganic free acid. 
Titrate 10 c.c. of the stomach contents with N/10 sodium alizarine 
sulphonate as the indicator. Titrate to a violet color. Determine 
and report the number of cubic centimeters of N/10 NaOH required 
by 100 c.c. of the juice. 
Combined Acidity. The difference between the total acidity and 
the free acidity is the combined acidity. This is also reported for 100 
c.c. of the stomach contents. 
Detection of Lactic Acid. Prepare Uffelmann's reagent by adding 
ferric chloride to a 1 per cent solution of phenol until a blue color results. 
Add 3 c.c. of this solution to 3 c.c. of the stomach contents. The pres- 
ence of lactic acid will be indicated by the formation of a yellow color. 
Organic Acids and Acid Salts. Subtract the free hydrochloric acid 
from the combined acidity and express the results in terms of 100 c.c. 
of gastric contents. 
Microscopical Examination. This may yield much valuable infor- 
mation. The microorganisms to be expected are yeasts, mold spores, 
rods and sarcinse. Other bacteria may be present under certain con- 
ditions. Other particles, such as cellulose epithelial cells, starch grains, 
etc., may also be present. 
Bacteria in Intestines and Feces. The bacteria which gain entrance 
to the intestines and feces are those which have passed through the 
stomach in the chyme or which may have been contributed, in rare cases 
from the blood stream. 
Relation of Bacteria to Life. This has been a much-debated ques- 
tion. Pasteur (1885) believed that bacteria were essential for normal 
life. Nuttal and Thierfelder (1895, 1896 and 1897) reared guinea pigs 
under aseptic conditions which had been delivered by Caesarian section. 
These increased in weight, but not as regularly as normal pigs. From 
these data, the authors concluded that bacteria were not necessary. 
Schotellius (1899, 1902) hatched chickens in a sterile environment and 
from the data which he secured concluded that chicks could not live 
in absence of bacteria. He stated also that sterilizing the grain may have 234 
INTESTINAL BACTERIA 
destroyed some essential substance. Levin (1899, 1904) found the in- 
testines of most Arctic animals completely sterile. He draws no con- 
clusions, but his work is of interest on account of normal growth without 
bacteria. Metchnikoff (1901) grew tadpoles in sterile bread and water 
and secured no transformation into frogs in either the sterile or unster- 
ilized tadpoles. The non-sterile grew larger than the sterile. Portier 
(1905) determined that larvae of certain lepidoptera lived aseptically. 
Moro (1905) confirmed the work of Schotellius which has been men- 
tioned above. He used turtles instead of chicks. Bogdanow (1908) 
concluded that bacteria were necessary for normal development because 
sterile larvae of piptera did not do as well as those kept in an unsterile 
environment. Wollman (1911) criticized the work of Bogdanow, 
stating that his sterilization was carried out at too high a temperature. 
He concluded from his work on flies that bacteria were not necessary 
for normal development. Cohendy (1912) thought that the digestive 
processes could proceed without bacteria, but that they aided digestion. 
Life was thought to be possible without bacteria, but bacteria are not 
indispensable. Kianizine (1915) produced data which he thought 
weakened the argument of Cohendy. With chickens, Kianizine believed 
that bacteria were of great aid in digestion. They carried on analytic 
and synthetic processes which were of much value to the host. In a 
later paper by the same author (1916) he continues his argument. He 
made guinea pigs breathe sterile air and eat sterile food and found that 
even after a few days they were greatly weakened. The bad results 
from the deprivation of bacteria was thought to be due to reduced 
oxidation and accumulation of leucomaines in the body. Loeb and 
Northrop (1916-1917) attempted to determine the ability of flies to 
synthesize their body proteins without the aid of microorganisms. 
Larvae grown on sterile media (banana) did not do well, while those 
placed in a sterile yeast culture developed normally. It was thought 
that yeasts were essential for this species of fly although the essential 
element could not be isolated. 
This rather extensive presentation of the literature gives the data 
from which our present conclusions must be drawn. Obviously much 
of the data which have been used cannot be applied to conditions in the 
human intestinal tract. 
Action of Bile on Bacteria. This juice is poured into the intestine 
just below the pylorus. It is a thick liquid with a decidedly bitter taste. 
It is made up of salts of glycocholic and taurocholic acids, pigments 
(bilirubin, biliverdin, etc.) fats, phosphatids and inorganic salts. 
The bile like most of the other body fluids is probably sterile when DIET AND BACTERIAL FLORA 
235 
secreted. Toida (1914) found that bile of man and dogs was normally 
sterile and that it was not a satisfactory medium for all bacteria. It 
was only slightly suited to Staphylococcus pyogenes aureus and Ps. 
pyocyaneus. Streptococcus pyogenes and Dip. pneumoniae were better 
adapted to it. The bactericidal action was found to vary with 
the origin. Just below the point of entrance into the intestine, the 
bacterial flora is much simpler. B. coli is often the predominating form. 
The regulation of carbohydrate fermentation by bile has been 
studied by Roger (1912, 1913, 1915). The hydrolysis of starch was 
increased up to 30-40 per cent but when smaller amounts (10 per cent) 
were used there was a retardation. Heating the bile did not destroy 
its activity. Moderate amounts of bile (5-20 parts per 100) retarded 
the action of intestinal bacteria on glycogen. Larger amounts were 
erratic, causing sometimes a retardation and sometimes an increase in 
the action. The same was found to be true with regard to glucose. 
Pure cultures of Bacillus colon were inhibited in their action on glucose, 
moderate amounts exerting a more consistent retarding effect than 
larger amounts. 
Bile also exerts a retarding effect on putrefaction. Roger (1913) 
found that the intensity of the biuret reaction of protein media inocu- 
lated with intestinal bacteria in the presence of different amounts of bile, 
showed that the attack on proteins was retarded. This retardation was 
evident up to concentration of 20 per cent after which the retardation 
decreases. Roger (1915) regards the bile as a factor tending to diminish 
the production of bacterial enzymes and neutralizing toxins. Boudielle 
(1913) reported that bile as well as bile salts had an inhibiting action on 
the fermenting power of B. coli on glucose. Moderate amounts had 
more pronounced effects than greater amounts. Lagane (1914) added 
bile to bouillon cultures and noticed no retardation in growth of intes- 
tinal bacteria. He claimed that it favored the development of B. coli 
at the expense of other species. 
Whether the bile is a germicide seems hardly to have been settled. 
However, most of the data which have been accumulated seems to show 
that bile exerts a selective action. Toida found that under normal 
conditions the bile to be sterile. In this connection, it is interesting to 
remember that gall stones may be started by bacteria and that very 
often typhoid carriers liberate the bacilli in the bile. In these instances 
the bile seems to exert no bactericidal action, but perhaps a selective 
action. 
Effect of Diet on the Intestinal Flora. It has long been considered 
that diet was one of the most important factors determining the intes- 236 
INTESTINAL BACTERIA 
tinal flora. Much work has been done by various investigators from 
which facts have been well established with regard to changes induced 
in the intestines by variations in the diet. The changes in the flora 
resulting from various diets have been measured in terms of certain 
large groups and rarely in terms of any one or few organisms. These 
groups which have thus been used may be enumerated as follows: 
I. Putrefactive Group 
Bacillus colon, B. Welchii 
II. Aciduric Group 
B. bulgaricus, B. acidophilus, B. bifidus 
III. Amylolytic Group 
Glycobacter amylolyticus 
Herter and Kendall (1908), working with monkeys, studied the effect 
on the intestinal flora of sudden changes in the diet. After the flora 
from a distinctly protein diet had been established the diet was changed 
to a milk and sugar diet. From this change they were able to detect 
a change in the intestinal flora and mental condition of the animal. 
Also in the urine, putrefactive products were quite evident. In the 
intestines the acidolphile group of organisms was changed to a proteo- 
lytic group. This was one of the first papers from which definite con- 
clusions could be drawn. The authors suggest that in a disease w'here 
either the acidophile or putrefactive group tends to become established, 
a rapid alteration in the diet would tend to prevent either group of 
bacteria from becoming established. The same conclusions were reached 
by Herter (1910) in a later paper. 
Blatherwick and Hawk (1914) have studied the effect of fasting and 
of low- and high-protein intake on the bacteria in the intestines. The 
concentration of bacteria was measured by determining the amount of 
bacterial nitrogen in the feces according to the method mentioned else- 
where in this chapter and the output of urinary indican. They found 
that there was no relation between the output of bacteria in the feces 
and amount of indican in the urine. By means of a seven-day fast 
(Blatherwick,and Hawk, 1914), the bacterial nitrogen excreted by a 
76 kg. man was reduced from 1.571 gms. to 0.101 gm. The actual 
weight of excreted bacterial substance was reduced from 14.336 to 0.920 
gm. per day. The output of bacterial nitrogen and bacterial sub- 
stance was about the same during a fast and on a low proten intake. 
Assumption of a high protein diet caused an increase in the above figures. FACTORS INFLUENCING INTESTINAL BACTERIA 
237 
Unfortunately a qualitative study of the bacteria in the feces was not 
made during this study and to secure such information it is necessary 
to consult the work of others. Herter and his colleagues, the work of 
whom has been summarized by Herter (1907), have given information 
to show that a protein diet induces the proteolytic group. Rettger and 
his colleagues have studied the effect of carbohydrate feeding. The 
experiments were made with laboratory animals and man. Hull and 
Rettger (1915) studied the effect of milk and carbohydrate feeding. 
They found that lactose when taken by adults along with their usual 
food, caused a shift in the flora to the acidophilli group of bacteria. 
B. acidophilus and B. bifidus appeared with the former more abundant. 
The most practical diet, however, to produce these results was a com- 
bination of milk and lactose. B. bulgaricus was not found which is in 
accord with the work of others. 
Fasting has a decided effect on intestinal bacteria. As quoted above 
Blatherwick and Hawk (1914), during fasting, noticed a decided reduc- 
tion in the amount of bacterial nitrogen and bacterial substance. Sisson 
(1917) has found that, after periods of starvation, there are definite 
changes in the number of bacteria in contrast to the usually vigorous 
growth. No change in the kind of organism after starvation was noted. 
The greatest decrease in the number of bacteria was noticed in the duo- 
denum. Sisson regards the condition produced by starvation as one of 
relative amicrobism. 
In this connection the influence of copious water drinking on fecal 
bacteria should be mentioned. Fowler and Hawk (1910) noticed a 
decrease in the total nitrogen appearing as bacterial nitrogen. The 
following weights of fecal bacteria were excreted per day during the 
periods mentioned: 
Preliminary 5.327 gms. per day 
Water period 4.579 gms. per day 
Final period •.... 3.280 gms. per day 
Hattrem and Hawk (1911) by means of copious water drinking 
obtained a decrease in putrefaction and stated that it was probably due 
to a decrease in the activities of indol-producing bacteria following 
accelerated absorption of products of protein digestion. Copious water 
drinking (1000 c.c.) caused a decrease of indican in the urine which 
was accompanied by an increase of ethereal sulphates. The drinking of 
distilled and softened waters were found by Sherwin and Hawk (1914) 
to cause a decrease in intestinal putrefaction as measured by urinary 
indican. 238 
INTESTINAL BACTERIA 
Number of Bacteria in Feces. The different investigators are not 
in agreement on this question. This may be in part due to different 
methods of analysis and to the variations in samples. The securing of 
concordant results is hardly to be expected. The data which are on 
hand vary from 5 to 50 per cent. Strasburger (1896) stated that one- 
third of the feces of an individual on a normal diet was bacteria. The 
quantity of daily bacterial wastes dried in adults was as follows: 
a. Eight gms. under normal conditions. 
b. In dyspeptic intestinal conditions on an average of 14 gms. and 
even as much as 20 gms. 
c. In chronic constipation 5.5 gms. and, at times, even as little as 
2.6 gms. 
The total number of bacteria eliminated by an individual in one day 
was believed by Strasburger to be 128,000,000,000. Among the deter- 
minations which have been made by different investigators are the fol- 
lowing collected by Mattill and Hawk (1911): 
Daily Excretion 
of Dry 
Bacteria. 
Percentage of 
Dry Bacteria. 
Strasburger 
8.0 gms. 
24.3 % 
Berger and Tsuchiya 
3.023 
12.60 
McNeal, Latzer and Kerr 
5.34 
26 90 
Mattill and Hawk 
8.27 
27 97 
Sato 
8.54 
24 39 
Schittenhelm and Tollens 
42 00 
Lissauer 
8.67 
Tobaya 
11.22 
Osborne and Mendel (1914) found that about 70 per cent of the 
nitrogen in feces of animals was caused by bacteria. 
Classification of Intestinal Bacteria. Since the intestinal flora is 
quite extensive it is not a simple matter to make a classification. The 
viewpoint and interests of the investigator might influence the kind of 
classification made. Ford classified the bacteria in the duodenum of 
infants. The following classification by Distaso (1912) is as satisfactory 
as any: INTESTINAL BACTERIA 
239 
THE INTESTINAL FLORA. BY A. DISTASO 
Lancet, 182 (1912), 496-98 
A. Gram Negative Bacilli. 
1. B. coli group. 
2. B. variabilis group. (Anaerobic bacteria with round extremity.) 
3. B. thetaiotaomicron group. (Bacilli very polymorphic, elliptical form.) 
4. B. rigidis group. 
5. B. preacutus group. (Bacillus swollen in the middle and pointed ex- 
tremities.) 
B. Gram Negative Cocci. 
1. Sarcina citrea group. (Very common in the mouth and feces, similar in 
appearance to the gonococcus.) 
2. Diplococcus obliculus group. (Strict anaerobes.) 
3. Parvulus group. (Strictly anaerobic, small cocci.) 
C. Gram-Positive Bacilli. 
1. B. Acetogenes group. The greater number of the gram-positive bacilli 
belong to this group. The bacillus acetogenes B is its chief repre- 
sentative, since the B. bifidus and B. acetogenes A are very rare 
in the feces examined in London. 
2. Streptobacillus group. This microbe always exists in the intestinal 
flora. 
3. Diploe, acximinatus group. An anaerobic bacillus. It produces burytic 
acid. 
4. B. perfrigens group. An anaerobic microbe. It produces enormous 
quantities of lactic acid. 
5. B. oedematic maligni group. With the B. sporogenes (Metchnikoff). 
6. B. rodella group. Anaerobic group with very long bacilli. 
7. A lemon-shaped bacillus, which is stained by iodine and is described by 
the author as being butyrous acid-producing microbe. It has not 
yet been obtained pure. 
D. Gram-positive Cocci. 
1. Enterococci group, in chains, less frequently diplococci. 
2. Small cocci group. Are also to be seen corresponding in size to the 
coccus Banani, to the Staphylococcus pyogenes, and to the Staph, 
asacch arolyticus. 
In addition three kinds of spores are to be found. 
1. Oval spores, rather large, which may belong to B. sporogenes and to 
other. 
2. Round spores, which may belong to B. putrificus (Bienstock-Tissier) 
or to the bacillus of Rodella and to the B. Alkaligines anaerobicus. 
3. Very small spores, strongly refractile, belonging to the B. perfrigenzs. 240 
INTESTINAL BACTERIA 
Fermentation in the Intestinal Tract. Pul refaction is used in this 
case to indicate the bacterial decomposition of proteins. There has 
been much discussion with regard to the definition of the terms putre- 
faction and fermentation. Kendall (1911) has given a good discussion 
of these terms in their relation to the digestive tract. He quotes therein 
the definitions which have been given to the processes of putrefaction 
and fermentation by various workers and accepts the definition for 
fermentation which was given by Alfred Fischer. Fischer defines fer- 
mentation as the biochemical decomposition of nitrogen-free, organic 
compounds, especially the carbohydrates induced by fermenting organ- 
isms. If this definition is accepted there may then be different types 
of the process, depending on the main product: lactic acid, butyric acid, 
alcoholic fermentations, etc. With regard to putrefaction more confu- 
sion exists. The term has been used in so many different ways that it is 
difficult to correlate them. Kendall states that the phenomena grouped 
together as putrefaction " represent a scries of symbioses, in which the 
initial superficial breakdown is brought about by the anaerobes, while 
the process is brought to its lowest terms by the facultative anaerobes." 
The Germans have made a distinction between " Faulniss" putrefaction 
in the popular sense and " Verwesung " eremecausis. This latter is 
supposed to be an aerobic process carried on by many bacteria. That 
fermentation should mean decomposition of carbohydrates and putre- 
faction, decomposition of proteins are the opinions of the biological 
chemists. 
Certain microbiologists, however, have not accepted this definition. 
They define fermentation as an intracellular process which furnishes 
energy to the cell and pay no attention to the products which are decom- 
posed. The decomposition of soluble nitrogenous compounds within 
the cell protoplasm is regarded as fermentation. This seems to be the 
best definition for the term as has been suggested, by Fischer (1902). 
He gives the following points as characteristic of such a conception of 
the term: 
1. Fermentation is an intracellular process. 
2. The products of the fermentation are essentially different from 
those in the substract and are not mere parts of these original substances. 
3. The products are useless to the cell and may be harmful. 
4. Vital energy is produced. 
According to this definition, then, and as it is stated by Fischer, the 
hydrolysis of starch to soluble sugars is not a fermentation, because it 
does not take place within the cell to yield energy to the cell. The 
burning of the sugar within the cell protoplasm, however, does consti- AEROBIC RESPIRATION 
241 
tute fermentation and the energy therefrom is available for cell use. 
Fermentation may then be regarded as respiration if the latter term is 
considered to cover those processes which yield energy. There are two 
kinds of respiration, aerobic respiration and anaerobic respiration. 
Aerobic Respiration. This involves the securing of energy from 
food substances by means of and in the presence of free oxygen. In 
order to present the facts required in a discussion of this nature, the 
carbohydrates lend themselves very well. It must be remembered, 
however, that there are certain well-known objections to the expression 
of bacterial changes by chemical equations and these should be borne 
in mind in considering the equations which follow. As a typical example 
of aerobic respiration the decomposition of dextrose may be taken 
CeHi2O6+6O2 = 6CO2 + 6H2O+674 cal. In this change molecular oxy- 
gen has been used to burn dextrose completely.* 
In the products carbon dioxide and water there is no energy which is 
available for bacteria. With nitrogenous substances the oxidation of 
glycocoll takes place as follows: 
7 o 
H-C-C-OH + 30 = 2CO2 + H20 + NH3 + 152 Cal. 
nh2 
In this equation allowance must be made for the combustion heat of 
ammonia since this compound may be further oxidized. Leucin is 
oxidized as follows: 
C6H13NO2+I5O-6O2+5H2O+NH3+755 cal. 
In this equation the same correction must also be made for the com- 
bustion heat of ammonia. 
Hydrogen sulphide is oxidized by the sulphur bacteria according to 
the following equation: 
H2S+2O2 = H2SO4+207 cal. 
or to neutral sulphur as follows: 
H2S+O = H2O+65 cal. 
* In all considerations of respiration, it must be remembered that the energy 
secured by complex molecules is latent in them, having been put into them by the 
organism which formed them. Such synthetic processes are endothermic for the 
organism which builds up these complex substances from the simpler compounds. 
The bacteria secure this energy when they split these complex compounds to simpler 
ones. 242 
INTESTINAL BACTERIA 
Anaerobic Respiration. The products of anaerobic respiration are 
usually left in an incompletely oxidized condition. If dextrose is taken 
again to show this, any of the following equations might serve. 
CH2OH(CHOH)4COH = CH3CHOH -COOH +15 cal. 
CH2OH(CHOH)4COH = 3CH3COOH+34 cal. 
CH2OH(CHOH)4COH = C2H5OH+2CO2+22 cal. 
The main product in each of these equations may be further oxidized 
to yield more energy. 
The amount of energy secured by aerobic and anaerobic bacteria 
from the same amount of food is striking. Take, for instance, the 
following equation as a typical one for aerobes: 
CH2OH(CHOH)4COH+6O2 = 12CO212H2O+674 cal, 
and the following as typical for anaerobic bacteria: 
CH2OH(CHOH)4COH = CH3CHOH C00H+15 cal. 
The proportion would then exist as follows: 
Aerobic : anaerobic = 674.15 
Aerobic : anaerobic = 45.1 
That is, from the same amount of food the aerobes by complete 
oxidation will get forty-five times as much energy. Looking at it from 
the standpoint of energy, the anaerobes have to have forty-five times 
as much food as the aerobes. This explains why the anaerobic bacteria 
are so destructive in soil, sewage, etc. 
Putrefaction in the Intestinal Tract. As stated above the biological 
chemists have reserved this term for the decomposition of nitrogenous 
substances. According to the definition of fermentation which has 
been given above, some decompositions of nitrogenous substances would 
be regarded as fermentations. The combustion in the protoplasm of 
those peptones which are diffusible would be regarded as fermentation 
while the hydrolysis of the proteins and proteoses which goes on outside 
of the cell wall would not be regarded as fermentation. 
The chemistry of the putrefactive changes which take place in the 
intestines is fairly well known. Straight chain acids are among the first ANAEROBIC RESPIRATION 
243 
compounds which are attacked and the changes may be indicated as 
follows: 
I. Deaminization. 
H H 
I I 
H-C-C-OH > H-C-C-OH + NH3 
nh2 h 
II. Decarboxylation. 
Glycocoll 
Acetic acid 
Ammonia 
H H 
I z° 1 
H-C-C-OH > H-C-H + C02 
NH2 nh2 
Glycocoll 
Methylamin 
Carbon dioxide 
III. Oxidation. 
H 
I ^0 
H-C-C-OH + 0 > C02 + H20 
H 
According to these reactions tyrosin may be putrefied as follows: 
CH2-CH-cZoH-CH2-CH2-COOH + NH3 
.J ^H2 
OH OH 
Tyrosin 
Para hydroxy phenyl propionic acid 
z\ O /\ 
/ CH2-C^-OH -z \-CH3 
' ^OH 
OH 
Hydroxy phenyl acetic acid 
Para cresol 
Phenol 
The oxidation stops with phenol. These substances have no well- 
marked location in the intestine although it occurs in the large intestine. 
Their formation is not limited to the intestines for they have been found 
in other parts of the body. For excretion phenols are detoxicated. 244 
INTESTINAL BACTERIA 
They are united with sulphuric acid in the liver to form ethereal sul- 
phate. 
x-OH H-Ox Z) ^0 
Z 1 + >Sf * 7 1~0 
H-OZ X) q/ 
\Z \Z 
This is usually excreted as a sodium or potassium salt. 
Putrefaction of Tryptophan. Hopkins and Cole have determined 
the steps in the decomposition of tryptophan by cells. 
C-CH2-CH- C-CH2-CH2^C-OH-> 
II I I II 
c nh2 I c 
H H 
Tryptophan 
Indol propionic acid 
C-CH2-CH3 C-CH3 -C-H 
! ii ! ii • ii 
\/\/C \/\/C \/\/C 
N NN 
N H H 
Indol acetic acid 
Methyl indol (skatol) 
Indol 
The final product is methyl indol or skatol. For excretion this may be 
united with sulphuric acid as follows: 
/\ z\ ^0 
Z \ _ C_Ch3 Z \ C-CH2OH / \ C-CH2-O-/SC . 
II I II II y 
| C -*1 C -> C 
\/\/ \/\/\ \Z\/\ Kz 
N N H N H 
H H H 
Indol is prepared for excretion in the same way. 
Putrefaction of Arginin. Putrecin in the end product and was 
supposed to be important in certain types of food poisoning. It is 
formed from arginin as follows: PUTREFACTION OF AMINO ACIDS 
245 
N-CH2-CH2-CH2-CH-C-OH -> 
C=NH NH2 
nh2 
Arginin 
nh2-ch2-ch2-ch2-chnh2-cooh 
Ornithin 
nh2-ch2-ch2-ch2-ch2-nh2 
Putrecin tetramethylene diamin 
Putrefaction of Lysin. Caclaverin is a common product of putre- 
faction. It was first observed by Brieger (1885). It is formed from 
lysin in the following manner: 
CH2NH2-CH2-CH2-CH2-CHNH2-C-OH -> 
ch2nh?-ch2-ch2-ch2-ch2nh2 
Pentamethylene diamin cadaverin 
Putrefaction of Cystine. 
s-;-s 
ch2 ch2 
chnh2 chnh2 
I I 
COOH COOH 
Cysteine first formed. 
H-C-|SH 
| >y|H 
H-C-N -* H2S 
• I 
COOH 
may form in this way. 
H 
H-C-SH -> CH3SH 
i 
■;I7* 
H-C-N 
COOH 
methyl mercaptan may form in 
this way. 246 
INTESTINAL BACTERIA 
According to Mathews cystine goes to cysteine, and then as: 
SH-CH2-CHNH2 -> SHCH2-CH2NH2 -> SHCH2-CH3 
Thioethyl amin 
Ethyl mercaptan. 
The action of bacteria and yeast-like fungi has been studied by 
Tanner (1917, 1918). These microorganisms were quite able to split 
hydrogen sulphid from cystine. 
Some of these decomposition products of the amino acids and pro- 
teins may be detected as follows: The solution to be tested may be 
either a filtered bacterial culture or a fecal suspension. 
Indol Determinations. A number of methods have been devised 
for the detection of indole. Most of these yield good results when 
applied to bacteriology. 
Nitroso-indol Nitrate Reaction. Acidify a portion of the substance 
under examination with concentrated nitric acid. The addition of a 
few drops of potassium nitrate will produce a red coloration if indol is 
present. The red compound is nitroso-indol nitrate. 
Nitro-prusside Reaction. Add a few drops of a freshly prepared solu- 
tion of sodium nitro-prusside to a filtered sample. Add sodium hydrox- 
ide and examine for the production of a violet color. This may be 
changed to a blue by the addition of a few cubic centimeters of strong 
acetic acid. 
Ehrlich's Para-dimethyl Amino Benzaldehyde Test. Add about 
one-third the volume of a 2 per cent solution of para-dimethyl amino 
benzaldehyde in aclohol to the sample under examination. Then 
slowly add dilute hydrochlorine acid until a red color is evident. This 
color may be deepened by the addition of a few drops of sodium nitrite 
solution. 
Bergeim's (1917) Method for Determination of Fecal Indol. Rub 
30 to 50 gms. of the fresh feces in a mortar with H2O. Transfer to a 
1 liter Kjeldahl flask and add H2O to 400 c.c. Add 5 c.c. 10 per cent 
KOH and 2 c.c. paraffine and distill with steam until 500 c.c. have been 
obtained, bringing the volume of the fecal suspension down to about 
100 c.c. Re-distill with steam after acidifying slightly with H2SO4 or 
else remove the NH3 with permutite. Mix an aliquot of the resulting 
solution (100 c.c.) in a 150-c.c. separatory funnel with 1 c.c. of a fresh 
2 per cent solution of NaB naphthoquinonesulphonate and 2 c.c. of 
10 per cent KOH. After fifteen minutes extract with CHCI3 using 
10 and 7 c.c. Dilute extract to 15 c.c. and mix. Compare color in the 
colorimeter with that of a standard similarly prepared, using 0.1 mg. 
indol. The error appears to be only - 1 per cent. BACTERIA IN FECES 
247 
Skatol Determinations. Skatol is a product of putrefaction which 
has much in common with indol. 
Herter's Test (1907). Add a few cubic centimeters of acid para- 
dimethyl amino benzaldehyde (5 gms. to 100 c.c. concentrated H2SO4) 
to about 6 c.c. of the filtered sample. After heating to boiling a bluish- 
lavender color is obtained. This may be deepened by adding a small 
amount of hydrochloric acid. 
Phenol Determinations. Ferric Chloride Test. By adding a few 
drops of ferric chloride solution to the solution under examination a 
blue color will be obtained if phenol is present. 
Nitric Acid Test. Add a few drops of nitric acid to the sample and 
heat. If phenol is present, a yellow color is formed due to the formation 
of picric acid. 
Bromine Water Test. When bromine water is added to a solution 
containing phenol, mono-, di-, and tribromophenols are formed. The 
first two possess a very sharp odor. Tribromophenol precipitates as 
yellow white needles. 
Methods for Determining Bacteria in Feces 
The accurate, rapid determination of the bacteria in feces is not a 
simple matter. Plating on ordinary media is known to give low results. 
On the other hand there are certain errors in the various microscopic 
methods. In these, the presence of pieces of organic matter hinder the 
counting. 
MacNeal's (et al.) Modification of the Winterberg Method. The 
first employment of the Thoma-Zeiss blood-counting chamber for bac- 
teriological technic was made in the one-cell dilution method of obtaining 
a pure culture. By this method the number of cells per unit volume 
could be ascertained and the dilution per unit volume of a suspension 
with one cell in two to five drops required to obtain a suspension with 
one cell in two to five drops calculated. In this way the first pure 
bacterial culture was obtained. Henrich Winterberg was the first to 
use this method of bacterial counting, and to test its accuracy both as 
regards suspensions of varying dilutions and in comparison with the 
microscopic plate-counting method. He considers the method more 
accurate than the plate-counting method, but as a quantitative pro- 
cedure he would consider it unimportant. He says his determinations 
were too low. Winterberg's counts were made with suspensions of 
living bacteria in bacteria-free distilled water. 
" The method, as used by MacNeal, Latzer and Kerr, is as follows: 248 
INTESTINAL BACTERIA 
a portion of the 1 : 100 suspension of feces is diluted ten times, and a 
portion of this is drawn up to the mark 1 in the capillary of a dilution 
pipette, ordinarily used in estimating the white blood cells. This is 
diluted to the mark 11 with a dilute solution of methylene blue in physio- 
logical-salt solution. (The staining solution consists of methylene 
blue, 1 gm.; glycerine, 25 c.c.; distilled water, 75 c.c. A few drops 
of this are mixed with 10 c.c. of 0.8 per cent salt solution until the mix- 
ture is well colored, but not too opaque. This mixture is used as the 
diluting fluid. A little practice will show the proper depth of color to 
be employed.) The suspension is thoroughly mixed in the bulb by 
shaking and rolling in the usual manner. Several drops are blown out 
and then a very small drop is placed in the center of the circular elevated 
portion of the slide, which has been previously thoroughly cleansed by 
washing in distilled water and alcohol. A clean, thin, ground cover-slip 
is made slightly moist by breathing upon it and is immediately placed 
upon the slide. Slight pressure upon it causes the Newton color rings to 
appear and these remain after the pressure is removed if the preparation 
has been properly made. The slide is allowed to stand one to two hours 
to allow the bacteria to settle. Then the bacteria in fifty small squares 
upon the marked scale are counted microscopically, the No. 7 Leitz 
objective and No. 3 ocular being used. The calculation is simple as an 
example will show. 
" Example. B252, Subject H, July 15, 1908. Fifty squares contain 
400 bacteria. Therefore the average per square is 8.0 bacteria. One 
small square = 1/4000 c.mn. One c.mn. contains 8X4000 = 32,000 
bacteria. One cubic centimeter contains 32,000X1000 = 32,000,000 
bacteria. Pipette dilution is 1 : 10. 32,000,000X 10 = 320,000,000 bac- 
teria per cubic centimeter of 1/1000 suspension. One cubic centimeter 
of 1/1000 suspension is equivalent to 1 mg. feces. Therefore, there are 
320,000,000 bacteria per milligram feces. 
" Counting by this method is an exacting process and much practice 
is required to see all the bacteria present. Careful adjustment of 
the light is important and best results have been obtained by illumina- 
tion with Welsbach light; constant focusing through the different layers 
is necessary. Perhaps the greatest source of error is the difficulty of 
distinguishing accurately the bacteria. Skill in this is acquired only by 
practice. The method has the advantage of simplicity. The results 
of the authors seem to conform to those of Winterberg in that high 
dilutions give a relativ.ely high count." 
MacNeal's (et al.) Modification of the Eberle-Klein Method for 
Determining the Number of Bacteria in Feces. Eberle, working in BACTERIA IN FECES 
249 
Escherich's laboratory, was the first to determine the quantity of fecal 
bacteria by counting them in stained films. His experiments were 
carried out with normal infant's stools. Coverglass preparations were 
made from fecal suspensions of known dilution and after being com- 
pletely dried were stained with freshly prepared aniline water fuchsin, 
then washed in water, air-dried, mounted in Canada balsam, and 
counted. The method was very much improved by Alex, Klein and 
Hehewerth. Klein had noticed that vegetative bacteria were more 
sensitive to disinfectants in a moist than a dry state, and this led him to 
believe that bacteria would also be stained more readily in a moist con- 
dition than after drying. Therefore he allowed the dye to act while 
the bacteria were suspended in a liquid and the coverglass preparation 
was made, dried, and fixed, only after the bacteria were stained. Later 
the same investigator modified the method still further, using gelatin to 
fix the stained bacteria to the coverglass. The aqueous solution of 
gelatin and the stained bacterial emulsion were put upon the cover- 
glass separately, then fixed and spread. When dry, the preparation 
was immediately mounted in Canada balsam without flaming. 
" The procedures as employed by MacNeal, Latzer and Kerr have 
been considerably modified. In the addition of the gelatin to the bac- 
terial suspension, in spreading the films, in selecting the fields to be 
counted, and in counting individuals, rather than groups, as units, the 
technic differs from Klein's method. Of the 1 : 100 suspension of the 
feces prepared as described above (p. 248), 2| c.c. are transferred to a 
clean, dry bottle, | c.c. of melted nutrient gelatin and 2 c.c. of aniline 
water gentian violet added, and the whole thoroughly mixed and allowed 
to stand for three to five minutes. Then, by means of a platinum loop, 
the carrying capacity of which has been determined with great care, a 
loopful of the mixture, well shaken immediately before, is transferred 
to a clean, flamed 20 mm. square No. 1 coverglass and deposited near the 
center of the glass. Immediately another coverglass of the same size 
is accurately placed on top of the first so that the two glasses are in 
contact over about three-quarters of their surfaces and the sides evenly 
fitted together. As soon as the liquid has spread evenly between the two 
coverglasses, they are quickly slipped apart and allowed to dry. The 
technic here is the same as ordinarily used in the preparation of cover- 
glass blood films. If the preparation does not appear evenly spread 
the process is to be repeated with two more coverglasses until a satis- 
factory result is obtained. The films are next accurately measured by 
a millimeter rule and then, without further treatment, mounted in Can- 
ada balsam upon one slide so that one diagonal of each rectangular 250 
INTESTINAL BACTERIA 
film is parallel with the long axis of the slide. By laying the slide over 
coordinate paper these diagonals are readily brought into the same line 
parallel with the edge of the slide. 
" The covers must be so mounted that these diagonals, now in a 
straight line, are those which crossed each other as the two coverglasses 
were originally put together in preparing the films. When properly 
made each diagonal measures almost exactly 25 mm. 
"For counting the bacteria in the preparation, the Leitz 1/12 oil 
immersion objective and the No. 3 ocular fitted with an Ehrlich ocular 
square to restrict the field to a convenient size, and a mechanical stage 
graduated in millimeters are employed. Beginning at the end of the 
diagonal of one coverglass the bacteria are counted in each of 25 fields 
1 mm. apart along this diagonal. In a similar way 25 fields are counted 
on the diagonal of the second coverglass, making a total of 50 fields, 
the average of which may be considered as representative of both films. 
The size of the square field is accurately measured by a stage microm- 
eter. From the data then at hand the number of bacteria per milligram 
feces is calculated. 
" Example. B252, Subject H, July 15, 1908. In the preparation 
made, as described, each film measures 16.5X17.5 mm., total film area 
therefore, 33X17.5 mm.; the field employed measured 0.0445 mm. 
square; the amount carried by the loop 2.01 mg.; the number of bac- 
teria counted in 50 fields was 559 and the original 1 : 100 suspension 
was used (diluted to 1 : 200 by dye and gelatin). From these data, 
n + • ir r 559X33X17.5X200 QO,nnnnan 
Bactena per milligram feees = gpx0 = 324,000,000. 
In this fraction all the members except the size of the films and the 
number of bacteria counted may be kept constant and the calculation 
simplified by the use of logarithmic tables. 
" With apparatus and reagents ready and the 1 : 100 suspension 
prepared, this entire estimation can be completed in about forty min- 
utes. The results cannot be considered very accurate as the platinum 
loop does not carry an exactly constant quantity. There is also some- 
times great difficulty in distinguishing micrococci from other fine par- 
ticles in the preparations. The concentration of the bacterial suspen- 
sion also influences the final result. The estimation is in general rela- 
tively higher when dilute suspensions are counted as we have observed 
in applying it to enumeration of bacteria in pure cultures." * 
Steele's Modification of the Strassburger Procedure for the Quanti- 
tative Enumeration of Fecal Bacteria. Steele describes the method as 
* From MacNeal, Latzer and Kerr. J. Inf. Dis. 6 (1909), p. 127. BACTERIA IN FECES 
251 
follows: " The possibility of separating the bacteria from the rest of 
the feces depends on the fact that the bacteria are so nearly of the same 
specific gravity as distilled water that they cannot be centrifugalized 
out of a watery suspension of the feces, but remain suspended in the 
supernatant fluid. Taking advantage of this, the bacteria can be 
removed by washing with the centrifuge. Then, if the specific gravity 
of the wash-water is lowered by the addition of large amounts of alcohol, 
the relation of the bacteria to the fluid is changed to such an extent that 
the microorganisms can be readily centrifugalized out, separated, and 
weighed. Unless the period of passage of the feces has been ascer- 
tained to be normal, it is better to mark the beginning and end of each 
period of examination by carmine. The use of the Schmidt diet is not 
necessary. The whole stool is saved. Unless the feces are liquid they 
are rubbed up with a known amount of distilled water until they are 
smooth and semi-liquid and as homogeneous as it is possible to make 
them. 
" Two portions of 5 c.c. are measured off with a pipette of large 
caliber, using for this purpose an ordinary 5 c.c. pipette with the tapering 
end cut off, and with the necessary correction made at the upper mark. 
One of these portions of 5 c.c. is put into a porcelain dish and dried over 
a water bath and later in a drying oven, in order to determine the dried 
weight. The addition of a little alcohol and thorough mixing will hasten 
the process of drying and prevent caking of the feces. 
" The second portion is washed free from bacteria. This is done as 
follows: The wash-water is 0.5 of 1 per cent HC1 solution in distilled 
water. The acid increases the solubility of the salts and soaps of the 
feces. One hundred c.c. of this solution is employed at the beginning 
of the washing The feces are thoroughly mixed with the wash solu- 
tion and then centrifugalized. The use of the water motor or electric 
centrifuge is almost essential. Each tube is centrifugalized for about 
1| minutes, then the cloudy supernatant liquid is poured through a 
layer of gauze. This fluid contains the bacteria in suspension. All of 
the mixture (the wash-water and the feces) is centrifugalized the same 
way, and then the residue in the tubes is shaken up with more of the 
wash-water and centrifugalized again. This is repeated until the super- 
natant liquid after the centrifugalizing is transparent, showing that 
approximately all the bacteria have been washed out. If smear is made 
of the residue at this point, it will be found that the bacteria are not 
entirely washed away, but are evidently very much reduced. They 
occur singly, while in the unwashed feces they are in great lumps and 
masses. The suspension of bacteria is then mixed with a liberal 252 
INTESTINAL BACTERIA 
portion of alcohol, and evaporated down slowly at a temperature of 
40° to 50° C. until it amounts to not more than 50 c.c. in all. This 
takes approximately twenty-four hours. It is then mixed with at least 
twice its volume of alcohol, preferably absolute alcohol, although this is 
not absolutely necessary. This lowers the specific gravity of the fluid 
to such an extent that now the bacteria readily centrifugalize out. 
The mixture is then centrifugalized until the supernatant liquid is 
quite clear. This takes thirty minutes or more for each tube. The 
residue, which consists of the bacteria, is washed with pure alcohol and 
is shaken up with ether to remove the fat; then it is again washed with 
alcohol. All of this washing is done by means of the centrifuge. The 
bacteria are next washed out of the tube with a little alcohol and evap- 
orated to dryness and dried in the oven at moderate heat, dried in the 
desiccator, and weighed. Smears of the final preparation show that 
it consists of bacteria with a very few minute particles of other material. 
These particles are only visible with high power, and are very few in 
number, perhaps two at each field of the 1/12-inch objective. They 
stain with methylene blue; Strasburger suggests that they are cellulose, 
which they may well be. At any rate, the error arising from the inclu- 
sion of these small particles in the dried weight of the bacteria must be 
very small, and is probably balanced by the bacteria that it is not pos- 
sible to wash out of the residue in the first washing. During the prep- 
aration of the bacteria the first portion of 5 c.c. has been dried and 
weighed. We then know the dried weight of 5 c.c., the weight of the 
dried bacteria in 5 c.c., the original volume of the stool, and the volume 
after the addition of a known amount of water. It is then easy to cal- 
culate the data that we desire, namely, the volume of the stool, its dried 
weight, 'the weight of the dried bacteria, and the percentage of bacteria 
in the dried weight." 
Mattil and Hawk's Method for Quantitative Determination of Fecal 
Bacteria. " The method is a simplification of MacNeal's adaptation of 
the Strasburger procedure. About 2 gms. of feces are accurately 
weighed and placed in a 50-c.c. centrifuge tube. To the feces in the 
tube a few drops of 0.2 per cent hydrochloric acid are added, and the 
material is mixed to a smooth paste by means of a glass rod. Further 
amounts of the acid are added with continued crushing and stirring 
until the material is thoroughly suspended. The tube is then whirled 
in the centrifuge at high speed for one-half to one minute. The sus- 
pension is found sedimented in not more or less definite layers, the upper- 
most of which is fairly free from larger particles. The upper and more 
liquid portion of the suspension is now drawn off by means of a pipette BACTERIA IN FECES 
253 
and transferred to a beaker. The sediment remaining in the tube is 
again rubbed up with a glass rod with the addition of further amounts 
of dilute acid, and again centrifugalized for one-half to one minute. The 
supernatant liquid is pipetted off and added to the first, the same 
pipette being used for the one determination throughout. A third por- 
tion of the dilute acid is then added to the sediment, which is again 
mixed by stirring and again centrifugalized. All the washings are added 
to the first one, and, during the process, care is taken to wash the 
material from the wall and mouth of the centrifuge down into it. Finally, 
when the sediment is sufficiently free from bacteria, the various remain- 
ing particles are visibly clean, and the supernatant liquid, after cen- 
trifugalization, remains almost clear. This is removed to the beaker 
in which are now practically all of the bacteria present in the original 
portion of feces, together with some solid matter not yet separated. In 
the centrifuge tubes there is a considerable amount of bacteria-free solid 
matter. 
" The suspension is now transferred to the same centrifuge tube, 
centrifugalized for a minute, and the supernatant liquid transferred to 
a clean beaker by means of the same pipette. The tube is then refilled 
from the first beaker and thus all the suspension centrifugalized a second 
time. The beaker is finally carefully washed with the aid of a rubber- 
tipped glass rod, the second sediment in the centrifuge tube is washed 
free of bacteria by means of this wash-water and by successive portions 
of the dilute acid, and the supernatant liquid after centrifugalization is 
added to the contents of the second beaker. The second clean sediment 
is added to the first. The bacterial suspension now in the second beaker 
is again centrifugalized in the same way and a third portion of bacteria- 
free sediment is separated. Frequently a fourth serial centrifugalization 
is performed-always if the third sediment is of appreciable quantity. 
At all stages of the separation, small portions of the dilute hydrochloric 
acid are used, so that the final suspension shall not be too voluminous. 
Ordinarily it amounts to 125 to 200 c.c. At the same time, the final 
amount of fluid should not be too small, as shown by Ehrenpfordt, 
because the viscosity accompanying increased concentration prevents 
proper and complete sedimentation. 
"To the final bacterial suspension an equal volume of alcohol is 
added and the beaker set aside to concentrate. A water bath at 50° to 
60° is very satisfactory. After two or three days, when the liquid is 
concentrated to about 50 c.c., the beaker is removed and about 200 c.c. 
of alcohol are added. The beaker is covered and allowed to stand 
at room temperature for twenty-four hours. At the end of this time 254 
INTESTINAL BACTERIA 
the bacterial substance is generally settled, so that most of the clear 
supernatant liquid, of dark brown color, can be directly siphoned off 
without loss of solid matter. The remainder is then transferred to 
centrifuge tubes, centrifugalized, and the remaining clear liquid pipetted 
off. The sediment consists of the bodies of the bacteria, and is trans- 
ferred to a Kjeldahl flask for nitrogen determination. This is the bac- 
terial nitrogen. Where a determination of bacterial dry substance is 
desired, the sediment of bacteria is extracted by absolute alcohol and 
ether in succession, transferred to a weighed porcelain crucible, and 
dried at 102° C. constant weight. This dried sample is then used in the 
nitrogen determination. Our procedure differs from that of MacNeal 
in that the bacterial dry matter is not determined. A saving of about 
seven days' time and of considerable labor is accomplished by this 
omission. 
" Inasmuch as it has been shown by various investigators that such 
bacteria as are present in the feces contain on the average about 11 per 
cent of nitrogen, the values for bacterial nitrogen as determined by our 
method may conveniently serve as a basis for the calculation of the 
actual output of bacterial substance." 
Examination of Feces for Bacillus Typhosus 
Lumsden and Stimson's Method. "Place about 5 gms. of the feces 
in a conical glass or other suitable vessel, add about. 15 or 20 c.c. of 
sterile physiological salt solution or bouillon and agitate. Let stand 
one-half to one hour cither at room temperature or, preferably, at 
incubator temperature (37° C.) in order to permit the heavy particles to 
settle. Deposit one or two drops of the prenatant fluid in the center 
of an Endo plate. With a right-angled glass rod distribute the drop 
over the entire surface of the plate and then rub the rod over the surface 
of a second, third, fourth and fifth Endo plate. By carrying the spreader 
over the surfaces of several plates in this way, one or two of the plates 
will furnish abundant but sufficiently isolated colonies to permit 
' fishing.' 
" After inoculation place the plates inverted in the incubator, leave 
there for twenty to twenty-four hours, and then examine. On the 
plates colonies of typhoid and paratyphoid bacilli will be transparent, 
colorless, dew-drop like and usually from 1 to 2 mm. in diameter; while 
colonies of colon bacilli will be deep red, showing sometimes on the sur- 
face a sheen from precipitation of the fuchsin, and measure usually from BACILLUS TYPHOSUS IN FECES 
255 
3 to 4 min. in diameter. If typhoid-like colonies appear on the plates 
fish five or six of them, and inoculate tubes of Russell's medium in the 
following manner: 
" Touch the colony with a sterilized platinum needle, make two 
streaks along the slanted surface of the medium in the tube and then 
stab the needle down through the center of the block of medium to the- 
bottom of the tube. Incubate the inoculated tubes of Russell's medium 
for twenty to twenty-four hours and then examine. 
Kendall and Day's Method. " The feces are collected preferably 
in a small rectal tube. A small portion of the feces (about a loopful) 
is thoroughly emulsified in 10 c.c. of sugar-free broth and preferably 
incubated one hour at 37° C. prior to the inoculation of the plates. This 
preliminary incubation does two things: the clumps of bacteria are 
thrown down, leaving a more uniform suspension of bacteria in the 
supernatant solution for inoculation and the bacteria undergo a slight 
development in a medium particularly suited for their growth. It is 
believed that better growth is secured if the feces is put through broth 
than if it is added directly to the solid media. It is essential that sugar- 
free broth be used. 
" The fecal suspension is then rubbed over the surface of the Endo 
agar plates in the usual manner by means of the sterile glass rods. 
These plates are then incubated at 37° C. for eighteen hours. At the 
end of this time small dewdrops are seen which may be removed entire 
to broth tubes. (1 c.c. broth which have been held at 37° C.) These 
are then incubated for two hours at the end of which time sufficient 
growth will have taken place, with which to make the agglutination 
tests." 
Holt-Harris-Teague (1916) Method for Isolating B. Typhi from 
Stools. These authors have devised a medium which they claim will 
give better results than Endo's medium. They claim that a greater 
percentage of colorless colonies on this medium turn out to be B. typhi 
than on Endo's medium. It is prepared as follows: 
Nutrient agar is made in the usual way, containing 1.5 per cent agar, 
1 per cent Witte's peptone, 0.5 per cent sodium chloride, and 0.5 per 
cent Liebig's meat extract, to the liter of distilled water. It is cleared 
with egg-white, placed in flasks, and sterilized in the Arnold sterilizer on 
three successive days. The reaction is brought to +0.8. The agar is 
melted and saccharose (.5 per cent) and lactose (.5 per cent) are added. 
The medium is then heated for ten minutes in the Arnold. To every 
50 c.c. of the medium are added 1 c.c. of 2 per cent yellowish eosin and 
1 c.c. of 0.5 per cent methylene blue. We always add the eosin first 256 
INTESTINAL BACTERIA 
and then the methylene blue. The mixture is shaken and plates are 
poured. The surface of the medium is (hied in the usual way before the 
plates are inoculated. We have also obtained excellent results by sub- 
stituting for Liebig's extract, meat infusion rendered free from sugar 
by incubation with B. coli. 
Stock solutions of 2 per cent eosin and 0.5 per cent methylene blue 
in distilled water are kept in the dark. We have not sterilized these 
solutions, as we found that they could be kept in the ice-box for weeks 
without causing contaminations of the medium. Ordinarily we do not 
heat the agar after the dyes are added, but we have demonstrated that 
the stained agar can be heated a half hour in the Arnold sterilizer without 
injury. 
Carnot and Halle's Sand Tube Method. This method has had much 
praise. The method has been described by Gautier (1915) and Levy 
(1916) somewhat as follows: A pipette tube 33 cm. long by about 5 to 6 
mm. in diameter is softened in the middle and the ends brought up to 
form a U. One of the arms thus formed is filled to a height of 10 cm. 
with very fine sand (passed through a No. 40 sieve). The other end is 
filled with hot bouillon and tinted with neutral red, which works up 
through the sand until the other tube is filled to about the same level. 
The arm in which there is no sand is inoculated with the sample and 
incubated for eighteen hours. The most motile bacteris will penetrate 
the sand and appear in the other arm. Gautier found that B. coli occa- 
sionally did this. Agglutination reactions should be made to confirm 
the results of the sand tube. Other adaptations of the sand-tube 
method have been reported by Bor zone and Carbone (1918) and Piazza 
(1916). 
Isolation of B. typhi from Feces. (Teague and Clurman's Method.) 
This method is especially adapted to specimens which must be kept 
for a short time before examination. The specimens are rubbed into 
a solution of G.6 per cent sodium chloride containing 30 per cent of 
glycerol. This environment seems to be very destructive to the colon 
bacilli and other bacilli in stools but not harmful to the B. typhi. After 
this, the stool is streaked on plates made from Teague and Clurman's 
special eosin-brilliant green agar the preparation of which has been 
mentioned in the chapter on the preparation of media. Beckler (1918) 
reports that this method has given very satisfactory results at the labora- 
tories of the Massachusetts State Board of Health. One specimen is 
reported which gave negative results on immediate examination but 
positive results after holding in the presence of glycerol as advised by 
Teague and Clurman. This work is also confirmed by Benians (1918). MICROSPIRA CHOLERA IN FECES 
257 
He claims that positive results are more likely after preserving the 
stool in glycerol especially in hot climates or countries. 
Isolation of Typhoid Bacilli from Urine. (Morishama and Teague's 
Method.) Streak two or three loops of the urine over an Endo or eosin- 
brilliant green medium plate; add to the urine about one-half its volume 
of nutrient broth and incubate the mixture over night. If the plate 
which has been inoculated directly with the urine is negative prepare 
dilution of the incubated urine next morning and streak on the above 
plates again. This latter procedure will furnish a higher percentage of 
positive results. 
Examination of Feces for Microspira Cholera 
Gedding's Method. It would be neither possible nor desirable to 
fix a technique limited by strict rules for the various operations of bac- 
teriological examinations, but the following general indications may be 
recommended as permitting in the great majority of cases a positive 
diagnosis within twenty-four to thirty-five hours: 
1. When mucous flakes are available for examination of microscopical 
investigation of the same, in stained preparations and in the hanging 
drop. 
2. The isolation of the vibrios, employing for the purpose, agar 
media, at a temperature of 37° C. 
(a) Plant plates of ordinary suitably alkalinized agar and of Dieu- 
donne's medium, using, for the latter, a risiform particle, or an equiva- 
lent quantity of feces. 
(6) Plant in 50 c.c. of peptone solution 1 c.c. of fecal matter. After 
a stay of six hours in the incubator (or twelve to eighteen hours, if need 
be), at 37° C. take several loopfuls from the surface and plant with them 
several plates of Dieudonne medium and ordinary agar. 
(c) Investigate the agglutination reaction, using drops for the pur- 
pose, from the isolated colonies, the properties belonging to cholera 
vibrios, and secure pure cultures. 
3. Demonstrate the character of the vibrios obtained in pure culture, 
by the reaction of agglutination or that of Pfeiffer. 
The conditions are much more favorable to the discovery of vibrios 
if pathological materials (feces or intestinal contents) are collected as 
early in the attack as possible, or secured from the cadaver as early as 
possible after death. Examinations made of the small quantity of 
material collected by a sound introduced into the rectum, in the living 
body, or from the cadaver are unreliable. It is sometimes possible to 258 
INTESTINAL BACTERIA 
recognize that a person even in good health has undergone an attack of 
cholera by determining whether his blood scrum gives with a genuine 
cholera vibrio the immunity reactions, viz., agglutination or the reaction 
of Pfeiffer. 
Teague and Travis' Method. Two pounds of beef are soaked in 
2 liters of water over night. In the morning filter through cloth, heat 
in the Arnold steam sterilizer and filter through paper. Adjust the 
reaction to neutrality to litmus by means of sodium hydroxide. Inoc- 
ulate with B. coli and incubate for a few days. Then prepare nutrient 
agar from it by adding 1 per cent of Witte's peptone, 0.5 per cent of 
sodium chloride and clear with egg white. Adjust the reaction to 0.5 
per cent alkaline, filter and add 0.25 per cent of nutrosc. To 50 per cent 
of this nutrose agar add 1 per cent of sucrose and 1 c.c. of a 3 per cent 
solution of bluish eosin and 2 c.c. of a 1 per cent solution of Bismarck 
Brown. Microspira choleras colonies will show brown centers after 
twenty-four hours. B. coli will show pale pink or yellowish colonies. 
Examination of Feces for B. Tuberculosis 
Petrof's Method. In order to remove the food particles, dilute with 
water and filter through gauze. Saturate the filtrate with sodium 
chloride and, at the end of a half hour, all of the bacteria will be found 
in this film. Collect the film and add sodium hydroxide, shake well and 
incubate at 22° C. for three hours. Then neutralize to sterile litmus 
paper, centrifuge and inoculate. 
Reh Method. Stir the lump of stool in an Esbach glass with suffi- 
cient sterile water to make a soft paste but solid enough not to flow when 
the glass is tilted. Add a little ether and shake after closing with a 
rubber stopper. Pour the ether into a centrifuge glass and centrifugalize. 
The ether is then decanted and the sediment examined after staining 
with Ziehl's method. 
Engleson's Method. Scrape the rectal mucosa with an ordinary 
sound. Make a smear and stain. 
Isolation of Yeasts from Feces 
Anderson's Method. Prepare plates of Sabouraud's agar. By 
means of a platinum wire which has been dipped into the feces emulsion, 
touch one of these plates in lines across the plate at distances of about 
4 mm. Make as many rows as possible. The yeast colonies which 
develop in this way should be subjected to further study. BIBLIOGRAPHY 
259 
BIBLIOGRAPHY 
Anderson, H. W. 1917. Yeast-like Fungi of the Human Intestinal Tract. 
Jour. Inf. Diseases, 21, 341-386. 
Bacon, R. F. 1916. The Germicidal Efficiency of Some Copper Cements used 
in Dental Work. Dental Cosmos, 58, 40-49. 
Barker, L. F. 1914. The Diet of Typhoid Fever. Jour. Amer. Med. Assn., 
63, 929-931. 
Basten, J. 1914. Methods of Determining the Bacterial Flora in Feces of 
Suckling Infants and our Knowledge of the Bacterial Types. Zeit. Hygiene, 
77, 282. 
Beckler, E. A. 1918. Use of Glycerine in the Examination of Feces for 
Typhoid Bacilli. Jour. Amer. Med. Assn., 70, 768. 
Benians, T. H. C. 1918. Preservation of Typhoid Bacilli in Stools. Lancet 
1, 255, 1918. Jour. A. M. A., 70, 961; Amer. Jour. Pub. Health, 8 (1918), 
396. 
Bergeim, O. 1917. The Determination of Fecal Indole. Jour. Biol Chem., 
32, 17-22. 
Blatherwick and Hawk. 1914. Fasting Studies XIII. The Output of 
Fecal Bacteria as Influenced by Fasting and by Low and High Protein Diet. 
Jour. Amer. Chem. Soc., 36, 147. 
Blatherwick, Sherwin and Hawk. 1912. intestinal Putrefaction and Bac- 
terial Development Accompanying Water Drinking and Fasting. Jour. 
Biol. Chem., 11, VIII Proceeding. 
Boas and Oppler. 1895. Zur Kenntnis des Mageninhalts beim Carcinoma 
ventriculi. Deutsch. Med. Wochenschr., 21, 73-75. 
Borzone and Carbonne, J. M. 1918. Sand Test of Typhoid Carriers. 
Jour. Amer. Med. Assn., 70, 895. 
Boudielle, T. 1913. Influence of Bile on the Fermenting Action of the 
Colon Bacillus. Comp. Rend. Soc. Biol., 72, 783-785. 
Brugnatelli. 1915. Etiology of Dental Caries. Zent. Biochem. u. Biophys., 
18, 96-97. 
Cadeac and Bouray. 1893. Rol. microbicide des sues digestifs et contagion 
par les matieres fecales. Cent. Bakt. Grig., 16, 672. 
Campo, G. 1902. Micro-organisms of the Mouth of the New Born. Dental 
Cosmos, 1.00. Cent. Bakt., 31, 496. 
Carnot, P. and Halle, B. W. 1915. A New Method of Rapid Bacteriologic 
Diagnosis of Typhoid Fever. Jour. Amer. Med. Assn., 66, 677. 
Collins, K. R. and Grund, M. 1907. Study of the Intestinal Flora. Col- 
lected Studies from the Research Laboratories, Department of Public 
Health, City of New York. 1907, p. 161. 
Debono. 1912. On Some Anaerobic Bacteria of the Normal Human Intestine. 
Cent. Bakt. Orig., 62, 229-234. 
Distaso, A. 1913-4. Ueber das Schicksal der per Os eingefiihrten Bacter.cn. 
Cent. Bakt. Abt. I. Ref., 60, 255. 260 
INTESTINAL BACTERIA 
Distaso, A. 1912. The Intestinal Flora. Lancet, 182, 496-498. 
Distaso, A. and Schiller, J. 1914. Sur l'acclimation des microbes Strangers 
a la flore intestinale. Comp. Rend. Soc. Biol., 76, 243-244. 
Dunon, E. 1903. Action of Tobacco Smoke upon Certain Microbes of the 
Mouth. Cent. Bakt., 32, 612. 
Ecker, E. E. 1918. The Inhibitive Action of Ox Bile on B. Typhosus. Jour. 
Inf. Diseases, 22, 95-100. 
Ford, W. W. Classification and Distribution of the Intestinal Bacteria in 
Man. Rockfeller Inst. Med. Res. 
Friedenwald, J. and Leitz, L. F. 1909. Experiments Relating to the 
Bacterial Content of the Feces, with some Researches on the Value of 
Certain Intestinal Antiseptics. Amer. Jour. Med. Sciences, 138, 653-661. 
Fuller, G. W. 1912. Sewage Disposal. McGraw-Hill Book Co., New 
York. Chapter II. Aerobic and anaerobic Decomposition of Sewage. 
Galt and Iles. 1914. A Study of the Boas-Oppler Bacillus. Jour. Path, 
and Bact., 19, 239-244. 
Garhart. 1916. Are there any Laws which Govern the Behavior of Dental 
Amalgama. Dental Register, 70, Chern. Abstracts, 10, 1379. 
Gautier, P. 1915. Nouveau proc6d6 de recherche du bacille typhique 
dans les selles. Rev. Medicale de la Suisse Romande, 34, 361-442. 
Geddings, H. D. 1912. The Bacteriological Diagnosis of Cholera. Reprint 
No. 75, Public Health Reports. 
Gies, W. G. 1915. Chemical Studies of the Relations of Oral Micro-organisms 
to Dental Caries I. The Importance of a Re-examination and an Exten- 
sion of the Available Knowledge Pertaining to the Kinds and Chemical 
Influences of Oral Bacteria in their Relation to the Teeth. Jour. Allied 
Dental Societies, 10, 137-140. 
Gordon. 1916. The Inhibitory Action of Saliva on the Growth of the Menin- 
gococcus. Brit. Med. Jour., 1916-1., 849-851. 
Gregersen. 1916. Untersuchungen fiber die antiseptische Wirkung des 
Magensaftes. Cent. Bakt. Orig., 77, 353-361. 
Grindley, H. S. and MacNeal, W. J. 1912. Bacteria and Chemistry of 
Feces of Healthy Men. Vols. 3, 4, and 5. Univ, of Illinois. 
Hatterm and Hawk. , 1911. Studies on Water Drinking. Intestinal Putre- 
faction During Copious and Moderate Water Drinking with Meals. Arch. 
Inter. Med., 7, 610. 
Haussen. 1912. Untersuchungen am Hund uber den Einfluss infizierter 
Milch auf das Bakterienwachstum in Verdauungstraktus speziell im Magen. 
Cent. Bakt., 62, 89-126. 
Hawk, P. B. 1916. Quantitative Determination of Fecal Bacteria. Prac- 
tical Physiological Chemistry, P. Blakiston's Son & Co. 
Heeter, C. A. and Kendall, A. I. 1908. The Use of the Fermentation Tube 
in Intestinal Bacteriology. Jour. Biol. Chern., 5, 283. 
Heniemann and Ecker. 1916. A Study of the Boas-Oppler Bacillus. Jour. 
Bact., 1, 443. BIBLIOGRAPHY 
261 
Herter, C. A. 1907. The Common Bacterial Infections of the Intestinal 
Tract. The Macmillan Co., New York. 
Holt-Harris, J. E. and Teague, 0. 1916. A New Culture Medium for the 
Isolation of Bacillus Typhosus from Stools. Jour. Inf. Dis., 18, 596-600. 
Hopkins and Cole. 1903. Constitution of Tryptophane and the Action of 
Bacteria on it. Jour. Physiology, 29, 451. 
Houston, A. C. 1902-3. Bacteriological Examination of Normal Stools 
of Healthy Persons. Local Gov. Bd. Med. Officer's Report 1902-3., 511; 
1903-4, 472. 
Houston, A. C. 1914. Study of Streptococci in Excremental Matter. Met- 
topolitan Water Board, 10th Rept. on Research Work, 1914, 22-23. 
Hull, T. G. and Rettger, L. F. 1915. The Influence of Milk and Carbo- 
hydrate Feeding on the Character of Intestinal Flora. Cent. Bakt., 75, 
365-388. Jour. Bact., 2, 47-71. 
Kendall, A. I. 1911-12. The Rapid Isolation of Typhoid, Paratyphoid and 
Dysentery Bacilli. Jour. Med. Res., 25, 95-99. 
Kendall, A. I. 1911. Activity of Bacteria in the Intestinal Tract. Jour. 
Med. Res., 25, 117-187. 
Kianowsky. 1891. Zur Frage uber die antimikrobiellen Eigengeshaft des 
Magensaftes. Cent. Bakt., 10, 236-237. 
Klein, A. 1900. Eine neue Mikroskopische Zahlungsmethode der Bakterien. 
Cent. Bakt. Grig., 27, 834. 
Klein, A. 1906. Ueber den Bakteriengehalt menschlicher und tierischer 
Feces. Arch. Hyg., 59, 283. 
Kligler, I. J. 1915. Chemical Studies of the Relations of Oral Micro- 
organisms to Dental Caries. IV. A Biochemical Study and Differentiation 
of Oral Bacteria with Special Reference to Dental Caries. Jour. Allied 
Dental Societies, 10, 445-448; 282-330. 
Kurloff and Wagner. 1889. Ueber die Einwirkung des menschlichen 
Magensaftes auf Krankheits erregende Keime. Cent. Bakt., 7, 448-450. 
Lagane, L. 1914. Action of Bile in Vitro on the Development of Intestinal 
Bacteria. Comp. Rend. Soc. Biol., 73, 242-243. 
Levy, M. D. 1916. The Use of the Sand Tube in the Isolation of the Typhoid 
Bacillus. Jour. Med. Assn., 66, 1022-1023. 
Lumsden, L. L. and Stimson, A. M. 1912. Examination of Excreta for 
Typhoid Bacilli. Reprint No. 80, Public Health Reports. 
MacConkey, A. 1907. Lactose Fermenting Bacteria in the Feces. Jour. 
Hyg., 7, 331; 5, 333. 
MacNeil, Latzer and Kerr. Fecal Bacteria of Healthy Men. Jour. Inf. Dis., 
6, 123-169; 571-609. 
Marshall. 1916. An Acidimetric Study of the Saliva and its Relation to 
Dental Caries. Dental Items of Interest, 38, 116-127. 
Mattill, H. A. and Hawk, P. B. 1911. A Method for the Quantitative 
Estimation of Fecal Bacteria. Jour. Exper. Med., 14, 433-443. 
Mattill and Hawk. 1911. Studies on Water Drinking. IX. Distribution 262 
INTESTINAL BACTERIA 
of bacterial and other forms of nitrogen and utilization of injected protein 
under the influence of copious and moderate water drinking with meals. 
Jour. Amer. Chern. Soc., 33, 1999-2032. 
Neucki, M., Sieber, N., and Schoumowa, E. 1898. Die Entgiftung der 
Toxine durch die Verdauungssafte. Cent. Bakt., 23, 840-847. 
Niedergesafs. 1915. Anatomical, Bacteriological and Chemical Investi- 
gations of the Production of Dental Caries. Arch. Hyg., 84, 220-260. 
Osborne, T. B. and Mendel, L. B. 1914. The Contribution of Bacteria 
to the Feces after Feeding Diets Free from Indigestible Components. 
Jour. Biol. Chern., 18, 177. 
Ozaki. 1912. Zur Kenntniss der Anaeroben Bakterien der Mundhole. Cent. 
Bakt., 62, 76-98. 
Petroff. 1915. A New and Rapid Method for the Isolation and Cultivation 
of Tubercle Bacilli Directly from the Sputum and Feces with the Aid of 
Sodium Hydrate and Gentian Violet Egg Meat Juice Media. Science, 
41, 661. 
Piazza. 1916. Sand Tube Method for Isolating Typhoid Bacilli from Stools. 
Jour. Amer. Med. Assn., 70, 1281-1282. 
Rahe. 1914. An Investigation into the Fermentation Activities of Aciduric 
Bacteria. Jour. Inf. Diseases, 15, 141-150. 
Rettger, L. F. 1915. The Influence of Milk Feeding on Mortality and 
Growth, and on the Character of the Intestinal Flora. Jour. Exper. Med., 
21, 365-388. 
Rettger, L. F. and Harton, G. D. A Comparative Study of the Intestinal 
Flora of White Rats Kept on Experimental and Ordinary Mixed Diet. 
Cent. Bakt. Abt. I., 73, 362-372. 
Rodella. 1908. Magen Carcinoma. Cent. Bakt. Abt. I., 47, 445-447. 
Roger, R. 1912. Influence on Microbic Fermentations of Carbohydrates. 
Arch. Med. Exp., 24, 461-488. 
Roger, R. 1913. Influence of Bile on the Putrefaction of Nitrogenous Mate- 
rial. Comp. Rend. Soc. Biol., 73, 274-276. 
Roger, R. 1913. Influence of Bile on Bacterial Fermentation. II. Fer- 
mentation of glycogen. Comp. Rend. Soc. Biol., 72, 544-545. 
Roger, R. 1913. Influence of Bile on Fermentation. HI. Fermentation 
of Glucose. Comp. Rend. Soc. Biol., 72, 603-604. 
Roger, R. 1913. Influence of Bile Extracts and Bile Salts on Bacterial 
Fermentations. Comp. Rend. Soc. Biol., 72, 656-657. 
Roger, R. 1915. Antiputrefactive Role of the Bile. Ann. Past. Inst., 29, 
545-550. 
Sandberg. 1904. Ein Beitrag zur Bakteriologie der Milchsauer Garung 
im Magen mit besonderer Beriicksichtigung der langen Bacillen. Zeit. f. 
klin. Med., 51, 80-94. Complete bibliography. 
Schmidt and Strassburger. 1905. Die Faeces des Menchen, Berlin. 
Schmitz, C. 1893. Zur Kenntniss der Darmfaulniss. Zeit. Physiol. Che., 17, 
401-403. BIBLIOGRAPHY 
263 
Schulz-Schultzenstein. 1908. Zur Kenntniss der Einwirkung des mensch- 
lichen Magensekrete auf Choleravibrionen. Cent. Bakt. Orig., 30, 785. 
Smirnow. 1915. Germicidal Action of Dental Cements. Dental Cosmos. 
57, 1209-1228. 
Steele, J. D. 1907. The Method of Determining the Total Amount of Fecal 
Bacteria by Weight and its Clinical Significance. Jour. Amer. Med. Assn., 
August 24, 1907. 
Stern. 1908. Ueber das Verhalten der Cholera-vibrionen dem menschlichen 
Mageninhalt gegeniiber. Cent. Bakt., 47, 561, Abt. I. 
Strassburger. 1902. Untersuchungen uber die Bakterienmengen in mensch- 
lichen Feces. Zeit. klin. Med., 46, 413. 
Strauss. 1909. Ueber die Abhangigkeit der Milch saner Gahrung voin HC1 
Gehalt des Magensaftes. Zeit. klin. Med., 28, 567-578. 
Tanner, F. W. 1917. Studies on the Bacterial Metabolism of Sulphur. I. 
Formation of hydrogen sulphid from certain sulphur compounds under 
aerobic conditions. Jour. Bact., 3, 565-593. 
Tanner, F. W. 1918. Studies on the Bacterial Metabolism of Sulphur. II. 
Formation of hydrogen sulphid from certain sulphur compounds by yeast- 
like fungi. Jour. Amer. Chern. Soc., 40, 663-669. 
Teague, O. and Clubman, A. W. 1916. A Method of Preserving Typhoid 
Stools for Delayed Examination and a Comparative Study of the Efficacy 
of Eosin Brilliant Green Agar and Endo Agar for the Isolation of Typhoid 
Bacilli from Stools. Jour. Inf. Diseases, 18, 653-671. 
Teague, O. and Clurman, A. W. 1916. An Improved Brilliant Green Cul- 
ture Medium for the Isolation of Typhoid Bacilli from Stools. Jour. Inf. 
Diseases, 18, 674-652. 
Teague, O. and Travis, W. C. 1916. A Differential Culture Medium for 
the Cholera Vibrio. Jour. Inf. Dis., 18, 601-605. 
Toida, R. 1914. Sterility of the Bile under Normal Conditions and its Bac- 
tericidal Action on Pathogenic Bacteria. Chern. Abstracts, 8, 2558-2559. 
Turesson, G. 1916. The Presence and Significance of Molds in the Intestinal 
Canal of Man and Animals. Exp. Sta. Rec., 35, 559-560. 
Washburn and Goadby. 1896. Some Points in Connection with the Bac- 
teria in the Mouth. Trans. Odontological Soc. Great Brit., 1896. 
Williams. 1899. A Contribution to the Bacteriology of the Human Mouth. 
Dental Cosmos. 1899. 
Wohman, F. 1912. The Amylolytic Bacteria of the Intestines. Am. Past. 
Inst., 26, 610-624. CHAPTER IX 
BACTERIAL EXAMINATION OF AIR 
The bacteriological examination of air has not been of much sanitary 
significance. Many investigations have been carried out and different 
apparatuses devised to remove the bacteria from a definite quantity of 
air. A review of these methods has been given by Besson and those 
wishing a rather complete historical survey should consult that treatise. 
Ruehle (1915) has devised the most satisfactory technique and probably 
his method represents the best which has been devised up to the present. 
Much of the matter presented in this chapter has been taken from his 
publication. This method involves the use of the aeroscope, which is 
regarded as an apparatus for gathering bacteria from the air. The 
other accessories to the procedure are not included in this procedure. 
Rettger (1910) made one of the first important advances in the 
bacterial examination of air. He used 5 c.c. of physiological salt solu- 
tion as the filtering medium in a special aeroscope described as follows: 
The entire apparatus consists of a glass tube with a small round bulb at the 
end. The bulb has eight or ten small perforations which serve the purpose of 
allowing the air to pass through at a rapid rate and divide the gas to such an 
extent that every particle of it is brought into close contact with the filtering 
fluid. This glass tube or aeroscope is fitted into a small, thick-walled test tube 
by means of a rubber stopper, which also bears, besides the aeroscope, a short 
glass tube bent at right angles. The upper end of the aeroscope is at an angle of 
about 45°, in order to prevent bacteria and particles of dust from falling into 
the open end of the tube, and still permit of the tube being drawn through the 
stopper without difficulty. 
The standard aeroscope has been described by Ruehle as follows: 
A 10 mm. layer of sand which has been passed through a 100-mesh sieve 
and has been retained by a 200-mesh sieve is supported within a cylindrical glass 
tube 70 mm. in length and 15 mm. in diameter upon a layer of bolting cloth 
folded over the end of a rubber stopper. Through a perforation in the stopper, 
there passes a tube 6 mm. in diameter and 40 mm. in length. This tube is 
attached to the aspirator bottle. The upper end of the cylindrical tube is closed 
by a perforated rubber stopper through which is passed a glass tube 40 mm. in 
264 AEROSCOPES 
265 
length and 6 mm. in diameter bent at an angle of 45° in order to prevent precip- 
itation of bacteria or dust particles into the aeroscope. In using this aeroscope 
a measured volume of air is filtered through the tube, the sand shaken out into 
10 c.c. of water and aliquot portions of this suspension plated on nutrient agar. 
The " modified " form of the aeroscope differs very little. The 
lower rubber stopper and the bolting cloth is eliminated by fusing a 
small tube into the large one. A layer of cotton supports the sand. 
Standard 
Aeroscope 
Modified Standard 
Aeroscope 
Rettger 
Aeroscope 
Fig. 57.-Types of Aeroscopes. (After Ruehle.) 
In order to render the apparatus capable of being sterilized by hot air, 
the upper stopper is made of cork. 
The Committee on Standard Methods for the Examination of Air 
of the American Public Health Association recommended the following 
procedure: 
The number of bacteria in the air does not appear to be a factor of any great 
significance. In special cases, however, as in the study of dairy conditions, it 
may be of interest. 
The sand filter method originally used by Petri has proved essentially sound 266 
BACTERIAL EXAMINATION OF AIR 
in principle. It was recommended in our 1909 report and again, after careful 
studies of altertive processes in 1912. 
One modification has been made in the apparatus since 1912 which is so 
clearly an improvement as to warrant immediate adoption. The layer of sand 
in the filters as previously used was supported on a perforated rubber stopper 
in a straight section of glass tubing, and the aspiration was applied to a small glass 
tube passing into the rubber stopper. It was clearly essential that the stopper 
should fit tightly, which meant that the apparatus must be sterilized in steam. 
Ruehle (1915) points out that the steam sterilization causes caking of the filter 
sand and thus introduced appreciable errors in analysis. Ruehle, therefore, 
made the suggestion that the rubber stopper should be eliminated and the small 
aspirating tube fused into the large one. 
We, therefore, recommend that for the study of bacteria in air there should 
be used a glass tube of 15 mm. in diameter and 70 mm. long with a smaller tube 
6 mm. in diameter and 40 mm. long fused into one end. On the shoulder of 
the joint between the tubes rests a plug of cotton supporting a layer of sand 
10 mm. deep. The sand should be capable of passing a 100-mesh sieve but not a 
200-mesh sieve. The opposite or inlet end of the larger tube is stoppered by a 
cork stopper (which need not be exactly tight) perforated by a glass tube 6 mm. 
in diameter and 40 mm. long bent at an angle of 45° to prevent direct precipi- 
tation of dust particles into the filter tube. 
Five cu. ft. of air should be drawn through the filter by the use of an aspira- 
tor of known volume, preferably one of the double or continuous type, or by the 
use of some form of pump or meter. A convenient and ingenious sampling pump 
was made by Wallace and Tiernan of New York City for the study of Basker- 
ville and Winslow (1913) of the air of New York City schools. After filtra- 
tion, the sand should be shaken out into 10 c.c. of sterile water, and, after thor- 
ough shaking, aliquot portions of the water should be plated on ordinary nutrient 
agar. 
Browne (1917) has proposed a tube which he claims has special 
advantages over the aeroscope used in standard methods. This con- 
sists of the filter tube and dilution tube fused together at right angles. 
During filtration the sand is placed over the stoppered end. After fil- 
tration the sand is shaken into the other end and the filter plug with the 
bolting cloth is replaced by a sterile stopper. Ten c.c. of sterile water 
are added and the sand thoroughly shaken. From this decimal dilutions 
are plated on standard media. 
BIBLIOGRAPHY 
Baskerville, C. and Winslow, C. E. A. 1913. The Air of New York City 
Schools. Report of the Committee on School Enquiry Board of Esti- 
mate and Apportionment, City of New York. Ill, 611. BIBLIOGRAPHY 
267 
Browne, W. W. 1917. Improved Technique in Bacterial Air Analysis. 
Amer. Jour. Pub. Health, 7, 52-53. 
Gordon, M. H. 1902-3. Report of a Bacterial Test for Estimating Air Pol- 
lution. Annual Report of the Local Government Board, containing the 
Report of the Medical Officer for 1902-3, 421. 
Rettger, L. F. 1910. A New and Improved Method for Enumerating Air 
Bacteria. Jour. Med. Res., 17, N. S., 461-468. 
Ruehle, G. L. A. 1915. Methods of Bacterial Analysis of Air. Jour. Ag. 
Res., 4, 343-368. 
Ruehle, G. L. A. and Kulp, W. L. 1915. Germ Content of Stable Air and its 
Effect on the Germ Content of Milk. Bull. 409, N. Y. Agric. Exp. Sta., 
Geneva. 
Winslow, C. E. A. and Browne, W. W. 1914. The Microbic Content of 
Indoor and Outdoor air. Monthly Weather Review, 42, 452. CHAPTER X 
WATER HYGIENE 
That water has a close connection with the spread of disease needs 
no special emphasis. Johnson (1916) has given ample proof of this by 
collecting statistics from the large registration cities of the United 
States. (Fig. 59.) 
TYPHOID FEVER AT PITTSBURGH PA. 
Oct. '08 Pop. supplied. 
Typhoid Deaths per 100,000 Pop. 
b-4 b-* 
Noy. '071, Filters installed. 
Is population supplied. 
Ill 
Met. '09, % Pop. si pplied. 
14 yop-«HU unsur>plie( . 
Fig. 58.-Showing the Effect of a Pure Water Supply on the Death Rate from a 
Typhoid Fever in Pittsburg. (After McLaughlin, 1912.) 
The " Mills-Reincke Phenomenon " and the " Hazen Theorem." 
In 1893, soon after the establishment of the Lawrence, Mass., filtration 
plant, Mills, the city engineer, noticed a reduction in the general death 
rate along with a reduction in the typhoid fever death rate. This was 
also noticed for Hamburg, Germany, by Reincke. When Sedgwick and 
268 SANITARY INSPECTION 
269 
MacNutt (1910) were studying the effect of water treatment on disease 
they gave the name " Mills-Reincke Phenomenon " to this observation. 
They stated that infant mortality was closely related to water supply. 
Also a decrease in tuberculosis was reported for an improvement in the 
water supply. Hazen (1910) reported somewhat the same fact that 
" where one death from typhoid fever has been avoided by the use of a 
pure water, a certain number of deaths, probably two or three, from 
other causes have been avoided." This has become known as the 
" Hazen Theorem." These statements have not been entirely borne 
out by a more careful study. Fink (1916) has pointed out that an error 
enters in comparing the total mortality with the typhoid death rate. He 
states that there are two factors which may cause a variation in the ratio 
between the total and the typhoid fever death rate. " One is the rate 
at which the general mortality is being lowered and the other is the 
height of the typhoid fever death rate before filtration and the propor- 
tion due to water-borne infection." 
The spread of typhoid fever by impure water has been so prevalent 
in the past that the disease is often referred to as a water-borne disease. 
Many data are on record to show a decided decrease in the typhoid fever 
death rate when an improved water supply has been made available to a 
community. Fig. 58 prepared by McLaughlin shows this reduction for 
Pittsburg. 
Sources of Water Supply. The sources of water supply may be 
classified as follows: 
A. Surface supplies 
(a) Lakes 
(6) Rivers 
(c) Upland gathering grounds 
(d) Impounding reservoirs 
B. Ground water supplies 
(a) Shallow walls 
(6) Deep wells 
1. Artesian 
2. Bored 
3. Drilled 
(c) Springs 
Sanitary Inspection. Too little attention is given to sanitary in- 
spection in water analysis. The analyst is quite inclined to regard his 
results as sufficient data for passing judgment on a water supply. 270 
WATER HYGIENE 
Millions of People Supplied With Filtered Water. 
Typhoid Fever Death Rate per 100,000. 
GROWTH OF 
WATER FILTRATION 
AND 
DECREASE OF 
TYPHOID FEVER DEATH RATE 
IN THE 
REGISTRATION CITIES OF 
UNITED STATES 
Fig. 59.-Relation of Water Filtration to Typhoid Fever. (After Johnson.) 
AVERAGE TYPHOID FEVER DEATH RATE 
FOR 
FIVE YEARS BEFORE AND FIVE YEARS AFTER FILTRATION 
IN 
ALL CITIES OF U.S. OVER 100,000 POPULATION IN 1915 
Pittsburg 
Columbus 
Albany- 
Philadelphia 
Louisville 
Cincinnati 
Washington 
Atlanta 
Reading 
Indianapolis 
New Haven 
New Orleans 
Toledo [ 
Paterson 
Scranton 
Providence 
Explanation 
Before Filtration 
After Filtration 
Average Reduction =61 # 
Average of Above 
Total Population of Above Cities 5,450,000 
Fig. 60.-Typhoid Fever Death Rate in American Cities. (After Johnson.) CHEMICAL VS. BACTERIAL EXAMINATION 
271 
Sawyer (1915) quotes an instance of a water-borne epidemic which could 
have been prevented had it been possible to carry out a sanitary inspec- 
tion. In a certain water supply indications of pollution appeared two 
months before the epidemic broke out. Could a sanitary inspection 
have been made to investigate the conditions indicated in the laboratory 
results, seven deaths and a large number of cases of typhoid fever could 
have been prevented. In many cases it would not be necessary to make 
laboratory examinations because the environmental factors would 
indicate a bad water. In such a case, the laboratory might be used to 
check up the remedial measures which had been carried out. The 
field investigations would also yield information with regard to the 
endemic disease which occurs in a community. Where possible, both 
field studies and laboratory studies should be made but where one 
is possible, the* sanitary inspection will make it unnecessary, very often 
to carry out the laboratory examination. 
Pure Water. Chemically water is a monoxide of hydrogen and is 
made chemically pure only with great difficulty. Only the greatest 
care in technique will yield a water which is fiee from all traces of 
metals and gases. 
Bacteriologically speaking, a water is pure when it contains no bac- 
teria which may cause disease or sickness. This is a difficult term to 
define since a water which is pure for one individual may be impure for 
another. The technique which is available for isolating certain path- 
ogenic bacteria is too crude to give accurate knowledge with regard to 
purity. Negative results may mean little more than that the certain 
bacteria sought for, were not found. Generally speaking, a pure water 
is one which will not cause any disturbance in those who partake of it. 
Relation of Chemical and Bacterial Examination. In the past there 
has been much discussion over the superiority of the chemical or bac- 
terial analysis of water. This has probably been quite useless since 
both are important and necessary for an accurate opinion with regard 
to the potability of a given water. The more data which an analyst 
has, the more accurate will his opinion be. Despite all this there are 
certain outstanding facts with regard to each analysis which might be 
emphasized. 
The chemical analysis may give more data with regard to the history 
and possibly the age of the water. It gives an idea with regard to the 
organic matter and the different stages in its decomposition. If most 
of the nitrogen is in the form of nitrates, it indicates possibly remote 
pollution and a partial purification. In giving an opinion which must be 
based on the chemical analysis only, the water analyst has to use the 272 
WATER HYGIENE 
organic matter in its different stages of oxidation. The chemical 
examination of water is important when a municipal supply is treated 
with alum since in this connection it is important to maintain sufficient 
alkalinity in the water to decompose all of this coagulant and not allow 
it to pass into the filtered water. Certain objectionable trade wastes 
may be detected in a water by chemical analysis. 
The bacteriological analysis is more delicate and direct. It is easy 
to conceive that a water which, according to chemical analysis was of 
good quality, might contain many pathogenic bacteria. These, unless 
they were present in enormous numbers could not be detected chem- 
ically. In the bacterial examination the analyst is searching for the 
factors which are most important. In the chemical analysis their pres- 
ence is inferred when other substances are present. While the isolation 
of specific pathogenic bacteria from water is not attended with much 
success, the bacterial examination yields more definite knowledge of 
their presence. In determining the efficiency of filters, this analysis is 
important. 
The purpose of water examinations are many. Mineral examina- 
tions are made by the chemist to determine the effect of any water 
on boilers, to determine the amount of pollution, presence of any of 
the heavy metals, etc. The bacteriologist is more interested in the 
number and kinds of bacteria which may be present. He has to con- 
tend with the disadvantage, unlike the chemist, that many of his con- 
clusions are drawn from circumstantial evidence. 
For our purposes, a sanitary water analysis may be regarded as 
made up as follows: 
L Sanitary chemical examination 
A. Physical (organo leptic) tests 
a. Turbidity, b. Color, c. Odor 
B. True chemical determinations 
1. Residue 
2. Chlorine 
3. Nitrogen as 
a. Nitrates 
b. Nitrites 
c. Free ammonia 
d. Albuminoid ammonia 
4. Alkalinity 
5. Oxygen consumed 273 
COLLECTION OF SAMPLES 
Collection of Samples. The collection of the sample of water is an 
important part of the analysis. If done carelessly, it may determine 
the results, which are secured in the later analysis. For chemical 
analysis, the samples should be collected in glass-stoppered bottles 
which are scrupulously clean. The stoppers should fit tightly and be 
protected during transportation by tying a piece of cloth over them. 
For bacteriological analysis, four-, six- or eight-ounce glass-stoppered 
bottles should be used. They should be cleaned as outlined before 
(Chapter I) and sterilized. After cooling and before shipment, the 
top of each bottle should be protected by a piece of cloth or tin foil. 
Specimens for bacteriological analysis should always be packed in ice 
during transportation. 
Interval before Analysis. The analysis should be started as soon 
as possible after collection of the sample. The composition of water 
undoubtedly changes and for this reason certain examinations may be 
conducted in the field. Any time which may be allowed to elapse be- 
tween collection and analysis of samples is entirely arbitrary. The 
committee on " Standard Methods " suggests the following as being 
fairly reasonable maximum limits. 
Physical and Chemical Analysis. 
Ground waters 72 hours 
Fairly pure surface waters 48 hours 
Polluted surface water 12 hours 
Sewage effluents 6 hours 
Raw sewage 6 hours 
Microscopical Examination. 
Ground waters 72 hours 
Fairly pure surface waters 24 hours 
Waters containing fragile organisms Immediate 
examination. 
Bacteriological Examination. 
Samples kept at less than 10° C 24 hours 
The directions for collecting samples of water are summed up in the 
following pages which are used by the Illinois State Water Survey. 274 
WATER HYGIENE 
STATE WATER SURVEY 
Department of Chemistry, University of Illinois 
Instructions for Collecting Water for Bacterial Examination 
THE SAMPLE OF WATER SHOULD BE COL- 
LECTED IMMEDIATELY BEFORE SHIPPING BY 
EXPRESS, SO THAT THE SHORTEST POSSIBLE 
TIME SHALL INTERVENE BETWEEN THE COL- 
LECTION OF SAMPLE AND ITS EXAMINATION. 
The accompanying "Certificate" must be filled out carefully and enclosed 
in envelope shipping tag. 
. The bottle for bacterial sample is packed in cotton in the small tin can. 
The bottle has been carefully cleaned and sterilized in the laboratory and is in 
perfect condition for the collection. The rubber cloth cap must not be removed 
from the bottle until you are actually at the spot and ready to take the sample. 
Then remove the rubber cap, being exceedingly careful not to touch the inside 
of the cap or lay it in a place where it will come in contact with anything. 
From a Stream, Pond or Reservoir. Immerse the bottle to a depth of about 
1 ft., then remove the stopper and allow the water to run in until the bottle is 
about two-thirds full. With the bottle still in the water, replace the stopper 
and do not again touch it, in or out of the water. Withdraw the bottle, replace 
the rubber cap, carefully avoiding contact with the inside of the cap, the stopper, 
or the mouth of the bottle. Next, put the bottle with proper certificate in enve- 
lope attached inside the tin can, put the cotton about it, and put the lid in place 
tightly. In order to keep the sample in good condition until it is actually 
delivered at the laboratory, it will be necessary to place the small can upright 
in the large galvanized iron box and fill the galvanized box with ice. 
. From a Pump or Tap. The water is allowed to flow freely for from five to 
ten minutes, then diminish the stream. Remove the stopper from the bottle, 
always careful that the part which is inserted in the mouth of the bottle does 
not come in contact with anything. Allow the water to flow into the bottle 
until it is from two-thirds to three-fourths full. Replace the stopper and pro- 
ceed as directed above. 
Important to Note Carefully 
First. Do not wash or rinse the bottle or in any way attempt to clean it. 
Second. Do not handle or touch the stopper to anything except the mouth 
of the bottle, only as it is necessary to do so to remove the stopper as directed 
above. 
Third. Replace the rubber cap with the same side next to the bottle as 
when it was removed. 
Fourth. The sample for Bacterial Examination must be accompanied by 
a gallon sample for chemical analysis. COLLECTION OF SAMPLES 
275 
STATE WATER SURVEY 
Department of Chemistry, University of Illinois 
Instructions for Collecting Samples of Water for Chemical Analysis 
1. From a Well. Water should be pumped out freely for a few minutes 
before it is collected. The bottle is then to be placed in such a position that the 
water from the spout may fall directly into it, and rinsed out with the water 
three times, pouring out the water completely each time. It is then again to be 
placed under the spout, filled to overflowing, and a small quantity poured out, 
so that an air space of about an inch shall be left under the stopper. The stopper 
must be rinsed off with flowing water, and inserted into the bottle while still 
wet, and secured by tying over it a clean piece of cotton cloth. The ends of 
string must be sealed on top of the stopper. Under no circumstances must the 
inside of the neck of the bottle or the stem of the stopper be touched by the hand or 
wiped with a cloth. 
2. From a Tap. Allow the water to flow freely from the tap for a few 
minutes and then proceed precisely as directed above. 
3. From a Stream, Pond or Reservoir. The bottle and stopper should be 
rinsed with the water, if this can be done without stirring up the sediment on 
the bottom. The bottle with the stopper in place should then be entirely sub- 
merged in the water and the stopper taken out at a distance of 12 in. or more 
below the surface. When the bottle is full, the stopper is replaced, below the 
surface if possible, and finally secured as above. It is important that the sam- 
ple should be obtained free from the sediment at the bottom of a stream and from 
the scum on the surface. If a stream should not be deep enough to admit of 
taking a sample in this way, the water must be dipped with an absolutely clean 
vessel and poured into the bottle after it has been rinsed. 
The sample of water should be collected immediately before shipping by express, 
so that the shortest possible time shall intervene between the collection of sample 
and its examination. Shipment on Monday or Tuesday insures prompt delivery 
and completion of the analysis before the end of the week. 
The accompanying "Certificate" must be filled out carefully and enclosed in the 
envelope shipping tag. 
The sample for chemical analysis must be accompanied by a sample for bac- 
terial examination, unless otherwise directed. 
Data Concerning the Sample. This is about as important to the 
analyst when giving an opinion with regard to the sanitary quality of the 
water as the data secured in the analysis. Some individuals have tried 
to " fool " an analyst by furnishing fictitious data for a sample of water 
and have caused much trouble when a perfectly harmless water was 
condemned. The following certificates which are used by the Illinois 
State Water Survey will give an idea with regard to what data should 
accompany a sample of water for analysis. 276 
WATER HYGIENE 
STATE WATER SURVEY 
Department of Chemistry, University of Illinois 
Use this Certificate for water whose original source is Well, Spring or Cistern. 
Sample of water from, Town County 
Report to be sent to 
Collected and sealed by 
Date, day and hour of collection 
Shipped by Express Company 
Date and hour of shipment 
Collected from 
Location 
State proximity of privy cesspool stable 
Feed lot dumping grounds for slops, dish water, wash water, etc., 
Is the drainage from all these places toward or from the Well, Spring or Cis- 
tern? 
If there is any other possible source of pollution, state it 
Has the water ever been considered unsafe? Why? 
If there have been any cases of Typhoid Fever among users of this water, state 
number of persons affected Date of illness 
Number of deaths 
What other diseases have been attributed to use of this water? 
State general condition of health to those using water 
Well-State depth 
Is it dug, bored, driven or drilled? 
State character and thickness of strata through which it is sunk 
State character of strata from which water is drawn 
Is it a flowing well? 
State approximate capacity and effect of dry or wet weather 
With what is it walled or cased? How is the well covered? 
Is the cover watertight? 
If cemented or cased with iron pipe, state depth to which cement or casing 
extends , 
Spring-What improvements has it? 
Character of stratum from which water issues 
Character of overlying strata 
Approximate capacity and effect of dry or wet weather 
Cistern-What form of filter, if any, is used? 
Does the Cistern leak? How long since last cleaned? 
Can small animals get into it at top? ' 
What care is taken in collecting and storing water? 
Laboratory No deceived 19............ M. DATA FOR SAMPLES 
277 
STATE WATER SURVEY 
Department of Chemistry, University of Illinois 
Use this Certificate for water whose original source is Stream, Lake or Pond. 
Sample of water from Town County 
Report to be sent to 
Collected and sealed by 
Date, day and hour of collection 
Shipped by Express Company 
Date and hour of shipment 
Collected from 
Location 
Has the water ever been considered unsafe? Why? 
If there have ever been any cases of Typhoid Fever among users of this water, 
state number of persons affected Date of illness 
Number of deaths ' What other diseases have been 
attributed to this water? 
Stream-Name of Stream 
Do sewers discharge into Stream above point of collection? 
State distance If the Stream is liable to pollution 
by drainage from privies, stables, feed lots, slaughter houses, factories or street 
washings, describe conditions 
Have there been recently any cases of Typhoid Fever on territory drained by 
Stream? 
Condition of Stream at present as affected by drought or recent rains or by tem- 
perature 
Lake-Name of Lake 
Do sewers discharge into the Lake at a point liable to pollute the water 
State distance from outfall of sewer to point of collection 
If this Lake is liable to pollution by drainage from privies, stables, feed lots, 
slaughter houses, factories, or street washings, describe conditions 
Have there recently been any cases of Typhoid Fever in territory drained into 
Lake? 
Condition of Lake at present as affected by drought or recent rains or by tem- 
perature 
Pond-Do tile drains discharge into it? Do washings from the vicinity 
of privies, stables or feed lots drain into it? 
Have there recently been any cases of Typhoid Fever in region draining into it? 
Condition of Pond at present as affected by drought or recent rains or by tem- 
perature 
Laboratory No Received 19 M. 278 
WATER HYGIENE 
Turbidity 
By turbidity is meant the suspended matter which is usually present 
in water from surface sources. This is gathered from the water shed 
and, as long as sufficient velocity is maintained, is carried along. Few 
people care for a turbid water although they may become educated to 
the appearance and use of a water having much matter in suspension. 
There seems to be little sanitary significance connected with the tur- 
bidity. This, of course, depends upon the kind of matter which makes 
up the turb'dity. 
Determination. Several methods are available for the determina- 
tion of turbidity. A convenient method is the comparison of the sample 
of water to a " silica standard." This " silica standard " is a 0.1 per 
cent suspension of diatomaceous earth in a liter of distilled water. This 
suspension has a turbidity of 1000 or contains 1000 parts per million of 
silica. This solution may be diluted with distilled water to give stand- 
ards of 5, 10, 15, 20, 30, 40, 50, etc. These standard solutions should 
be stored in clear glass bottles, preferably of the same type in which the 
samples are taken and should be thoroughly shaken before using. The 
sample and the standard should be compared by viewing any dark object 
and noting the details of each. 
Color 
Color is of little sanitary significance. It usually comes from 
organic matter and often is removed from water with difficulty. Very 
often other substances such as iron may color a water. 
Determination. The platinum-cobalt method for determining color 
is the standard. The standard solution is made by dissolving 1.246 
gms. of potassium platinic chloride (PtCb-2KCl) containing 0.5 gm. 
platinum and 1.00 gm. crystallized cobalt chloride (CaCh-GHaO) 
containing 0.25 gm. cobalt, with 100 c.c. of concentrated hydrochloric 
acid in distilled water and diluting it to a liter. Such a solution is the 
standard having a color of 500. It should be diluted to give standards 
of from 5 to 80. These may be kept in 50 c.c. Nessler tubes which should 
be carefully protected when not in use. 
The sample of water should be shaken and 50 c.c. poured into a 
clean 50 c.c. Nessler tube. Compare this with the standards in a color- 
imeter. Standard methods prescribes the recording of results as follows: 
Color between 1 and 50 recorded to nearest unit. 
Color between 51 and 100 recorded to nearest 5. 
Color between 101 and 250 recorded to nearest 10. 
Color between 251 and 500 recorded to nearest 20. DETERMINATION OF ODOR 
279 
Odor and Taste 
Odor and taste are probably the most important of any of the 
organo-leptic tests. A chemically pure water is odorless and tasteless. 
It also has a decidedly insipid flavor as may be determined by tasting 
distilled water. Most people demand a water with some taste. 
Determination of Odor. Whipple's (1899) method is the standard. 
The quality of the odor may be expressed in the following terms: 
a. aromatic 
C. free chlorine 
d. disagreeable 
f. fishy 
g. grassy 
m. moldy 
M. musty 
P. peaty 
s. sweetish 
S. hydrogen sulphide 
v. vegetable 
The intensity of the odor may be expressed in numerical terms as 
follows: 
Numerical 
Value. 
Term. 
Approximate Definition. 
0 
None 
No odor perceptible. 
1 
Very faint 
An odor that would hot be ordinarily detected by the 
average consumer, but that could be detected in 
the laboratory by an experienced observer. 
2 
Faint 
An odor that the consumer might detect if his atten- 
tion were called to it, but that would not otherwise 
attract attention. 
3 
Distinct 
An odor that would be readily detected and that 
might cause the water to be regarded with disfavor. 
4 
Decided 
An odor that would force itself upon the attention 
and that might make the water unpalatable. 
5 
Very strong 
An odor of such intensity that the water would be 
absolutely unfit to drink. (A term to be used only 
in extreme cases.) 
Residue 
The residue is obtained by evaporating a known amount of the water 
to dryness. The amount of the residue depends upon the character of 
the water. One having a high content of carbon dioxide will dissolve 
much inorganic matter. The relation of the residue to health depends 
upon their character. Certain salts cause decided physiological dis- 
turbances in some individuals which accounts for the therapeutic value 
of certain natural waters. 280 
WATER HYGIENE 
Determination. Place 100 c.c. of the sample in a tared platinum dish 
and evaporate to dryness over a water bath. Then heat the dish in an 
oven at 103° C. or 180° C. for one hour. The weight of the residue mul- 
tiplied by 10 gives the parts per million in the sample. 
Chlorine 
Chlorine in a water is often a good index of pollution since it is usually 
high in sewage. A normal urine may contain in a day from 10-15 gms. 
of chloride calculated to sodium chloride. The interpretation of 
chlorine results demands a knowledge of the local conditions about the 
source of the sample. The amount of chlorine which is normal for the 
district from which the sample was taken must be known. 
Determination. Chlorine may be determined by titrating the water 
with silver nitrate in the presence of potassium chromate as an indicator. 
Add 1 c.c. of the indicator to 50 c.c. of the sample and titrate with the 
standard solution of silver nitrate. A standard of 1 c.c. of indicator 
in 50 c.c. of distilled water should be kept for comparison. The number 
of cubic centimeters of silver nitrate solution required multiplied by 10 
gives the titrated chloride (Cl) in parts per million. 
The solutions which are required in the determination of chlorine 
in water may be prepared as follows: 
1. Potassium Chromate. Dissolve 50 gms. of potassium chromate 
in a small amount of distilled water. Add sufficient silver nitrate 
to produce a slight red precipitate, filter and dilute to 1 liter with dis- 
tilled water. 
2. Standard Sodium Chloride Solution. Dissolve 16.48 gms. of pure 
fused sodium chloride in distilled water. One hundred c.c. of this 
stock solution should be diluted to a liter in order to secure a solution 
which will contain 0.001 gm. of chloride. 
3. Standard Silver Nitrate Solution. Dissolve 2.40 gms. of silver 
nitrate in a liter of distilled water. Standardize this with the above 
standard sodium chloride solution and adjust the volume so that each 
cubic centimeter is equivalent to .0005 gm. of chloride. 
Nitrogen 
Nitrogen furnishes the chemist a convenient index of bacterial 
activities in water. There were times in sanitary work when waters 
were condemned for, what was considered then, excessive amounts of 
the various nitrogen compounds. At one time nitrites were regarded 
as an infallible index of pollution. These different determinations of NESSLERIZATION 
281 
nitrogen in water trace the changes through which the organic matter 
is made to pass by the bacteria. The free and albuminoid ammonias 
indicate the organic matter while the nitrites and nitrates indicate the 
amount of oxidation which has taken place. 
Nesslerization. This may be done directly or after distillation de- 
pending upon the amount which is present in the sample. The following 
solutions are required: 
1. Standard Ammonium Chloride Solution. Dissolve 3.82 gms. of 
ammonium chloride in 1 liter of ammonium free water. Dilute 10 c.c. 
of this to 1 liter with ammonia free water. One c.c. of this latter solu- 
tion equals 0.00001 gm. N. 
2. Nessler Solution. Dissolve 50 gms. of potassium iodide in a little 
cold water. To this is added a saturated solution of mercuric chloride 
until a slight precipitate persists permanently. Add 400 c.c. of a 50 
per cent solution of potassium hydroxide. This should be a clear solu- 
tion made by allowing it to settle. Dilute to a liter and, after allowing 
it to settle, decant. This should give the required color with ammonia 
within five minutes and should not give a precipitate with small amounts 
in two hours. 
Folin and Dennis' Modified Nessler Reagent. Double iodide solu- 
tion is made by dissolving 75 gms. of potassium iodide in 500 c.c. of 
warm water, adding 100 gms. of mercuric iodide and stirring. The 
solution will be complete in a few minutes but possibly not quite clear 
on account of impurities. Dilute with 400 to 500 c.c. of water and make 
the filtrate up to a liter. 
To 300 c.c. of the double iodide solution add 200 c.c. of 10 per cent 
sodium hydroxide, 500 c.c. of water, and mix. Fifteen c.c. of this mod- 
ified Nessler reagent rapidly added from a measuring cylinder will 
yield crystal clear mixtures with as large amounts of ammonia nitrogen 
as are usually involved (0.7 to 1.6 mg. ammonia nitrogen). 
This has the advantage over the standard Nessler reagent that it 
may be used very soon after preparation and will not give a precipitate. 
For " standards " a series of Nessler tubes is prepared by adding 
increasing amounts of the standard ammonium chloride solution and 
diluting with ammonia free water to the 50 c.c. mark. Standard 
methods recommends the following standards, 0.0, 0.1, 0.3, 0.5, 0.7, 1.0, 
1.4, 1.7, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 6.0 c.c. These standards 
should be Nesslerized by adding 1 c.c. of Nessler solution to the tubes 
and allowing them to stand for 10-20 minutes. The sample whether 
Nesslerized directly or after distillation is treated the same way and 
compared with the standards. If the amount of ammonia is known to 282 
WATER HYGIENE 
be high the distillate may be caught in a flask (200 c.c.) and an aliquot 
portion Nesslerized. Usually four Nessler tubes are sufficient. The 
last one should contain no nitrogen. When Nessler tubes are used the 
following example will serve to illustrate the computation. 
First tube =3.0 c.c. of standard NH4CI solution. 
Second tube = 2.6 c.c. of standard NH4CI solution. 
Third tube =1.0 c.c. of standard NH4CI solution. 
Fourth tube = 0.0 c.c. of standard NH4CI solution. 
Total 6.6 c.c. of standard NH4CI solution. 
6.6X0.00001 = 0.000066 gm. N. 
If 500 c.c. of the water were taken this amount must be multiplied by 
2 to get the amount of N in a liter of the water. 
0.000066X2 = 0.000132 gm. N. in a liter = .132 part per million. 
If an aliquot portion from a flask is Nesslerized the calculation is 
about the same. 
Free Ammonia. Five hundred c.c. of the sample should be intro- 
duced into a 800 c.c. Kjeldahl flask fitted to a condenser so that the dis- 
tillate may be received in four Nessler tubes. Heat so that not more 
than 10 c.c. nor less than 6 c.c. comes over in a minute. If the nitrogen 
is known to be high, catch the distillate in a flask. Nesslerize, as 
described above. 
Albuminoid Nitrogen. This is determined on the same sample after 
the free ammonia has been distilled off. Alkaline potassium perman- 
ganate, prepared as follows is required for this determination. Pour 
1200 c.c. of distilled water into a large evaporating dish and boil for ten 
minutes. Remove the flame and add 16 gms. of potassium perman- 
ganate. Dissolve and add 800 c.c. of a 50 per cent solution of potassium 
hydroxide. Add a little distilled water and boil down to 2000 c.c. 
After the distillate for free ammonia nitrogen has been received, 
remove the flame and add 50 c.c. or more of alkaline potassium per- 
manganate to the flask and collect 200 c.c. of distillate in four Nessler 
tubes. Nesslerize as described above. 
The free ammonia determined above probably represents nitrogen 
which is present as some inorganic salts. These may be split by boiling. 
It may represent a stage in denitrification resulting from deaminization 
of amino acids. As such, free ammonia has little significance but it 
does represent changing organic matter. The albuminoid ammonia is 
of more sanitary significance. It indicates recent organic pollution. NITRITES AND NITRATES 
283 
Nitrites. This determination is carried out on a 50 c.c. sample of 
the water in a Nessler tube. A series of standards is also prepared by 
putting the following amounts of the standard solution of sodium nitrite 
in 50 c.c. Nessler tubes and diluting to the mark with nitrogen-free 
water: 0.0, 0.1, 0.2, 0.4, 0.7, 1.0, 1.4, 1.7, 2.0, and 2.5 c.c. add 1 c.c. 
of sulphanilic acid and 1 c.c. of the naphthalamin acetate or hydro- 
chloride solutions to each of the tubes including the sample. Mix and 
after standing for fifteen minutes compare with the standards. When a 
50 c.c. sample is used the standard matched times 0.01 equals the parts 
per million of nitrites. 
The following reagents are required: 
1. Sulphanilic Acid Solution. Dissolve 8 gms. of the purest sulphan- 
ilic acid in 1 liter of 5N acetic acid (sp. gr. 1.04) or in 1000 c.c. of water 
containing 50 c.c. of concentrated hydrochloric acid. This will be 
practically a saturated solution. 
2. a-naphthylamin Acetate or Hydrochloride. Dissolve 5 gms. of 
solid and naphthylamin in 1000 c.c. of 5N acetic acid or in 1000 c.c. of 
water containing 8 c.c. of concentrated hydrochloric acid. Filter 
through alundum or washed absorbent cotton. 
3. Stock Sodium Nitrite Solution. Dissolve 1.1 gms. silver nitrite 
in nitrite free water; precipitate the silver with sodium chloride solution 
and dilute the whole to a liter. 
4. Standard Sodium Nitrite Solution. Dilute 100 c.c. of solution 
3 to 1 liter; then dilute 50 c.c. of this solution to a liter with nitrite free 
water, add 1 c.c. chloroform, and preserve in a sterilized bottle. One 
c.c = 0.0005 mg. nitrogen. 
Nitrates. (Reduction method.) Place 100 c.c. of the sample 
in a casserole and, after adding 2 c.c. of the sodium hydroxide solution, 
reduce the volume by boiling to 20 c.c. Pour this into a 100 c.c. test 
tube (16X3 cm.) along with two or three rinsings with nitrogen free 
water. Put a strip of aluminum foil and close the tube by means of a 
rubber stopper carrying a 5 m.m. glass tubing which will extend below 
the surface of distilled water in another tube. The hydrogen is thus 
allowed to escape. The nitrogen which is lost in this procedure may be 
neglected. Allow this to stand over night (at least four hours). At the 
end of this time pour the contents of the tube into a distilling flask and 
dilute to 200-300 c.c. with nitrogen free water. Distill, collecting 
the distillate in Nessler tubes or in 200 c.c. flasks if the amount of nitrates 
is known to be high. If flasks are used to receive the distillate, an ali- 
quot portion must be Nesslerized. Nesslerization may be done directly 
when the supernatant liquid in the reduction tube is clear. 284 
WATER HYGIENE 
The following reagents are required: 
1. Sodium or Potassium Hydroxide Solution. Dissolve 250 gms. of 
hydroxide in 1.25 liters of distilled water. Add several strips of alumi- 
num foil and allow the evolution of hydrogen to continue over night. 
Concentrate to a liter by boiling. 
2. Aluminum Foil. Strips of pure aluminum about 10 cm. long, 
6 mm. wide and weighing about 0.5 gm. should be used. 
Nitrates represent the end products of nitrification. They repre- 
sent stable forms of nitrogen and are desired in the various methods of 
sewage treatment. They indicate that there was originally in the 
water a relative amount of organic matter which has been changed by 
ammonification and nitrification to completely oxidized forms. 
Nitrites give indication that nitrogen is being oxidized. This in 
turn represents great bacterial activity on organic matter. All deter- 
minations of nitrogen represent the various stages in the oxidation and 
reduction of nitrogenous compounds. 
Oxygen Consumed. Place 100 c.c. of the sample in a flask. Add 
10 c.c. of the sulphuric acid solution and 10 c.c. of the standard per- 
manganate solution and digest the solution for thirty minutes on a 
water bath. The flasks should be placed in the water bath so that the 
level of the water is kept above the level of the water in the flask. If 
the amount of permanganate is not sufficient for the oxidation of the 
sample, repeat the digestion with a larger sample. At least 5 c.c. excess 
of permanganate should be present when the oxalate is added. Remove 
the flask and add 10 c.c. ammonium oxalate solution, and titrate until a 
faint pink color appears and remains with the permanganate solution. 
If 100 c.c. of water is used the number of cubic centimeters of potassium 
permanganate solution in excess of the number of cubic centimeters of 
ammonium oxalate solution is equal to the parts per million of oxygen 
consumed. 
Reagents. 1. Dilute Sulphuric Acid. Dilute 1 part of concentrated 
sulphuric acid with three parts of distilled water and free the solution 
of oxidizable matter by adding potassium permanganate until a faint 
pink color persists after the solution has stood for several hours. 
2. Standard Ammonium Oxalate. Dissolve 0.888 gm. of the pure 
salt in a liter of distilled water. One c.c. is equivalent to 0.1 mg. of 
oxygen. An equivalent quantity of oxalic acid may be used. 
3. Standard Potassium Permanganate. Dissolve 0.4 gm. of crys- 
tallized salt in a liter of distilled water. Add 10 c.c. of the dilute sul- 
phuric acid and 10 c.c. of this solution of potassium permanganate to 
100 c.c. of distilled water, and digest thirty minutes. Add 10 c.c. of ALKALINITY 
285 
the ammonium oxalate solution and the potassium permanganate till a 
pink coloration appears. This destroys the oxygen consuming capacity 
of the water used. Now add another 10 c.c. of ammonium oxalate 
solution and titrate with potassium permanganate. Adjust the potas- 
sium permanganate solution so that 1 c.c. is equivalent to 1 c.c. of the 
ammonium oxalate solution or 0.1 mg. of available oxygen. 
Alkalinity 
Determination. Add 4 drops of phenolphthalein to 50 c.c. of water 
and titrate with N/50 sulphuric acid solution until the coloration dis- 
appears. The number of cubic centimeters of N/50 sulphuric acid 
times 20 will indicate the phenolphthalein alkalinity in parts per million. 
This same sample may be used for the methyl orange alkalinity by 
adding 2 drops of methyl orange and continuing the titration to a faint- 
pink appearance. The methyl orange alkalinity is equal to the total 
number of cubic centimeters of N/50 sulphuric used multiplied by 20. 
The alkalinity of water is due to the content of inorganic salts such 
as, carbonates, bicarbonates, hydroxides, silicates, etc. The relations 
between the phenolphthalein and methyl orange alkalinities are ex- 
pressed in the following table; 
Table XXV 
RELATIONS BETWEEN ALKALINITY TO PHENOLPHTHALEIN AND 
METHYL ORANGE IN PRESENCE OF CARBONATE, BICAR- 
BONATE AND HYDROXIDE 
(Standard Methods, 1917) 
Result of Titration. 
Value of Radial Expressed in Terms of Calcium Carbonate. 
Bicarbonate. 
Carbonate. 
Hydroxide. 
P = 0 
T 
0 
0 
T = ±T 
T-2P 
2P 
0 
P = %T 
0 
2P 
0 
P = }T 
0 
2 (T-P) 
2P-T 
P = T 
0 
0 
T 
T = total alkalinity in presence of methyl orange. 
P = alkalinity in presence of phenolphthalein. 286 
WATER HYGIENE 
Classification of Water Bacteria 
The water bacteriologists have been primarily interested in the pres- 
ence of certain bacteria which are of sanitary significance. They have 
done very little with the other bacteria that may be in water with which 
they are dealing but have confined their attention to some ten organisms 
which indicate pollution. Only after, the other branches of the science 
had become well advanced did water bacteriologists give any attention 
to classification. This may have been due to the fact that the flora 
of a water is heterogeneous and that a few bacteria are probably secured 
from each material with which the water comes in contact. The en- 
vironment of a water determines its bacterial flora. One coming from 
an unpolluted water shed will have quite a different flora than a water 
flowing from a river which is made to carry the wastes of industrial and 
domestic life. Each of these examples would have a constant flora but 
one characteristic for the case mentioned. With the exception of pos- 
sibly ten bacteria or groups whose presence is supposed to indicate 
pollution, very little is known about the large number of other bacteria 
which may be present. More accurate deductions might be made with 
regai d to the sanitary character of a sample of water could more be 
known concerning its bacterial flora. This question has been discussed 
in Standard Methods for the Examination of Water and Sewage for 
1912, page 106. " In certain cases the determination of species may play 
directly a useful part in water analysis. It sometimes happens that the 
bacteria present in a filtered water are different in character from those 
found in the raw water and unless the facts are known, erroneous infer- 
ences may be drawn as to the efficiency of the filters. The determination 
of particular species is sometimes of importance in proving the identity 
of a water from a particular source. At times also, the presence of cer- 
tain species in water may be indicative of pollution." 
Bacterial control of filter operations is very important when the 
health of the public depends upon this factor. A knowledge of the 
general flora of the raw water might, at certain times, be a valuable 
factor in determining the efficiency of the filters. This would tell 
whether the bacteria in the effluent were passing through the filters 
from the raw water or whether they were growing in the under drains. 
Certain bacterial standards have been formulated that place the limit 
of 100 per cubic centimeter for bacteria in the filtered water. The 
character of the bacteria in those effluents which contain more than 100 
per cubic centimeter would be very important. CLASSIFICATION OF WATER BACTERIA 
287 
In order to make some definite bases for describing cultures the 
committee of the American Public Health Association suggested the 
following scheme which has been outlined by Savage (1906) as follows: 
I. Source and habitat. 
II. Morphological characters. 
Form. Manner of grouping, dimensions, staining reactions 
motility, spores, capsules, involution forms. 
III. Cultural characteristics. 
Mode of growth in and upon nutrient broth, gelatin plates, 
gelatin tubes, agar plates, agar tubes. 
IV. Biochemical reactions. 
Action upon milk, carbohydrates, nitrates; production of indol; 
relation to free oxygen; temperature relations; pigment 
formation; liquefaction of gelatin. 
Such a scheme as outlined above tends toward greater uniformity in 
the examination of bacteria in water. It seems to be the general ten- 
dency at the present time to arrange bacteria into groups and this is the 
best method for handling the flora of a substance like water. Tanner 
(1918) has shown that the fluorescent bacteria in water compose a closely 
related group. Wyatt Johnston (1894) early called attention to the 
grouping of water bacteria. In his paper, he discussed the tests which 
should be applied to separate bacteria into these groups. He analyzed 
235 described organisms and reached the following conclusions: That 
points of difference are given more attention than resemblances. In 
the grouping of bacteria a single strongly marked characteristic peculiar 
to a few species is of more value than a number of minor details but the 
members of any group should not differ unduly in regard to minor 
points. About this time the American Public Health Association real- 
ized the necessity of uniform procedures for the study of bacteria and 
appointed a committee to study methods for that purpose. This com- 
mittee reported in 1897 and recommended the following procedures: 
Necessary Information'. 
I. Source and habitat. 
II. Morphological characters. 
Form, dimensions, grouping, staining, capsule, flagella spores, 
pleomorphism, involution forms. 288 
WATER HYGIENE 
III. Biological characters. 
A. Cultural growth in or on nutrient broth, gelatin plates, 
gelatin tubes, agar plates, agar tubes, potato, milk and 
blood serum. 
B. Biochemical. 
Temperature relations, relation to oxygen, acidity of 
medium action on gelatin, protein, carbohydrates, 
nitrates, indol, pigments and odor. 
C. Pathogenesis. 
Optional Tests: 
I. Morphological. 
Staining reactions, study of flagella by special stains, perma- 
nency of characters. 
II. Physiological. 
A. Cultural. 
Growth-litmus gelatin, blood serum, synthetic media, 
photograph. 
B. Biochemical. 
Maximum, minimum and optimum temperature of 
growth, growth in special gases, limit of acid and 
alkali, chemical properties of pigments. 
C. Pathogenesis. 
Inoculation, toxins, etc. 
At the end of the report are given charts for recording the characters of 
bacteria. This is one of the first attempts in which the organized efforts 
of a society were brought to bear in systematizing methods for the study 
of the bacterial cell. 
Marshall Ward (1897) in studying the bacterial flora of the Thames 
river arranged his strains into the following groups: 
I. Forms identical with Bacterium urece. 
II. Violet bacteria. 
III. B. fluorescens liquefaciens group. 
IV. B. fluorescens non-liquefaciens group. 
V. Typical B. coli communis. 
VI. Series of forms centering around B. proteus. 
VII. Like VI except that a yellow pigment is formed. 
VIII. Bacteria between VII and IX. Characters changed. 
IX. Golden yellow liquefying forms. CLASSIFICATION OF WATER BACTERIA 
289 
X. Yellow non-liquefying forms. Probably related to IX. 
XI. Colorless capsulated forms. 
XII. Yellow capsulated forms. 
XIII. B. prodigiosus types. 
XIV. Rapid liquefiers, colorless, pronounced putrefactive tendencies. 
XV. B. subtilis like group. 
XVI. Yellow sarcina-like bacteria. 
XVII. Rose-colored micrococci. 
XVIII. Fungus forms. 
XIX. White micrococci (M. candicans). 
XX. Lemon yellow gelatin liquefiers. 
XXL Short oval cocci forming a red pigment. 
That this classification of water bacteria is artificial was realized by 
Ward for he made the following statement: " My work goes to show that 
species cannot be made out but that the limits of the species are, in most 
cases, far wider than is assumed in descriptions-in other words that 
many so-called species in books are merely variated forms, whose char- 
acters, as given, are not constant but depend on treatment. How far 
this is true for any given case will have to be tested on the particular 
form in question." 
Fuller and Johnson (1899) studied water bacteria from a river in 
America and arranged their strains into thirteen groups. They realized 
weaknesses in their system and state that too much importance was 
given to morphological data and too little to cultural studies. Dif- 
ficulty was also experienced in separating short rods from cocci. This 
classification has formed the basis for much systematic work in bac- 
teriology. Their groups are given below: 
I. Fluorescent forms. 
II. All red chromogenic forms. 
III. All orange chromogenic forms. 
IV. All yellow chromogenic forms. 
V. All violet chromogenic forms. 
VI. All non-fluorescent, non-chromogenic, gelatin liquefying bacteria, 
forming proteus-like colonies on gelatin. 
VII. All non-fluorescent, non-chromogenic, gelatin liquefying bacteria, 
forming subtilis-like colonies on gelatin. 
VIII. All non-fluorescent, non-chromogenic, non-proteus like non- 
subtilis-like bacteria which liquefy gelatin and ferment car- 
bohydrates with production of gas. 290 
WATER HYGIENE 
IX. All bacteria conforming to specified characteristics of VIII, except 
that fermentation of carbohydrates takes place without 
formation of gas. 
X. All bacteria conforming to group VIII except that no fermenta- 
tion of carbohydrates occurs. 
XI. All non-fluorescent, non-chromogenic, non-liquefying bacteria 
which ferment carbohydrates with the production of gas. 
XII. All bacteria conforming to specified characteristics of XI except 
that carbohydrates are fermented without gas. 
XIII. All bacteria conforming to specified characteristics of group XI 
except that ilo fermentation of carbohydrates occurs. 
Jordan (1901) studies 543 strains from the Illinois, Mississippi and 
Missouri rivers and developed the following classification: 
I. B. coli communis. 
II. B. lactis aerogenes. 
III. B. porteus. 
IV. B. enteritidis. 
V. B. fluorescens liquefaciens. 
VI. B. fluorescens non-liquefaciens. 
VII. B. subtilis. 
VIII. Non-gas formers, non-fluorescent, non-spore forming bacilli 
which liquefy gelatin and acidify milk. 
IX. Similar to group VIII save that milk is rendered alkaline. 
X. Similar to group VIII save that gelatin is liquefied. 
XI. Similar to group IX, save that gelatin is not liquefied. 
XII. Similar to group XI, save that the reaction of milk is not altered. 
XIII. Chromogenic bacteria not included above. 
XIV. Chromogenic staphylococci. 
XV. Non-chromogenic staphylococci. 
XVI. Sarcinse. 
XVII. Streptococci. 
The Bacterial Count. In early sanitary history, this was supposed 
to be a reliable index of pollution. Many standards were proposed in 
which it was stated that a water should not contain over a certain num- 
ber of bacteria per cubic centimeter to be regarded a good drinking 
water. One of the most elaborate of these was proposed by Miquel 
(1891). 
Less than 10 cells per cubic centimeter excessively pure 
10-100 cells per cubic centimeter very pure 37° C. VS. 20° C. COUNT 
291 
100-1000 cells per cubic centimeter pure 
1000-10,000 cells per cubic centimeter mediocre 
10,000-100,000 cells per cubic centimeter impure 
Over 100,000 very impure 
Such a standard would be followed with difficulty to-day. Obvi- 
ously, according to our present information, such a scheme for grading 
waters is untenable. Probably Koch's old standard of not over 100 
bacteria per cubic centimeter on gelatin is as accurate as any. It is 
evident that a very few cells of a certain variety would be far more sig- 
nificant than thousands of another variety. In natural waters, however, 
it has become established that a high content of organic matter usually 
means a high content of bacteria. High organic content usually means 
pollution and, in this way, the count itself may mean something. Pos- 
sibly a water with a large number of bacteria, according to the law of 
probability, might contain some objectionable forms. 
Relation of the 37° C. Count to the 20° C. Count. This has re- 
ceived the attention of a number of bacteriologists and their opinions 
sum up our knowledge with regard to the two counts. 
Whipple (1913) made the following comment: 
" That the 37° count more nearly represents the sanitary quality of 
water than the 20° count is probably true in a general way, but it is not 
necessarily so." He cites counts made at the Watertown, N. Y., fil- 
tration plant and states: 
" The difference between the two counts is striking. The 20° counts 
are very high in the winter and spring but are low during the summer, 
whereas the 37° counts are low during the winter and spring and high 
during the summer, at which season the sewage which enters the stream 
is less diluted." He calls attention to the fact that this seasonable 
difference gives a varying ratio, and suggests that it would seem advis- 
able to procure more comparative data before making the recommended 
change. Caird (1913) thinks it advisable to continue the use of gelatin 
to determine the efficiency of water-treatment plants, and he appends 
to his paper discussions by various waterworks men, whose opinions 
seem about evenly divided. Baton (.1914) also protests against the 
recommended change. He concludes that if only one medium is to be 
used it should be gelatin as it is more reliable and serviceable. 
Gaub (1913) at the Washington, D. C., filtration plant found a vari- 
able ratio between counts on agar and on gelatin plated from the un- 
filtered water of the Potomac River. He also found a seasonal ratio 
which seemed to remain constant for several months at a time and then 292 
WATER HYGIENE 
changed. With the filtered water there was a less constant seasonal 
ration, which was much lower than that with the unfiltered water. This 
was not due to treatment of the water with calcium hypochlorite. The 
efficiencies in removal of B. coll correspond better to the counts on gela- 
tin, but Gaub does not state how these efficiencies were computed. 
Gaub forms no conclusion and asks, " Which is the correct medium to 
use in order to show the efficiency of a plant?" Prescott and Winslow 
(1915) state that the decomposition of organic matter may be measured 
either by the material decomposed or by the number of organisms en- 
gaged in carrying out the process of decomposition; and that the latter 
method has the advantage of far greater delicacy since bacteria respond 
by enormous multiplication to very slight increases in their food supply; 
thus the count on standard gelatin at 20° C. is roughly proportionate 
in not too heavily polluted waters to the free ammonia and oxygen 
consumed by chemical analysis. Savage asserts that many water bac- 
teria are unable to grow at 37° C. though most organisms associated 
with excreta grow readily at 37° C.; unfortunately, many harmless soil 
organisms also grow readily at 37° C. An experiment by Savage illus- 
trates the limitations of this enumeration. He plated on agar and gel- 
atin a pure tap water collected in a sterile bottle; he then added 1 gm. 
of soil to 500 c.c. of the sample and plated on agar and gelatin and emul- 
sion thus obtained. The agar plates were incubated at 37° C. for forty 
hours and the gelatin plates at 21° C. for three days. The soil con- 
tained no B. coli in 3 gms. The water contained 3 bacteria per cubic 
centimeter by the agar count and 76 by the gelatin count. The emul- 
sion contained 1360 bacteria per cubic centimeter by the agar count 
and 1970 by the gelatin count. Savage concludes that the count at 
37° C. is an index of the presence of bacteria other than those natural to 
pure water, but not necessarily harmful ones. 
Harrison expresses the following opinion: " The number of organ- 
isms which develop on beef peptone agar incubated at blood heat, com- 
monly termed the 1 agar ' or ' blood-heat ' count, is perhaps more 
important than the gelatin count, as many water bacteria do not grow 
at blood heat, whereas sewage and soil organisms grow readily at this 
temperature. The agar count eliminates the water flora, but obscures 
the sanitary results by reason of the presence of soil bacteria." Muir 
and Ritchie state that the count on gelatin plates incubated at 20° C. 
gives an idea of the number of bacteria which grow at summer heat and 
that the count on agar plates " incubated at 37° C. gives an idea of the 
number of those which grow at blood heat." The following comments 
are added: " As the pathogenic and intestinal bacteria grow at this 37° C. VS. 20° C. COUNT 
293 
temperature (37° C.), the determination of the numbers of blood-heat 
bacteria is important. The counts on the two media usually differ as 
each is favorable to the growth of its own group of organisms." The 
same authors note that the presence of liquefying bacteria on the gelatin 
plates may make it necessary to count the plates at the end of twenty- 
four hours, or at the end of a shorter time than is directed in the com- 
monly accepted procedure. Most bacteriologists apparently believe 
that the agar count at 37° C. indicates more nearly the bacteria of sewage 
origin and that it consequently is more important in ascertaining the 
potability of water. The results of Savage's experiment show, however, 
that erroneous conclusions will be drawn if the count at that tempera- 
ture is considered to furnish indication of possible sewage pollution, 
because soil may contribute large numbers of harmless bacteria that 
grow at 37° C. 
The advantages and disadvantages of each medium are well known 
to all bacteriologists. Agar is better for field work in warm weather 
because it can be transported with less trouble either in test tubes or 
in Petri dishes; great care must be used to prevent liquefaction if gelatin 
is used. As gelatin has to be shipped in cooled containers in warm 
weather it can seldom be used in the field. A less elaborate incubator is 
required for agar because no special precaution has to be taken to keep 
the temperature below a certain maximum. In some filter plants in 
which agar is used to control the operation agar plates are left at room 
temperature for two days before counting. This could not be done with 
gelatin plates, for which constant low temperature must be maintained. 
The count on agar is made at the end of twenty-four hours whereas the 
count on gelatin is made at the end of forty-eight hours. Spreaders 
often develop on agar plates but any inconvenience from these is offset 
by liquefiers on gelatin plates. The two media themselves differ in 
their properties; gelatin is good for bacteria, but no bacteria have been 
described that can metabolize agar. Both media are subject to wide 
variations in chemical content, but agar is more easily purified. 
Recent work by Cummings (1916) indicates that in the summer the 
agar and gelatin counts are nearly equal. In February the gelatin 
count, on Potomac River water, became sixteen times as great as the 
agar count. The gelatin count seems to follow the turbidity readings. 
Where possible as Tanner (1916) has pointed out, both media should be 
used where it is convenient. Race (1916) has stated that in water 
analysis no one medium will give a count that will possess any constant 
ration to the excremental bacteria as shown by B. coli content. The 
37° C. count is said to be nearest to the B. coli content. The highest 294 
WATER HYGIENE 
count for the shortest incubation period is secured at 27° C. Gelatin 
was found to yield the highest count. Hill (1908) and Breed and Dot- 
terer (1916) have given some interesting data to show that a good agar 
plate should not contain over 200 bacteria when counts are to be made. 
These investigators show that a plate which has less than about 20 and 
over about 200 bacterial colonies will give a count which is not accurate. 
Indicators of Pollution 
As soon as sanitary science had demonstrated that diseases were 
spread by polluted water and foods, it became necessary to find some 
method of detecting the presence of fecal matter. With this effort 
originated the Bacillus colon test which has passed through many stages 
to reach its present status. Other " indicator " organisms have been 
used in bacteriology such as the streptococci and Bacillus enter idiiis 
Gaertner. Indicators of pollution in bacteriology have certain definite 
characteristics. 
1. They should be present in water and on foods which have received 
sewage pollution. 
2. They should be absent from foods which have not been in contact 
with sewage. 
3. They should be easily detected in the shortest time consistent 
with accurate results. 
4. They should possess constant characteristics and not be subject 
to either morphological or physiological changes. 
From the above, it is apparent that an ideal indicator does not exist. 
Admitting this, the analyst has to resort to the next best thing and take 
an indicator which best fulfills the necessary requirements. The relative 
amount of the indicator has come to be the important factor and not the 
mere presence of the indicator. 
The Streptococci. The examination of water for streptococci is 
not used in America for the routine examination. It would probably 
be of much value were it used in this country. Houston (1898 9) 
originally emphasized its value. Possibly one reason why the strep- 
tococci are not used in routine analysis is the lack of methods for dif- 
ferentiating fecal and non-fecal streptococci. Winslow (1902) and 
Fuller and Armstrong (1913) pointed out that the amounts of acid 
formed in carbohydrate media might be used as bases for differentiation. 
Armstrong and Fuller found that human fecal streptococci were char- 
acterized by high acidities in glucose, lactose, and mannite broths and a 
low acidity in raffinose broth. On the other hand equine and bovine STREPTOCOCCI IN WATER 
295 
types were characterized by a low acidity in glucose, lactose and man- 
nite and a higher acidity in raffinose. The following tables are quoted 
from Houston's work: 
Table XXVI 
PERCENTAGE OF STREPTOCOCCI FERMENTING THE SUGARS 
STATED 
F. A. = Fuller and Armstrong. W. = Winslow 
Human Feces. 
Horse Feces. 
Cow Feces. 
F. A. 
w. 
F. A. 
w. 
F. A. 
w. 
Glucose 
95 
89 
75 
65 
81 
84 
Lactose 
94 
62 
24 
8 
77 
52 
Saccharose 
90 
46 
75 
Mannite 
65 
28 
2 
2 
3 
6 
Raffinose.. . 
0 
6 
12 
4 
73 
28 
Table XXVII 
PERCENTATE NUMBER OF STREPTOCOCCI FERMENTING SUGARS 
MENTIONED 
Houston's Data. 672 Specimens 
Human Feces (400 Specimens). 
Milk, 
172 
Specimens. 
Cow 
Dung 
100 
Specimens. 
Series I. 
100 Speci- 
mens. 
Series 
II. 
Series 
III. 
Average 
of Series 
I, II and 
III. 
i 
Series IV. 
In this 
series only 
lactose + 
specimens 
were 
accepted. 
100 speci- 
mens. 
Glucose 
Lactose 
Not tested 
66 
78 
85 
76.34 
100 
97 
85 
Saccharose 
89 
84 
86 
86.34 
49 
90 
89 
Mannite 
7 
27 
39 
24.34 
4 
20 
None 
Raffinose 
62 
8 
26 
32.00 
100 
19 
74 
Salicin 
96 
91 
91 
29.67 
97 
60 
93 
Inulin 
12 
None 
2 
4.67 
Not tested 
21 
13 
It is well known that streptococci are present in the intestinal tract 
of man and animals. They are essentially parasitic in their charac- 
teristics and, generally speaking, indicate recent pollution when found 296 
WATER HYGIENE 
in water. The evidence, however, is less convincing than that of B. coli 
since it is probable that the methods which exist for differentiating 
between fecal and non-fecal streptococci are unreliable. Their short life 
in water points out that a negative result for their examination may not 
indicate the absence of pollution. More data with regard to the strep- 
tococci as indicators of pollution are needed. Savage and Reed (1916) 
in some work on this subject found that neutral red broth was the best 
medium. They regard the estimation of the streptococci as of un- 
doubted value but that a positive result has much more meaning than a 
negative result. The same author with the collaboration of Wood 
(1917) compared the viability of B. coli and streptococci in natural 
waters. The streptococci were not able to live longer than two weeks 
while B. coli showed a distinct multiplication. In one instance the 
bacilli increased to fifty times the original number in eleven weeks. 
The presence of the streptococci in a water would indicate recent pollu- 
tion and, therefore, of a more dangerous nature. The greatest objec- 
tion to the use of the streptococci as indicators of pollution is the lack of 
a rapid method for detecting them in a sample of water. Savage has 
recommended the use of neutral red media but this does not exert a 
sufficiently selective action to render the test easily and quickly carried 
out. The author has found that it was necessary to verify the suspected 
streptococci with hanging drop determinations in many of the cases 
where neutral red was used. 
Houston's Method for Streptococci in the Feces. Drigalski and 
Conradi plates are inoculated from an emulsion of the feces, made in 
sterile water. After incubation at 37° C., the minute colonies are sub- 
cultured in " litmus lemco, lactose peptone " medium. After twenty- 
four hours the tubes showing no acidity are discarded; those showing 
acidity are examined microscopically and if results were satisfactory 
further subcultures were made into a series of lemco peptone sugar 
media untinted with litmus. After three days incubation at 37° C. 
the acidity produced is estimated with N/20 NaOH, using phenol- 
phthalein as the indicator. Control tubes are also inoculated. (Mtr. 
Water Bd. 10th Am. Rept., p. 25, 1914.) 
The streptococcus colonies on Drigalski and Conradi plates are small 
colonies with a tinge of violet. They are to be distinguished from col- 
onies of B. typhosus which possess larger colonies. 
Detection of Sewage Streptococci. " For this purpose many proc- 
esses have been devised, but the simplest and best appears to be the 
examination of the bile-salt glucose broth used for the ' presumptive 
test ' for colon bacilli. The incubation should, however, be prolonged STREPTOCOCCI IN WATER 
297 
to forty-eight hours, and, at the end of that period a loopful from the 
surface of each tube should be examined as a hanging drop under the 
microscope. The broth should then be carefully poured off so as to 
leave at the bottom any precipitate which has been produced. Add a 
few drops of dilute sodium carbonate solution, to dissolve the bile salts 
and examine again in the hanging drop. If distinct chains are not found, 
streptococci may be regarded as absent." This method has been found 
by Thresh to yield the best results. 
Savage's Method for Isolation of Streptococcus from Water, etc. 
Add 0.1 and 1 c.c. of the sample (water) to tubes of glucose neutral 
red broth and 10 c.c. of the sample to a tube of double strength 
neutral red broth. Incubate the tubes at 37° C. for forty-eight hours 
and then examine microscopically in hanging drop for chains of cocci. 
The broth tube containing the quantity of water next below the one 
giving a positive result is examined several times in fresh preparations. 
Where it is doubtful whether chains are present, a little of the fluid is 
centrifugalized and stained with methylene blue. The streptococci are 
best isolated from glucose broth tubes by adding a platinum loopful to 
a litmus lactose plate and distributing over its surface. All suspicious 
strains must be subcultured and worked out. 
Method for Separation of B. coli and Streptococci in Water. " Inoc- 
ulate the desired quantity of water, preferably 1 c.c. into dextrose broth 
fermentation tube. Incubate at 37° C. Examine after six to twelve 
hours. Within two to three hours after gas formation has appeared, 
plate the broth into litmus lactose agar, incubating for twelve to eigh- 
teen hours at 37° C. If, at the end of this time, no acid-producing 
colonies are present, it is probably safe to assume that there were no 
colon bacilli present." 
After the first plating from dextrose broth, replace the fermentation 
tube for thirty-six hours and plate in litmus lactose agar. This culture 
should give a nearly pure culture of streptococci, if these organisms 
were originally present in the water. 
Bacillus Enteritidis Sporogenes. This organism was discovered by 
Klein in 1895 in diarrhoea stools. Its use as an indicator of pollution 
rests on the fact that it is present in feces and, therefore, in sewage. 
This organism should be regarded as one of a group of bacteria the 
members of which are very closely related. Table XXVIII quoted from 
Thresh will indicate the essential differences between certain members of 
this group. Bacillus welchii (Bacillus aerogenes capsulatus) is probably 
identical with Klein's Bacillus enteritidis sporogenes. Welch describes 
his bacillus as non-motile but otherwise the descriptions and the changes 298 
WATER HYGIENE 
produced in milk are identical. The nomenclature of this group appa- 
rently is in a chaotic state. 
Table XXVIII 
SHOWING THE ESSENTIAL CHARACTERISTICS OF SOME MEMBERS 
OF THE ANAEROBIC GAS FORMING GROUP OF BACTERIA 
(After Thresh, 1913) 
I. Bacillus 
Entertidis sporagenes. 
II. Bacillus 
Butyricus. 
III. Bacillus 
Cadaveris sporogenes. 
1. Cylindrical rods, on the 
average 2.5-3.5m long, 
0.8-1.25m broad, stains 
well by Grain's method 
some individual motile. 
1. Same as I. 
1. Cylindrical and thread- 
like, thinner and lon- 
ger than I and II, very 
motile, stains by 
Gram's method. 
2. Spores oval; stain after 
the several methods; 
situated in the middle 
of the rods more or less, 
1.6m long, Im broad. 
2. Same as I. 
2. Spores oval, terminal 
drumsticks, stain after 
usual methods 1.6m 
long, Im broad. 
3. On gelatin grows well, 
softens rapidly and 
slowly liquefies. 
3. Grows well on the sur- 
face of ordinary gela- 
tin as a translucent 
mass of convoluted 
threads; does not liq- 
uefy the gelatin. 
3. Rapidly liquefying, pu- 
trid odor, numerous 
spores formed. 
4. In stab gelatin, much 
gas produced; spheri- 
cal colonies without 
filamentous projec- 
tions; slowly lique- 
fying. 
4. Forms spherical colon- 
ies with numerous hori- 
zontal filament pro- 
jections; not lique- 
fying; forms much gas. 
4. Much gas, rapidly lique- 
fying, and putrid odor. 
5. On the surface of agar, 
gray round flat colon- 
ies, few crenations, no 
spores. 
5. Gray round flat colo- 
nies, margin thin and 
much crenate, no 
spores. 
5. Thready branched col- 
onies, with or without 
finely granular plate, 
rapidly forming spores. 
6. Stab in agar, little tend- 
ency for forming lat- 
eral branching, much 
gas, no spores. 
6. Forms characteristic 
bundles of threads pro- 
jecting laterally from 
the growth in the 
stab, much gas, no 
spores. 
6. Much gas, rapidly form- 
ing spores, conspicu- 
ous masses of threads 
growing out of the 
stab. 
7. In milk, rapid separa- 
tion of acid whey and 
flocculi of casein, small 
of butyric acid, no 
spores, much gas. 
7. Same as I. 
7. Milk is slowly decom- 
posed, putrid odor 
much gas, rapidly 
forming spores. 
8. Grows well on serum, 
slowly liquefying, some 
spores found. 
8. Grows well on serum, 
very slow softening. 
8. Rapidly liquefying, pu- 
trid odor, rapidly form- 
ing spores. 
9. Virulent for rodents. 
9. Not pathogenic for ro- 
dents. 
9. Not pathogenic for ro- 
dents. 
Isolation of Bacillus Enteritidis Sporogenes. In examining water 
for this organism, it may be necessary to concentrate it either by fil- TESTS . FOR B. COLI 
299 
tration or evaporation under reduced pressure at low +emperatures. 
Decimal dilutions of this concentrated sample should be made and 
1 c.c. quantities added to sterile litmus milk. It is a good plan to boil 
the tubes of milk for about five or ten minutes before inoculating in 
order to expel as much air as possible. After the tubes have been 
inoculated they should be heated on a water bath at 80° C. for twenty 
minutes which will kill all vegetative forms. They are then incubated 
under anaerobic conditions either in a Novy jar or made anaerobic by 
the Wright method. 
The Bacillus Colon Test. Escherich isolated Bacillus colon soon 
after the discovery of solid media by Koch. Escherich was attempting 
to find the causal organism of cholera. At first it was thought that he 
had found the specific bacterium which caused cholera because Bacillus 
colon was found to be present in the intestinal tract of each patient. 
This, however, was soon found to be untrue for the bacillus was found 
in the intestinal tract of normal individuals. Later investigations 
indicated that it was always present in the feces and from these facts 
bacteriologists attempted to use it as the indicator of the presence of 
fecal matter in foods even though the amount was very small. For 
some time it was accepted as a reliable factor in bacteriology but as 
knowledge was supplemented with more data it became apparent that 
some limitations had to be placed in interpreting this test. Much of 
our work since this time has been concerned with studies to find out the 
limits in the value of Bacillus colon in this connection. 
Different definitions have been given for the Bacillus colon group. 
Standard Methods (1917) recommended that the B. coll group be con- 
sidered as including all non-spore-forming bacilli which ferment lactose 
with gas formation and grow aerobically on standard solid media. 
The value of the B. coli test has been discredited by some of the prac- 
tical waterworks men. They argue from the several researches which 
have shown that the organism may not at all times be restricted to 
sources which have received sewage pollution. Rogers, Clark and Evans 
(1915), Prescott (1906), Metcalf (1905), Smith (1905), and others have 
reported the organism or B. coli like organisms from grain fields and 
grains which had not received sewage pollution. Their work was of 
such import that it became necessary to distinguish between fecal and 
non-fecal B. coli. The English and American bacteriologists have 
regarded the B. coli test with favor while some German bacteriologists 
have fought its use. Kruse (1894), for instance, believes that B. coli 
may be found in any water no matter from what source, provided a large 
enough sample is taken. Chick, representing the English opinion, believes 300 
WATER HYGIENE 
that B. coli is not present in a water except it has received sewage pol- 
lution. These, however, may be extreme opinions and may not repre- 
sent the opinions of the sanitarians in each country. Race (1914, 1916) 
argues that B. coli is a reliable indicator of pollution and states that those 
cities which have many B. coli in their water usually have a high typhoid 
fever death rate. He warns that B. coli should be regarded as merely an 
inferential indicator. The fact that the presence of B. coli in a sample 
of water may not be sufficient evidence of serious pollution, it is, how- 
ever, a danger signal which must not be overlooked. Houston (1915) 
representing the English practice regards B. coli as the best index for the 
control of purification processes. Winslow (1916), in tracing the history 
of this organism as an indicator has advised the accumulation of as much 
data as possible to more definitely determine whether B. coli is purely a 
fecal form and B. aerogenes a saprophytic form. 
The testing of water for this organism has taken some recent advances. 
Thanks to the research of Rogers and his co-workers in the laboratories 
of the Bureau of Animal Industry, we have now fairly reliable methods 
for differentiating between fecal and non-fecal B. coli. The methods of 
analysis which are given in the following pages have been taken with 
permission from the 1917 report of the committee on water and sewage 
analysis of the American Public Health Association. 
American Public Health Association Method for Determining the 
Presence of Members of the B. coli Group. It is recommended that 
the B. coli group be considered as including all non-spore-forming bacilli 
which ferment lactose with gas formation and grow aerobically on 
standard solid media. 
The formation of 10 per cent or more of gas in a standard lactose 
broth fermentation tube within twenty-four hours at 37° C. is presump- 
tive evidence of the presence of members of the B. coli group, since the 
majority of the bacteria which give such a reaction belong to this 
group. 
■ The appearance of aerobic lactose-splitting colonies on lactose-litmus- 
agar or Endo's medium plates made from a lactose-broth fermentation 
tube in which the gas has formed confirms to a considerable extent the 
presumption that gas-formation in the fermentation tube was due to 
the presence of members of the B. coli group. 
To complete the demonstration of the presence of B. coli, as above 
defined, it is necessary to show that one or more of these aerobic plate 
colonies consists of non-spore-forming bacilli which, when inoculated 
into a lactose-broth fermentation tube, forms gas. 
It is recommended that the standard tests for the B. coli group be TESTS FOR B. COLI 
301 
either (A) the Presumptive, (B) the Partially Confirmed, or (C) the 
Completed test, as hereafter defined, each test being applicable under 
the circumstances specified. 
A. Presumptive Test 
1. Inoculate a series of fermentation tubes with appropriate gradu- 
ated quantities of the water to be tested. 
2. Incubate these tubes at 37° C. for forty-eight hours. Examine 
tube at twenty-four and forty-eight hours, and record gas-formation. 
The records should be such as to distinguish between: 
(a) Absence of gas-formation. 
(6) Formation of gas occupying less than 10 per cent (10%) of the 
closed arm. 
More detailed records of the amount of gas formed, though desirable 
for purposes of study, are not necessary for carrying out the standard 
tests prescribed. 
3. The formation within twenty-four hours of gas occupying more 
than 10 per cent (10%) of the closed arm of fermentation tube con- 
stitutes a positive presumptive test. 
4. If no gas is formed in twenty-four hours, or if the gas formed is 
less than 10 per cent (10%), the incubation shall be continued to forty- 
eight hours. The presence of a gas in any amount in such a tube at 
forty-eight hours constitutes a doubtful test, which, in all cases, requires 
confirmation. 
5. The absence of gas-formation after forty-eight hours' incubation 
constitutes a negative test. (An arbitrary limit of forty-eight hours' 
observation doubtless excludes from consideration occasional members 
of the B. coli group which forms gas very slowly, but for the purposes of a 
standard test the exclusion of these occasional slow gas-forming organ- 
isms is considered immaterial.) 
B. Partially Confirmed Test 
1. Make one or more Endo's medium or lactose-litmus-agar plates 
from the tube which, after forty-eight hours' incubation, shows gas- 
formation from the smallest amount of water tested. For example, if 
the water has been tested in amounts of 10c.c., 1 c.c., and 0.1 c.c., and 
gas is formed in 10 c.c., and 1 c.c., and not in 0.1 c.c., the test need be 
confirmed only in the 1 c.c. amount.) 
2. Incubate the plates at 37° C., eighteen to twenty-four hours. 302 
WATER HYGIENE 
3. If typical colon-like red colonies have developed upon the plate 
within this period, the confirmed test may be considered positive. 
4. If, however, no typical colonies have developed within twenty- 
four hours, the test cannot yet be considered definitely negative, since 
it not infrequently happens that members of the B. coli group fail to 
form typical colonies on Endo's medium or lactose-litmus-agar plates, 
or that the colonies develop slowly. In such cases, it is always neces- 
sary to complete the test as directed under C 2 and 3. 
C. Completed Test 
1. From the Endo's medium or lactose-litmus-agar plate, made as 
prescribed under B, fish at least two typical colonies, transferring each 
to an agar slant and a lactose broth fermentation tube. 
2. If no typical colonies appear upon the plate within twenty-four 
hours, the plate should be re-incubated another twenty-four hours, 
after which at least two of the colonies considered to be most likely 
B. coli, whether typical or not, shall be transferred to agar slants and 
lactose broth fermentation tubes. 
3. The lactose broth fermentation tubes thus inoculated shall be 
incubated until gas formation is noted; the incubation not to exceed 
forty-eight hours. The agar slants shall be incubated at 37° C. for 
forty-eight hours, when a microscopic examination shall be made of at 
least one culture, selecting one which corresponds to one of the lactose 
broth fermentation tubes which has shown gas-formation. 
The formation of gas in lactose broth and the demonstration of 
non-spore-forming bacilli in the agar culture shall be considered a sat- 
isfactory completed test, demonstrating the presence of a member of 
the B. coli group. 
The absence of gas-formation in lactose broth or failure to demon- 
strate non-spore-forming bacilli in a gas-forming culture constitutes a 
negative test. 
Application of Presumptive, Partially Confirmed, and Completed 
Tests. 
A. The Presumptive Test. 
1. When definitely positive, that is showing more than 10 per cent 
(10%) of gas in twenty-four hours, is sufficient. 
(a) As applied to all except the smallest gas-forming portion 
of each sample in all examinations. 
(b) As applied to the smallest gas-forming portion in the 
examination of sewage or of water showing relatively TEST FOR B. COLI 
303 
high pollution, such that its fitness for use as drink- 
ing water does not come into consideration. This 
applies to the routine examinations of raw water in 
connection with the control of the operation of puri- 
fication plants. 
2. When definitely negative, that is, showing no gas in forty-eight 
hours, is final and, therefore, sufficient in all cases. 
3. When doubtful, that is, showing gas less than 10 per cent (10%) 
(or none) in twenty-four hours, with gas either more or 
less than 10 per cent in forty-eight hours, must always be 
confirmed. 
B. The Partially Confirmed Test. 
1. When definitely positive, that is, showing typical plate colonies 
within twenty-four hours, is sufficient. 
(a) When applied to confirm a doubtful presumptive test in 
cases where the latter, if definitely positive, would 
have been sufficient. 
(6) In the routine examination of water-supplies where a 
sufficient number of prior examinations have estab- 
lished a satisfactory index of the accuracy and sig- 
nificance of this test in terms of the completed test. 
2. When doubtful, that is, showing colonies of doubtful or negative 
appearance in twenty-four hours, must always be com- 
pleted. 
C. The Completed Test. 
The completed test, is required as applied to the smallest gas- 
forming portion of each sample in all cases other than those 
noted as exceptions under the " presumptive " and the " par- 
tially confirmed " tests. 
The completed test is required in all cases where the confirmed 
test has been doubtful. 
In order that tests for B. coli may have a quantitative significance, 
the following general principles and rules should be observed: 
Ordinarily not less than three portions of each sample should be 
tested, the portions being even decimal multiples of fractions of a cubic 
centimeter; for example, 10 c.c., 1 c.c., 0.1 c.c., .01 c.c., etc. It is 
essential that the dilutions should be such that the largest amount 
gives a positive test (unless the water is such as to give negative tests 
in 10 c.c.), and the smallest dilution, a negative result. To insure this 
result, it is often necessary to plant four or five dilutions, especially in 304 
WATER HYGIENE 
the examination of a sample of entirely unknown quantity. The quan- 
titative value of a series of tests is lost, unless all or, at least, a large 
portion of the smallest dilutions tested have given negative results. 
In reporting a single test, it is preferable merely to record results as 
observed, indicating the amounts tested and the result in each, rather 
than to attempt expression of the result in numbers of B. colt per cubic 
centimeter. In summarizing the results of a series of tests, however, 
it is desirable for the sake of simplicity to express the results in terms 
of the numbers of B. coll per cubic centimeter or per 100 c.c. To con- 
vert the result of fermentation tests to this form, the result of each test 
is recorded as indicating a number of B. coli per cubic centimeter equal 
to the reciprocal of the smallest decimal or multiple fraction of a cubic 
centimeter giving a positive result. For example the result: 10 c.c. 
plus; 1 c.c. plus; 0.1 c.c. minus would be recorded as indicating one 
B. coli per cubic centimeter. An exception should be made in the case 
where a negative result is obtained in an amount larger than the smallest 
portion giving a positive result; for example, in a result such as 10 c.c. 
plus; 1 c.c. minus; 0.1 c.c. plus. In such a case, the result should be 
recorded as indicating a number of B. coli per cubic centimeter equal to 
the reciprocal of the dilution next larger than the smallest one giving a 
positive test, this being a more probable result. 
Where tests are made in amounts larger than 1 c.c. giving an average 
of results less than one B. coli per cubic centimeter, it is convenient to 
express results in terms of the number of B. coli per 100 c.c. 
The following table illustrates the method of recording and aver- 
aging results of B. coli tests. 
Result of Tests in Amounts Designated. 
Indicated No. of B. coli. 
10 c.c. 
1 c.c. 
0.1 c.c. 
.01 c.c. 
per C.C. 
per 100 c.c. 
plus 
minus 
minus 
minus 
0.1 
10. 
plus 
* plus 
minus 
minus 
1.0 
100. 
plus 
plus 
plus 
minus 
10.0 
1000. 
plus 
plus 
plus 
plus 
100.0 
10,000. 
plus 
plus 
minus 
plus 
10.0 
1,000. 
121.1 
12,110 
24.0 
2,422 
The above method of expressing results is not mathematically alto- 
gether correct. The average number of B. coll per cubic centimeter, as 
thus estimated is not precisely the most probable number calculated by ROUTINE PROCEDURES 
305 
application of the theory of probability. To apply this theory to a 
correct mathematical solution of any considerable series of results 
involves, however, mathematical calculations so complex as to be im- 
practicable of application in general practice. The simpler method 
given is, therefore, considered preferable, since it is easily applied and 
the results so expressed are readily comprehensible. 
In order that the results as reported may be checked and carefully 
valuated, it is necessary that the report should show not only the aver- 
age number of B. coli per cubic centimeter, but also the number of sam- 
ples examined; and for each dilution, the total number of tests made, 
and the number (or per cent) positive. 
Routine Procedures for Examination of Samples of Water 
First Day. 
1. Prepare dilutions as required. 
2. Make two (2) gelatin plates from each dilution, and incubate at 
20° C. 
3. Make two (2) agar plates from each dilution, and incubate at 37° C. 
4. Inoculate lactose broth fermentation tubes with appropriate 
amounts for B. coli tests, inoculating two (2) tubes with each 
amount. 
Note. Where repeated tests are made of water from the same 
source as is customary in the control of public supplies, it is 
not necessary to make duplicate plates or fermentation tubes 
in each dilution. It is sufficient, in such circumstances, to 
make duplicate plates only from the dilution which will most 
probably give from 25 to 250 colonies per plate. 
Second Day. 
1. Count the agar plates made on the first day. 
2. Record the number of additional fermentation tubes which show 
10 per cent (10%) or more of gas. 
Note. In cases only the presumptive test for B. coli is required 
fermentation tubes showing more than 10 per cent (10%) 
of gas at this time may be discarded. 
Third Day. 
1. Count gelatin plates made on the first day. 
2. Record the additional number of fermentation tubes which show 
10 per cent or more of gas. 306 
WATER HYGIENE 
3. Make a lactose-litmus-agar of Endo's medium plate from the 
smallest portion of each sample showing gas. Incubate plate 
at 37° C. 
Note. In case the smallest portion in which gas has been formed 
shows less than 10 per cent (10%) of gas it is well to make a 
plate also from the next largest portion so that in case the 
smallest portion gives a negative end result, it may still be 
possible to demonstrate B. coli in the next larger dilution. 
Fourth Day. 
1. Examine Endo's medium on lactose-litmus-agar plates. If typical 
colonies have developed, select two and transfer each to a 
lactose-broth-fermentation tube and an agar slant, both of 
which arc to be incubated at 37° C. 
2. If no typical B. coli colonies arc found, incubate the plates another 
twenty-four hours. 
Fifth Day. 
1. Select at least two colonics, whether typical or not, from the 
Endo's medium or lactose-litmus-agar plates which have been 
incubated an additional twenty-four hours; transfer each to a 
lactose-broth-fermcntation tube and an agar slant, and com- 
plete the test as for typical colonies. 
2. Examine-lactose-broth fermentation tubes inoculated from plates 
on the previous day. Tubes in which gas has been formed may 
be discarded after the result has been recorded. Those in 
which no gas has formed should be incubated an additional 
twenty-four hours. 
Sixth Day. 
1. Examine lactose-broth-fermcntation tubes reincubated the previ- 
ous day. 
2. Examine microscopically agar slants corresponding to lactose-fer- 
mentation tubes inoculated from plate colonies and showing 
gas formation. 
Methyl Red Test. As sanitary knowledge increased and it became 
apparent that B. coli or B. coZf-like bacteria were present on substances 
which had not received fecal pollution, studies were made to detect 
those B. coli which came from fecal sources. To this end Rogers (1915) 
and his co-workers found that the gas ratio separated B. coli of fecal 
origin from B. coli of non-fecal origin. B. coli from bovine feces fer- 
mented dextrose with the formation of equal volumes of carbon dioxide VOGES-PROSKAUER REACTION 
307 
and hydrogen while those from grains formed two or three times as 
much carbon dioxide. The next step was to find a simple procedure 
for determining this ratio or finding some procedure which was correlated 
with it since the determination of the gas ratio is too exacting for routine 
analyses. Clark and Lubs (1915) working in the same laboratory dis- 
covered a constant correlation between the gas ratio and the hydrogen 
ion concentration. The B. coll from bovine feces were found to possess 
a higher hydrogen ion concentration than those from grains or non- 
fecal origins. A study of indicators yielded the information that methyl 
red was yellow with the high ratio group and red or acid with the low 
ratio group. Levine (1916) correlated this test with the Voges-Pros- 
kauer reaction. 
Voges-Proskauer Reaction. This has been studied by Levine who 
correlated it with the methyl red test. He found the bacteria which 
give the Voges-Proskauer reaction were rarely found in feces and that 
the Voges-Proskauer reaction, like the high gas ratio and the alkalinity 
to methyl red, is characteristic of non-fecal strains and, therefore, of 
much sanitary significance. Harden (1901), in his various publications, 
has reported his studies on the chemistry of this reaction which are well 
reviewed by Levine. The red color which results is due to a definite 
end product in the fermentation of glucose. Voges and Proskauer 
(1898), in their original publication, describe the test as follows: 
" On addition of caustic potash, we observed a new and interesting 
color reaction. If the tube be allowed to stand twenty-four hours and 
longer at room temperature, after the addition of the potash, a beautiful 
fluorescent color somewhat similar to that of a dilute alcoholic solution 
of eosin forms in the culture fluid particularly at the open end of the 
tube exposed to the air. We have investigated a few of the properties 
of this coloring substance which is not produced by the action of the 
alkali on the sugar and have found that it is fairly resistant to the action 
of the external air. After a time, however, it becomes paler, and 
finally gives place to a dirty greenish brown." 
Harden and Walpole (1905-6) could not account for all of the car- 
bon in the dextrose by the ordinary products (organic acids, alcohol, 
etc.) along with these substances was secured a glycol which was made 
up of 2 : 3 butylene glycol. 
H H H H 
Illi 
H-C-C C C-H 
H OH OH H 308 
WATER HYGIENE 
This was oxidized to acetyl-methyl-carbinol. 
H H OH 
II II I 
H-C-C C-C-H 
H OH H 
When this was mixed with potassium hydroxide, in the presence of 
peptone, an eosin-like color was obtained after a period. Walpole (1910) 
found that under aerobic conditions Bacillus aerogenes gave a greater 
amount of acetyl-methyl-carbinol. The coloration was not secured when 
butylene glycol or acetyl-methyl-carbinol were mixed with potassium 
hyrdoxide. Harden (1905) later explained this by finding that the 
acetyl-methyl-carbinol was oxidized to diacetyl CH3CO-CO-CH3. 
This, in some way, acted with the pepton to give a pink color. Harden 
and Norris (1911) later secured the same pink color between diacetyl 
and arginine, dicyanamide, creatin and gaunidine acetic. They state 
that the reaction depends upon the group NH=C-N=HR, R has not 
I 
NH2 
been determined. 
Levine demonstrated that the Voges-Proskauer reaction had much 
sanitary significance and that it would distinguish between fecal and 
non-fecal strains of Bacillus colon. He states that the natural habitat 
of those strains which form the acetyl-methyl-carbinol is probably the 
soil. Bacillus coli from non-fecal origins would then be methyl red and 
V-P plus. 
Standard Methods gives the following procedure for the differen- 
tiation of fecal from non-fecal members of the B. coli group. 
American Public Health Association Method for the Differentiation 
of Fecal and Non-fecal B. coli. (1) At least ten cultures should be 
used. If possible these should be sub-cultured from plates made direct 
from the water since all the cultures obtained by plating from fermenta- 
tion tubes may be descendants of a single cell in the water. If cul- 
tures from water plates are not available those obtained from the plates 
made as prescribed under B may be used. 
(2) Inoculate each culture into dextrose potassium phosphate broth,* 
* The preparation of this medium has been described in the chapter on media. 
Since devising this medium, these investigators (Clark and Lubs, 1917) have de- 
scribed the preparation of a substitute. The new medium is a synthetic solution 
without Witte's peptone. Aspartic acid is used in its place, thus satisfying the 
nitrogen requirements and acting as a buffer. The preparation of this new medium 
is also given in the chapter on media. FECAL AND NON-FECAL B. COLI 
309 
adonite broth and gelatin. For additional confirmatory evidence 
inoculation may be made into tryptophane broth and saccharose broth. 
The dextrose broth must be incubated at 30°. Other sugar broths may 
be incubated at 30° or 37° as convenient. Gelatin should be incubated 
at 20°. 
(3) After forty-eight hours record gas formation in adonite and 
saccharose broths. Determine indol formation in tryptophane broth 
by adding, drop by drop, to avoid mixing with the medium, about 1 c.c. 
of a 2 per cent alcoholic solution of p-dimethyl amido-benzaldehyde, 
then a few drops of concentrated hydrochloric acid. The presence of 
indol is indicated by a violet color. 
(4) After five days apply methyl red test and Voges-Proskauer test 
to dextrose broth. 
Methyl Red Test 
Indicator Solution. Dissolve 0.1 gm. methyl red in 300 c.c. alcohol 
and dilute to 500 c.c. with distilled water. 
Procedure in Test. 1. To 5 c.c. of each culture add five drops of 
methyl red solution. 
2. Record distinct red color as methyl red+, distinct yellow color 
as methyl red - , and intermediate colors as ?. 
Voges-Proskauer Test 
To the remaining 5 c.c. of medium add 5 c.c. of a 10 per cent solution 
of potassium hydroxide. Allow to stand over night. A positive test 
is indicated by an eosin pink color. 
(5) Gelatin tubes should not be pronounced negative until they 
have been incubated at least fifteen days. 
The following group reactions indicate the source of the culture 
with a high degree of probability. 
Methyl red+ 
V oges-Proskauer- 
Gelatin - 
Adonite - 
Indol, usually - 
Saccharose, usually- 
B. coli of fecal origin WATER HYGIENE 
310 
Methyl red - 
V oges-Proskauer+ 
Gelatin - 
Adonite+ 
Indol, usually + 
Saccharose+ 
B. aerogenes of fecal origin 
Methyl red - 
V oges-Proskauer+ 
Gelatin - 
Adonite - 
Indol, usually- 
Saccharose+ 
B. aerogenes, probably not of fecal origin 
Methyl red - 
V oges-Proskauer+ 
Gelatin+ 
Adonite+ 
Indol, usually - 
Saccharose+ 
B. cloacae, or may not be of fecal origin 
The Treasury Department Standard for the Examination of Water 
on Interstate Common Carriers. The following method for the exam- 
ination of water on Interstate common carriers has been formulated 
by a committee of prominent sanitarians. The permissible limits of 
bacteriological impurity are stated as follows: 
1. The total number of bacteria developing on standard agar plates, in- 
cubated twenty-four hours at 37° C., shall not exceed 100 per cubic centimeter; 
provided, that the estimate shall be made from not less than two plates, showing 
such numbers and distribution of colonies as to indicate that the estimate is 
reliable and accurate. 
2. Not more than one out of five 10 c.c. portions of any sample examined 
shall show the presence of organisms of the Bacillus coli group when tested as 
follows: 
(a) Five 10 c.c. portions of each sample tested shall be planted, each in a 
fermentation tube containing not less than 30 c.c. of lactose peptone broth. 
These shall be incubated forty-eight hours at 37° C. and observed to note gas 
formation. 
(6) From each tube showing gas, occupying more than 5 per cent of the 
closed arm of fermentation tube, plates shall be made after forty-eight hours' 
incubation, upon lactose litmus agar or Endo's medium. 
(c) When plate colonies resembling B. coli develop upon either of these plate 
media within twenty-four hours, a well-isolated characteristic colony shall be TREASURY DEPARTMENT STANDARD 
311 
fished and transplanted into a lactose-broth fermentation tube, which shall be 
incubated at 37° C.- for forty-eight hours. 
For the purposes of enforcing any regulations which may be based upon 
these recommendations the following may be considered sufficient evidence of 
the presence of organisms of the Bacillus coli group. 
Formation of gas in fermentation tube containing original sample of water (a). 
Development of acid-forming colonies on lactose-litmus-agar plates or bright 
red colonies on Endo's medium plates, when plates are prepared as directed 
above under (6). 
The formation of gas, occupying 10 per cent or more of closed arm of fer- 
mentation tube, in lactose peptone broth fermentation tube, inoculated with 
colony fished from twenty-four-hour lactose litmus agar or Endo's medium plate. 
These steps are selected with reference to demonstrating the presence in 
the samples examined of aerobic lactose-fermenting organisms. 
3. It is recommended, as a routine procedure, that, in addition to five 10 c.c. 
portions, one 1 c.c. portion, and one 0.1 c.c. portion of each sample examined be 
planted in a lactose peptone broth fermentation tube in order to demonstrate 
more fully the extent of pollution in grossly polluted samples. 
4. It is recommended that in the above-designated tests the culture media 
and methods used shall be in accordance with the specifications of the Committee 
on Standard Methods of Water Analysis of the American Public Health Asso- 
ciation, as set forth in Standard Methods of Water Analysis (A. P. H. A., 1912). 
This standard has been applied to the examination of water from 
trains by Bartow (1916), Creel (1914), Hanford (1916), and to water 
from boats by Cobb, Williams and Letton (1916). Bartow confirmed 
the presence of B. coli in 83 per cent of the samples examined. Creel 
found that an anaerobic bacillus which formed gas was responsible for 
gas in the presumptive test in 91 out of 421 samples. Such data indicate 
the significance of the confirmatory test. More data is required before 
it may be determined whether this method is the best. Letton (1917) 
has later stated that the requirement of " not more than 100 colonies 
per cubic centimeter on agar " of the Treasury Department standard 
is very lenient. He regards the limit of permissible B. coli (not more 
than two per 100 c.c.) as not too low. 
Lactose Bile in Water Analysis. Lactose bile was advocated for 
some time for use in the presumptive test for B. coli. It was supposed 
to inhibit other bacteria and a few of the weaker types of B. coli. For 
that reason it was deemed superior for the presumptive test than the 
ordinary carbohydrate broths. Jordan (1915) found that the typical 
as well as the typical B. coli were inhibited. The work of Cummings 
(1917) for the United States Public Health Service is interesting in this 
connection. He found, in his stream pollution studies on the Potomac 312 
WATER HYGIENE 
river that lactose broth was the better medium. In 1851 parallel sets of 
lactose broth and lactose bile tubes, lactose broth gave higher results, 
confirmed as well as presumptive, than lactose bile. It was granted, 
however, by Cummings that, in the lower reaches of the river, remote 
from pollution, the bile tubes were more reliable. The opinion of other 
bacteriologists has been that for highly polluted waters the lactose 
broth was the better medium, and for slightly polluted waters lactose 
bile gives the better results. This is attributed to the presence of spore- 
forming lactose fermenting anaerobic bacteria which are not manifest 
when the number of B. coli are large. Obst (1916) also found that the 
bile inhibited about one-half of the typical B. coli. The general use, 
then of lactose broth in place of lactose bile seems to be justified. 
Hauser (1917) has described the method used for isolating and iden- 
tifying B. coli at the Cincinnati filtration plant. This procedure is 
interesting and valuable because it is representative of the practice in 
one of the largest water-treatment plants. 
The B.coli group may be defined as aerobic non-spore forming Gram negative 
bacilli, fermenting lactose with production of gas, and not liquefying gelatin. 
At the Cincinnati filtration plant the attempt is made to carry out the examina- 
tions so that the above definition is fulfilled as completely as possible, consistent 
with the speed and facility necessitated by routine water analysis. 
The particular water samples under examination are planted into lactose 
broth. Lactose broth is used rather than lactose bile, which is the medium 
recommended in the 1912 edition of the Standard Methods of Water Analyses of 
the A. P.H. A., for the reason that, not only at this laboratory, but at the labora- 
tory of the Ohio River Investigation of the United States Public Health Service 
and other laboratories, it has given consistently a higher percentage of con- 
firmed end-results from original inoculations. In planting the water sample 
decimal dilutions are used to facilitate computations of the B. coli index. In 
routine examinations, at least three dilutions are used in order to obtain, if pos- 
sible, a negative and a positive test. The formation of gas in any amount after 
forty-eight hours' incubation at 37° C. is recorded as positive and considered as 
presumptive positive. The smallest quantity of water giving gas is then 
confirmed. 
A loopful of culture from the tube furnishing the presumptive test is stroked 
upon Endo's medium previously poured in plates. The plates are incubated 
at 37° C. for twenty-four hours. If no aerobic colonies (sterile plate) develop, 
the respective gas-formation is considered due to anaerobes, and B.coliis recorded 
as not present. If aerobic colonies develop, the most typical of B. coli is trans- 
planted into lactose broth and gelatin and a microscopical examination made 
directly. As there are several varieties of typical B. coli colonies, experience 
alone will determine the choice. 
If in the lactose broth transplant, no gas is formed after the forty-eight CINCINNATI PROCEDURE 
313 
hours at 37° C., the result is considered negative, and B. coli is recorded as not 
present. If gas is formed the result is considered positive for B. coli, subject to 
the results of the gelatin transplant and a microscopical examination. If after 
forty-eight hours' incubation at 37° C. the gelatin transplant will solidify on 
cooling, the culture is considered as non-liquefying. If the gelatin does not 
solidify on cooling, and gas is formed in the lactose broth transplant and the 
microscopical examination demonstrates a bacillus, the organism is called B. 
cloaca: and included in the B. coli numerical estimation as it has the same sani- 
tary significance. 
The microscopical examination consists of a smear made according to the 
method of Gram from the Endo plate colony selected for transplanting. A 
Varying Quantities (0.01-0.1-1.0 c .c .etc.) 
Planted into 
Lactose Broth 
Gas Formation-Presumptive Positive 
Smallest Quantity giving gas 
is Plated on Endo's Medium 
Sterile Plate 
Negative 
Typical or Atypical Colony 
Fished and Planted into 
Lactose Broth 
B 
I Gelatine | 
No Gas Formed 
Negative 
Gas Formed 
Liquefaction 
X Negative 
Microscopical 
Examination 
Non- 
Liquefaetion 
Not A - Bacillus 
Gram-negative 
Non-spore former 
Negative 
A ■ Bacillus 
Gram-negative 
Non-spore former 
B. Coli-Confirmed 
Method Followed at Cincinnati Plant for Identification of B. coli. 
Gram negative non-spore-forming bacillus obtained following the above pro- 
cedure fulfills in every particular the definition for the Bacillus coli. If other 
than a gram-negative non-spore-forming bacillus is obtained, it is not considered 
B. coli and is so recorded, but such a result is unlikely and has not been en- 
countered in experiences at Cincinnati. The liquefaction of gelatin and dis- 
cordant results in the microscopical examination accompanying gas production 
in the lactose broth transplant are likely to be due to mixed cultures, in which 
case separation should be made by replating from the lactose broth transplant. 
" Estimation of the B. coli index is made according to Phelps' method 
-that is, the reciprocal of the smallest volume of water which gave a positive 
test is taken as the approximate number of B. coli per cubic centimeter and is 
recorded as such. In case of an anomaly it is assumed that the positive result 
should have been negative and the negative result positive and is so considered 314 
WATER HYGIENE 
in obtaining the B. coll index. If a negative confirmation test is obtained, 
B. coli is considered present in the next larger quantity without confirmation. 
If a negative result is obtained from the largest quantity planted, either by no 
gas formation in the original planting or by a negative confirmation test, B. coli 
is considered present in the next larger amount that would have been planted, 
except that a negative result in 100 c.c. is recorded at zero. The assumption 
is value only in the case of waters such as are encountered in water purification 
plants where the approximate coli content of the water is known. Such assump- 
tions are rarely necessary as the quantities of water chosen for examination can 
ordinarily be changed in sufficient time to always obtain a positive test. It is 
probable that this assumption causes less error than would be introduced by 
either omitting the figure from the averages or calling it zero. 
"The laboratory work above described with the numerical estimations of 
B. coli derived, are recorded for the month under headings of waters in the 
consecutive stages of purification. The form used is convenient and compact 
and lends itself readily to a grasp of conditions throughout the plant at any 
time. Five days from the collection of the water sample, B. coli may, following 
the above scheme, be satisfactorily and conclusively isolated and identified 
to conform to the definition. 
The following table will show the number of samples examined, according 
to this method, from January to October, 1916, and the percentage of samples 
confirmed as B. coli. 
River 
Water. 
Settled 
Water. 
Applied 
Water. 
Filtered Water. 
Effluent from 
Filters. 
Outlet Clear 
Water 
Reservoir. 
Samples tested 
271 
257 
266 
258 
194 
Percentage confirmed. . 
97.4 
91.0 
96.6 
*94.9 
*86.1 
* Includes one sample that showed B. Cloacae. 
The B. coli Index. This was proposed by Phelps and was incor- 
porated in a report of the Committee of Water Purification Plants. 
The following is taken from that report. The B. coli index is the approx- 
imate number of B. coli per cubic centimeter as determined from qual- 
itative tests made on different quantities of water. For any individual 
sample, it may be taken as the reciprocal of the smallest volume of 
water used in the test which gave a positive result. Thus, if a sample 
gave a negative test with 0.1 c.c. and a positive test with 1.0 c.c. and 
10.0 c.c., the B. coli index would be 1 - 0.1 = 10. The B. coli index for a 
single sample is not very accurate. The index becomes more accurate 
as the square root of the number of tests increases. INTERPRETATION OF RESULTS 
315 
The following is an example: Write down the percentage of plus 
( + ) tests for the quantities examined expressed as decimals of 100. 
Take the differences between these percentages. Multiply each of 
these differences by the reciprocal of the quantity corresponding to the 
larger of the two percentages from which such difference is taken. The 
sum of these products will be the B. coli index. In the following exam- 
ple, the B. coli index may be taken as 0.019-equivalent to 19 B. coli 
per cubic centimeter 
Quantity of 
Water 
Examined. 
Per Cent 
Positive 
Tests. 
Expressed as 
Decimals 
of 100. 
Differences. 
Reciprocals of 
Quantities. 
Product. 
100 
13.8 
0.1380 
.1090 
.01 
.00109 
10 
2.9 
0.0290 
.0260 
.10 
.00260 
1 
0 3 
0.0030 
.0026 
1.00 
.00260 
0.1 
0.04 
0.0004 
.0003 
10.00 
.00300 
0.01 
0.01 
0.0001 
.0001 
100.00 
.01000 
In this B. coli index the committee has made no provision for per- 
centage reduction. It recommends that it be omitted for various 
reasons. The bacteria in an effluent from a filter are not derived from 
the raw water. It required quite a little time for a water to pass through 
a filter. The percentage removal is, to a certain extent, a function of 
the number in the raw water and does not vary with the number left 
in the filtered water. Other objections are also mentioned by the 
committee. 
Interpretation of Results. The accurate interpretation of results 
of analysis is a part of sanitary work which is often taken too lightly. 
This problem is rendered more difficult by the lack of an ideal indicator 
of pollution. No organism has been described which is always and only 
present in water and on foods which have received sewage pollution. 
Thresh (1913) gives an interesting discussion with regard to what should 
be regarded as polluted water. He sent questions to Houston and Wins- 
low seeking their opinions on the subject. He submitted the following 
questions to Houston of the Metropolitan Water Board: What bac- 
teriological proof would you consider conclusivae as to the pollution of a 
water with sewage or manurial matter? ' Houston did not give a definite 
answer and stated that one was not possible. He advises that the final 
verdict should rest on data secured from all analyses. His experience 
seems to' indicate that B. coli should be absent from 100 c.c. quantities 
of the water in a majority of representative samples, preferably not less 316 
WATER HYGIENE 
than 10. What bacteriological proof would you consider conclusive that 
a water is free from such pollution or so free that it is safe for drinking 
purposes? Houston regards the absence of B. coli from 100 c.c. of the 
deep well water as reasonably sufficient evidence of safety and the greater 
the number of these negative results, the more satisfactory is the water. 
A water which never contains B. coli in 100 c.c. quantities is to be 
regarded as a safe drinking water. A water which contains B. coli in 
less than half of the samples examined occupies an intermediate posi- 
tion but is probably reasonably safe. One which contains B. coli in more 
than half should be regarded with some preliminary disfavor. 
Winslow replied to these questions and stated that the presence of 
colon bacilli in all of ten duplicate 10 c.c. samples, is reasonable proof 
of the presence of sewage or manurial matter while the absence of colon 
bacilli from such samples would be reasonable proof of the absence of 
such sewage. Winslow would carry out a confirmative test to show 
that the fermentations were due to true B. coli. 
McLaughlin (1914) regards a safe water as one which contains no 
pathogenic bacteria. According to his opinion a water containing no 
more than 2 B. coli per 100 c.c. is satisfactory. A filter plant that can- 
not furnish such an effluent is either inefficiently operated or is handling 
a raw water which imposes an unreasonable load on the filters. 
In light of the recent contributions of Rogers, Clark and Lubs on 
the differentiation of the members of the colon group, in which they 
have shown that B. coli of fecal origin is methyl red plus (+) careful 
extensive work must be done in the future to work out the relation of 
the methyl red test to the analysis and control of water supplies. Clark 
(1918) has advised this. Orchard (1918), in view of the lack of uni- 
formity concerning standards has recommended that the societies con- 
cerned with water analysis appoint committees to study this problem. 
This would probably be the quickest and most reliable method of 
handling the question. 
Wording of Reports on the Results of Bacteriological Examination of 
Water. This is often a difficult procedure for the analyst especially if 
the certificate accompanying the sample is not filled in. An investiga- 
tion of this question reveals the lack of uniformity in reports from dif- 
ferent laboratories on the same sample of water. This was realized 
among the English analysts to such an extent that a committee of the 
Royal Institute of Public Health was appointed to go over the subject. 
They reported as follows: When the source and surroundings are un- 
known, it is undesirable to report as follows: 
" This water is grossly polluted and is dangerous for drinking " or PATHOGENIC BACTERIA IN WATER 
317 
" This water is of superlative quality and is absolutely safe for con- 
sumption." 
This committee recommended the use of guarded terms as follows: 
" This particular sample of water as judged by the above tests 
yields unsatisfactory results (this may be amplified) and suggests the 
necessity of inquiring into the local conditions with regard to the source 
of contamination together with consideration of the desirability of 
further samples being examined." 
Satisfactory results may be reported as follows: 
" This particular sample of water, as judged by the above tests 
yielded satisfactory results " (may be amplified) " but in the absence of 
knowledge of local conditions, it is impossible to say that the supply 
will always attain the same standard of purity indicated by the present 
results." 
From the above report some valuable suggestions may be secured 
with regard to the proper wording of reports. The layman usually 
must depend on the analyst for a correct interpretation of his results. 
Too strong opinions should be avoided since they may cause the lay- 
man to secure the wrong impression. Some laboratories simply use the 
words " good " and " bad." It is the case in some laboratories that this 
part of the work is handled by a person who has had too little experience 
or who has not informed himself with regard to the prevailing standards. 
No part of the analysis should have more attention than the reporting 
of the results and their interpretation to the clients of the laboratory. 
Detection oe Pathogenic Bacteria in Water 
There are a few diseases that have been spread so much by water 
that they have become known as water-borne diseases. In some of these 
the isolation of the causal organism has been atended with little suc- 
cess even if it is present. 
Bacillus typhosus. The isolation of Bacillus typhosus from water 
has not been attended with much success. When attempts have been 
made on polluted water known to contain the bacilli, there has been no 
certainty in the results. Those instances in the literature where it is 
claimed that this has been done, are lacking in sufficient confirmatory 
evidence. It is possible that by means of recent advances in technique, 
the isolation of B. typhi will be attended with more success than in the 
past. Most of the methods which are available for isolating the organ- 
ism from water, have been devised for isolating the organism from other 318 
WATER HYGIENE 
material such as feces and urine. The reader is also referred to the 
methods developed for the isolation of B. typhi from feces. 
Drigalski and Conradi Method (1902). The preparation of the 
medium for this method has been described in the chapter on the 
media. The medium is solidified in Petri dishes which are allowed to 
become thoroughly dry before use. This may be accomplished by 
allowing the plates to stand at room temperature with the covers sus- 
pended above or in the 37° C. incubator. The surface is then streaked 
with the sample by means of a sterile bent glass rod. It is often a good 
plan to concentrate the water, either by reduced pressure of filtration. 
Bacillus colon forms red opaque colonies on this medium while Bacillus 
typhosus forms a blue and often transparent colony. Suspicious col- 
onies must be subcultured into other media and a complete cultural 
and morphological study made. Identification must be confirmed by 
agglutination reactions. 
Endo's Medium. The preparation of this medium has been described 
in another chapter. The sample of water is streaked over the surface 
and the plates incubated at 37° C. for about fifteen hours. At the end 
of this time the Bacillus colon colonies will be red with possibly a metallic 
appearance, while the Bacillus typhosus colonies will be colorless. The 
same will be true with regard to the paratyphoid colonies. These sus- 
picious colonies should be subcultured into other media and a con- 
firmatory test made by means of the agglutination reactions. 
Kligler (1917) has shown that a differentiation in the typhoid- 
paratyphoid group may be made by means of hydrogen sulphid forma- 
tion and gas formation in the following medium which is semi-solid. 
1. Meat infusion agar containing | or 1 per cent of agar (depending 
on the moisture content of the agar shreds). 
2. 0.1 per cent of glucose. 
3. 0.05 per cent lead acetate. 
The agar is prepared in the usual way except that the amount of agar 
is reduced. The sugar and lead acetate are dissolved in water sepa- 
rately and added to the sterile agar. The agar should be cooled to 60° C. 
in order not to precipitate the peptones. Stab inoculations are made. 
Strains of B. typhi brown the medium along the line of inoculation. 
B. paratyphi B. causes browning and gas formation; B. paratyphi A. 
causes gas but no browning. This has been confirmed by Jordan and 
Victorson (1917) who used a medium containing 1.5 per cent of agar. 
Morishima (1918) also described a similar method. The differentiation 
is secured in about the same way as with Kligler's method. 
Russell's medium may be used to good advantage with any of the RUSSELL'S MEDIUM 
319 
above methods for identifying members of the colon-typhoid group. 
Russell has described the preparation and use of this medium in the fol- 
lowing manner: 
" Enough 5 per cent aqueous solution of litmus (3 to 5 per cent) is 
added to plain agar (2 to 3 per cent) which usually has a reaction of 
about .8 per cent to phenolphthalein, to give it a distinct purple violet 
color, the amount of the litmus depending on the original color of the 
agar; dark requiring more than light, and the reaction then adjusted 
by adding sodium hydrate until the mixture is neutral to litmus. Next, 
and last, 1 per cent of lactose and of 1 per cent of glucose dissolved in 
a small amount of hot water is added and the medium tubed for slants. 
The sterilization is done in the Arnold and, because of the danger of 
breaking down the lactose, must not be carried too far; if the tubes are 
packed loosely in the sterilizer basket to allow good circulation of steam, 
ten minutes on the first and fifteen on the second day has been time 
enough. The tubes are then slanted and stored in small quantities in 
a dark place. 
" On this double sugar tube, the typhoid bacillus gives, after an 
incubation period of from eight to eighteen hours, an extremely char- 
acteristic appearance; the surface growth is filiform and colorless, the 
upper part of the tube is unchanged in color but the lower part, the butt, 
is a brilliant uniform red. The entire point of the medium rests upon 
the difference in the changes produced by the growth of the typhoid 
bacillus under aerobic and under the imperfect anaerobic conditions 
found in the butt of the tube, where the bacillus obtains its oxygen by 
breaking down the glucose with the liberation of considerable acid; on 
the surface, however, in the presence of free oxygen, no acid is formed. 
" The colon bacillus, which is often slow in producing acid on the 
Endo plate, shows abundant gas and acid formation on this medium. 
The tube is reddened throughout, both above and below, and since the 
abundant lactose is attacked equally with the glucose there is exuberant 
gas formation. . . . 
" The bacillus Jecalis alkaligenes and other alkali formers leave 
the medium unchanged or slightly bluer. The staphylococcus reddens 
the tube above but leaves it blue below; the streptococcus intestinalis, 
when it grows well, gives a beaded growth and reddens the tube slightly 
throughout. B. subtilis, which is commonly found in feces, usually 
leaves the medium unchanged but may redden it below without pro- 
ducing gas, yet the heavy, rough surface growth suffices for its differ- 
entiation. B. pyocyaneus gives a greenish blue surface growth and leaves 
the color of the medium unchanged. B. proteus produces small gas bub- 320 
WATER HYGIENE 
bles in the depth and reddens and then decolorizes the butt very early, 
while the upper part of the tube is unchanged except for the spreading 
surface growth. 
" All dysentery bacilli alter the medium in the same manner as 
typhoid, yet the quantity of acid-produced is small and the reddening 
is usually confined to the line of inoculation. This reaction is so char- 
acteristic that we use this medium regularly in isolating dysentery bacilli; 
in fact, the same media and technique are used for both typhoid- and 
dysentery. 
" The paratyphoids leave the upper part of the medium unchanged, 
the surface growth is like typhoid but in the butt of the tube in addition 
to the reddening are found a few small gas bubbles. The only organ- 
isms which may simulate the paratyphoids are slow colons and these 
must be thrown out by agglutination tests and further observation of 
the sugar tube." 
Kligler (1917) has stated that by adding 0.5 per cent of basic lead 
carbonate to Russell's medium, a good differentiation between B. para- 
typhi A and B. paratyhpi B may be secured. Krumwiede and Kohn 
(1917), by adding 1 per cent of sucrose to Russell's medium found that a 
large percentage of 11 intermediates " giving a typical paratyphoid 
reaction could be excluded. 
Bacillus Dysenteriae. This organism may be isolated by means 
of Endo's medium plates. It grows as colorless colonies which 
distinguishes it from other bacteria. After the use of Russell's 
medium, agglutination reactions should be employed to confirm the 
results. 
Agglutination. The serum of a person who has had typhoid fever 
or has been immunized against this disease will cause the agglutination 
of the typhoid bacillus. This reaction is not specific, however, unless 
high enough dilutions are used to prevent the agglutination of other 
members of the colon-typhoid group. In low dilutions of serum the 
reaction is not specific for closely related bacteria. Citron (1914) 
reports the following data with regard to a strongly agglutinating typhoid 
and strongly agglutinating cholera serum. 
Agglutination Titre. 
Of Typhoid 
Serum. 
Of Cholera 
Serum. 
Against typhoid 
1-2000 
1-10 
Against paratyphoid 
1-100 
1-10 
Against B. coli 
1-25 
1-10 
Against cholera 
1-10 ' 
1-3000 AGGLUTINATION 
321 
Macroscopic Agglutination. By this procedure the presence of 
agglutinins is demonstrated without the use of a microscope. To carry 
out this procedure, a series of small tubes are necessary. Mixtures of 
physiological salt solution, a twenty-four-hour broth culture of B. typhi 
and serum are used. Dilutions of 1-20, 1-40, 1-80 and 1-160 should be 
prepared. These may be secured as follows: The dilution of serum 1-10 
may be secured by drawing the serum up to 1.0 mark on the white blood 
corpuscle pipette and then filling the bulb to the mark with sterile salt 
solution or bouillon. Transfer 1 c.c. of this 1-10 dilution to the second 
test tube and add 1 c.c. of the twenty-four-hour broth culture. This 
will give the 1-20 dilution. The other dilutions may be prepared in 
the same way. These tubes should be incubated at 37° C. and exam- 
ined at frequent intervals for the presence of a precipitate. 
Microscopic Agglutination. This requires the use of a microscope 
but has the advantage of requiring less time. The same dilutions 
should be prepared as were mentioned above under macroscopic agglu- 
tination. Instead of putting them into small test tubes hanging drop 
mounts are made which are examined for agglutination after thirty 
minutes. Agglutination in a dilution of 1 to 80 after thirty minutes 
constitutes a positive reaction. 
Identification of Bacteria by Agglutination. The technique is essen- 
tially the same as for the detection of the presence of agglutinins. In 
this case the bacteria are the unknown factors while the serum is known 
to be specific for a certain organism. The bacterium should be grown 
in broth for twenty-four hours and tested against dilutions of the serum. 
Coles' Method for the Microscopic Agglutination of Bacteria. The 
author claims that the method is easy, rapid and requires less blood than 
some of the other methods. 
1. Draw a line across the middle of two slides at right angles to their 
long axes. 
2. Spread a film of blood to be examined on one-half of each slide, 
and, when dry, spread film of blood from a person who has not had 
typhoid fever or received protective inoculation as a control over the 
other half. Dry the smears carefully over a flame. 
3. By means of a platinum needle place a small drop of an emulsion 
of killed typhoid bacilli on the center of each half of both slides. Run 
the drop well over the smears taking care not to pass from one-half to 
the other without sterilizing the needle. 
4. On one slide carefully place a cover glass on each half of the slide 
taking care that they are well separated by a wax pencil mark. 
5. Place the other slide on a wet piece of blotting paper and cover 322 
WATER HYGIENE 
with a Petri dish to prevent evaporation. Allow to stand for fifteen to 
twenty minutes. At the end of this time, stain and examine for clump- 
ing of the bacteria. 
Examine both slides for agglutination of the bacteria. 
Isolation of Microspira Cholerae from Water; Metschnokoff's 
Method after Besson. Prepare a series of flasks and add 200 c.c. of 
the water under examination. To each flask add the following solution: 
Water 50 c.c. 
Peptone 2 gms. 
Common salt 2 gms. 
Gelatin , 4 gms. 
Sodium carbonate Sufficient to make alkalin 
Sterilize this in the autoclave. Then add 150 c.c. of the water under 
examination and incubate at 37° C. If the water contains microspira 
a thin film will form on the surface in about eight hours. This film 
should then be examined under the microscope and if microspira are 
present they must be proven to be the pathogenic Microspira cholera. 
Besson gives the following " classical characteristics of the M. cholera." 
1. Characteristic appearance on gelatin plates and in gelatin stab. 
2. The presence of and the number of flagella. 
3. The nitroso-indol reaction in pepton water. 
4. Virulence for guinea pigs. 
5. The immunity reaction (Pfeiffer's reaction). 
6. Agglutination by anti-cholera serum. 
The agglutination reaction is probably the most reliable since this 
is quite specific. Besson mentions that the other characteristics of 
Microspira cholera are not fixed and are thus of little use in identifying 
a suspected organism. 
The Cholera-red Reaction. This is a reliable test for the diagnosis 
of Microspira cholera. Not many other organisms. The test is carried 
out by adding a little sulphuric acid to a broth culture of the organism. 
A red color results due to the presence of both indol and nitrites which 
are formed by the organism. Some care must be used in controlling 
the factors involved in the test. The pepton must be proven to yield 
the test when a known organism is used. The cholera-red test is of 
much value in identifying this organism in water. 
Detection and Isolation of Bacillus Anthracis from Water. Pasteur 
used the resistance of the spores as a means of isolating the bacillus from 
different substances. Heat a small quantity of the water in a water 
bath for twenty minutes at 80-85° C. At the end of this time, make POLLUTION OF WATER COURSES 
323 
agar plates using one or two decimal dilutions and incubate at 20° C. 
for several days. All suspicious colonies must be studied morphologi- 
cally, culturally and microscopically. The cultures which pass through 
this treatment should be injected into healthy guinea pigs. Koch's 
postulates must be satisfied before the suspicious colonies are accepted 
as anthrax. 
Bacillus Tuberculosis. The search for this organism in water has 
not been carried out to any great extent. If acid-fast bacteria 'are 
isolated from water, it is necessary to confirm the findings by animal 
inoculation. 
Brown's et al. Method. Brown and his collaborators used this 
method to detect the tubercle bacillus in river water. Near the source 
of pollution (sewer outlet) large bottles of water were collected. Further 
down the stream different methods were used. Cheesecloth bags were 
stretched across the surface of the stream and allowed to remain in 
position from one to two hours, after which they were put into pails 
and carried to the laboratory. According to this method, they should 
then be carefully washed with sterile water and the wash water put 
into wide-mouth bottles, saturated with sodium chloride and allowed 
to stand in the dark for several hours; at the end of this time, the 
scum should be collected and transferred to 50 c.c. centrifuge tubes. 
Ten c.c. of NaOH are added to each tube and the tube incubated for 
one-half hour. Neutralize the contents of the tube with NHC1, cen- 
trifugalize, and study the sediment microscopically, inoculate part into 
egg media and inject part into guinea pigs. 
Tracing Pollution in Water Courses. Different methods have been 
used and also different materials such as, sodium chloride, lithium salts 
fluorescin, and cultures of B. prodigiosus. 
Salt was used by McCallie (1904), in Georgia, for the purpose of 
demonstrating the effects of discharging sewage into deep wells. A 
large amount of salt was put into a 124-ft. drilled well, and the content 
of chloride in the water of the surrounding wells was determined. 
Increased content of chloride showed that there was underground con- 
nection between the wells and springs in the vicinity. 
Dole (1906) summarized his investigations on the use of fluorescein 
in the following statements: 
1. In determining the sanitary value of a well or spring it is more important 
to study the underground flow than to analyze the water itself. 2. Foreign sub- 
stances put into the aquifer and traced from point to point are of great value in 
this study. 3. With the fluorescope one part of fluorescein can be detected in 
10 billion parts of water. 4. Fluorescein is a particularly valuable flow indi- 324 
WATER HYGIENE 
cator for fissured or cavernized rocks. 5. It is also available in gravels, where 
it has been used with success. 6. It progresses at a slightly lower rate than the 
water in which it is suspended. 7. It is not decolorized by passage through 
sand, gravel, or manure; it is slightly decomposed by calcareous soils. 8. It is 
entirely decolorized by peaty formations and by free acids, except carbonic acid. 
Trillat (1899) states and claims that fluorescein can be detected 
in dilutions of 1 part in 2,000,000,000, but that before the dye is used a 
study of the soils should be made with regard to the presence of matters 
which decompose it. Marboutin (1901) comes to somewhat similar 
conclusions. Martel (1903) shows that fluorescein even in very con- 
centrated solutions decolorizes rather quickly after being kept in the 
sunlight. When it is kept in complete darkness, which would be the 
case in the earth, it did not change even after long periods. 
Gehrmann (1913) reports that Alba Orlandi and Roudelli, who used 
a suspension of B. prodigiosus, found that cultures of this organism 
poured on the ground passed through soil 200 meters, and Pfulil found 
that it took the same organism a short time to pass through 24 ft. of 
gravel. Gehrmann also reports a place at which wells 200 to 300 ft. 
deep too near an old canal were subject to entrance of contaminated 
water; no experimental data are given. 
Methods of Treatment 
After attempting to use a great many different compounds and 
methods the use of calcium hypochlorite and liquid chlorine is well 
established. 
Traube (1894) proposed the addition of bleaching powder to water. 
Since that time the use of hypochlorite has greatly extended in sanitation. 
Water to which this compound is added in the usual amounts is quite 
harmless. Professor Hulett, testifying in the Jersey City case, that in 
the Jersey City water to which had been added 10 lbs. of bleach per 
million gallons of water he was unable to detect the presence of free 
chlorine. Theoretically, it was possible for free chlorine to be present 
to the extent of 6.4 parts per trillion. It was also revealed that for a 
person to get a medicinal dose such as given in typhoid fever it would 
be necessary to drink 2,500,000 gals, of water so treated. This, with 
other data, indicates that there can be little objection to using a water 
which has received the usual amount of calcium hypochlorite. The 
waterworks officials have received all kinds of objections against the use 
of this chemical for water treatment. Some even claim that they can- 
not raise flowers of a certain tint on account of the chlorine in the water. "BLEACH " IN WATER TREAMTENT 
325 
Others have claimed that it was impossible to make tea with such 
water. 
Advantages and Disadvantages of " Bleach " in Water Treatment. 
These have been given as follows by Johnson (1910, 1913): 
Advantages 
1. Destruction of bacteria, especially intestinal forms. 
2. Reliability of chemical and ease of application. 
3. Absence of poisonous chemical compounds formed. 
4. Cheap cost of chemical. 
5. Rapid speed of reaction-no storage basins required. 
6. Saves coagulating chemicals. 
7. Allows much higher rate of filtration. 
8. Reduces clogging of filters and lengthens the runs. 
Disadvantages 
1. Does not destroy all spore forms. 
2. Does not get bacteria inside of suspended matter. 
3. Does not remove turbidity. 
4. Does not take color out or remove vegetable stain. 
5. Inability to remove organic matter. 
6. Leaves swampy tastes and odors. 
7. Does not soften the water. Increases the hardness slightly. 
Bleach usually contains a little CaO which takes out some CO2. 
8. Care must be used to allow for reducible compounds such as 
nitrites or oxidizable iron. 
The quantity of " hypo " to add to a water places the sanitarian 
between two fires-tastes in the filtered or final product and destruc- 
tion of the pathogenic bacteria. The absolute amount varies greatly. 
The bacteriological analysis together with turbidity determinations 
are probably the best indicators to tell whether the amount is sufficient. 
This must be determined for each separate water supply. Under 
ordinary conditions when added to the filtered water from 7-12 lbs. per 
million gallons is sufficient for disinfection. This will give from .28 
to .48 p.p.m. of available chlorine (based on 30 per cent bleach). Many 
factors influence the amount of bleach required by a water but tur- 
bidity is one of the most important especially when this is largely 
organic matter. The amount of bleach added to a water supply must, 
therefore, vary with the turbidity. Where storage basins are used, less 326 
WATER HYGIENE 
attention has to be given to the amount of bleach being added because 
one important function of storage, as Houston has pointed out, is 
equalization. 
For a time, there was some discussion with regard to whether the 
bleach should be added to the raw or filtered water. At present it 
seems to be the consensus of opinion that it should be added to the 
filtered water. The following facts bear on the consideration: 
1. When it is added to the raw water, a greater amount is required. 
2. The life in the " schmutzdecke " on the filter may be destroyed. 
3. Reactions between the organic matter and chlorine may yield 
compounds imparting a bad taste. 
4. The addition to the filtered water is easier since there would be 
no cumulative effect. 
The method of adding it to the filtered water has been standardized. 
It is usually mixed to a thick cream and later diluted with water. After 
settling, it is fed to the water supply through standard orifices by which 
the amount applied is regulated. This is an important part of the pro- 
cedure since overdosing must be avoided. 
Race (1918) has shown that by using a little ammonia along with the 
bleach, the amount of the bleach may be materially reduced. He 
regards the possibility of chloramine being formed as fairly conclusive. 
On Ottawa River water at flood period, when the water contained 
from 500 to 1000 B. coli per cubic centimeter, satisfactory results were 
obtained with 0.60 p.p.m. of available chlorine, and 0.13 p.p.m. of 
ammonia. This method of treatment is recommended for those waters 
where the margin between the dose required for satisfactory treatment 
and successful purification is small. It is well known among certain 
practical waterworks men that such is often the case. The character of 
the raw water may demand an amount of bleach which is very close 
to the amount which may be tasted in the water. 
The question of tastes has caused much trouble to practical water- 
works men. Some of the essential oils excreted by microorganisms 
resemble very much the odor of chlorine. This has caused undue blame 
to be put on the bleach. Lederer and Bachmann (1912) found that 
under experimental conditions the average amount of available chlorine 
that could be detected was around 0.6 p.p.m. while 0.5 p.p.m. could 
often be detected. Van Brunt placed the amount that could be tasted 
at 0.6 p.p.m. While the chemical nature of these compounds which 
cause tastes in water treated with chlorine has not been carefully inves- 
tigated, it is generally admitted that they may be related to the chlora- 
mines. APPLICATION OF " BLEACH " 
327 
Different means may be used to determine the amount of available 
chlorine which is being added to a water supply. The " bleach " may 
be analyzed for available chlorine to determine its strength in per cent. 
Calculations may then be made to find how much should be added 
to the mixing tanks. Lewis (1912) reports the following scheme used 
at Evanston, Ill.: 
jV = No. of gallons pumped per minute. 
1,000,000 ,, . . . , , . nn_ ___ . 
--- = M, or number of minutes to pump 1,000,000 gals.; 
C = No. of pounds of " hypo " to be added to each 1,000,000 gals, 
of water. 
P. or pounds of " hypo " to be added each minute. 
P 
- = No. of gallons of liquid to be added per minute. 
X = pounds of " hypo " in 1 gal. of solution. 
Changes in the rate of pumpage demand a change in the amount of 
chemical which is applied. This is easily accomplished by changing the 
size of the orifice. 
If the strength of the bleach solution is determined from that in the 
orifice box the following scheme will indicate how much is being applied 
to the water. 
7?X7O = p.p.m. available chlorine; 
Z^ = No. of cubic centimeters of N/10 Na2S20a used per 50 c.c. 
of bleach solution. 
Parts per million of available chlorine in the filtered water equals 
Gal. bleach solution bed 
p.p.m. Qa|jong 0£ wa£er pumped* 
Analysis of the dry bleaching powder which is necessary in the above 
method, is accompanied by certain errors. To obviate these, the bleach 
solution may be mixed in the usual way and samples removed directly 
from the orifice box. This procedure has the advantage that it is more 
direct-indicating the actual amount of available chlorine which is 
being applied to the water. The following calculations may be used: 328 
WATER HYGIENE 
Evaluation of Bleaching Powder 
Available Chlorine. Weigh 7.09 ms. of the sample from a weighing 
bottle into a mortar. Add water and rub into a smooth cream. Add 
more water, and after settling, decant the clear liquid into a liter flask. 
Add more water, grind and decant until all of the powder has been 
transferred to the flask. Dilute to the mark. Aliquot portions of this 
may be removed for analysis but in each case the flask should be thor- 
oughly shaken. 
Great care must be exercised in the sampling of bleach. It rapidly 
decomposes in the presence of air. Very often samples taken from the 
center of a carboy will contain a larger amount of available chlorine 
than a sample which is removed from the surface. 
A. Thiosulphate Method. 
Solution required: 
1. N/10 sodium thiosulphate (^28263). 
Dissolve 24.83 gms. of sodium thiosulphate in 1 liter of boiled, cooled 
distilled water. Boiling the distilled water decreases the error caused 
by the presence of CO2. The solution may be checked against standard 
iodine solution. It keeps very well on standing. 
2. Starch solution. 
Rub a gram of starch to a smooth paste and pour into boiling water 
with constant stirring. Decant after settling into test tubes putting 
about 5 c.c. into each tube. Plug and sterilize in the autoclave. 
3. Potassium iodide. 
Dissolve 25 gms. potassium iodide in 500 c.c. of distilled water. 
Procedure. Transfer 50 c.c. of the bleach solution (either that pre- 
pared from the powder or that from the orifice box) to an Erlemneyer 
flask by means of a pipette. Dilute with 50 c c. distilled water, add 
10 c.c. of the potassium iodide solution, a little acetic acid and titrate 
with the N/10 ^28263 until a faint yellow color remains. Add 1 c.c. 
of the sterile starch solution and continue addition of N/10 Na2S2O3 
until the blue color is discharged. The number of cubic centimeters of 
N/10 sodium thiosulphate used is equivalent to percentage of available 
chlorine in the sample. 
1 c.c. N/10 Na2S20s = .003545 gm. chlorine. 
1 c.c. N/10 Na2S2O3-1 per cent available chlorine. 
B. Arsenious Acid Method. 
Solutions required: 
1. N/10 arsenious acid. Transfer 4.95 gms. of pure arsenious acid 
to a beaker, add water and 107 gms. of Na2HPO4-I2H2O. Stir AVAILABLE CHLORINE 
329 
and beat until all of the arsenious acid is in solution. Cool and make 
up to 1 liter with distilled water. 
2. Iodized starch paper. 
Dip pieces of filter or other bibulous paper into a starch solution 
containing a very small amount of potassium iodide. Moisten when 
using. 
Procedure. Transfer 50 c.c. of the bleach solution (either that pre- 
pared from the powder or that taken from the orifice box) to an Erlen- 
meyer flask. Add very slowly the N/10 arsenious acid solution until a 
drop of the solution causes no dark coloration on the moist iodized 
starch paper. The number of cubic centimeters of arsenious acid 
solution used is equivalent to the percentage of available chlorine in the 
bleach. 
1 c.c. N/10 arsenious acid = .003545 gram cl. 
The computation to determine how much available chlorine may 
be made as follows: Take 50 c.c. of bleach solution from the orifice box 
and titrate with N/10 Na2S20s. One c.c. of this is equivalent to 
0.003545 gm. chlorine. If the 50 c.c. of bleach solution requires 30 c.c. 
of N/10 Na2§203 then 30 X 0.003545 X 20 = 2.127 gms. chlorine per liter. 
One liter=1000 gms., then the solution contains .2127 per cent 
available chlorine. A cubic foot of the solution weighs about 28,320 
gms. In this case then 1 cu. ft. of the solution will contain 59.094 
gms. of available chlorine. 
Examination of Water for Available Chlorine. The Committee on 
the Analysis of Water and Sewage of the American Public Health Asso- 
ciation recommended the following procedure. This has not been 
accepted by the Canadian Public Health Association. They have 
recommended that it be eliminated from standard methods. (Amer. 
Jour. Pub. Health 8. (1918) 320.) 
In waters that have been treated with calcium hypochlorite or liquid chlorine 
it is frequently advisable to ascertain the presence or absence of available 
chlorine. As the reagents which have been proposed for its detection are not 
specific for chlorine but give similar or identical reactions with oxidizing agents 
or reducible substances, care must be exercised in interpreting the results of such 
tests; nitrites and ferric salts are of common occurrence, and chlorates also may 
lead to misinterpretation in waters treated with calcium hypochlorite. 
Reagents.-1. Tolidin solution. One gram of o-tolidin, purified by being 
recrystallized from aclohol, is dissolved in 1 liter of 10 per cent hydrochloric 
acid. 
2. Copper sulphate solution. Dissolve 1.5 gms. of copper sulphate and 1 c.c. 
of concentrated sulphuric acid in distilled water and dilute the solution to 200 c.c. 330 
WATER HYGIENE 
3. Potassium bichromate solution. Dissolve 0.025 gm. of potassium 
bichromate and 0.1 c.c. of concentrated sulphuric acid in distilled water and 
dilute the solution to 200 c.c. 
Procedure.-Mix 1 c.c. of the tolidin reagent with 50 c.c. of the sample in 
a Nessler tube and allow the solution to stand at least five minutes. Small 
amounts of free chlorine give a yellow and larger amounts a yellow-green color. 
For quantitative determination, comparison of the color is made with that 
of standards in similar tubes prepared from the solutions of copper sulphate and 
potassium bichromate. The amounts of solution for various standards are 
indicated in Table XXVIII. 
Concentrations greater than 0.10 part per million of chlorine require use of a 
stronger bichromate solution, containing 0.25 gm. of potassium bichromate and 
1 c.c. of concentrated sulphuric acid dissolved in distilled water and diluted to 
200 c.c. The proper amounts of solution for stronger permanent standards are 
given in Table XXVIII. 
Table XXVIII 
PREPARATION OF PERMANENT CHLORINE STANDARDS FOR LOW 
CHLORINE CONTENT 
Value in Chlorine. 
Copper Sulphate 
Solution. 
Potassium Bichrom- 
ate Solution. 
Parts per million 
C.C. 
C.C. 
0.01 
0.0 
0.8 
.02 
.0 
2.1 
.03 
.0 
3.2 
.04 
.0 
4.3 
.05 
.4 
5.5 
.06 
.8 
6.6 
.07 
1.2 
7.5 
.08 
1.5 
8.7 
.09 
1.7 
9.0 
.1 
1.8 
10.0 
Dissolved Oxygen 
Reagents.-1. Sulphuric acid. Concentrated (sp. gr., 1.83-1.84). 
2. Potassium permanganate. Dissolve 6.32 gms. of the salt in water and 
dilute the solution to 1 liter. 
3. Potassium oxalate. A 2 per cent solution. 
4. Manganous sulphate. Dissolve 480 gms. of the salt in water and dilute 
the solution to 1 liter. 
5. Alkaline potassium iodide. Dissolve 700 gms. of potassium hydroxide 
and 150 gms. of potassium iodide in water and dilute the solution to 1 liter. 
6. Hydrochloric acid. Concentrated (sp. gr. 1.18-1.19). LIQUID CHLORINE 
331 
7. Sodium thiosulphate. A N/40 solution. Dissolve 6.2 gms. of chemically 
pure recrystallized sodium thiosulphate in water and dilute the solution to 1 liter 
with distilled water. Each cubic centimeter is equivalent to 0.2 mg. of oxygen 
or to 0.1395 c.c. of oxygen at 0° C. and 760 mm. pressure. Inasmuch as this 
solution is not permanent it should be standardized occasionally against N/40 
solution of potassium bichromate. The keeping qualities of the thiosulphate 
solution are improved by adding to each liter 5 c.c. of chloroform and 1.5 gms. 
of ammonium. 
Table XXIX 
PREPARATION OF PERMANENT STANDARDS FOR HIGH CHLORINE 
CONTENT 
Value in Chlorine. 
Copper Sulphate 
Solution. 
Stronger Potassium 
Bichromate Solution. 
Parts per million 
C.C. 
C.C. 
0.10 
1.8 
1.0 
.20 
1.9 
2.0 
.30 
1.9 
3.0 
40 
2.0 
3.8 
.50 
2.0 
4.5 
.60 
2.0 
5.1 
.70 
2.0 
5.8 
.80 
2.0 
6.3 
.90 
2.0 
6.7 
1.0 
2.0 
7.2 
2.0 
2.0 
12.0 
3.0 
2.0 
21.0 
4.0 
2.0 
30.0 
5.0 
2.0 
39.0 
6.0 
2.0 
46.0 
7.0 
2.0 
56.0 
8.0 
2.0 
63.0 
9.0 
2.0 
70.0 
10.0 
2.0 
75.0 
Liquid Chlorine. To some extent, this chemical has replaced cal- 
cium hypochlorite in water treatment. It has certain distinct advan- 
tages over " bleach " as has been outlined by Dutton (1917) and Jen- 
nings (1915) as follows: 
1. A more accurate means of control. 
2. An elimination of dirt and the odor of chlorine. 
3. An appreciable decrease in labor. 
4. A means of storing the chemical so as to eliminate any decrease in 
strength. 332 
WATER HYGIENE 
5. An elimination of chlorine taste in the treated water which is so 
often found where hypochlorite is used. 
6. A decrease in the cost of the chemical required. 
7. The change in the rate of application is quickly and more easily 
accomplished. 
8. Absorption of the gas is more rapid. 
Fig. 61.-Automatic Control Chlorinator. (Wallace-Tiernan Co.) 
Dutton (1917) reported that with hypochlorite at 7 cents per 
pound and liquid chlorine at 20 cents, it would cost $1.07 to treat a 
million gallons of water with bleach and 28 cents to use liquid chlorine. 
With such data at hand, it is evident that the liquid chlorine is the 
more efficient chemical to use. 
The advantages of softening a water supply with lime are becoming 
more and more apparent. Houston has shown that lime is a bac- 
tericidal agent and he proposed the addition of this chemical for ster- APPLICATION OF LIQUID CHLORINE 
333 
ilization. Experiments on the raw Thames river water showed that 
B. coll were killed in from 5 to 24 hours in a concentration of 1 to 5000. 
Hoover and Scott (1914) claim that if enough lime is added to the water 
to absorb the free and half-bound carbonic acid and the precipitate the 
magnesium, B. colon and B. typhi are killed in 48 hours after being so 
Fig. 62.-Automatic Control Chlorinator Direct Feed. (Wallace-Tiernan Co.) 
treated, if the water does not contain large quantities of organic matter. 
An effective bactericidal action when the lime was added in from | to 
1 part per million in excess of that which is needed to reduce the tem- 
porary hardness. Intestinal bacteria will not live in a water containing 
no free or half-bound carbonic acid. When a lime-softened water is 
inoculated with typhoid bacteria or crude sewage, it soon becomes bac- 334 
WATER HYGIENE 
teria free. These authors further state that the action is selective in 
that certain harmless bacteria grow in this water. Table XXX will show 
some of the results secured by Hop ver and Scott. Haller (1914) con- 
Back Pressure Gage 
Blow-off Valve- 
Manometer Filling Screw 
Glass Orifice 
Glass Orifice Cap 
Manometer - -Scale 
Chlorine Control ValveT^i: 
Pressure Compensator-- 
Compensator Cap 
Contact Spring 
Tank Pressure Gage^2Z22 
Contact Arm ---1111 
Pressure Equalizing Valve 
Pressure Pipe Valves- 
Flexible Tank Connections^ 
Tank Valves 
Auxiliary Tank Valvea^^~-- 
Valve Yokes 
Chlorine Line 
Check Valve 
DiffusorTube - Silver 
. Open Conduit 
/. Diffusor 
Chlorine Tanks- -- 
' Venturi Pressure Pipes - 
/ Venturi Tube 
Fig. 63.-Automatic Control Chlorinator, Direct Feed, Type B, Venturi Operated. 
(Wallace-Tiernan Co.) 
firms the statements of Hoover and Scott and also maintains that intes- 
tinal bacteria cannot live in a water from which the CO2 has been 
removed. He regards water which has been treated with calcium oxide 
safe for consumption very soon even though it was previously contam- BACTERIOLOGY OF ICE 
335 
mated with typhoid bacilli. Watt (1913) reported that three weeks' 
treatment of river water with three parts of lime per 100,000 reduced the 
number of B. coli and gave generally satisfactory results with the excep- 
tion of a slight coloring and increased alkalinity. This investigator 
recommends the " excess lime method " as satisfactory for epidemics of 
typhoid fever where it is necessary to quickly improve the water supply. 
Table XXX 
CHEMICAL ANALYSIS IN PARTS PER MILLION WITH THE ACTION 
OF LIME-TREATED WATER ON TYPHOID BACTERIA 
(Hoover and Scott, 1914) 
Oct., 1912. 
Total 
Alkalinity. 
Bicarbonate 
Alkalinity. 
Normal 
Carbonate 
Alkalinity. 
Excess 
Lime 
Caustic 
Alkalinity. 
Bacterial Analysis of 
Softened 
Water 
Control. 
Softened Water Inocu- 
lated with Typhoid 
Bacilli. 
Initially. 
After 
24 Hours. 
. 20 
47 
5 
42 
0 
0 
180 
0 
21 
52 
8 
44 
0 
0 
20 
0 
23 
59 
7 
52 
0 
0 
50 
6 
24 
58 
12 
46 
0 
0 
165 
2 
25 
64 
8 
56 
0 
0 
390 
0 
25 
58 
2 
56 
0 
0 
360 
1 
25 
50 
2 
48 
0 
14 
3600 
1 
28 
46 
0 
42 
4 
0 
300 
0 
29 
48 
0 
48 
0 
1 
3600 
0 
Bacteriology of Ice 
The harvesting of natural ice and the manufacture of artificial ice 
have become well-established industries. The growth of large metro- 
politan centers which are well removed from the source of food supplies 
and rural districts, have made these industries important ones. 
Kinds of Ice. There are two kinds of ice, artificial and natural. 
Natural ice is secured, in the northern climates, from natural bodies of 
water. It is cut into blocks which are convenient for handling and 
delivered to the consumer after storage. Such ice may be contaminated 
either from being taken from too badly polluted bodies of water or by 
being handled in an unsanitary manner. 
Artificial ice is produced mostly by the can method. These are 
usually about the size of an ordinary cake and, before freezing are 336 
WATER HYGIENE 
filled with distilled water; if ordinary water with a high mineral content 
is used, the cake of ice, after it has been frozen, will have a white core 
through the center. This is formed from the dissolved and suspended 
matter and will also contain the bacteria which were present in the 
original water. 
Properties of Ice. Ice is crystallized water which is made to form 
by the removal of heat. The temperature at which this change from 
the liquid to the solid phase takes place is dependent upon a number 
of different factors. The presence of dissolved air, inorganic salts 
pressure, etc., are all concerned. It is well known that salt water 
freezes at a lower temperature than pure water. During the freezing 
process, the water contracts until it reaches 4° C. when it begins to 
expand and becomes lighter than water. Since the formation of ice is a 
process of crystallization, it naturally follows the laws of crystallization. 
Substances which crystallize from solution tend to come out in the pure 
state. The chemist uses this method for purifying his reagents. The 
foreign matter is excluded unless a small amount may be occluded 
during the process between the crystals. The same thing takes place 
when water crystallizes. The bacteria and suspended matter are 
forced out and do not appear in the ice. Ice made from dirty water by 
the can method usually shows the dirt in a core. This will be found 
to contain many bacteria; more are found in this core than in the ice 
at the surfaces of the can. 
Bacteriology of Ice. Ice does not furnish the essentials of a good 
medium and consequently there is no multiplication of bacteria. The 
question resolves itself into determining how many bacteria may sur- 
vive in such an environment. The following causes contribute to the 
inhibitory action on bacteria: 
1. Temperature. 
2. Lack of oxygen. 
3. Absence of moisture. 
The longevity of bacteria in ice has received some attention from 
bacteriologists and sanitarians. Frankland (1894) reports some data 
which were secured by Prudden. Culture of Bacillus prodigiosus, 
Bacillus proteus vulgaris, Staphylococcus pyogenes aureus a fluorescent 
bacillus and Bacillus typhosus were put into samples of water and 
exposed to temperatures of from 14° to 30° F. for 103 days. Bacillus 
prodigiosus decreased from 6300 in 4 days to 3000; in 37 days the number 
went to 22 and after 51 days all of the cells had died. Bacillus proteus 
vulgaris disappeared in 51 days. At the end of 11 days there were ANALYSES OF ICE 
337 
{ Lab. 
No. 
Source. 
Nitrogen as 
Tur- 
bidity. 
Color. 
Odor. 
Resi- 
due. 
Chlor- 
ine. 
Oxy- 
gen 
Cons. 
Free 
Amm. 
Alb. 
Amm. 
No. 2. 
No. 3. 
Alka- 
linity. 
Bac- 
teria 
per c.c. 
10 c.c. 
1 c.c. 
.1 c.c. 
Indol. 
21769 
Lake Rice, i mile east of 
Galesburg 
12,000 
1 + 
2 -4- 
21771 
Ice, Lake Rice, J mile east 
"T 
of Galesburg 
125 
1 
22921 
Pool opposite channel, 
Moline 
8 
50 
IE 
183 
2.4 
9.1 
.072 
.152 
.004 
.680 
144 
520 
1 + 
2 + 
2 - 
+ 
22925 
Ice, Moline Ice Co., Moline 
2 
0 
0 
11 
.0 
5.6 
.154 
.364 
.000 
.000 
1 
3 
1 - 
2- 
2- 
- 
22922 
Pool opposite Moline Ice 
Co,'s plant, Moline. . . . 
7 
50 
IE 
194 
2.3 
7.9 
.080 
. 150 
.004 
.680 
144 
675 
1 + 
2 + 
2 + 
+ 
22926 
Ice from Sylvan Ice Co., 
Moline 
2 
0 
0 
10 
. 1 
2.6 
.096 
. 128 
.000 
.000 
3 
6 
1 - 
2- 
2- 
- 
22961 
Lake Rice, east of Gales- 
burg 
7 
20 
IE 
277 
3. 
4.6 
.274 
.310 
.005 
1.12 
180 
1,400 
1 + 
2 + 
2- 
+ 
22963 
Ice, Lake Rice, Galesburg 
3 
0 
0 
10 
.2 
1.5 
. 118 
. 156 
.000 
.040 
1 
16 
1 
1±1 - 
2- 
+ 
23002 
Rock River, Dixon 
T 
2 + 
4- 
23004 
Ice, Rock River, Dixon.. . 
2 
0 
1 
34 
.8 
1.8 
.096 
. 104 
.002 
.200 
4 
22 
1- 
2- 
2- 
- 
Table XXXI 
ANALYSES OF WATER AND OF ICE FROM THAT WATER 
(After Bartow) 338 
WATER HYGIENE 
1,000,000 cells of Bacillus typhosus and 7000 after 103 days. Alternate 
freezing and thawing were very detrimental to bacteria. Experiments 
with anthrax vegetative and spore cells indicated that the spores resisted 
for some time while the vegetative cells were rather quickly destroyed. 
The cholera vibrio is reported to have been destroyed in from 6 to 10 
days. 
Table XXXII 
SHOWING DURATION OF LIFE OF TYPHOID BACILLI AND COLON 
BACILLI IN ICE KEPT IN A SHED IN WINTER WEATHER 
- 
- 
- 
- 
Typhoid 
Bacilli. 
Colon Bacilli. 
- 
- 
- 
Number of 
Typhoid 
Bacilli in Ice. 
Percentage 
of Typhoid 
Bacilli 
Living. 
Number of 
Colon Bacilli 
in Ice. 
Percentage 
of Colon 
Bacilli 
Living. 
Before freezing 
214,000,000 
100 
269,000,000 
100 
Frozen 8 days 
145,000 
.067 
7,000,000 
3.0 
Frozen 14 days 
54,000 
.025 
5,000,(XX) 
1.85 
Frozen 22 days 
220 
.0001 
1,020,000 
.38 
Frozen 31 days 
0 
0 
Frozen 36 days 
10,700 
.0039 
Frozen 45 days 
1,950 
.00072 
Frozen 46 days 
0 
0 
Reudiger (1911) gives in Table XXXII data secured by him on B. 
coli and B. typhosus. From this it will be seen that 99.9 per cent of 
the B. typhi die during the first eight days. Park also found similar data. 
Jordan, Russell, and Zeit (1904) found that in filtered water B. typhi 
died rapidly. Two organisms were found after the first day and none 
later. Hilliard et al. (1915) reported that 99 per cent of B. coli wore 
destroyed by freezing in tap water for three hours. B. subtilis showed 
a less uniform reaction. No apparent difference was noticed by these 
investigators between alternate freezing and continued freezing. Cream 
with 30 per cent of milk fat afforded a decided protection to the bacteria. 
Ice and Typhoid Fever. The general consensus of opinion among 
sanitarians is that ice is not an important epidemiological factor in the 
spread of typhoid fever. Hutchins and Wheeler (1903) reported an ice- 
borne epidemic of typhoid fever but Hill (1910) believes their evidence to 
be inconclusive. He mentions the following characteristics of ice- 
borne epidemics of typhoid fever: 
1. They should occur before June. 
2. Bulk of the cases should be on one iceman's route. SELF IMPROVEMENT OF STREAMS 
339 
3. Ice should have come from grossly polluted sources. 
4. It should be young ice-recently frozen. 
5. Cases should develop among ice-water drinkers chiefly. 
6. Ice should have been placed in and not around the water. 
Hill further states: 11 Ice, were it equally subject to contamination as 
water and without natural purification processes, could not, on account 
of its relative small use, constitute a factor accounting for over f of 1 
per cent of water-borne typhoid (which, of course, would be a much 
smaller proportion of the total typhoid of* the country) even if its use 
were as general as the use of water." 
Jordan (1911) states, "There has never been much danger from 
the use of impure ice; there need be none. Certified ice of a purity 
beyond reproach can be much more readily attained under existing 
conditions than pure water, and very much more readily than pure 
milk." About the same opinion has been expressed by other sani- 
tarians. Bartow (1916) has stated, " Nature certainly does her share 
toward a pure natural ice. If reasonable precautions are taken so 
that no ice is taken from grossly polluted ponds or rivers and the 
surface of the ice is protected, there need be no difficulty in placing a 
pure ice on the market." 
Methods for the Bacterial Examination of Ice. After the ice has 
been sampled and the sample is melted, the methods of analysis, chem- 
ical and bacterial are no different than those used in water analysis. 
The sampling of the ice for the bacteriological analysis does, however, 
require some special attention. Care must be exercised to secure a 
sample which is not contaminated. This may be accomplished in dif- 
ferent ways. The block of ice should be carefully cleaned and washed 
with distilled water. Then, it should be copiously rinsed with sterile 
distilled water. After this it should be split if it is a large block and 
small pieces chipped off and transferred to sterile containers by means 
of sterile instruments. If this is carefully carried out no contamina- 
tion will result. The block may also be bored out with a sterile auger 
and the shavings collected in a sterile bottle. Greenfield (1916) has 
described an instrument for sampling ice. It is constructed after the 
cheese sampler. 
Self Purification of Streams 
This is a subject which is becoming more and more important as 
population increases. It is very closely related to the disposal of 
sewage by dilution. Directly it may concern the viability of patho- 340 
WATER HYGIENE 
genic bacteria in water but indirectly it is concerned with nuisances 
which almost certainly result when a stream is made to carry too much 
waste matter. In America the question is being given some official 
attention by the Federal government and most States have statutes 
governing the pollution of streams. These have been reviewed by 
Montgomery and Phelps (1917). The United States Public Health 
Service has investigated the pollution and sanitary conditions of 
the Potomac watershed and a very complete report has been prepared 
by Cumming (1916). In this interesting sanitary survey it was found 
that the Potomac River " even during the period of lowest stream flow 
the river in the area of heaviest pollution . . . has, at all times, suffi- 
cient oxygen available for the sewage now discharged into the river and 
enough to take care of that which will probably be added for several 
years to come." Investigations are also being conducted on the Ohio 
river but the results are not available. 
Table XXXIII 
SHOWING THE EXTENT OF SELF PURIFICATION IN THE ILLINOIS 
RIVER 
(After Jordan, 1900) 
Collecting Stations. 
Distance from 
Bridgeport. 
Chlorine, 
P.p.m. 
Bacteria. 
Number of 
Analyses. 
Bridgeport, Illinois 
0 
119.2 
1,245,000 
19 
Lockport 
29 
117.4 
650,000 
30 
Desplaines R. at Lockport 
7.9 
9,180 
28 
Joliet 
33 
104.8 
486,000 
28 
Kankakee River 
3.4 
5,000 
28 
Morris 
57 
68.1 
439,000 
26 
Ottawa 
81 
58.5 
27,400 
26 
Fox River at Ottawa 
5.0 
6,510 
29 
Big Vermillion at LaSalle 
61.2 
7,970 
30 
LaSalle 
95 
46.1 
16,300 
31 
Henry 
123 
44.2 
11,200 
29 
Avery ville 
159 
40.9 
3,660 
30 
Wesley City 
165 
40.1 
758,000 
22 
Pekin 
175 
38.4 
492,000 
29 
Havana 
199 
36.2 
16,800 
26 
Beardstown 
231 
29.3 
14,000 
26 
Kempsville 
288 
22.9 
4,800 
19 
Grafton ' 
318 
18.3 
10,200 
28 
Mississippi River at Grafton 
2.8 
7,600 
29 
One of the most instructive cases of stream pollution is that of the 
Illinois River, which is made to carry the sewage of Chicago. The facts SELF IMPROVEMENT OF STREAMS 
341 
of the case were brought out when the State of Missouri and the city of 
St. Louis tried to secure an injunction against the Sanitary District of 
Fig. 64.-Chicago Drainage Canal and the Illinois River Basin. (From Kinnicut, 
Winslow and Pratt's Sewage Disposal, John Wiley and Sons, Inc.) 342 
WATER HYGIENE 
Chicago to prevent the pollution of this river. It was argued that the 
water supply of St. Louis was endangered. The testimony heard in 
Parts per Million 
Parts per Million 
DISSOLVED OXYGEN 
ILLINOIS RIVER 
DISSOLVED OXYGEN 
ILLINOIS RIVER 
Parts per 2 
Million 
Parts per 
Million 
Fig. 65.-Showing Dissolved Oxygen Concentration in Different Parts of the 
Illinois River. (From Illinois State Water Survey Bulletin.) 
this case has been summarized by Leighton (1907). Both sides of the 
case presented some very interesting opinions and data. Jordan (1900) 
who did valuable work for the defendants reported some of his data in PATHOGENIC BACTERIA IN RIVERS 
343 
a separate publication. Table XXXIII is taken from that paper. It will 
be seen from this table that there is a steady decrease in the chlorine 
content of the water and also in the bacteria content. The rise in the 
count at Wesley City may be explained by the fact that at Peoria large 
amounts of distillery slops and animal refuse were dumped into the 
river. These would have a greater effect on the bacteria count than on 
the chlorine. Dissolved oxygen studies have been carried out by the 
Illinois State Water Survey, some of the data of which are presented 
in Fig. 65. From these data, it will be seen that there is evident puri- 
fication in the Illinois River. 
Pathogenic Bacteria in River Water. There are many data available 
in the literature but in looking it over one is impressed with the diversity 
of opinion. Much of this disagreement may probably be explained by the 
different conditions under which each of the investigators worked. It is 
evident that the factors in the environment influence, to a certain extent, 
the longevity of bacteria in nature. These factors will not be taken up 
here, but those who arc interested will find discussions in texts on water 
bacteriology. From the evidence which was presented in the Illinois River 
case, we know that the typhoid bacilli die rather rapidly in nature. Prob- 
ably after three or four weeks the water which contained them would be 
safe. Jordan, Russell and Zeit (1904) studied this question in both pure 
and polluted waters and their data are taken as the bases upon which 
our present knowledge rests. They suspended the bacteria in collodion 
sacs in both pure and polluted waters. In pure Lake Michigan water, 
the bacilli did not die out until after a week, but three days seemed 
to be the limit for the polluted Chicago Drainage Canal water. In 
1906 Russell and Fuller confirmed these results using pure Lake Men- 
dota water and sewage as the substrates. In this experiment the bacilli 
lived for about ten days in Lake Mendota water and three days in sew- 
age. Houston has studied this question in England. He points out 
that 1 c.c. samples were used by the American workers in most of the 
cases, and from these small samples conclusions were drawn with regard 
to the longevity of B. typhi. When no organisms were found in 1 c.c. 
samples, it was stated that the organism had died out. Probably 
American bacteriologists have been too much accustomed to use small 
amounts of water in analytical work. Houston carried on experiments 
using Thames, Lee and New River waters, using 100 c.c. quantities of 
the water as the maximum amounts examined. For 100 c.c. quantities 
in eighteen experiments, it required nine weeks for the total disappear- 
ance of B. typhi. Ninety-nine per cent of them disappeared in one week 
under laboratory conditions of the experiment. The above data of 344 
WATER HYGIENE 
both American and English bacteriologists point out the value of 
storage. The rate of death of B. typhi and B. coli in pure natural 
water has been reported by Rahn and Hinds (1914) to follow the mono- 
molecular law. Under these conditions the rate increased with the 
temperature. Oxygen was found to be harmful to B. coli but bene- 
ficial to B. typhi. 
The viability of Microspira cholerce has also been investigated by 
Houston using the same technique as for B. typhi. Ninety-nine per 
cent of the cholera organisms perished in three days under the labora- 
tory conditions of the experiment. This is a much shorter period than 
required for B. typhi. Gelarie (1916) studied the longevity of Micro- 
spira cholerce in water of New York Ray. He stated that the survival 
of these organisms in water depends upon the strain which is used, the 
number of bacteria seeded and other factors. The organisms are said 
to live in native bay water from 7 to 45 days and in sterilized bay water 
up to 285 days. In sterile tap water they lived for from 1 to 18 days 
and in native tap water from 1 to 3 days. In competition with the other 
water organisms the life of the cholera organisms was found to be short. 
The other phase of the stream pollution question has been con- 
cerned with nuisances resulting from overloading the stream with 
sewage or organic industrial wastes. It is now fairly well established 
that rivers do purify themselves but it is more often a difficult question 
to determine the length of time required. Practically every river is 
able to handle a certain amount of organic matter. Phelps (1914) has 
stated that three factors are involved-the oxidizing bacteria, organic 
matter and oxygen. Since the organic matter and bacteria are always 
present, " the questions of stream purification and prevention of nui- 
sance reduce to one of oxygen supply." 
Solution of Atmospheric Oxygen in Water. The amount of oxygen 
in the water which is available for the oxidizing bacteria is the important 
question in stream purification. Especially so is the extent to which 
the oxygen content may be reduced before putrefaction sets in. As 
long as there is sufficient oxygen, the bacteria will carry to completion 
the oxidation of organic matter but when this oxygen is reduced to a 
certain point, putrefaction sets in and this is always accompanied by 
foul odors. As long as the available oxygen is sufficient, the bacteria 
will carry it over to the organic matter always driving the reaction in 
that direction and not allowing an equilibrium to be established. The 
greater the speed of this reaction, the greater will be the amount of re- 
aeration which will take place and Phelps (1916) has stated that this is 
the important factor in stream purification and that dams and rapids DISSOLVED OXYGEN 
345 
increase this by a mixing action. The balance between the available 
oxygen and the required oxygen must probably be established for 
each stream. 
Determination of Dissolved Oxygen. Different methods have been 
in use for determining the oxygen content of a water. The Winkler 
method has been included in standard methods and has certain distinct 
advantages over the Levy method which has been advocated by some. 
It is given in standard methods as follows: 
Reagents. 1. Sulphuric acid, concentrated. (Sp. Gr. 1.83-1.84.) 
2. Potassium permanganate. Dissolve 6.32 gms. of the salt in 
water and dilute the solution to a liter. 
3. Potassium oxalate. A 2 per cent solution. 
4. Manganous sulphate. Dissolve 480 gms. of the salt in water 
and dilute the solution to 1 liter. 
5. Alkaline potassium iodide. Dissolve 700 gms. of potassium 
hydroxide and 150 gms. of potassium iodide in water and dilute to a 
liter. 
6. Hydrochloric acid, concentrated. (Sp. Gr. 1.18-1.19.) 
7. Sodium thiosulphate. A N/40 solution. Dissolve 6.2 gms. of 
chemically pure recrystallized sodium thiosulphate in water and dilute 
the solution to 1 liter with freshly boiled distilled water. Each cubic 
centimeter is equivalent to 0.2 mg. of oxygen or to 0.1395 c.c. of oxygen 
at 0° C. and 760 mm. pressure. Inasmuch as this solution is not per- 
manent it should be standardized occasionally against a N/40 solution 
of potassium bichromate. The keeping qualities of the thiosulphate 
solution are improved by adding to each liter 5 c.c. of chloroform and 
1.5 gms. of ammonium carbonate before diluting to the prescribed 
volume. 
8. Starch solution. Mix a small amount of clean starch with cold 
water until it becomes a thin paste and stir this mass into 150 to 200 
times its weight of boiling water. Boil for a few minutes, then sterilize. 
It may be preserved by adding a few drops of chloroform. 
Collection of the Sample. Collect the sample in a narrow-necked 
glass-stoppered bottle of 250 to 275 c.c. capacity. The following pro- 
cedure should be followed in order to avoid entrainment or absorption 
of atmospheric oxygen. In collecting from a tap, fill the bottle through 
a glass or rubber tube extending well into the tap and to the bottom of 
the bottle. To avoid air bubbles allow the bottle to overflow for several 
minutes, and then carefully replace the glass stopper so that no air 
bubble is entrained. In collecting from the surface of a pond or tank 
connect the sample bottle to a bottle of 1 liter capacity. Provide each 346 
WATER HYGIENE 
bottle with a two-hole stopper having one glass tube extending to the 
bottom and another glass tube entering, but not projecting, into the 
bottle. Connect the short tube of the sample bottle with the long tube 
of the liter bottle. Immerse the sample bottle in the water and apply 
suction to the outlet of the liter bottle. To collect a sample at any 
depth arrange the two bottles so that the outlet tube of the liter bottle 
is at a higher elevation than the outlet of the sample bottle. Lower 
the two bottles in any convenient form of cage properly weighted, to the 
desired depth. Water entering during the descent will be flushed 
through into the liter bottle with a glass stopper in such a manner as 
to avoid entraining bubbles of air. 
Procedure. Remove the stopper from the bottle and add, first, 0.7 
c.c. of the concentrated sulphuric acid, and then 1 c.c. of the potassium 
permanganate solution. These and all other reagents should be intro- 
duced by pipette'under the surface of the liquid. Insert the stopper 
and mix by inverting the bottle several times. After twenty minutes 
have elapsed, destroy the excess of permanganate by adding 1 c.c. of 
the potassium oxalate solution, the bottle being at once restoppered 
and its contents mixed. If a noticeable excess of potassium perman- 
ganate is not present at the end of twenty minutes again add 1 c.c. of 
potassium permanganate solution. If this is still insufficient use a 
stronger potassium permanganate solution. After the liquid has been 
decolorized by the addition of potassium oxalate add 1 c.c. of man- 
ganous sulphate solution and 3 c.c. of the alkaline potassium iodide 
solution. Allow the precipitate to settle. Add 2 c.c. of the hydro- 
chloric acid and mix by shaking. 
The procedure to this point must be carried out in the field, but 
after the acid has been added and the stopper replaced there is no 
further change, and the rest of the test may be performed within a 
few hours as convenient. Transfer 200 c.c. of the contents of the 
bottle to a flask and titrate with N/40 sodium thiosulphate, using a 
few cubic centimeters of the starch solution as an indicator toward 
the end of the titration. Do not add the starch solution until the 
color has become a faint yellow and titrate until the blue color 
disappears. 
The use of potassium permanganate is made necessary by high 
nitrite or organic matter. The procedure outlined must be followed on 
all work on sewage and partly purified effluents or seriously polluted 
streams or samples whose nitrite nitrogen exceeds 0.1 part per million. 
In testing other samples the procedure may be shortened by beginning 
with the addition of the manganous sulphate solution and proceeding DISSOLVED OXYGEN 
347 
from that point as outlined, except that only 1 c.c. of alkaline potas- 
sium iodide need be added. 
Calculation of Results. Oxygen shall be reported in parts per million 
by weight. It is sometimes convenient to know the number of cubic 
centimeters per liter of the gas at 0° C. temperature and 760 mm. 
pressure and also to know the percentage which the amount of gas 
present is of the maximum amount capable of being dissolved by dis- 
tilled water at the same temperature and pressure. If 200 c.c of the 
sample is taken the number of cubic centimeters of N/40 thiosulphate 
used is equal to parts per million of oxygen. Corrections for volume of 
reagents added amount to less than 3 per cent and are not justified except 
in work of unusual precision. To obtain the result in cubic centimeters 
per liter, multiply the number of cubic centimeters of thiosulphate used 
by 0.698. To obtain the result in percentage of saturation consult 
the table on page 68 of the 1917 edition of Standard Methods for the 
Examination of Water and Sewage, and divide the number of cubic 
centimeters of thiosulphate used by the figure in this table opposite 
the temperature of the water and under the proper chlorine figure. 
The last column of this table permits interpolation for intermediate 
chlorine values. At elevations differing considerably from mean sea 
level and for accurate work, attention must be given to barometric 
pressure, the normal pressure in the region being preferable to the 
specific pressure at the time of sampling. The term " saturation " 
refers to a condition of equilibrium between the solution and an 
oxygen pressure in the atmosphere corresponding to 158.8 mm., or 
approximately one-fifth atmosphere. The true saturation or equilib- 
rium between the solution and pure oxygen is nearly five times this 
value and consequently values in excess of 100 per cent saturation fre- 
quently occur in the presence of oxygen-forming plants. 
When the oxygen concentration becomes too low, the stream becomes 
putrescible. This same condition may be duplicated in a bottle by 
allowing it to stand full of sewage tightly stoppered. The following 
things will be noticed: formation of evident amounts of hydrogen 
sulphid; heavy deposits of black sediment; disappearance of dissolved 
oxygen. These are conditions which are not desired in streams and 
they indicate that the available oxygen is insufficient to care for the 
organic matter. 
Stability and Relative Stability. A stream is stable when its oxygen 
demand has been satisfied; the products resulting from the decomposi- 
tion of the organic matter should be completely oxidized. Phelps has 
stated that this condition obtains when the available oxygen exceeds the 348 
WATER HYGIENE 
required oxygen. He stated that available oxygen is used up according 
to the following order: Dissolved oxygen, nitrates, nitrites, sulphates, 
phosphates, etc. Since all streams are not stable it is necessary to use 
other terms to fit their condition. For such conditions the term relative 
stability is used. Stable streams have a relative stability of 100 since 
they have all of the oxygen that is demanded by the organic matter. A 
relative stability of 60 means that a stream has 60 per cent of the oxygen 
necessary to make it stable. This is determined in the following manner: 
Reagent. Methylene blue solution. A 0.05 per cent aqueous solu- 
tion of methylene blue, preferably the double zinc salt or commercial 
variety. 
Collection of Sample. Collect the sample in a bottle holding approxi- 
mately 150 c.c. If the dissolved oxygen is low, observe precautions 
similar to those used in collecting samples for dissolved oxygen (p. 162). 
Use rubber stoppers or a cork stopper of good grade which has been 
boiled in water. 
Procedure. Add 0.4 c.c. of the methylene blue solution to the sample 
in the 150 c.c. bottle. As methylene blue has a slightly antiseptic prop- 
erty be careful to add exactly 0.4 c.c. Add the methylene blue solution 
preferably below the surface of the liquid after filling the bottle with the 
sample. If the methylene blue is added first do not allow the liquid to 
overflow as coloring matter will thus be lost. Incubate the sample at 
20° C. for ten days. Four days' incubation may be considered as 
sufficient for all practical purposes in routine plant-control work. If 
quick results are desired incubate the sample at 37° C. for five days 
using suitable stoppers to prevent the loss and reabsorption of dissolved 
oxygen. The bacterial flora at 37° C. is different from the flora at 20° C. 
The lower temperature is more nearly the average temperature of sur- 
face waters and, therefore, the higher temperature should be used only 
when quick approximate results are essential. Observe the sample 
at least twice a day during incubation. Give a sample in which the 
methylene blue becomes decolorized a relative stability corresponding to 
the time required for reduction (see Table XXXIV). For routine filter 
control ordinary room or cellar temperature will give fairly satisfactory 
results. For accurate studies, room temperature incubation is very 
undesirable, as the fluctuations in temperature which are ordinarily 
not noticed are responsible for appreciable deviations from the true 
values of relative stability. If the samples are incubated less than 
ten days at 20° C. and are not decolorized, place a plus sign after the 
stability value in order to indicate that the stability might have been 
higher if more time had been allowed. In applying this test to river DISSOLVED OXYGEN 
349 
waters it often happens that the blue coloring matter is precipitated 
either partly or completely through absorption by the clay which many 
rivers carry in suspension. True relative stabilities cannot be obtained 
for such waters except by determining the initial available oxygen at the 
start and the biochemical oxygen demand on incubation at 20° C. for 
ten days. Germicides, such as calcium hypochlorite, if present in suf- 
ficient quantity vitiate the results. If a sample contains free chlorine, 
therefore, store it about two hours, or until the chlorine is gone, and 
then add methylene blue. 
Table XXXIV gives the relation between the time of reduction in 
days at 20° C. and the relative stability number. 
Table XXXIV 
RELATIVE STABILITY NUMBERS 
ho 
s 
4'20 
s 
0.5 
11 
8.0 
84 
1.0 
21 
9.0 
87 
1.5 
30 
10.0 
90 
2.0 
37 
11.0 
92 
2.5 
44 
12.0 
94 
3.0 
50 
13.0 
95 
4.0 
60 
14.0 
96 
5.0 
68 
16.0 
97 
6.0 
75 
18.0 
98 
7.0 
80 
20.0 
99 
L 
- 
- 
- 
- 
>S = Relative stability or ratio of available oxygen to oxygen required for equilib- 
rium. Expressed in per cent. 
£20 = Time in days to decolorize methylene blue at 20° C. 
The theoretical relation is, 
>8 = 100(1-0.794) t10. 
The relation between the time of reduction at 20° C. and that at 37° 
C. is approximately two to one. It is desirable that each observer work 
out his own comparative 37° C. table or factor, but results should be 
reported in terms of 20° C. stability numbers. 
A relative stability of 75 signifies that the sample examined con- 
tains a supply of available oxygen equal to 75 per cent of the amount 
of oxygen which it requires in order to become perfectly stable. The 
available oxygen is approximately equivalent to the dissolved oxygen 
plus the available oxygen of nitrates and nitrites. The nitrites in 
sewage are usually so low as to be negligible. 350 
WATER HYGIENE 
The theory of the test is unique. Methylene blue is very sensitive 
to products of putrefaction. Such compounds as hydrogen sulphide 
mercaptans, etc., and other reducing substances quickly reduce it to its 
colorloss leuco base. Consequently when this is mixed with a polluted 
water, it will change from blue to white as soon as any of these com- 
pounds are formed. They are present when putrefaction sets in and, 
therefore, cause the change in the color of the dye. Phelps has stated 
that this occurs when the nitrites are used up. This reducing time rep- 
resents the time 11 required for the exhaustion of available oxygen." 
Lederer (1914, 1915) who, as bacteriologist and chemist for the 
Sanitary District of Chicago has had ample opportunity to study this 
subject, has pointed out that great care must be used in the test to 
secure comparable results. He has devised some unique methods for 
the study of required oxygen. They are given in Standard Methods as 
follows: 
Biochemical Oxygen Demand of Sewage and ETiuents (Lederer, 
1914). Relative Stability Method. The relative stability method may 
be employed to obtain a measure of the putrescible material in sewages 
and effluents in terms of oxygen demand. 
Procedure for Effluents. Divide the total available oxygen, including 
the oxygen of nitrites and nitrates, by the relative stability expressed 
as a decimal. 
Procedure for Sewages. Make one or two dilutions with fully aerated 
distilled water of known dissolved oxygen content. Tap water may be 
employed if it is free from nitrates. Vary the relative proportions of 
sewage and water to be employed to give a relative stability of from 50 
to 75. Unless proper seals are employed bring the water as well as the 
sewage to the temperature at which the mixtures are to be incubated 
before preparing the dilutions. During the manipulation avoid aeration. 
Having made the proper dilutions, determine the relative stability of each. 
Calculate the oxygen demand in parts per million by the following 
formula: 
n j j • O(l-p) 
Oxygen demand is ■- 
Rp 
In this formula 0 is the initial dissolved oxygen of the diluting water; 
p is the proportion of sewage; and R is the relative stability of the mix- 
ture. Ordinarily the available oxygen in crude sewages, septic tank 
effluents, settling tank effluents, and tradewastes can be neglected. 
Sodium Nitrate Method. For the determination of the biochemical 
oxygen demand the sodium nitrate method may be used. The method BIOCHEMICAL OXYGEN DEMAND 
351 
is based on the biochemical consumption of oxygen from sodium nitrate 
by a sewage or polluted water during an incubation period of ten days 
at 20° C. A reasonable excess of sodium nitrate does not give a higher 
oxygen demand, as do higher dilutions with aerated water. The oxygen 
absorbed from the air in applying the method to sewages is negligible. 
Reagent. Sodium nitrate solution. Dissolve 26.56 gms. of pure 
soditim nitrate in 1 liter of distilled water. One c.c. of this solution in 
250 c.c. of sewage represents fifty parts per million of available oxygen. 
The strength of the sodium nitrate solution may be varied to suit con- 
ditions. 
Procedure for Sewages. Ordinarily disregard the initial available 
oxygen as it is very small compared with the total biochemical oxygen 
demand. Add measured amounts of the sodium nitrate solution to the 
sewage in bottles holding approximately 250 c.c'which have been com- 
pletely filled and stoppered. Incubate for ten days at 20° C. A seal 
is not required during incubation. The appearance of a black sediment 
and the development of a putrid odor during incubation indicates that 
too little sodium nitrate has been added. Methylene blue solution in 
proper proportion may be added at the start to serve as an indicator 
during the incubation. Domestic sewage usually varies in its oxygen 
demand from 100 to 300 parts per million, approximately 30 per cent 
of which is used up at 20° C. in the first twenty-four hours. At the 
end of the incubation period determine the residual nitrite and nitrate. 
Determine the nitrate by the aluminium reduction method, followed 
by direct Nesslerization. To convert the nitrogen into oxygen equiv- 
alents, multiply the nitrite nitrogen by 1.7 and the nitrate nitrogen 
by 2.9. The difference between the available oxygen added as sodium 
nitrate and that found as nitrite and nitrate at the end of the incuba- 
tion period is the biochemical oxygen demand. 
Procedure for Tradewastes. Employ the same procedure using larger 
quantities of the sodium nitrate solution. Make the reaction alkaline 
to methyl orange and acid to phenolphthalein. Adjust an acid reaction 
with sodium bicarbonate, and a caustic alkaline reaction with weak 
hydrochloric acid. If the liquid is devoid of sewage bacteria seed it 
with sewage after adjusting the reaction. 
Procedure for Polluted River Waters. Determine the initial available 
oxygen. Unless the river water is badly polluted add ten parts per 
million of sodium nitrate oxygen. Collect carefully to avoid aeration, 
three samples in 250 cc. bottles. To one sample add a definite quan- 
tity of sodium nitrate solution and incubate. Incubate the other two 
samples for the determination of the residual free oxygen, nitrite and 352 
WATER HYGIENE 
nitrate. If there is free oxygen left, the bottle containing the sodium 
nitrate solution may be discarded. If then' is no free oxygen determine 
residual nitrite and nitrate as directed under the procedure for sewage, 
and calculate the oxygen demand. (Standard Methods of Water 
Analysis.) 
Examination of Sewage. The chemical examination of sewage and 
sewage effluents is probably more valuable than the bacterial examina- 
tion. The problem involved in controlling a sewage treatment plant 
is quite different than that involved in the control of a water treatment 
plant. As Lederer and Bachmann (1911) have stated the effluent from 
a water treatment plant is subjected to different requirements than 
the effluent from a sewage treatment plant. In the first case the bac- 
teriologist is concerned in the presence of certain types of bacteria and 
their concentration. Little attention is given to the daily variations in 
the chemical content of the effluent. With the sewage effluent, however, 
the question is a different one. It is necessary to watch carefully the 
variations in the chemical content and keep the organic matter reduced 
to a minimum. The question of stream pollution is involved if the 
effluent is to be emptied into a stream. It must not be putrescible and 
thus cause the nuisances which result from too heavily polluting a 
stream. Lederer and Bachmann have stated some of the fallacies 
connected with the bacterial control of sewage plants. They have 
stated that " to try to obtain reliable information on the number of 
bacteria in effluents from various devices in a large plant would require 
a staff of trained men entirely out of proportion to the value of the 
results obtained." The difficulties in securing careful sampling and the 
necessity of immediate plating since bacteria in sewage die very rapidly 
when stored at icebox temperatures, are also mentioned. For these 
reasons and others more attention is given to the determination of the 
organic matter content and the biochemical oxygen demand according 
to the methods which have been given before. 
The deposits from rivers and sewage sludges are examined in the 
following manner according to Standard Methods for the Examination 
of Water and Sewage, 1917. 
Analysis of Sewage Sludge and Mud Deposits 
Collection of the Sample. Collect a representative sample. In 
general, more than one sample should be taken from a spot and a large 
number of samples should be collected rather than a few large samples. 
If the surface layer is darker and a lower layer consists of pure clay ANALYSIS OF SLUDGE 
353 
sample only the surface layer. Samples may be analyzed either sep- 
arately or as composites of careful mixtures. After the sample has 
settled a few minutes roughly drain or siphon the excess water. Allow 
sewage sludge to stand for one hour before draining it free from excess 
water unless it is essential to determine the moisture content of the 
sample originally collected. If sludge cannot be analyzed within 
twenty-four hours it is better not to use air-tight bottles and to add 
small quantities of chloroform to retard decomposition. At the time 
of collection carefully examine mud from the bottom of surface water 
for evidence of sewage pollution and macroscopic and microscopic 
animal and plant organisms. Record the predominant species. Note 
the physical appearance of the material, particularly its color, odor, and 
consistency. Express all analytical results in percentage on a dry basis. 
Specific Gravity. Weigh to the nearest tenth of a gram a wide- 
mouthed flask of 100 to 300 c.c. capacity, according to the quantity of 
material available. Then completely fill the flask with distilled water 
to the brim, and weigh it again. Empty and fill the flask completely 
with fresh sewage sludge or mud. If the material is of such consistency 
that it flows readily, fill the flask to the brim and weigh. The specific 
gravity is equal to the weight of the sludge or mud divided by the 
weight of an equal volume of distilled water. 
If the material does not flow readily, fill the weighed flask as com- 
pletely as possible without exerting pressure during the procedure. 
Weigh and then fill the flask to the brim with distilled water. Let it 
stand for a few minutes, until trapped air has escaped, then add more 
water if necessary and weigh. The specific gravity is equal to the 
weight of the material divided by the weight of the distilled water less 
the weight of the water added. Record the specific gravity only to the 
second decimal place. 
Moisture. Heat approximately 25 gms. of sludge or mud in a 
weighed nickel dish on the water bath until it is fairly dry. Dry the 
residue in an oven at 100° C., cool, and weigh. Repeat to approximate 
constant weight. The loss in weight is moisture. 
Volatile and Fixed Matter. Ignite, in a hood, the residue from the 
determination of moisture until all the carbon has disappeared. Cool 
the residue in a desiccator and weigh it. The residue is the fixed matter. 
The volatile matter is the difference in weight between the original dried 
sludge and the ignited sludge. 
Total Organic Nitrogen. Preparation of Sample. For the deter- 
mination of organic nitrogen and fat dry approximately 50 to 75 gms. 
of the sludge or mud in a porcelain dish first on the water bath and 354 
WATER HYGIENE 
finally in the hot-water oven until all the moisture has disappeared. 
Grind the dry material to a fine powder and keep it in a glass-stoppered 
bottle. 
Reagents. 1. Sulphuric acid. Concentrated, nitrogen free. 
2. Copper sulphate solution. Ten per cent. 
3. Potassium permanganate. Crystals. 
Procedure. Weigh accurately 0.5 gm. of dried sludge or 5.0 gms of 
dried mud and put it in a 500 c.c. Kjeldahl flask. Digest it with 20 c.c. 
of sulphuric acid, or more if necessary, and 1 c.c. of copper sulphate 
solution to assist the oxidation. Boil for several hours until the liquid 
becomes colorless or slightly yellow. Oxidize the residue with 0.5 gm. 
of potassium permanganate, and follow the " Procedure for Sewage " 
(p. 165). 
The following method is convenient for routine work at sewage 
disposal plants. Digest 0.5 gm. of dried sludge or 5.0 gms. of dried mud 
with 20 c.c. of strong sulphuric acid and 1 c.c. of the copper sulphate 
solution in a 300 c.c. Kjeldahl flask. After digestion for several hours, 
cool, transfer to a glass-stoppered 100 c.c. flask, dilute with distilled 
water to 100 c.c., and mix well. Transfer 50 c.c. with a pipette into 
another 100 c.c. volumetric flask, and make this portion alkaline with 
50 per cent sodium hydroxide, testing a drop of the liquid on a porcelain 
plate with phenolphthalein to insure neutralization. The formation of 
a floc usually indicates that the neutralization is complete. Pour the 
solution into a small glass-stoppered bottle and permit it to stand until 
the next day. Nesslerize an aliquot portion of the clear, supernatant 
liquid, and calculate the percentage of nitrogen in the material. 
Fats. Fats are usually determined only on sewage sludge, but some 
mud deposits contain small quantities due to the presence of trade 
wastes. 
Procedure. Weigh, according to the quality of the sewage or mud, 
0.5 to 25 gms. of dry material. Add water to the weighed portion in a 
porcelain dish and acidify the mixture with N/50 sulphuric acid in the 
presence of litmus tincture or azolitmin solution indicator. Avoid 
adding too much acid as an excess gives too high results on account of 
fatty acid residues. Evaporate the acidified mixtures to dryness on 
the water bath, and heat it in the hot-air oven at 100° C. for two to three 
hours. Extract the dry residue with boiling ether, rubbing the sides 
and bottom of the dish to insure complete solution of the fat. Three 
extractions with ether are usually sufficient. Filter the ether solution 
through a 5 cm. filter paper into a small flask. Evaporate the ether 
slowly, dry the fatty extract for half an hour at 100° C., cool in a des- ANALYSIS OF SLUDGE 
355 
iccator and weigh. If it is desirable, particularly with certain trade 
wastes, to determine the quantity of soap fat, determine the fats with 
and without the addition of acid. The difference between the amounts 
found by the two determinations is the amount of soap fat present. 
Ferrous Sulphide. The liberation of hydrogen sulphide on adding 
dilute hydrochloric acid to a sludge indicates the presence of ferrous 
sulphide. As ferrous sulphide quickly oxidizes on exposure to air, a 
quantitative determination of this constituent must be made imme- 
diately after collection of the sample. 
Procedure. Heat a definite portion of the sludge with hydrochloride 
acid in a flask. Pass the liberated gas through bromine water or hydro- 
gen peroxide. Determine gravimetrically the sulphate in the oxidizing 
solution, and calculate the equivalent of ferrous sulphide by multiplying 
the weight of barium sulphate by 0.376. 
Biochemical Oxygen Demand. The quantity of river mud most 
suitable for the determination of the biochemical oxygen demand ranges 
within certain limits, largely according to the amount of deoxygenating 
matter present. For examinations of river mud prepare a 1 per cent 
stock solution in distilled water or tap water saturated with oxygen 
and free from nitrate; use in the test a dilution of this stock solution 
equivalent to a concentration of 1 to 10 gms. per liter of mud. For 
examinations of fresh sewage sludge prepare a 1 per cent stock solution 
in a similar manner, but use in the test a dilution equivalent to only 0.1 
to 1.0 gm. per liter of wet material. For examinations of dried sludges, 
which have undergone more or less mineralization, higher concentrations 
may be required. 
Procedure. Place a measured portion of the sample, or the proper 
amount of the 1 per cent stock solution of the sample, in a 300 c.c. 
narrow-mouth, glass-stoppered bottle, and dilute it to the desired dilu- 
tion with water saturated with oxygen. Determine the oxygen content 
at 20° C. of the waters that are used for dilution. This determination 
must be made before the mud or sludge is added, because iron sulphide 
in the mud or sludge rapidly consumes part of the dissolved oxygen. 
Incubate at 20° C. for five days. 
Shortly before the determination of the oxygen remaining in solution 
at the end of five days rotate the bottle once or twice to mix its contents 
and allow sedimentation for about thirty minutes. Siphon the greater 
part of the liquid through a narrow-bore siphon into a 150 c.c. bottle, 
which has been filled with carbon dioxide. Reject the first 25 c.c. of 
the siphoned liquid and allow a little to overflow at the end of siphoning. 
Determine the oxygen content of the solution in the bottle in the usual 356 
WATER HYGIENE 
way. Report the oxygen demand in grains of oxygen per 100 gms. of 
dried mud or sludge. 
BIBLIOGRAPHY 
Bartow, E. 1916. Examination of Drinking Water on Railway Trains. 
Univ, of Ill. Bull. No. 13. Water Survey Series 12 (1916), 173-182; Jour. 
Am. Water Works Assn., 2, 74-82. 
Baton, W. U. C. 1914. Investigation into the Advisability of Substituting 
• Agar for Gelatin as a Medium for the Determination of Bacteria Counts 
in Water Analysis. Jour. Am. Water Works Assn., 1, 11-23. 
Breunig. 1894. Bakteriologische Untersuchungen des Trinkwassers der 
Stadt Kiel im August und September, 1887. Kiel (1888); quoted from 
Frankland, in Micro-organisms in Water, London. 
Burling, H. A. 1918. Concentration of Glucose and Lactose and Viability 
of Coli-like Bacteria. Amer. Jour. Pub. Hdalth, 8, 306-307. 
Caird, J. M. 1913. The Bacterial Count on Gelatin and Agar Media and its 
Value in Controlling the Operation of Water Purification Plants. Pro- 
ceedings American Water Works Association, 1913, 325-351. 
Chick, H. The Distribution of Bacterium Coli Commune. Thompson- 
Yates Lab. Repts. No. 3, 1. 
Clark, W. M. 1918. The Differentiation of Bacteria of the Colon-a'rogenes 
Family. Jour. Amer. Water Works Assn., 5, 26-35. 
Clark, W. M. and Lubs, H. A. 1917. Improved Chemical Methods for 
Differentiating Bacteria of the Coli-arogenes Family. Jout. Biol. Chern., 
30, 209-234. 
Cobb, Williams and Letton. 1916. Drinking Water on Interstate Carriers. 
Reprint No. 368, Public Health Reports, 31, 2845-67. 
Coles, A. C. 1916. An Easy and Rapid Method of Doing Widal's Reaction 
for Typhoid Fever. Brit. Med. Journal, 1916-1, 684. 
Committee. 1898. Procedures Recommended for the Study of Bacteria 
with Special Reference to Greater Uniformity in the Description and 
Differentiation of Species. Being the report of a committee of bacteri- 
ologists to the committee on the pollution of water supplies of the Amer- 
ican Public Health Association. Amer. Pub. Health Assn. Proceedings, 
1898, 60-100. 
Creel, R. H. 1914. Examination of Drinking Water on Railway Trains. 
Bull. 100, Hyg. Lab., Wash. 
Cumming, H. S. 1914. Safe Ice. Public Health Reports, 29, 3. 
Cumming, H. S. 1916. Investigations of the Pollution and Sanitary Con- 
ditions of the Potomac River. Bull. 104, Hyg. Lab., Wash. 
Dole, R. B. 1906. The Use of Fluorescein in the Study of Underground 
Waters. U. S. Geological Survey, Water Supply Paper, 160. 
Dorange. 1898. Epideinie de fievre typhoide du a 1'ingestion de glace 
impure. Rev. d'Hygiene, 20, 295. BIBLIOGRAPHY 
357 
Dutton, M. S. 1917. Sterilization by Liquid Chlorine and Hypochlorite of 
Lime. Jour. Amer. W. W. Assn., 4, 228-230. 
Dyer, H. G. arid Keith, Jr., S. C. 1893. Notes on Normal Intestinal Bacilli 
of the Horse and of Certain other Domesticated Animals. Tech. Quarterly, 
6, 256-257. 
Eijkmann. 1904. Die Garungsprobe bei 46° als Hilfsmittel bei Trinkwasser- 
untersuchung. Cent. Bakt., Abt. I. Grig., 37, 74. 
Eyre, J. W. H. 1904. On the Distribution of Bacillus Coli in Nature. Lan- 
cet, 1, 648-649. 
Fink, E. B. 1917. American Mortality Statistics and the Mills-Reincke 
Phenomenon. Jour. Inf. Diseases, 21, 62-94. 
Flint, J. M. 1896. Notes on the Distribution of Bacillus Coli Communis. 
Jour. Amer. Med. Assn., 26, 410-411. 
Frankland, Percy and Frankland, G. C. 1894. Micro-organisms in Water, 
105-6, London. 
Frost, W. H. 1916. Confirmation Tests for B. coli in Routine Water Exami- 
nations. Amer. Jour. Pub. Health, 6, 585-8. 
Frost, W. H. 1914. The Functions and Value of Epidemiological Studies 
of Infectious Diseases in Sanitary Administration. Proceedings Indiana 
Sanitary and Water Supply Assoc., 1914, 95-102. 
Fuller and Johnson. 1899. On the Differentiation and Classification of 
Water Bacteria. Jour. Exp. Med., 4, 609-626. American Pub. Health 
Assn. Proceedings, 25, 580-586. 
Gaub, J. 1913. Some Results from the Use of Gelatin and Agar. Proc. Ill. 
Water Supply Assn. 1913, 155-159. 
Greenfield, M. 1916. A New Ice Sampler. Journal Bacteriology, 1, 623. 
Haller, C. 1914. The Sterilization of Water with Lime. Statisch Tiefblau, 
4, 299-303. Chern. Abstracts, 8, 2206. 
Hanford, W. W. and Bartow, E. 1916. Quality of the Drinking Water 
Obtained in Illinois by Common Carriers. Illinois State Water Survey 
Bulletin, 13, 256-271. 
Harden, A. 1901. Action of B. communis on Dextrose. Trans, of the Chemi- 
cal Society, 1901, 604. 
Harden, A. 1905. Action on Glucose of Lactose Fermenting Bacteria of 
Feces. Jour. Hygiene, 5, 488. 
Harden, A. 1905-1906. On the Voges-Proskauer Reaction for Certain Bac- 
teria. Proc. Roy. Soc., 77, 424. 
Harden, A. and Norris, D. 1911-1912. The Bacterial Production of Acetyl- 
methyl-carbinol and 2, 3, Butylene-glycol from Various Substances. 
Proc. Royal Soc. Series B, 84, 492; 85, 73. 
Harden, A. and Norris, D. 1911. The Diacetyl Reaction for Proteins. 
Journal of Physiology, 47, 332. 
Harden, A. and Walpole, S. G. 1905-1906. Chemical Action of B. lactis 
Aerogenes (Escherich) on Glucose. Production of 2, 3, Butylene, Glycol 
and Acetyl Methyl Carbinol. Proc. Royal Soc. Series B, 77, 399. 358 
WATER HYGIENE 
Harrison, F. C. 1912. Microbiology of Water and Sewage, (in Micro- 
biology, edited by C. E. Marshall), 204. 
Hauser, S. J. 1917. Methods Used at Cincinnati Filtration Plant for Iso- 
lating and Identifying Bacillus Coli. Eng. and Contractor, 48, 296-297. 
Also published in Amer. Jour. Pub. Health, 7, 1050-1051. 
Hazen, A. 1904. The Hazen Theorem. Trans. Amer. Soc. Civil Eng., 54, 
Pt. D., 131. 
Hill, H. W. 1910. Non-relation of the Natural Ice Supply to Typhoid Fever 
and Dysentery. Cold Storage and Ice Trade Jour., 40, 33-35. 
Hilliard, C. M., Torissian, C., and Stone, R. P. 1915. Notes on the 
Factors Involved in the Germicidal Effect of Freezing and Low Tempera- 
tures. Science, 42, 770-1. 
Hoover, C. P. and Scott, R. D. 1914. Advantage of the Use of Lime in 
Water Purification. Monthly Bull. Ohio State Board of Health, December, 
1914. 
Houston, A. C. First Report on Research. The Vitality of the Typhoid 
Bacillus in Artificially Infected Samples of raw Thames, Lee and New 
River Waters, with Special Reference to the Question of Storage. Metro- 
politan Water Board, London. 
Houston, A. C. Fourth Report on Research Work. The Vitality of the Chol- 
era Vibrio in Artificially Infected Samples of raw Thames, Lee and New 
River Waters, with Special Reference to the Question of Storage. Metro- 
politan Water Board. 
Hutchins and Wheeler. 1903. An Epidemic of Typhoid Fever Due to 
Impure Ice. Amer. Jour. Science, 126, 680. 
Johnson, G. A. 1904. Isolation of B. coli communis from the Alimentary 
Tract of the Fish and the Significance Thereof. Journal of Infectious 
Diseases, 1, 348-354. 
Johnson, G. A. 1910. Hypochlorite Treatment of Water Supplies: Its Sig- 
nificance and Limitations. Eng. Record, 62, 321. 
Johnson, G. A. 1913. The Purification of Public Water Supplies. U. S. 
Geological Survey, Water Supply Paper No. 315. 
Johnson, G. A. 1916. The Typhoid Toll. Jour. Amer. Water Works Assn., 
3, 249-326. 
Johnston, W. 1894. On the Grouping of Water Bacteria. Proc. Amer. 
Pub. Health Assn., 20, 445-449. 
Jordan, E. O. 1915. The Bacterial Examination of Water. Trans. XV. 
Intern. Cong. Hyg. and Demog., 2, 47-50. 
Jordan, E. O. 1900. The Self-purification of Streams. Jour.Exper. Med., 5,302. 
Jordan, E. O. 1903. The Kinds of Bacteria Found in River Water. Jour- 
nal Hygiene, 3, 1-27. 
Jordan, E. O., Russell and Zeit. 1904. The Longevity of Typhoid Bacillus 
in Water. Jour. Inf. Diseases, 1, 641. 
Jordan, E. O. and Victorson, Ruth. 1917. Differentiation of the Para- 
typhoid Enteritidis Group II. Jour. Inf. Diseases, 21, 554-555. BIBLIOGRAPHY 
359 
Klein, E. 1895. Ueber einen pathogenen anaeroben Darmbacillus, Bacillus 
enteritidis sporogenes. Cent. Bakt., 18, 737. 
Klein, E. 1897-8. Morphology and Biology of Bacillus Enteritidis Sporo- 
genes; on Association of this Microbe with Infantile Diarrhoea and 
with Cholera Nostras; on its Relation with Milk and with Manure. Local 
Government Board; Medical Officer's Report, p. 210. 
Klein, E. 1898-9. Further Report on Bacillus Enteritidis Sporogenes. 
Local Government Board, Medical Officer's Report, p. 312. 
Klein, E. 1899. Ein Beitrag zur Bakteriologie der Leichenverwesung. 
Cent. Bakt., 25, 278. 
Klein, E. 1901-2. Differentiation of the Several Anaerobic Microbes Com- 
monly Present in the Intestinal Contents of Man and Other Animals. 
Local Government Board; Medical Officer's Report, p. 404. 
Kligler, I. J. 1917. A Simple Medium for the Differentiation of Members of 
the Typhoid Paratyphoid Group. Amer. Jour. Pub. Health, 7, 1042-1044. 
Krumwiede, C. and Kohn, L. A. 1917. A Triple Sugar Modification of the 
Russell Double Sugar Medium. Jour. Med. Research, 37, 225-227. 
Kruse, W. 1894. Kritische und Experimentale Beitrage zur Hygienischen 
Beurtheilung des Wassers. Zeit. Hyg., 17, 1894. 
Lederer, A. and Bachmann, F. 1913. Some Interesting Observations on the 
Disinfection of Lake Water with Calcium Hypochlorite. Proceedings 
Illinois Water Supply Assn., 1913, 184-201. 
Lederer, A. 1914. A Serious Fallacy of the "Standard" Methylene Blue 
Putrescibility Test. Amer. Jour. Pub. Health, 4, 241-248. 
Lederer, A. 1914. A New Method for Determining the Relative Stability of 
Sewage Effluent or Polluted River Water. Jour. Inf. Diseases, 14, 482-497. 
Lederer, A. 1915. Notes on the Practical Application of the "Saltpeter" 
Method for Determining Strengths of Sewage. Amer. Jour. Pub. Health, 
5, 354-361. 
Lederer, A. 1915. Determination of the Biochemical Oxygen Demand by 
the Saltpeter Method in Stockyards, Tannery and Corn Products Wastes. 
Jour. Ind. Eng. Chem., 7, 514, 6. 
Leighton. 1907. Pollution of the Illinois and Mississippi Rivers by Chicago 
Sewage. A digest of the testimony taken in the case of the State of Mis- 
souri vs. the State of Illinois and Sanitary District of Chicago. U. S. 
Geological Survey Water Supply Paper No. 194. 
Letton, H. P. 1917. The Treasury Department Standard for Drinking Water: 
Its Value and Enforcement. Jour. Amer. Water Works Assn., 4, 478-481. 
Levine, M. 1916. The Significance of the Voges-Proskauer Reaction. Jour. 
Bact., 1, 153-164. 
Levine, M. 1916. The Correlation of the Voges-Proskauer Reaction and 
Methyl Red Reactions in the Colon-a?rogenes Group of Bacteria. Jour. 
Inf. Diseases, 18, 358-367. . 
Levine, M. 1918. Presumptive Test for Bacillus Coli. Eng. Contractor, 49, 
34. Also see Jour. Inf. Dis., 23, 43-47. 360 
WATER HYGIENE 
Lewis, W. L. 1912. Evanston's Experience with Hypochlorite. Proc. 
Illinois Water Supply Association, 1912, 124-139. 
McCallie, S. W. 1904. Experiment Relating to Problems of Well Contam- 
ination at Quitman, Ga. U. S. Geol. Survey, Water Supply Paper, 110, 
45-54. 
McLaughlin, A. J. 1912. The Necessity for Safe Water Supplies in the 
Control of Typhoid Fever. Proceeding Illinois Water Supply Association, 
1912, 64-84. 
McLaughlin, A. J. 1914. What is a Safe Drinking Water? Public Health 
Reports, 29, June 26, 1914. 
MacNeal, W. J., Latzer, I. and Kerr, J. 1909. The Fecal Bacteria of 
Healthy Men. Jour. Inf. Diseases, 6, 571. 
Marboutin, F. 1901. Contribution a 1'Etude des Eaux Souterraines. Comp. 
Rend., 132, 365-368. 
Maroni. 1910. Bakterium Coli Commune. Arch. Exper. Med., 22, 261. 
Martel, E. A. 1903. Sur l'application de la fluorescein a 1'hydrologie 
souterraine. Compt. Rend., 137, 225-227. 
Metcalf, H. 1905. Organisms on the Surface of Grains with Special Refer- 
ence to Bacillus coli. Science, 22, 434-441. 
Montgomery, S. D. and Phelps, E. B. 1917. Stream Pollution; a Digest 
of Judicial Decisions and a Compilation of Legislation Relating to the 
Subject. U. S. Public Health Service, Public Health Bulletin, 87. 
Moore, V. A. and Wright, F. R. A. 1900. A Comparison of B. Coli Com- 
munis from Different Species of Animals. Jour. Bost. Med. Soc.; 4, 175. 
Morishima, K. A Simple and Practical Medium for Differentiating B. Typho- 
sus, B. Paratyphosus, A. B. Paratyphosus B and B. Enteriditis. Journal 
of Bacteriology, 3, 19-22. 
Muir, R. and Ritchie, J. 1914. Manual of Bacteriology, New York, 5th 
Edit. 
Nankivell, A. T. 1911. The Sand Filtration and Purification of Chalk 
Waters. Journal of Hygiene, 11, 235-258. 
Nichols, A. H. 1876. Report of an Outbreak of Intestinal Disorder Attrib- 
utable to the Contamination of Drinking Water by Means of Impure Ice. 
7th Annual Report State Board of Health, Massachusetts, 467. 
Obst, M. M. Bile Compared with Lactose Broth for Determining the Presence 
of B. Coli in Water. Jour. Bact., 1, 73-79. 
Orchard, W. J. 1918. Water Supply Standards and their Improvement. 
Jour. Am. Water Works Assn., 5, 49-58. 
Park, W. H. 1907. The Importance of Ice in the Production of Typhoid 
Fever. Jour. Amer. Med. Assn., 49, 731-732. 
Prescott, S. C. 1906. The Occurrence of Organisms of Sanitary Signific- 
ance on Grains. Biol. Studies of Pupils of W. T. Sedgwick, 208-222, 
Boston. 
Prescott, S. C. and Winslow, C. E. A. 1915. Elements of Water Bacteri- 
ology. New York. 3d Edit., 217. BBILIOGRAPHY 
361 
Phelps, E. B. 1909. The Disinfection of Sewage and Sewage Filter Effluents, 
with a Chapter on the Putrescibility and Stability of Sewage Effluents. 
U. S. Geol. Survey, Water Supply Paper 229. 
Phelps, E. B. 1914. Studies on the Self-purification of Streams. Public 
Health Reports, 29, August 14, 1914. 
Phelps, E. B. 1916. Re-aeration Held to be Important in the Self-purifi- 
cation of Streams. Eng. Record, 74, 617-618. 
Race, J. 1914. The Significance of B. Coli in Water Examination. Amer. 
Jour. Pub. Health, 26, 758-759. 
Race, J. 1914. Bacillus Coli in Water Supply. Municipal Eng., 47, 
12-14. 
Race, J. 1916. Bacterial Counts in Water Examination. Amer. Jour. Pub. 
Health, 6, 488-496. 
Race, J. 1918. Chlorination and Chloramine. Jour. Amer. Water Wks. 
Assn., 5, 63-68. 
Reynolds. 1902. Report of Stream Examination, Chemic and Bacteriologic, 
of the Water Between Lake Michigan and the Mississippi River at St. 
Louis, Chicago, 1902. 
Rogers, L. A., Clark, W. M. and Evans, A. 1915. Science, 41, 624. 
Royal Institute of Public Health. Committee Report. Jour. State Med., 
22, 559-567. 
Savage, W. G. 1906. The Bacteriological Examination of Water. Chap. 9, 
Philadelphia. 
Savage, W. G. and Read, W. J. 1916. Significance of Streptococci in Water 
Supplies. Jour. Hyg., 5, 334-351. 
Savage, W. G. and Wood, D. R. 1917. The Vitality and Viability of Strep- 
tococci in Water. Jour. Hygiene, 16, 227, 239. 
Sedgwick, W. T. and Winslow, C.-E. A. 1902. Experiments on the Effects 
of Freezing and Other Low Temperatures upon the Vitality of the Bacillus 
of Typhoid Fever with Consideration Regarding Ice as a Vehicle of Infec- 
tious Disease. Mem. Amer. Acad. Arts and Sciences, 12. 
Sedgwick, W. T. and MacNutt, J. S. 1910. Study on the Hazen Theorem 
Jour. Inf. Diseases, 7, 489. . 
Smith, E. G. B. 1905. Notes on the Occurrence on Grain of Organisms 
Resembling Bacillus Coli Communis. Science, 21, 710-711. 
Tanner, F. W. 1916. Comparison of the Number of Water Bacteria Growing 
on Agar at 37° C. and on Gelatin at 20° C. University of Illinois, Bulletin 
No. 13. Water Survey Series, No. 12, 242-8. 
Thresh, J. C. 1913. Examination of Water and Water Supplies. Blakistons, 
Philadelphia. 
Traube, M. 1894. Einfaches Verfahren Wasser in grossen Mengen Keim- 
frei zu machen. Zeit. Hyg., 149-188. 
Treasury Department. Bacteriological Standards for Drinking Water. 
Pub. Health Reports, 29, 2959-2966. Jour. Amer. Water Works Assn., 4, 
927-946. 362 
WATER HYGIENE 
Trillat, A. 1899. Sur 1'emploi matieres polourantes pour la recherche de 
1'origine des sources et des eaux d'infiltration. Comp. Rend., 128, 698- 
700. 
Tuller, C. A. and Armstrong, V. A. 1913. The Differentiation of Fecal 
Streptococci by their Fermentative Reactions in Carbohydrate Media. 
Jour. Inf. Diseases, 13, 442-462. 
Van Brunt, G. A. 1911. The Action of Bleaching Powder in Water Puri- 
fication. University of Illinois Bulletin, Water Survey Series No. 8, 53. 
Ward, M. 1897. Fifth Report to the Royal Society Water Research Com- 
mittee. Proc. Royal Soc., London, 61, 415-423. 
Watt, J. 1913. Purification of Water Supplies by the Excess Lime Method. 
Jour. State Medicine, 21, 489-499. 
Weissenfeld, J. 1900. Der Befund des Bakterium Coli im Wasser und das 
Thiere Experiment sind keine brauchbaren Hiilfmittels fiir die hygienische 
Beurtheilung des Wassers. Zeit. Hyg., 35, 78-86. 
Whipple, G. C. 1913. The 37° Bacterial Count. American Journal of Pub- 
lic Health, 3, 36-43. 
Winslow, C.-E. A. and Hunnewell, M. P. 1902. Streptococci Charac- 
teristics of Sewage and Sewage Polluted Waters. Science, 15, 827. 
Winslow, C.-E. A. and Hunnewell, M. P. 1902. A Study of the Distri- 
bution of the Colon Bacillus of Escherich and of the Sewage Streptococci 
of Houston in Polluted and Unpolluted Waters. Journal of Medical 
Research, 8, 502. 
Winslow, C.-E. A. 1916. Tests for Bacillus Coli as an Indicator of Water 
Pollution. Jour. Amer. Water Works Assoc., 3, 927-946. 
Wolman, A. 1917. The Quality of Water and- Confirmatory Tests for B. 
Coli. Jour. Amer. Works Association, 4, 200-205. CHAPTER XI 
MILK AND MILK PRODUCTS 
Milk is the special secretion of the mammary glands and is generally 
used for food by man. Fresh milk is amphoteric to litmus with a spe- 
cific gravity heavier than water-1.030. It has a yellowish color and a 
characteristic odor. On account of its chemical composition, which is 
mentioned later, it is susceptible to many undesirable changes. Many 
of these are brought about by microorganisms and, therefore, may be 
controlled by holding the milk in an environment, the factors of which 
are detrimental to bacterial development, 
Chemical Constituents of Milk 
The constituents of milk have been classified by Van Slyke and 
Bosworth (1914) in the following manner: 
1. Milk constituents in 
true solution in 
milk serum. 
a. Sugar 
b. Citric acid 
c. Potassium 
d. Sodium 
e. Chlorine 
2. Milk constituents part- 
ly in solution and 
partly in suspen- 
sion or colloidal 
solution. 
a. Albumin 
b. Inorganic phos- 
phate 
c. Calcium 
d. Magnesium 
3. Milk constituents en- 
tirely in suspen- 
sion or colloidal 
solution. 
a. Fat 
b. Casein 
Bosworth and Van Slyke (1915) report the composition of cow's, 
goat's and human milk, shown in Table XXXVI. From this presenta- 
tion of the chemical composition of milk, it will be seen that it is an 
excellent medium for bacterial growth. 
Van Slyke and Bosworth (1914) studied the composition of milk 
serum after the other constituents had been removed by filtration 
through a Pasteur-Chamberland filter. The analysis of the serum is 
given in Table XXXVII. 
363 364 
MILK AND MILK PRODUCTS 
Table XXXVI 
COMPOUNDS IN COW'S, GOAT'S, AND HUMAN MILK 
(After Bosworth and Van Slyke, 1915) 
Compounds. 
Cow's Milk. 
Goat's Milk. 
Human Milk. 
Fat 
Per cent 
3.90 
Per cent 
3.80 
Per cent 
3.30 
Milk sugar 
4.90 
4.50 
6.50 
Proteins combined with calcium 
3.20 
3 10 
1.50 
Salts : 
0.901 
0.951 
0.313 
Di calcium phosphate 
0.175 
0.092 
0.000 
Tri calcium phosphate 
0.000 
0.062 
0.000 
Mono magnesium phosphate 
0.103 
0.000 
0.027 
Di magnesium phosphate. 
0.000 
0.068 
0.000 
Tri magnesium phosphate 
0 000 
0.024 
0.000 
Mono potassium phosphate.. . . ^ 
0.000 
0.073 
0.069 
Di potassium phosphate 
0.230 
0 000 
0.000 
Potassium citrate 
0.052 
0.250 
0.103 
Sodium citrate 
.0.222 
0.000 
0.055 
Potassium chloride 
0.000 
0.160 
0 000 
Sodium chloride 
0.000 
0.095 
0.000 
Calcium chloride 
0.119 
0.115 
0.059 
Table XXXVII 
COMPOSITION OF MILK SERUM 
(After Van Slyke and Bosworth, 1914) 
Constituents- 
Sample No. 1. 
Sample No. 2. 
Original 
Milk, 
100 c.c . 
Milk 
Serum, 
100 c.c. 
Percentage 
of Milk 
Constitu- 
ents in 
Serum. 
Original 
Milk, 
100 c.c. 
Milk 
Serum, 
100 c.c. 
Percentage 
of Milk 
Constitu- 
ents in 
Serum. 
Grams 
Grams 
Per cent 
Grams 
Grams 
Per cent 
Sugar 
5.75 
5.75 
100.00 
Casein 
3.35 
0 
00 
0 00 
3.07 
0 00 
0.00 
Albumin: 
0.525 
0 
369 
70.29 
0.506 
0.188 
37.15 
Nitrogen in other compounds 
. . . 
0.049 
0.049 
100.00 
Citric acid 
0.237 
0.237 
100.00 
Phosphorus (organic and in- 
organic) 
0.125 
0 
067 
53.60 
Phosphorus (inorganic) 
0.096 
0 
067 
70.00 
0.087 
0.056 
64.40 
Calcium 
0.128 
0 
045 
35.16 
0.144 
0.048 
33.33 
Magnesium 
0.012 
0 
009 
75.00 
0.013 
0.007 
53.85 
Potassium 1 
* 0.354 
*0 
352 
99.44 / 
0.120 
0.124 
100.00 
Sodium ) 
0.055 
0.057 
100.00 
Chlorine 
0.081 
0.082 
100.00 
0.076 
0.081 
100.(XI 
Ash 
0.725 
0.400 
55.17 
* As chlorides. CHANGES IN MILK 
365 
Milk Proteins. According to the table from Bosworth and Van 
Slyke cow's milk has a larger content of protein than goat's or human 
milk. Data from other sources confirm this fact. The following 
proteins are found in milk: 
Casein / 
Lact-albumin 
Lact-globulin 
Changes in Milk 
Milk is subject to many types of changes. One of the most common 
and one which occupied the attention of chemists for a long time is the 
coagulation or clotting of the casein. The natural state of this milk 
constituent may be changed either by acid or the presence of a specific 
enzyme. Other proteins may be coagulated in the same way by enzymes. 
This change in the state of the casein is the foundation of the whole 
cheese industry. 
Souring of Milk. This usually refers to the normal changes in milk 
which are brought about by the continued growth of lactic acid bacteria. 
The lactic acid which is thus formed removes the calcium from the 
calcium caseinate. The casein thus formed is insoluble in the milk 
serum and is precipitated. 
The chemical changes which take place in the soluble and insoluble 
constituents of milk have been studied by Van Slyke and Bosworth 
(1916). The milk was inoculated with a culture containing Bacterium 
lactis acidi and B. lactis aerogenes and allowed to stand at room tempera- 
ture for sixty hours. At the end of this time it was filtered through a 
porcelain filter and subjected to analysis. A blank was made by pre- 
serving milk with chloroform and separating the serum in the same way. 
The changes which took place in sixty hours are given in Table XXXVII I. 
Coagulation by Rennin. It was believed for a long time that the 
coagulation of milk was due only to acid. Fremy, in 1839, one of the 
earliest workers on the subject, supposed that a special enzyme was 
present in the lining of calves' stomachs which formed acid from the 
lactose. Hammarsten (1872) proved that the production of acid from 
the lactose was not the cause of coagulation by rennin but that it was 
brought about by a special enzyme, which acted directly on the casein. 
The phenomenon involved in the action of rennin is the change of 
casein in milk into soluble paracasein along with another compound. 
However, before actual clotting takes place an insoluble calcium para- 
caseinate must be formed from calcium salts in the milk. Van Slyke 366 
MILK AND MILK PRODUCTS 
and Bosworth (1913) have shown that the molecular weight of casein 
is 8888 and that of paracasein 4444. The valence of the molecule in 
basic caseinates is 8 and in basic paracaseinates 4. Bosworth (1913). 
in another paper states that these facts indicate three things: 
Table XXXVIII 
CHANGES IN CONDITION OF MILK CONSTITUENTS AS A RESULT 
OF SOURING 
(After Van Slyke and Bosworth) 
Serum from 
Fresh Milk. 
Serum from 
Sour Milk. 
Constituents. 
Original 
Milk, 
100 c.c. 
Serum 
100 c.c. 
Percent- 
age of 
Milk Con- 
stituents 
in the 
Serum. 
Serum, 
100 c.c. 
Percent- 
age of 
Milk Con- 
stituents 
in the 
Serum. 
Grains. 
Grams. 
Per Cent. 
Grams. 
Per Cent. 
Sugar 
5.75 
5.75 
100.00 
4.48 
Casein 
3.07 
0.00 
0.00 
0.00 
0.00 
Albumin , 
0.506 
0.188 
37.15 
0.506 
100.00 
Nitrogen in other compounds 
0.049 
0.049 
100.00 
0.049 
100.00 
Citric acid 
0.237 
0.237 
100.00 
0.000 
0.00 
Phosphorus (inorganic) 
0.087 
0.056 
64.40 
0.090 
100.00 
Calcium 
0.144 
0.048 
33.33 
0.148 
100.00 
Magnesium 
0.013 
0.007 
53.85 
0.014 
100.00 
Potassium 
0.120 
0.124 
100.00 
0.120 
100.00 
Sodium 
0.055 
0.057 
100.00 
0.058 
100.00 
Chlorine 
0.076 
0.081 
100.00 
0.079 
100.00 
Ash. 
0.725 
0.4(H) 
55.17 
0.690 
95.17 
Lactic acid 
0.000 
0.000 
0.00 
1.124 
First, that rennin action consists of the hydrolytic splitting of the 
casein molecule into two similar molecules of paracasein; perhaps in 
somewhat the same manner that maltose is split into two molecules of 
dextrose. 
Second, that, as a consequence of this cleavage, it would seem to be 
doubtful if Hammarsten's whey-protein could be one of the products of 
rennin action. 
Third, that rennin is not, strictly speaking, a coagulating ferment, 
the coagulation of paracasein being due to the fact that calcium para- 
caseinates are less soluble than the calcium caseinates, especially in 
the presence of soluble salts of calcium barium or strontium. CHEMICAL EXAMINATION 
367 
The action of rennin is then proteolytic since it destroys casein. 
It might be regarded as a preliminary process in digestion, Bosworth 
(1913) states that probably the action attributed to rennin may be 
produced by any proteolytic enzyme. In the bacterial world this 
has been known for some time that an organism which will split casein 
will also probably split gelatin and other proteins. This is especially 
well borne out in the following table compiled by Frost and McCamp- 
bell and taken from their text. 
From this table one might infer that there was a specificity even for 
the different proteins. 
Table XXXIX 
ACTION OF CERTAIN BACTERIA ON CERTAIN PROTEINS 
(Frost and McCampbell) 
Name of Organism. 
Milk. 
Gelatin. 
Serum. 
Egg 
Albumin. 
Fibrin. 
Coag. 
Digest. 
Bact. anthracis 
+ 
+ 
+ 
- 
+ 
+ 
Microspira comma 
+ 
+ 
+ 
+ 
+ 
+ 
M. pyogenes aureus 
+ 
+ 
+ 
- 
- 
- 
Pseudomonas pyocyaneus. 
+ 
+ 
+ 
+ 
+ 
+ 
Pseudomonas violacea.. . . 
- 
- 
+ 
- 
- 
- 
Bacillus mycoides 
+ 
+ 
+ 
+ 
- 
- 
Bacillus prodigiosus 
- • 
+ 
+ 
+ 
+ 
+ 
Aspergillus niger 
+ 
+ 
- 
- 
- 
- 
Asoergillus oryza; 
- 
+ 
+ 
+ 
+ 
- 
Chemical Examination 
The functions of the chemical and bacteriological examinations may 
be entirely different although, in many cases, they must go along 
together. A sanitarian must have all the knowledge which it is pos- 
sible to secure and should not exclude either data if he would come to 
an accurate conclusion. In the chemical examinations that are made on 
milk, data may be secured with regard to the normal or abnormal occur- 
rence of any milk constituent. The presence of preservatives may be 
directly determined while, in the bacteriological examination, they may 
be indirectly inferred if the milk does not sour when it is placed in a 
favorable environment for this change. 
Sampling of Milk. To secure accurate results from the chemical 
or bacterial examination of milk requires good sampling. Too much 368 
MILK AND MILK PRODUCTS 
attention cannot be paid to this important step. The larger portion 
from which the sample is taken should be thoroughly stirred. If the 
sample is collected in a bottle, the content^ should be very thoroughly 
mixed. 
Specific Gravity. This may be determined by any of the usual 
methods. Hydrometers are convenient and, in this form, are known as 
lactometers. They are of different types. The New York Board of 
Health has an arbitrary scale. This is divided into 120 parts with zero 
equal to the specific gravity of water and 120 equal to the specific gravity 
of 1.029. The length is usually about 12 ins. The Quevenne lactometer 
is graduated in accordance with the specific gravity scale with the first 
two figures omitted for sake of convenience (1.029 sp. gr. = 29 Que- 
A 
B 
C 
Fig. 66.-Types of Lactometers. 
A, New York Board of Health Type; B, Same as A Except with Thermometer; C, Quevenne 
Type. 
venne). They are now made with blue and yellow columns. The 
fractions in the blue column indicate the percentage of water in the 
skimmed milk, and in the yellow column, in whole milk. 
Determination of Ash in Milk. Weigh about 20 gms. of milk in a 
tared dish and, after adding 6 c.c. of nitric acid, evaporate to dryness, 
ignite just below redness. 
Determination of Fat in Milk. Babcock Test. This rests on the 
fact that concentrated sulphuric acid will dissolve the solids not fat 
in milk. The fat is unchanged and may be separated in a centrifuge 
as follows: Special bottles are necessary and into each should be placed 
18 gmfe. (17.6 c.c.) of the well-shaken sample of milk. By means of a 
special pipette add 17.5 c.c. of commercial sulphuric acid with a spe- 
cific gravity of about 1.83. During the addition of the sulphuric acid, 
the contents of the bottle should be constantly shaken. The bottle 
should now be placed in a centrifuge and whirled for five minutes at 
about 1000 r.p.m. After this, boiling water should be added to the 
Babcock bottle, in order to raise the fat column into the neck of the FAT DETERMINATIONS 
369 
bottle. The bottle should be whirled again for about a minute and then 
the percentage of fat read by means of a pair of calipers. This test is 
adapted to ice cream and cream with the exception that a larger bottle is 
used. 
Leffmann-Beam Test. The distinctive feature is the use of fusel 
oil, the effect of which is to produce a greater difference in surface ten- 
sion between the fat and the liquid in which it is suspended, and thus 
promote its readier separation. This effect has been found to be 
heightened by the presence of a small amount of hydrochloric acid. 
The test bottles have a capacity of about 30 c.c. and are provided 
with a graduated neck, each division of which represents 9.1 per cent by 
weight of butter fat. 
Fifteen centimeters of the milk are measured into the bottle, 3 c.c. of a 
mixture of equal parts of amyl alcohol and strong hydrochloric acid 
added and mixed. Then 9 c.c. of concentrated sulphuric acid is added 
in portions of about 1 c.c.; after each addition the liquids are mixed by 
giving the bottle a gyratory motion. If the fluid has not lost all of its 
milky color by this treatment, a little more concentrated acid must be 
added. The neck of the bottle is now immediately filled at about the 
zero point with one part sulphuric acid and two parts water, well mixed 
just before using. Both the liquid in the bottle and the diluted acid 
must be hot. The bottle is then placed at once in the centrifugal ma- 
chine; after rotation from one to two minutes, the fat will collect in 
the neck of the bottle and the percentage may be read off. 
Gerber's Test. This test is applied as follows: The test bottles are 
put into the stand with the mouths uppermost; then, with the pipette 
designed for the purpose, or with an automatic measurer, 10 c.c. of 
sulphuric acid are filled into the test bottle, care being taken not to 
allow any to come in contact with the neck. The few drops remaining 
in the tip of the pipette should not be blown out. Then 11 c.c. of milk 
are measured with the proper pipette and allowed to flow slowly onto 
the acid, so that the two liquids mix as little as possible. Finally, the 
amyl alcohol is added. (It is important to use the reagents in the 
proper order, which is-sulphuric acid, milk, amyl alcohol. If the sul- 
phuric acid is followed by amyl alcohol and the milk last, then the result 
is sometimes incorrect.) A rubber stopper, which must not be dam- 
aged, is then fitted into the mouth of the test bottle, and the contents 
are well shaken, the thumb being kept on the stopper to prevent it 
coming out. As a considerable amount of heat is generated by the 
action of the sulphuric acid on the milk, the test bottle should be wrapped 
in a cloth. 370 
MILK AND MILK PRODUCTS 
The shaking of the sample must be done thoroughly and quickly, 
and the test bottle inverted several times, so that the liquid in the neck 
becomes thoroughly mixed. By pressing in the rubber stopper the height 
of the liquid can be brought to about the zero point on the scale. 
If only a few samples have to be analyzed and the room is warm, 
the test bottles can be put into the centrifuge without any preliminary 
heating, otherwise the test bottles must be warmed for a few minutes 
(not longer) in the water bath at a temperature of 60° to 65° C. When 
the temperature rises higher than this, say above 70° C., the rubber stop- 
per is liable to be blown out of the test bottle. After the test bottles 
have been heated they are arranged symmetrically in the centrifuge and 
whirled for three to four minutes at a speed of about 1000 r.p.m. When 
the centrifuge has a heating arrangement attached to it, the preliminary 
warming is not, of course, necessary. When the test bottles are taken 
out of the centrifuge, they are again placed in the water bath at a tem- 
perature of 60° to 65° C., and left there for'several minutes before being 
read; where the centrifuge is heated, the tubes can be read off as taken 
from the centrifuge. 
By carefully screwing in the rubber stopper, or even by pressing it, 
the lower limit of the fat column is brought onto one of the main 
divisions of the scale, and then, by holding the test bottle against the 
light the height of the column of fat can be accurately ascertained. 
The lowest point of the meniscus is taken as the level when reading the 
upper surface of the fat in a sample of whole milk, and the middle of the 
meniscus for separated milk. 
If the column of fat is not clear and sharply defined, the sample 
must be again whirled in the centrifuge. 
Each division on the scale is equivalent to 0.1 per cent, so it is very 
easy to read to 0.05 per cent, or, with a lens, to 0.025 per cent. If the 
number which is read off is multiplied by 0.1, then the percentage quan- 
tity of fat in the milk is obtained; e.g., if the number on the scale was 
36.5, then the percentage of fat is 3.65. (Milk and Dairy Products, 
Barthel; translated by Goodwin, p. 71.) 
Arla-Jensen (1916) advises the use of chemically pure amyl alcohol. 
Proteins in Milk 
Procedure. . Measure 5 c.c. of the milk into a 500 c.c. Kjeldahl flask 
delivering it to the bottom of the flask. Add 25 c.c. of concentrated 
sulphuric acid and 0.7 gm. mercuric oxide or the equivalent of pure 
mercury. Heat the flask directly over a free flame in a hood until the TESTS FOR PRESERVATIVES 
371 
contents are yellow or white. After the flask has cooled add about 200 
c.c. of ammonia-free water, 20 c.c. of a potassium sulphide solution (40 
gms. potassium sulphide per liter of water) and a small piece of zinc. 
Connect the flask to a condenser and, after adding carefully 50 c.c. of a 
60 per cent of sodium hydroxide solution, distill, receiving the distillate 
in standard acid solution. Titrate the excess of acid with standard 
alkali, using methyl orange and compute the amount of nitrogen in the 
milk. This percentage of nitrogen multiplied by 6.25 will roughly give 
the percentage of protein. 
Test for Coloring Matter in Milk. Shake about 10 c.c. of the milk 
with 5 or 10 c.c. of ether. Fresh milk will give a colorless supernatant 
ether layer, while, if coloring matter has been added to the milk, this 
ether layer will be colored. 
Detection of Preservatives. The refusal of milk to sour when 
placed in a warm place is evidence that a preservative has been added. 
The following preservatives are often found in milk and their detection 
may be carried out as follows: 
Benzoic Acid. Add a few drops of lime water to 300 c.c. of the sam- 
ple and reduce the volume to about 75 c.c. Mix powdered calcium 
sulphate with this to a paste and evaporate to dryness. When dry, 
grind and moisten with dilute sulphuric acid; shake out in 50 per cent 
alcohol. Repeat this three times and reduce the acid alcohol solution 
to small volume on the water bath after adding barium hydroxide to 
neutrality. Acidulate with sulphuric acid and extract with ether. 
When the ether is evaporated, the benzoic acid is left behind. This 
precipitate may be subjected to further study. 
Boric Acid. Add sodium hydroxide to 75 or 100 c.c. of the milk and 
evaporate to dryness. Fuse and dissolve the ash in water. Add a 
little hydrochloric acid and filter. Moisten a strip of turmeric paper 
with the filtrate and dry on a watch glass. A red color indicates boric 
acid or its salts. 
Salicylic Acid. Add a little sulphuric acid to 15 or 20 c.c. of the milk 
and shake out with ether. Evaporate to dryness and treat the residue 
with alcohol and ferric chloride. The presence of salicylic acid is indi- 
cated by a violet color. 
Formaldehyde. To 5 c.c. of the milk add 5 c.c. of distilled water 
and 10 c.c. of hydrochloric acid, containing a little ferric chloride. 
The presence of formaldehyde is indicated by a violet color. 
Detection of Heated Milk. Storch's Method. Five c.c. of milk 
are poured into a test tube; a drop of weak solution of hydrogen dioxide 
(about 0.2 per cent) which contains about 0.1 per cent of sulphuric acid 372 
MILK AND MILK PRODUCTS 
is added, and two drops of a 2 per cent solution of paraphenylendiamin 
(solution should be renewed quite often), then the fluid is shaken. If 
the milk or cream becomes, at once, indigo blue or the whey violet or 
reddish brown, then this has not been heated, or, at all events, it has 
not been heated higher than 78° C.; if the milk becomes a light 
bluish gray immediately or in the course of half a minute, then it 
has been heated to 79° to 80° C. If the color remains white, the 
milk has been heated at least to 80° C. In the examination of sour 
milk or sour buttermilk, lime water must be added as the color 
reaction is not shown in acid solution. (Report of the Commission 
on Milk Standards.) 
Arnold's Guaiac Method. A little milk is poured into a test tube 
and a little tincture of guaiac is added, drop by drop. If the milk has 
not been heated to 80° C. (176° F.) a blue zone is formed between the 
two fluids; heated milk gives no reaction, but remains white. The 
guaiac tincture should not be used perfectly fresh, but should have 
stood a few days and its potency have been determined. Thereafter it 
can be used indefinitely. These tests for heated milk are only active in 
the case of milks which have been heated to 176° F. or 80° C. (Jensen's 
Milk Hygiene, Pearson's translation, p. 192.) 
Microscopic Test for Heated (Pasteurized) Milk. Frost and 
Ravenel (1911). About 15 c.c. of milk are centrifuged for five minutes, 
or long enough to throw down the leucocytes. The cream layer is then 
completely removed with absorbent cotton and the milk drawn off with 
a pipette, or a fine-pointed tube attached to a Chapman air pump. 
Only about 2 mm. of milk are left above the sediment which is in the 
bottom of the sedimentation tube. 
The stain, which is an aqueous solution of safranin 0, soluble in water, 
is then added very slowly from an opsonizing pipette. The important 
thing is to mix stain and milk so slowly that clotting does not take place. 
The stain is added until a deep opaque rose color is obtained. After 
standing three minutes, by means of the opsonizing pipette, which has 
been washed out in hot water, the stained sediment is then transferred 
to slides. A small drop is placed at the end of each of several slides and 
spread by means of a glass spreader, as in Wright's method for opsonic 
index determinations. 
In an unheated milk the polymorphonuclear leucocytes have their 
protoplasm slightly tinged or are unstained. 
In heated milk the polymorphonuclear leucocytes have their nuclei 
stained. In milk heated to 63° C. or above, practically all of the leu- 
cocytes have their nuclei definitely stained. When milk is heated at a TESTS FOR HEATED MILK 
373 
lower temperature the nuclei are not all stained above 60° C. The 
majority, however, are stained. 
Frost (1915) has pointed out some of the difficulties of the above 
method and devised a new one. 
Procedure. Add one part of aqueous methylene blue (7 gms. of 
Gruebler's dry dye to 100 c.c. distilled water) to five parts of milk. 
This should be added slowly to prevent coagulation of the milk. Allow 
the stain to remain in contact with the milk for from fifteen to thirty 
minutes. Centrifuge and spread the sediment on a slide. This may 
be done by using the edge of another slide. Frost reports the following 
appearance in raw and pasteurized milk. Raw: The entire field is 
stained a light blue, the depth of the stain depending on the thickness 
of the film. In this blue background may be clear areas. These may 
be either leucocytes or fat globules. The leucocytes are practically 
always colorless. Heated Milk: Smears made from heated milk are not 
as deeply stained as those made from raw milk. The leucocytes are 
always more deeply stained than the background and appear as dark 
blue areas in a blue field. The background immediately about the 
leucocytes frequently shades off into the color of the background, thus 
forming a halo about them. Under the oil immersion objective the 
leucocytes are less regular in outline than those in raw milk. The nuclei 
are distinctly stained. 
Balaz's Test for Boiled Milk. To 5 c.c. of the milk, add 2 c.c. of 
copper sulphate solution which contains 69.26 gms. of copper sulphate per 
liter. Shake and filter. Then, to five drops of the transparent filtrate 
add Adamkiewicz's reagent (one part of sulphuric acid and two parts of 
acetic acid). Warm carefully without boiling, shake, and allow to 
stand for a while. The serum of the boiled milk remains colorless, while 
the raw milk shows a violet red color. Its greatest intensity occurs 
within ten or fifteen minutes. 
The " Commission on Milk Standards Appointed by the New York 
Milk Committee " has made a careful study of the production of safe 
milk. It is composed of the foremost authorities on the relation of 
milk to the public health. Realizing the lack of uniformity with regard 
to milk standards, the committee made the following recommendations. 
Chemical Standards. The following are proposed and represent 
those which are enforced in many cities and States. 
The Babcock test makes easily practicable the determination of 
fat and solids-not-fat in milk. Such examinations of milk can be readily 
adopted and executed by any health board laboratory at a very moderate 
expense. It is believed that such chemical standards as are suggested 374 
MILK AND MILK PRODUCTS 
will help to raise the standards of dairying in this country, and that the 
provision regarding substandard milk is a liberal one. 
Cow's Milk. Standard milk should contain not less than 8.5 per 
cent of solids-not-fat and not less than 3.25 per cent of milk fat. 
Skim Milk. Standard skim milk should contain not less than 8.75 
per cent of milk solids. 
Cream. Standard cream should contain not less than 18 per cent of 
milk fat, and should be free from all constituents foreign to normal 
milk. The percentage of milk fat in cream over or under that standard 
should be stated on the label. 
Adjusted Milks. On the question of milks and creams in which the 
ratio of the fat to the solids-not-fat has been changed by the addition 
to or subtraction of cream or milk fat the commission has hesitated to 
take a position. On the one hand they are in favor of every procedure 
which will increase the market for good milk and make the most profit- 
able use of every portion of it. On the other, they recognize the 
sensitiveness of milk, the ease with which it is contaminated, and the 
difficulty of controlling such processes as standardizing, skimming, homo- 
genizing, souring, adjusting, etc., so as to prevent contamination and 
the use of inferior materials. On this subject the commission passed a 
resolution presented by a special committee, as follows: 
The committee believes that it is probably necessary to admit standardized 
and adjusted milk. They believe that such manipulation should be controlled 
and that such milk should be distinctly labeled as to its modifications. 
Milk in which the ratio of the fat to the solids-not-fat has been changed by 
the addition to or subtraction of cream should be labeled "adjusted milk"; the 
label should show the minimum guaranteed percentage of fat and should comply 
with the same sanitary or chemical requirements as for milk not so standardized 
or modified. 
The committee very carefully considered the subject of the agita- 
tion which has taken place regarding percentage of solids-not-fat due 
to the fact that in some large cities much of the milk contains less than 
8.5 per cent solids-not-fat. While the commission is disposed to admit 
that these conditions may exist, yet it believes that these conditions can 
be remedied, if not immediately at least gradually. On the other hand, 
experience has shown that to lower the standard would, in a few' years, 
result in the lowering of the general quality of the milk placed on the 
market, since commerce always tends to approach the minimum stand- 
ard. The commission, therefore, thinks it is unwise to reduce the 
standard for solids-not-fat below the percentage of 8.5. In those com- 
munities where such a standard can not be rigidly enforced at the BACTERIAL EXAMINATION 
375 
present time the commission suggests that the standard be gradually 
applied. 
The Bacterial Examination of Milk 
The value of the bacterial examination of milk is somewhat depend- 
ent upon the application of the results and the opinions of those who are 
interpreting them. The Commission on Milk Standards regards the 
bacterial content as being due to dirt, temperature and age. The bac- 
terial examination, then is supposed to yield information bearing on 
these three factors. 
Initial Contamination. Milk is such an excellent medium for bac« 
teria that the initial infection determines the number which will be 
present after a time. The factors which influence this may be men- 
tioned as follows: 
I. Cow 
a. Udder. 
b. Coat, etc. 
II. Barn Conditions 
III. Milker 
IV. Utensils 
a. Pails, cans, etc. 
b. Special apparatus 
1. Separator 
2. Clarifier 
3. Pasteurizer 
4. Cooler 
5. Bottler 
It is fairly well established that milk as it is excreted from the udder 
is not sterile. Harding and Wilson (1913) and other workers to whom 
they refer in their paper furnish much information to confirm this state* 
ment. An examination of 1230 samples from the udders of 78 cows 
showed an average of 428 bacteria per cubic centimeter. Evans (1916) 
studied 192 samples of milk from 161 cows in dairies which were widely 
separated. Three types of bacteria were found commonly present in 
milk from the five dairies which were studied: streptococci, staphy- 
lococci and bacilli. Streptococcus lacticus (Kruse) was not found in any 
of the samples. This author states that " there is a definite udder 
flora comprising bacteria which belong to parasitic types. It is not 376 
MILK AND MILK PRODUCTS 
surprising that the majority of udder bacteria should be of the same 
type as those common on the skin and mucous membranes of man and 
animals. The majority of the staphylococci on the skin are of the non- 
virulent variety which fails to produce pigment and fails to ferment 
mannite. But pathogenic varieties are also found on the skin where 
they ordinarily cause no trouble. . . . Whatever the variety may be, 
conditions in the udder are favorable to multiplication and frequently 
large numbers are eliminated in the milk." Rogers and Dahlberg 
(1914) in studying the origin of streptococci in milk decided that they 
were contributed by the feces; infected udders and the animals' mouths 
were also mentioned. Licking of the flanks and udders allow the bac- 
teria to reach the milk. Sherman (1915) found 19 out of 142 cows from 
6 different herds that gave a milk with over 10,000 bacteria per cubic 
centimeter. Two of them had a bacterial content of 100,000 bacteria 
per cubic centimeter in their fresh milk. Sherman states that all of 
the cows were in a normal healthy condition but he does not state what 
data were secured to arrive at this conclusion. In 48 cows, in which 
there were no indications of disease, there were streptococci in the milk 
of 15. He regards the streptococci of milk as of little significance, 
which is a very interesting statement in light of the work on septic sore 
throat. Colwell (1917) studied the influence of high count and gargety 
cows on the number of bacteria in milk. Seventy-two per cent of the 
cows examined produced a milk with less than 10,000 bacteria per cubic 
centimeters. The other 28 per cent were classed as high-count animals. 
An interesting fact was established that, where one or more of the 
quarters were infected with an organism, the same organism could be 
isolated from the other quarters. Such data have much significance in 
the question of the relation of milk to septic sore throat; it points out 
that all of the milk from an animal with an infected udder must be 
excluded from the milk supply. - 
From the literature which has beeen presented above, it will be seen 
that some difference of opinion exists about the udder flora. The pre- 
dominating organisms seem to be micrococci and, as Evans pointed out, 
may come from the cow's body. These enter the teat and reproduce 
since they are in a favorable environment. With regard to high-count 
cows too little information is available to definitely understand them. 
They are excluded from certain herds when certified milk is being pro- 
duced. No exacting study is usually made to determine whether they 
are really healthy. If nothing is outwardly the matter with them, they 
are usually considered healthy without any other examination. It is 
possible that the large numbers of bacteria in the milk of certain cows, FACTORS INFLUENCING BACTERIAL CONTENT 
377 
could be explained by a more careful investigation with regard to the 
condition of the animal. 
Barn Conditions. Some difference of opinion still exists with regard 
to the effect of barn conditions on the bacteria in milk. Harding et al. 
(1913) found that many procedures about the barn which were sup- 
posed to limit the number of bacteria contributed to milk might, under 
some conditions, increase the count. Huehle and Kulp (1915) have 
shown that dusty air in a stable might increase the bacteria in milk 
but the increase, as such, would be of little sanitary importance. If 
these added bacteria developed, the increased count, at a later time, 
would come within sanitary considerations. Savage (1909-10) found a 
general agreement between the cleanliness of cows' stables and the 
number of bacteria in milk. This also was borne out by the work of 
Brainerd (1911). 
Utensils. Recent work points to the conclusions that utensils 
may be one of the most important factors determining the number of 
bacteria in milk. Pease (1916) claims that, where high counts have 
been obtained in a dairy which has operated for a long time, they are due 
to inefficiently cleaned apparatus and by incubation of the bacteria on 
the moist surfaces of the cans, pails, etc. Prucha, Harding and Weeter 
(1915) reported some interesting data to substantiate this contention 
of Pease. With sterile utensils and sterile bottles, milk leaving the 
barn was found to contain 2588 bacteria per cubic centimeter and the 
bottled milk 3875. Where the utensils were simply washed and with 
only the bottles sterile, there were increases due to the pails of 57,077, 
up to the clarifier of 15,353, due to the clarifier of 172,763, due to cooler 
of 19,841 and due to the bottler of 247,611. More data on the same 
subject have been since reported by Prucha, Weeter and Chambers 
(1918). Ayers, Cook and Clemmer (1918) in a careful study of the 
factors involved in the production of a milk of low bacterial content, 
state as follows: " Three simple factors were necessary for the pro- 
duction of milk with a low bacterial content, namely, sterilized utensils, 
clean cows with clean udders and teats, and the small top pail." A 
fourth factor, holding the milk at a temperature near 10° C. or lower is 
necessary in order to keep the bacterial count low. Even cow manure 
was found to be of less importance in influencing the bacterial count 
than utensils. 
Age. The bacterial count is supposed among other things to yield 
information with regard to the age of the milk. Up to certain limits 
this is true, but soon a limit is reached beyond which no further growth 
may take place unless something happens to disturb the conditions. 378 
MILK AND MILK PRODUCTS 
The number of bacteria is probably not proportional to the age of the 
milk. 
Temperature. The effect of temperature on the bacteria in milk 
has received attention from several standpoints. No single extensive 
piece of work has been carried out on this subject and consequently our 
knowledge must come from the various separate pieces of work. Lux- 
wolda (1911) found that milk kept at low temperatures contained an 
enormous number of bacteria. These did not cause an acid reaction 
in the milk but the changes were regarded as more dangerous than if 
the acid bacteria had been present. At 20° C. lactic acid bacteria 
appeared and exerted a restraining action on the other forms. This 
restraining action was steadily diminished as the temperature was 
lowered. Reed and Reynolds (1916) inoculated sterile flasks of milk 
with pure cultures and incubated them at four different temperatures 
for six weeks. The results are shown in Table XL. 
Table XL 
EFFECT OF AGE AND TEMPERATURE ON BACTERIAL GROWTH IN 
MILK 
(After Reed and Reynolds) ■ 
Name of Organism. 
Age in Days, of Milk kept at Various Tempera- 
tures, which Gave Maximum Counts on Nutrient 
Agar. 
Incubator, 
35° C. 
Room, 
15-28° C. 
Water Tank, 
13° C. 
Cold Stor- 
age, - 1° C. 
Bacterium lactis acidi 
1 
3 
5 
3 
Sarcina lutea 
1 
42 
42 
21 
Bacillus coli 
3 
5 
5 
2 
Bacillus cyanogenes 
2 
21 
4 
42 
Bacillus proteus vulgaris 
2 
2 
4 
3 
Bacillus aerogenes 
3 
5 
42 
3 
Bacillus fluorescens liquefaciens. . . 
2 
5 
42 
3 
Bacillus puditum 
3 
4 
5 
21 
Microspira tyrogena 
3 
21 
42 
42 
Bacillus subtilis 
3 
42 
42 
21 
Micrococcus citricus 
21 
5 
4 
4 
Oidium lactis 
42 
42 
5 
42 
Bacillus prodigiosus 
3 
42 
42 
42 
Reed, II. S. and Reynolds, R. R. Some effects of temperature upon the growth and activity 
of bacteria in milk. Virginia Ag. Exp. Sta. Bull. (Tech.), 10, 1916. 
Conn and his co-worker Esten (1902, 1903, 1904) gave much atten- 
tion to this subject. Their work was some of the earliest on this sub- 
ject. In general, they found that at 20° C. the ordinary lactic acid "QUALITY" IN MILK 
379 
bacteria predominated. At the end of forty hours, when the milk 
had curdled, Bacillus acidi lactici constituted about 90 per cent of the 
total bacteria. At 37° C., B. lactis aerogenes predominated over B. 
acidi lactici as did B. coli when it was present. At 10° C. all types of 
bacteria developed uniformly. Neutral bacteria grew more rapidly 
and liquefiers became abundant. Conn and Esten found little differ- 
ence in the flora at 10° C. and 1° C. Growth, however, was more 
rapid at 10° C. Ravenel et al. (1910) have studied the changes in milk 
at -9° C. and 0° C. At -9° C. there was no increase in the number of 
bacteria as determined on agar and gelatin plates. At 0° C. there was a 
decided increase. This was accompanied by an increase in acidity 
and other changes incident to marked bacterial development. The 
lactic acid bacteria were inhibited at 4 his temperature but putrefactive 
bacteria were not. 
Pennington et al. (1908,1913) in their studies on colci storage reported 
that milk stored at 0° C. underwent marked proteolysis which was 
noticeable at the end of two weeks. In a later paper it was stated that 
proteolysis of casein was due primarily to bacteria while the lact- 
albumin was destroyed by the enzymes in the milk. The numbers of 
bacteria greatly increased and this increase was most striking in the 
raw, untreated milk. The freezing-point was gradually lowered with 
the decomposition. Market milk held below 0° C. increased from 15,956 
to 376,000,000 at the end of five weeks. These investigations tend to 
indicate that milk and other foods may not be held indefinitely even at 
low temperatures. Changes may eventually take place to render the 
food unfit for consumption. 
" What is Meant by Quality in Milk?" Quality in any food sub- 
stance may be defined in different ways. The definition often depends 
entirely on the viewpoint of the definer. The farmer may have quite 
another conception of quality in milk than the sanitarian; the chemist 
may define milk quality from the standpoint of food value while the 
bacteriologist is concerned with disease. It is a far easier proposition 
to define milk quality than to devise methods for measuring it. One 
could scarcely find a field in hygiene which is more complicated than 
that of dairy and milk sanitation. The difficulty of finding practical 
methods for measuring quality in milk has caused much contention 
among health authorities. Probably little difficulty would be experi- 
enced in reaching a definition for safe milk. A milk to be safe must be 
clean. In fact cleanliness is the slogan of modern sanitation. 
A number of different methods have been used or proposed for 
measuring quality in milk. Each one has its own special advantages and 380 
MILK AND MILK PRODUCTS 
objections. Sonic have been discussed before in this book and will not 
be given further attention. The following methods have been pro- 
posed at one time or another: 
1. Sanitary inspection of the dairies; 
2. Bacterial examination of the milk; 
3. Dirt or sediment test. 
Harding et al. (1917) consider that the elements of quality in city 
milk to consist of food value, healthfulness, cleanliness and keeping 
quality. The discussion of these factors leads these authors to the 
following combination of them to form a basis for grading milk. Park 
Grade 
Element of Quality 
Food value 
Healthfulness 
Cleanliness 
Keeping quality 
Fat content as stated on package 
Medical supervision of health of men and 
animals, or proper pasteurization 
Sediment, not more than a trace 
Excellent 
Degree of Excellence 
Special milk 
Food value 
Healthfulness 
Cleanliness 
Keeping quality 
Fat content as stated on package 
Properly pasteurized 
Sediment, not more than a small amount 
Good 
Table milk 
Food value 
Healthfulness 
Cleanliness 
Keeping quality 
Fat content as stated on package 
Boiled 
May not be sufficient for table grade 
May not be sufficient for table grade 
Cooking milk 
(1918) does not approve some of the claims in this report. He calls 
attention to the fact that Harding and his collaboratois are inter- 
ested in the dairy instead of the infant. He further states, " The 
experts . . . were considering the best methods of grading milk and 
not the question as to whether excessive numbers of bacteria in 
market milk were wholesome or not. It would be, to my mind, a step 
backward to accept the views expressed in the body of the editorial." 
(From a letter discussing this report.) Park and his colleagues at the 
Research Laboratories of the New York City Department of Health 
have given much study to the relation of milk bacteria to disease in 
children. 
The Dairy Score Card. No attempt will be made to present in this 
place a complete discussion of the dairy score card. The score card had DAIRY SCORE CARD 
381 
its origin in 1904 when Doctor W. C. Woodward prepared one for use 
in Washington A year later Pearson at Cornell University devised a 
similar card. The score card received such approbation by sanitarians 
that the National Association of Dairy Instructors appointed a com- 
mittee which had much to do with the present official score card. Much 
has been written in favor of and against the card. It is regarded by some 
milk hygienists as one of the most important factors in the production 
of clean milk. Others, even though they represent large cities do not 
regard the card as of any great help in milk control. It must be empha- 
sized that the score card was not devised to give information with regard 
to bacteria in milk but merely to rate the production plants. It has 
been established, generally speaking, that a high scoring dairy will have 
a low number of bacteria in its milk. This is to be expected. Some of 
the recent work in dairy hygiene has emphasized the fact that the 
present score card lays too much stress on factors which are not directly 
concerned with the production of milk with few bacteria. Brew (1915) 
made a comparative study of the bacteria in the milk and the scores of 
thirty-four commercial dairies. He found no correlation between these 
two factors. The apparent reason for this was that a large number of 
items called for on the score card have little or no effect on the bacteria 
in the milk. ' Too much emphasis is placed on procedures which do not 
affect the milk. This study indicates that the score card needs revision 
if it is to be an index of the bacteria in milk. As indicated by recent 
studies, within certain limits, the environment of the cow does not seem 
to exert much influence on the milk. Prucha, Harding and Weeter 
(1915) also confirm the contention of Brew that the score card needs 
revision. They found that utensils were important in relation to the 
number of bacteria in milk. These are not given an important place 
on the card. Ayers, Cook and Clemmer (1918) reach the same con- 
clusion. Brainerd and Mallory (1914) in studying the Richmond, 
Virginia, milk supply by means of the bacterial count and score card 
found the count to be cumbersome-a high count not always being char- 
acteristic of a dangerous milk. For some reason which was not deter- 
mined, they found that the highest scoring dairy showed a larger count 
than the lowest scoring dairy. North (1917) believes that the life of 
the present score card is threatened if it is not changed to give a reason- 
ably close indication of the character of the product of dairies. North 
has presented a revised score card on which he has tried to place 
emphasis on factors which are concerned in keeping the number of 
bacteria in milk low. This to a certain extent may change the purpose 
of the card. 382 
MILK AND MILK PRODUCTS 
BOARD OF HEALTH 
Racine, Wis. 
Sanitary Inspection of Dairies 
Dairy Score Card 
Owner or lessee or farm 
P. O. Address State 
Total Number of cows Number milking 
Gallons of milk produced daily 
Product is retailed by producer in 
Sold at wholesale to 
For milk supply of 
Permit No Date of Inspection , 191.. 
Remarks • 
{Signed) 
Inspector. DAIRY SCORE CARD 
383 
Equipment. 
COWS 
Health 
Score. 
Methods. 
COWS 
Score. 
Perfect. 
6 
2 
2 
2 
o 
4 
4 
3 
3 
1 
1 
3 
1 
1 
Allowed. 
Perfect. 
8 
6 
6 
2 
2 
3 
8 
9 
1 
2 
2 
3 
3 
Allowed. 
Apparently in good health 
6 
If tested with tuberculin 
once a year and no tu- 
berculosis is found, or 
if tested once in six 
months and all reacting 
animals removed. ... 5 
(If tested only once a year 
and reacting animals found 
and removed, 2.) 
Comfort 
STABLES 
Cleanliness of stables 
Floor 2 
Walls 1 
Ceiling and ledges 1 
Mangers and partitions 1 
Windows 1 
Stable air at milking time. . 
Freedom from dust.... 3 
Freedom from odors. . . 3 
Barnyard clean and well 
drained 
Removal of manure daily 
to field or proper pit 
(To 50 ft. from stable, 1.) 
MILK ROOM 
Cleanliness of milk room. . . 
UTENSILS AND 
MILKING 
Care and cleanliness of uten- 
sils 
Bedding 1 
Temperature of stable. 1 
Food (clean and wholesome) 
Water 
Clean and fresh 1 
Convenient and abundant 
1 
STABLES 
Location of stable 
Well drained 1 
Free from contaminating 
surroundings 1 
Construction of stable 
Tight, sound floor and 
proper gutter 2 
Smooth, tight walls and 
ceiling 1 
Proper stall, tie, and man- 
ger 1 
Means of light: 4 sq.ft, of 
glass per cow 
(Three sq.ft., 3; 2 sq.ft., 
2; 1 sq.ft., 1. Deduct for 
uneven distribution.) 
Ventilation: Automatic sys- 
tem 
Adjustable windows 1 
Cubic feet of space for cow: 
500 to 1000 ft 
Thoroughly washed.... 2 
Sterilized in live steam 
for thirty minutes... 3 
(Placed over steam jet, or 
thoroughly scalded with 
boiling water, 2.) 
Inverted in pure air 3 
Cleanliness of milking 
Clean, dry hands 3 
Udders washed and dried 
6 
1 (Udders cleaned with 
mois.t cloth, 4; cleaned with 
dry cloth at least fifteen min- 
utes before milking, 1.) 
HANDLING THE MILK 
Cleanliness of attendants in 
milk room 
Milk removed immediately 
from stable 
Prompt cooling (cooled im- 
mediately after milking 
each cow) 
Efficient cooling: below 50° 
F 
(Less than 500 ft., 2; 
less than 400 ft., 1; less than 
300 ft., 0; over x000 ft., 0.) 
UTENSILS 
Construction and condition 
of utensils 
Water for cleaning 
(Clean, convenient, and 
abundant.) 
Small-top milking pail 
Facilities for hot water or 
steam 
(Should be in milk house 
not in kitchen.) 
Milk cooler 
(51° to 55°, 4; 56° to 60°, 
2.) 
Storage: below 50° F 
(51° to 55°, 2; 56° to 60°, 
1) 
Transportation: iced in sum- 
mer 
. . .... 
(For jacket or wet blan- 
ket, allow 2; dry blanket or 
covered wagon, 1.) 
Clean milking suits , 
MILK ROOM 
Location of milk room 
Free from contaminating 
surroundings 1 
Convenient 1 
Construction of milk room. 
Floor, walls, and ceiling, 1 
Light, ventilation, screens 
1 
Total 
1 
2 
2 
40 
■ ' 1 1 - - - 1' -II 
- 
DETAILED SCORE 
Equipment 4- Methods = Final score. 
Note 1.-If any filthy condition is found, particularly dirty utensils, the total score shali be 
limited to 49. 
Note. 2.-If the water is exposed to dangerous contamination, or there is evidence of the 
presence of a dangerous disease in animals or attendants, the score shall be 0. 384 
MILK AND MILK PRODUCTS 
Dirt or Sediment in Milk. The estimation of dirt in milk has been 
regarded by many as a reliable index of milk quality. Most attention 
has been given to insoluble dirt or that which may be removed from the 
milk by some mechanical means. Schroeder (1914) has given a resume 
of the different methods of determining dirt in milk. He stated that the 
amount of dirt in fresh milk approximates the number of bacteria. This 
was borne out by the work of Reed and Reynolds whose data bearing 
on this question have been presented in Table XLI. Campbell (1916) 
carried out an interesting study on the relation of the number of bac- 
teria to the amount of dirt and stated that the amount of dirt which 
appeared on the disks was no criterion of the number or kind of bacteria 
in the milk. Ayers, Cook and Clemmer (1918) found that*the " sedi- 
ment test bore a somewhat close relation to the number of bacteria in 
fresh, unstrained milk handled in sterilized utensils." The kind of dirt 
is, of course, important in this connection. Cow dung would probably 
be an objectionable type. Ayers and his colleagues, however, state 
that cow dung (fresh) " though an important source of contamination in 
general, is not so great a factor as unsterilized utensils in causing high 
bacterial counts." Kinyoun (1914) regards cleanliness in production 
as the most important factor in milk hygiene. From an examination 
of 3000 samples of milk he states that in good milks, there is 1 colon 
bacillus to every 50,000 bacteria while in dirty milk the ratio is 1 to 555. 
Table XLI 
RELATION BETWEEN THE AMOUNT OF DIRT IN MILK AND THE 
BACTERIAL CONTENT 
(After Reed and Reynolds) 
Amount of Dirt in 100 c.c. of Milk. 
Number of Bacteria per 
Cubic Centimeter of Milk. 
10 milligrams 
4,833 
20 milligrams 
6,750 
30 milligrams 
12,(XXI 
40 milligrams 
17.625 
50 milligrams 
18,375 
100 milligrams 
32,875 
The available data seem to indicate that milk may be produced 
under decidedly filthy conditions and yet have a low bacteria content. 
The utensils seem to be the contributing factor. These data should 
not be taken to prove that barns may be kept dirty. Milk, which is 
produced in dirty plants, is more liable to contain dirt; this dirt may be SIGNIFICANCE OF BACTERIAL CONTENT 
385 
very objectionable. It is reasonable that a farmer who is careful about 
the condition of his cows will be just as careful about the other features 
which are known to affect milk quality. 
Schroeder (1914) has classified the methods of determining dirt in 
milk as follows: 
Group I. (a) Type in which the sediment is obtained by gravity fil- 
tration. 
Conn's filter paper method. 
The Lorenz or Wisconsin tester. 
The Stewart tester. 
The Gerber tester. 
The Schroeder tester, or multiple filter. 
(6) Type in which pressure or suction is used. 
The Lorenz improved. 
The Wizard. 
The Gooch crucible. 
Group II. Type in which sediment is obtained by means of the cen- 
trifuge. 
The Babcock. 
The Gerber. 
The Stewart-Slack. 
Conn's centrifugal method. 
Significance of the Bacterial Count. There is no agreement on this 
important question. The Commission on Milk Standards makes the 
following statement on the subject: 
Bacterial Counts and Decency. On this subject the commission passed the 
following resolutions: 
(a) Because high bacterial counts indicate milk is either warm, dirty, or 
stale, the bacterial count is an indicator of decency in milk character, entirely 
apart from its significance as an indicator of the safety of milk. 
(6) In determining the sanitary character of milk and the grade in which it 
belongs, decency must be considered as desirable for its own sake, entirely apart 
from the consideration of safety. Decency is important as a characteristic of 
foods and drinks, because it gives pleasure to the consumption of food, while 
the lack of decency means distaste, displeasure, and even disgust. 
(c) The bacterial count is a sufficiently accurate measure of decency to justify 
the health officer in condemning milk with a high bacterial count because it is 
lacking in this characteristic. 
Bacteriological Laboratory Testing of Milk. On the subject of laboratory 
examinations of milk for bacteria the commission believes that the interest of 386 
MILK AND MILK PRODUCTS 
public health demand that the control of milk supplies, both as to production 
and distribution, should include regular laboratory examinations of milk by 
bacteriological methods. They stated by resolution that- 
Among present available routine laboratory methods for determining the 
sanitary quality of milk, the bacterial count occupies first place, and that bac- 
terial standards should be a factor in classifying milk of different degrees of 
excellence. 
The adoption and enforcement of bacterial standards will be more effective 
than any other one thing in improving the sanitary character of public milk 
supplies. The enforcement of these standards can be carried out only by the 
regular and frequent laboratory examinations of milk for the numbers of bacteria 
it may contain. 
It is of the utmost importance that standard methods should be adopted by 
all laboratories for comparing the bacterial character of milks, since by this 
means only is it possible to grade and classify milks and properly enforce bac- 
terial standards. 
Concerning the methods which should be used by milk laboratories for 
determining the numbers of bacteria the commission unanimously resolved: 
That there be adopted as standards for making the bacterial count the stand- 
ard methods of the American Public Health Association Laboratory Section. 
One of the chief objections raised against pasteurization is the claim that it is 
frequently employed to cover filthy methods, the milk producer using less care 
in his methods if he knows that the milk is to be subsequently pasteurized. To 
meet this objection the commission believes there should be bacterial standards 
for raw milk as well as bacterial standards for pasteurized milk. In the case of 
pasteurized milk, standards should be required of the-milk before pasteurization 
as well as after pasteurization. 
Reliability of Bacterial Tests. The commission has considered the numerous 
criticisms that have been raised as to the unreliability of bacteriological analyses, 
and has made extensive inquiry as to the force of these criticisms. An opinion 
concerning the reliability of laboratory tests for numbers of bacteria has been 
reached based on voluminous statistics secured for the most part by groups of 
observers working together, as well as by individuals. One of these researches 
alone carried out by members of the commission in cooperation with others 
included the testing of over 20,000 samples of milk. In other instances repeat- 
edly the same sample of milk was tested one hundred times. Some variations 
in the analysis of duplicate samples are inevitable, due to the fact that the bac- 
teria are not in solution, but are floating in the milk more or less clustered to- 
gether in clumps, each of which will count only as a single colony. Under such 
conditions only an approximate agreement can be expected. 
The results of extensive study justify the commission in the conclusion that 
the analysis of duplicate samples of milk made by routine methods in different 
laboratories may be expected to show an average variation of about 28 per cent, 
with occasional samples of wider variation. In some good laboratories the varia- 
tion may not be greater than 10 per cent. Variations in results diminish w'ith RELIABILITY OF BACTERIAL COUNTS 
387 
the numbers of samples analyzed. If five samples of the same milk are tested 
the results may be relied upon as fairly accurate, and always sufficiently accurate 
to place any particular milk supply unhesitatingly in Grade A, B, or C. The 
object of bacterial tests of milk samples for the numbers of bacteria should be 
primarily to determine the sanitary character of the milk supply from which 
the sample is taken, rather than the character of a single sample of milk. It is 
strongly urged by this commission that no grading of milk should be made upon 
the analysis of single samples, and that no prosecutions or court cases should be 
brought upon the bacterial analysis of a single sample of milk. 
Interpretation of Bacterial Tests. The commission has put its opinions on 
this subject in the form of resolutions, as follows: 
Whereas milk is one of the most perishable foods, being extremely suscep- 
tible to contamination and decomposition; and 
Whereas the milk consumer is justified in demanding that milk should be 
clean, fresh, and cold, in addition to having the element of safety; and 
Whereas milk which is from healthy cows and is clean, fresh, and which has 
been kept cold, will always have a low bacterial count; and 
Whereas milk that is dirty, stale, or has been left warm, will have a high 
bacterial count; therefore it is resolved: 
First. That the health officer is justified in using the bacterial count as an 
indicator of the degree of care exercised by the producer and dealer in securing 
milk from healthy cows and in keeping the same clean, fresh, and cold; and 
Second. That the health officer is justified in condemning milk with a high 
bacterial count as being either unhealthy or decomposed, or containing dirt, 
filth, or the decomposed material as a result of the multiplication of bacteria 
due to age and temperature. 
Third. That the health officer is justified in ruling that large numbers of 
bacteria are a source of possible danger, and that milk containing large numbers 
of bacteria is to be classed as unwholesome, unless it can be shown that the bac- 
teria present are of a harmless type, as, for example, the lactic acid bacteria in 
buttermilk or other especially soured milks. 
Grading by the Bacterial Count. Concerning the number of tests which 
should be made in order to determine the grade of a milk supply, the commission 
recommends that the grade into which a milk falls shall be determined bac- 
teriologically by at least' five consecutive bacterial counts, taken over a period 
of not less than one week, nor more than one month, and that at least four out 
of five of these counts (80 per cent) must fall below the limit or standard set for 
the grade for which classification is desired. 
The grading of milk has necessarily been based on its sanitary character, 
primarily as determined by the bacterial test. The enforcement of grading, 
therefore, requires the application of the bacterial test in a manner sufficiently 
comprehensive to fairly determine the sanitary character of milk so that it may 
be assigned to the grade in which it belongs. Such an administrative system 
greatly modifies the former conception of milk inspection by public health 
officials. The inspection service under the grading system becomes subordinate 388 
MILK AND MILK PRODUCTS 
to the bacterial laboratory, or at least must look to the bacterial laboratory as a 
guide. If bacterial tests are recognized as an indication of the sanitary charac- 
ter of milk, then the bacterial laboratory tests should precede the dairy inspec- 
tion since they will point out to the dairy inspector the location of unsanitary 
milk. In the enforcement of the grading system, therefore, the milk inspection 
service should be reorganized in such a manner that the bacterial laboratory 
makes its tests first, in order to determine the sanitary character of the various 
milks offered for sale on the city market, and the inspection service then takes 
up the task of discovering the location and causes of the defects which the 
laboratory has discovered and of remedying them. The laboratory service and 
inspection service consequently must be centralized under one head and their 
work thoroughly coordinated in order to give the greatest economy and effi- 
ciency. 
It is quite apparent from the above rather lengthy quotation from 
the report of the Commission on Milk Standards, that they regard 
the bacterial count as of much importance. Contrary to this is the 
opinion of Harding (1917) and a few others who maintain that the 
number of bacteria in milk has no relation to the conditions under which 
the milk was produced. In order to back up this stand the following 
argument has been used. " The most offensive, and, at the same time, 
a typical form of filth that may get into milk is cow dung. The bac- 
terial plate count resulting under standard methods of milk analysis 
gives rather less than 5,000,000 per gram as the germ count of fresh cow 
dung. A gram of such material added to a liter (approximately 1 qt.) 
of milk would, accordingly, increase the germ count but 5000 per cubic 
centimeter. Hence, otherwise low-content milk might carry approxi- 
mately 2 gms. of cow dung per quart and still not exceed the ordinary 
limit of 10,000 per cubic centimeter for certified milk. Likewise a quart 
of Grade A milk, which is understood to be clean milk, according to the 
present accepted standards might contain 12 gms. of cow dung per 
quart and still be legal Grade A milk. ... In establishing bacterial 
count standards as an index of cleanliness, we are lending official sanc- 
tion to conditions which would outrage public decency and are creating 
a false sense of security and of cleanliness." It is common knowledge 
that fresh cow dung has a very variable bacteria content. Usually 
it is much higher than 5,000,000 per gram and may even approach or 
exceed a billion. Ayers et al. (1918) found an average bacterial content 
of 50,000,000 in 57 samples of fresh cow dung. This is probably 
dependent upon what the cow eats, for with human beings the diet has 
much to do in determining the bacterial flora. Then, again, fresh cow 
dung does not ordinarily gain entrance to milk. It is the dry dung BACTERIAL COUNTS 
389 
which happens to fall into the milk from the cow's body and this has 
a bacteria content which is far different from fresh cow dung. In this 
connection, Taylor (1918) has shown that 85 per cent of fresh cow dung 
will dissolve in milk. His studies were carried out with the fresh (wet) 
cow feces and it required a little restriction to apply his results directly 
to dirt in milk. Fresh cow dung contains about 85 per cent of moisture 
and this water ought not to be considered as cow feces. Prucha, 
Weeter and Chambers (1918) carried out a series of investigations on 
this subject using dried feces. In discussing this question of bacterial 
counts in milk Pease (1913, 1916) claims that " none of the bacteria 
found on our counting plates are even recognized by bacteriologists as 
belonging to the groups of those microorganisms which produce the well- 
known specific diseases." His general opinion seems to be that the 
dairy score card representing sanitary inspection of the dairy should 
come before the bacterial count and that any attempt to make it 
of secondary importance to the count is a decided step backwards. 
North (1913, 1916) has stated that confusion exists over the question 
of dirt and disease and that this has caused some misunderstanding. 
North (1917) has presented a revised score card and has tried to obviate 
some of the discrepancies inherent in the present one. In his earlier 
papers North states that there has been an undercurrent of competition 
between dairy inspection and bacterial testing and suggests that a 
system of cooperation be worked out between these two factors. One 
cannot help but be impressed with the fair-minded stand of this inves- 
tigator. 
Rogers (1915) claims that, how much a high bacterial count is due 
to contamination and how much is due to multiplication is uncertain. 
He suggests that it may become necessary to distinguish between 
measures to reduce the bacterial count and measures in the interest of 
decency and cleanliness. Conn (1917) has well stated the question. 
" The bacteriological analysis of milk is not to be taken as indicating in 
itself either a condition of safety or a condition of danger, but only as a 
warning. Good, clean, fresh milk will have a low bacterial count, and a 
high bacterial count means dirt, age, disease or temperature. A high 
bacterial count is, therefore, a danger signal and justifies the health 
officer in putting a source with a persistently high bacterial count among 
the class of unwholesome milk." Rosenau (1912) makes the following 
statement on this subject: 
The enumeration of bacteria in milk is, therefore, one of the readiest and 
cheapest methods at the disposal of the health officer to determine the general 
sanitary quality of the market milk supply. The laboratory results serve not 390 
MILK AND MILK PRODUCTS 
only as a guide to direct the efforts of the health officer, but to confirm the con- 
clusions arrived at from an inspection of the dairies and dairy farms. 
A review of the literature with regard to the bacterial analysis of 
milk indicates a confused condition. There seem to be two distinct 
views with regard to the significance of the number of bacteria in milk. 
That side supporting the view that the bacterial content should not be 
used is made up of those who are interested in the production of milk and 
possibly the " protection " of the farmer. The other side is supported 
by well-known physicians and public health officials who have been 
engaged for some time in protecting the consuming public from dirty 
foods. Park and Williams (1910) have shown a close relationship 
between infant death rate and the number of bacteria in milk. These 
data were secured after careful experimentation and observation. 
Numerous data have been secured in this connection when comparing 
the value of raw and pasteurized milk in infant feeding. In essentially 
all of the cases, the pasteurized milk has been shown to be superior and 
this is probably not all due to destruction of certain harmful bacteria. 
A milk or any other food which contains large numbers of bacteria, is 
more liable to contain undesirable forms than one with a low count. 
If the streptococci are of any sanitary significance and there are data 
on record which support the theory that they are, they are more liable 
to be present in milks with a large number of bacteria. Breed and 
Brew (1917), in studying the control of market milk by the microscopic 
examination, found that one-fifth of all the milk which contained 
bacteria in excess of 1,000,000 per cubic centimeter contained large 
numbers of streptococci. They state that, " So far as was deter- 
mined, all of the milk of this type was originally infected from 
the udders of some one or more cows in a herd." In this case, then, 
the number of the bacteria in the milk was closely related to undesira- 
ble conditions at the source of production. The greater part of the 
argument against the bacterial count seems to rest upon data which 
have been produced at several experiment stations to show that many 
of the barn conditions which have, in the past, been supposed to be 
very important, are not related to the number of bacteria in the milk 
when it reaches the consumer. These investigators argue that the 
quality of city milk is too complex a problem to be solved by the con- 
sideration of any one factor. They, therefore, put down the four 
essential factors as food value, healthfulness, cleanliness and keeping 
quality. From a bacteriological viewpoint the element of cleanliness 
is the most important because the others may depend upon this one. 
This element is supposed to be measured by the sediment or visible dirt. GRADES OF MILK 
391 
No attention is given to the " invisible dirt " which may be present. 
The sediment test of Weld (1907) detects only one kind of dirt-the 
insoluble or visible. It does not show the amount of invisible dirt or 
insoluble foreign matter, such as urine; this must be regarded as filth 
in the same way as other matter. Special mention is made of the 
" extreme sensitiveness of the public in this matter." It is open to 
question whether the public is sensitive to dirt in milk or any other food 
product. If it was so the filthy establishments where food is on sale 
would not be open for business very long. The absence of dirt in milk 
may not indicate that the milk has been produced under favorable 
conditions. That no dirt is visible, does not indicate a safe milk. This 
question, doubtless, needs more consideration from all standpoints. 
Grades of Milk. The commission believes that all milk should be 
classified by dividing it into three grades, which shall be designated by 
the letters of the alphabet. It is the sense of the commission that the 
essential part is the lettering and that all other words on the label are 
explanatory. In addition to the letters of the alphabet used on caps or 
labels, the use of other terms may be permitted so long as such terms 
are not the cause of deception. Caps and labels shall state whether 
milk is raw or pasteurized. The letter designating the grade to which the 
milk belongs shall be conspicuously displayed on the caps of bottles or 
the labels of cans. 
The requirements for the three grades shall be as follows: 
Grade A. Raw Milk. Milk of this class shall come from cows free from 
disease as determined by tuberculin tests and physical examinations by a qual- 
ified veterinarian, and shall be produced and handled by employees free from 
disease as determined by medical inspection of a qualified physician, under 
sanitary conditions, such that the bacterial count shall not exceed 10,000 per 
cubic centimeter at the time of delivery to the consumer. It is recommended 
that dairies from which this supply is obtained shall score at least eighty on 
the United States Bureau of Animal Industry score card. 
Pasteurized Milk. Milk of this class shall come from cows free from dis- 
ease as determined by physical examinations by a qualified veterinarian, and 
shall be produced and handled under sanitary conditions, such that the bac- 
teria count at no time exceeds 200,000 per cubic centimeter. All milk of this 
class shall be pasteurized under official supervision, and the bacteria count shall 
not exceed 10,000 per cubic centimeter at the time of delivery to the consumer. 
It is recommended that dairies from which this supply is obtained shall score at 
least 65 on the United States Bureau of Animal Industry score card. 
Grade B. Milk of this class shall come from cows free from disease as 
determined by physical examinations, of which one each year shall be by a 
qualified veterinarian, and shall be produced and handled under sanitary con- 392 
MILK AND MILK PRODUCTS 
ditions, such that the bacteria count at no time exceeds 1,000,000 per cubic 
centimeter. All milk of this class shall be pasteurized under official supervision, 
and the bacterial count shall not exceed 50,000 per cubic centimeter when deliv- 
ered to the consumer. 
It is recommended that dairies producing grade B milk should be scored, and 
that the health departments or the controlling departments, whatever they may 
be, strive to bring these sources up as rapidly as possible. 
Grade C. Milk of this class shall come from cows free from disease, as 
determined by physical examinations, and shall include all milk that is pro- 
duced under conditions such that the bacteria count is in excess of 1,000,000 per 
cubic centimeter. 
All milk of this class shall be pasteurized, or heated to a higher temperature, 
and shall contain less than 50,000 bacteria per cubic centimeter when delivered 
to the consumer. 
Whenever any large city or community finds it necessary, on account of the 
length of haul or other peculiar conditions, to allow the sale of grade C milk, its 
sale shall be surrounded by safeguards such as to insure the restriction of its use 
to cooking and manufacturing purposes. 
Milk Standards 
If it is difficult and intricate to formulate standards for various 
foods, it is especially true with regard to milk. To a great extent, this 
is due to the various number of standpoints from which the milk ques- 
tion may be considered. The farmer is interested more in securing a 
reasonable financial return on the investment in his milk production 
plant. The sanitarian, however, often gives little attention to the 
farmer's side of the question and considers only the effect of the milk 
on the health of the consumer. Undoubtedly, a broader consideration 
by any one of these specialists would greatly clear up some of the mis- 
understanding with regard to certain phases of the milk question. 
Milk standards are essential but the question with regard to who 
shall formulate them is still an open one. If a study is made of the 
milk standards which are being enforced by many of our large cities, the 
great discrepancy is apparent. There is essentially no agreement 
whatever in many of these standards. The Commission on Milk Stand- 
ards in their third report has attempted to establish a simple system for 
grading milk and to distinguish between milks which are different in 
their sanitary and other characteristics. The variation in bacterial 
standards for milk analysis is emphasized very well in Reprint No. 
192, May 15, 1914, from the Public Health Reports 29. The standards MILK STANDARDS 
393 
for many cities in the United States having a population of 10,000 or 
over are tabulated. 
The procedure for the bacteriological examination of milk has been 
standardized in the same way as has the bacteriological examination 
of water and sewage. Standard Methods of Bacteriological Analysis 
of Milk prepared by the Laboratory Section of the American Public 
Health Association are now used by all bacteriologists making routine 
analyses of milk and wishing their results to be comparable to those 
secured in other laboratories. For that reason, much of the following 
is adapted or copied, with permission, from that report: 
Routine Milk Analysis. This type of analysis is designed for the control of 
the public milk supply and for the purpose of grading milk. For routine analysis 
the work must be capable of being done quickly and cheaply, and in such a way 
as to give the most speedy results possible and by methods having the smallest 
possible expenditure of time and money consistent with fair uniformity of 
result. The demand for rapid methods, with the smallest possible expenditure 
of time and money has been forced on different laboratories in the past few 
years, and has brought about various short cuts from the standard methods as 
previously formulated. The necessity for such rapid and inexpensive methods 
must be recognized. But it is also evident that they should be uniform in' dif- 
ferent laboratories. 
Research Methods. Collection of Samples. All collecting apparatus, glass- 
ware, pipettes, collecting tubes, bottles, etc., shall be sterilized at a temperature 
of at least 175° C. for one hour. Each sample shall consist of at least 10 c.c. of 
milk. Before taking the sample the milk shall be mixed as thoroughly as pos- 
sible. If the original container can be inverted the mixing of the milk should 
be done by inverting it several times. If this is impossible, the milk should be 
stirred with some sterile stirrer. Any stirrer already in the container may be 
used. If there is none in the container, the sampling pipette (or any other 
sterile article) may be used; but it should be used for one container only until 
it is again sterilized. 
A sample simply poured from a large can is not a fair sample unless the milk 
in the can be thoroughly stirred. The sample shall be taken from the cans by 
means of a tube with straight sides long enough to reach to the bottom of the 
original container, and inserted not too rapidly, with the bottom of the tube left 
open. This will result in the tube containing a cylindrical section of the milk 
from top to bottom of the can. The finger then placed on the top of the tube 
will make it possible to withdraw the tube full of milk and transfer it to the 
sampling bottle. The sample bottle shall be large enough to hold the entire 
contents of the tube, all of which must be reserved as the sample. Each tube shall 
be used for collecting a single sample only and must be washed and sterilized 
again before being used again. An aluminum tube of the diameter of | in. and 
21 ins. long is very convenient. If the sample is to be taken from the bottle, 394 
MILK AND MILK PRODUCTS 
the bottle should be first shaken to insure thorough mixing and then the milk 
may be poured into the sample bottle, although it is better here to use a sampling 
tube. 
If the temperature of the milk is desired, it should be taken from a separate 
sample that should then be discarded. All records shall be made immediately 
after taking the sample. The milk sample shall be placed in a properly labeled 
bottle. The most convenient kind of sample bottles are glass stoppered, or those 
closing with a cork-lined screw cap. Cotton plugs are not a satisfactory method 
of closure. The sample bottles shall be placed at once in a carrying case contain- 
ing cracked ice, so that the milk is cooled at once to near the freezing-point. 
The samples shall be transferred to the laboratory as quickly as possible, and 
shall be plated at once. If the samples can be placed in melting ice and water, 
they may be kept for several hours (12) without an increase in bacteria. If the 
plates are not made within four hours of the time of collection, the number of 
hours that have elapsed should be stated on the report. If the milk is kept at 
40° F. a slight increase may be found in twelve to twenty hours. Up to twenty 
hours this will not be more than 20 per cent. 
Media. This is essentially identical with that used for water analysis 
and has been fully treated in the chapter on that subject. Sherman 
(1916) has demonstrated that lactose agar gives a much higher count 
than plain agar plates for enumerating bacteria in milk. Lactose agar 
also of some value in differentiating the types of bacteria which are 
present. Sherman (1915) has recommended that the reaction of agar 
media used in the bacterial analysis of milk should be 0.5 per cent plus. 
Plating. 9 For miscellaneous samples the character of which is not known, 
three dilutions shall be made 1-100,1-1000 and 1-10000. Where the character of 
milk is known the number of dilutions may be reduced. If the milk is pas- 
teurized, certified or known to be fresh, and of high grade, the 10,000 and 100 
dilutions may be omitted; if the milk is known to be old, the 100 dilution may be 
omitted. In no case shall less than two plates be made of each sample. Any 
convenient method of making dilutions may be used always using pipettes and 
sterile water blanks. The water for dilutions may be placed in dilution bottles 
(99 c.c. and 9 c.c. are convenient sizes) and sterilized for one hour in an auto- 
clave at 15 lbs. pressure. 
These standard methods do not give the maximum number of bac- 
teria. Some research workers in order to secure the maximum number 
of bacteria incubate their plates at 37° C. for five days and at 20° C. for 
two days. Such an incubation period of seven days' duration is obvi- 
ously too long for routine work. Slack (1917) has advised the incuba- 
tion of agar plates made from milk in routine analysis for forty-eight 395 
MICROSCOPIC METHOD OF MILK ANALYSIS 
hours rather than twenty-four hours because on an average the count 
is doubled or trebled. In this connection he advises meat infusion 
agar in place of meat extract agar. Sherman (1916) recommended the 
addition of dextrose or lactose to agar used in the examination of 
milk. A much higher count was obtained and the colonies grew 
much larger. 
Incubation. Only one period of incubation and one temperature 
is regarded as standard, forty-eight hours at 37.5° C. In crowded 
incubators ventilation shall be provided. 
Counting. If among the different dilutions, there are plates contain- 
ing from 30 to 300 colonies these should be counted, and the number 
multiplied by the dilution, be reported as the final count. All colonies 
on such plates should be counted and the numbers averaged. If there 
are no plates within these limits the one that comes nearest to 300 
is to be counted, unless it happens that there are no other plates with a 
larger number of colonies, or, unless the numbers in the plates check 
with other dilutions. If the number of colonies on the plate is over 300, 
a part of the plate may be counted and the whole plate averaged. 
Counting shall be done with a lens magnifying 2.5 diameters (or what 
opticians call a 2.5 X lens. Near-sighted persons shall use their glasses 
in counting while far-sighted persons shall remove them. In case it is 
doubtful whether certain objects are colonies or dirt specks they should 
be examined under a compound microscope. 
Reports. In making reports it must be borne in mind that with 
high numbers obtained by routine methods, only an approximation to 
accuracy can be obtained. Only the left-hand figures of the final 
numbers are significant. It is best, therefore, to report only the two 
left-hand figures of the results, in order to avoid an unwarranted im- 
pression of accuracy. For example, where the numbers are in the mil- 
lions no.figures smaller than millions have any significance in the routine 
analysis of milk. In making the report raise the number to the next 
highest round number, but never lower it. In no case shall the count 
of a single plate be regarded as sufficient for the purpose of grading milk. 
If a single sample of milk only is to be tested, there should be at least 
three plates counted before a report is made. 
Microscopic Method of Analysis. This method has been brought 
to such a stage by Breed and his colleagues that it is now included 
in the Standard Methods of Bacteriological Milk Analysis of the Labora- 
tory section of the American Public Health Association, 1917. Brew 
has summed up the advantages and disadvantages of the plate and 
direct microscopic method as follows: 396 
MILK 4ND MILK PRODUCTS ' 
ADVANTAGES AND DISADVANTAGES OF THE DIRECT MICROSCOPIC 
METHOD AS COMPARED WITH THE PLATE METHOD 
Disadvantages 
Direct Microscopic Method 
1. Difficult to measure accurately such 
a small quantity of milk. 
2. The sample measured is too small to 
be representative. 
3. Dead bacteria may be counted. 
4. Error of count is great where bacteria 
are few or many. 
5. Cannot be used for quantitative, 
work when bacteria are few in 
number. 
6. Many fields must be counted, because 
of the uneven distribution, if an 
accurate count is desired. 
7. Large compact clumps cannot be 
counted. 
8. Bacteria may be lost in the process of 
preparing slides. 
Plate Method 
1. All bacteria do not grow on the plates 
because of changes in food tempera- 
ture relations, or other conditions 
of environment. 
2. The difficulty of breaking up the 
clumps in the milk affects the ac- 
curacy of the count. 
3. Requires from two to five days' in- 
cubation period. 
4. Different species require different in- 
cubation temperatures. 
5. Gives no idea of the morphology of 
the bacteria present. 
6. More apparatus required, therefore 
more expensive. Technique com- 
plicated and difficult for the trained 
bacteriologists to use in such a 
way to secure consistent results. 
Direct Microscopic Method 
1. Less apparatus required, therefore 
less expensive. Technique simple. 
2. The results on a given sample may 
be reported in a few minutes. 
3. Shows the cell content, the presence 
or absence of streptococci and other 
important things necessary in esti- 
mating the sanitary quality of milk. 
4. Gives a better idea with regard to the 
actual number of germs present. 
Advantages 
Plate Method 
1. Is necessary for isolation of pure cul- 
tures. 
2. Gelatin shows the liquefiers and if 
litmus is used, the acid producing 
bacteria. 
3 Shows the character of growth. 
4. Shows living organisms only. 
Samples. Milk samples collected, as above described, may be pre- 
served by icing and handled as in the case of the plate method. All 
samples on which the cream has risen to the surface must be vigorously 
shaken before preparations are made from them. 
Apparatus. In addition to a microscope and ordinary microscopic 
slides, stains, etc., the only special apparatus required is a pipette PREPARATION OF SMEARS 
397 
which measures 1/100 c.c. The most convenient form of pipette is the 
straight capillary pipette, calibrated to deliver 1/100 
c.c., the graduation mark being 1| to 2| ins. from 
the tip. Such pipettes are now for sale by manu- 
facturers, and can be easily obtained. The cali- 
bration should be tested by weighing with chemical 
balances the amount of milk discharged from the 
tube. Only a single pipette is needed in making a 
series of tests, provided this is kept clean while in 
use. In this kind of work cleanliness rather than 
sterilization is required. Clean towels may be used 
for wiping the exterior of these pipettes, while their 
bores may be kept clean by rinsing them in clean 
water between each sample. The small amount of 
water left in the tube may be rinsed out into the 
milk sample under examination. This method of 
procedure, while adding a small number of bacteria 
to each sample, introduces only a theoretical error, 
tests showing that such bacteria cannot subsequently 
be detected, and make no difference in the final 
result. 
Preparation of Smears. One one-hundredth c.c. 
of milk or cream is deposited upon a clean glass slide 
by means of a pipette described above. By the use 
of a clean, stiff needle this drop of milk is spread over 
an area of 1 sq. cm. This may be most conveniently 
done by placing the slide upon any glass or paper 
ruled into areas 1 cm. square. These marks show- 
ing through the glass serve as guides. After uniform 
spreading the preparation is dried in a - warm place 
upon a level surface. In order to prevent noticeable 
growth this drying must be accomplished within 
five to ten minutes; but excessive heat must be 
avoided or the dry films may crack and peel from the 
slides in later handling. 
After drying, the slides are to be dipped in 
xylol (gasoline may be used) for one minute, then 
drained and the slides dried. They are then 
immersed in 90 per cent grain or denatured 
alcohol, for one minute or more, an J then trans- 
ferred to a fresh aqueous solution of methylene blue. Old or 
A 
B 
Fig. 67. - Breed's 
Capillary Pipettes. 
Several types of pi- 
pettes have been de- 
vised. Type A gives 
as much satisfaction 
as any. Pruchaatthe 
Illinois Agricultural 
Station uses ordinary 
capillary tubing and 
standardizes the 
amount of milk deliv- 
ered by weighing it on 
an analytical balance. 398 
MILK AND MILK PRODUCTS 
unfiltered stains are to be avoided, as they may contain troublesome 
precipitates. The slides remain in this solution for five seconds 
to one minute or longer, depending upon the effect desired, and are 
then rinsed in water to remove the surplus stain, and decolorized in 
alcohol. The decolorization takes several seconds to a minute, during 
which time the slide must be under observation in order that the decol- 
orization may not proceed too far before they are removed from the 
alcohol. When properly decolorized the general background of the 
film should show a faint blue tint. Poorly stained slides may be decol- 
orized and restrained as many times as necessary, without any apparent 
injury. After drying the slides may be examined at once, or they may 
be filed away and preserved for further reference. 
Standardization of the Microscope. The microscope to be used 
must be adjusted in such a way that each field of the microscope covers a 
certain known fraction of the total square centimeter's area. This 
procedure is simple, with the proper materials at hand. The micro- 
scope should have a 1.9 mm. (1/12 in.) oil immersion objective, and an 
ocular giving approximately the field desired, and should preferably 
be fitted with a mechanical stage. To standardize the microscope, place 
upon the stage a stage micrometer, and by the selection of oculars or 
adjusting the draw tube, or both, bring the diameter of the whole 
microscopic field to .205 mm. When so adjusted, the microscope field 
will cover almost exactly 1/300,000 of a cubic centimeter of the milk 
(actually 1/302,840). This means that if . the bacteria in one field only 
are counted, the number should be multiplied by 30,000 to give the 
total number for a cubic centimeter. If the bacteria in a hundred 
fields are to be counted, the total should, of course, be multiplied 
by 3000. 
Inasmuch as it is difficult to count bacteria lying near the margin 
of the microscopic field, it is much better to have an eyepiece micrometer, 
with a circular ruling, 8 mm. in diameter, and divided into quadrants. 
This will give, in the microscopic field, a smaller area within which the 
bacteria may be seen most sharply, and which may be more easily 
counted. Such eyepiece micrometers arc now manufactured by labora- 
tory supply houses, and may be easily obtained. In the use of this eye- 
piece micrometer the inner circle, by the adjustment of the draw tube, 
should be made to cover a circle with a diameter of .146 mm. In this 
case this inner circle will cover 1/600,000 of a cubic centimeter of milk, 
meaning, of course, that the number of bacteria in a single field should 
be multiplied by 600,000, or, if a hundred fields are counted, by 6000, 
to obtain the number per cubic centimeter. Plate I.-Drawings of Milk Smears as Seen under the Microscope. 
(After Brew, 1914.) 
All prepared in su< h a way that each bacterium or tissue cell seen is equivalent to 400,000 per 
cubic centimeter. 
GOOD QUALITY MILK 
Fig. 1.-No Bacteria Seen. 
Two tissue cells. Cell count =800,000 per cubic centimeter. 
MILK SOURING NORMALLY 
Fig. 3.-Milk which is Nearly Sour. 
The majority of the bacteria are lactic acid bacteria. One tissue cell. Bacterial count - 
80,000,000 per cubic centimeter. Cell count =400,000 per cubic centimeter. 
To face page 39S. Platt. 1a.-Drawings of Milk Smears as Seen under the Microscope. 
(After Brew, 1914.) 
All prepared in such a way that each bacterium or tissue cell seen is equivalent to 400,000 
per cubic centimeter. 
MILK OF FAIR QUALITY 
Fig, 2.-Two Pairs of Lactic Acid Bacteria and One Single Bacterium. 
One tissue cell. Bacterial count =2,000,000 per cubic centimeter. Cell count =400,000 per 
cubic centimeter. 
POOR QUALITY MILK 
Fig. 4.-Milk which is both nearly Sour and Suspicious in Sanitary Quality. 
Seven tissue cells. Bacterial count =100,000,000 per cubic centimeter. Cell count =2,SOO,000 
per cubic centimeter. 
To face page 398. INTERPRETATION OF RESULTS 
399 
The number of microscopic fields to be counted will depend some- 
what upon the kind of data that are desired. If this method is to be used 
simply for the purpose of dividing milk into grades, it will in most cases 
be unnecessary to do the actual counting, since a Grade A milk will show 
field after field without any bacteria at all, while a Grade C milk will 
show' the field crowded with bacteria. In all doubtful cases, however, 
counting should be done, and there should never be less than thirty fields 
counted in order to have reliable results. Counting thirty fields is not 
so tedious a task as would seem to be, since, in ordinary milk, the num- 
ber of bacteria in each field is small, and the counting may be done 
very rapidly. 
Counting. Counting the bacteria in such a smear may be done in 
two ways: 1. The number of groups of one or more bacteria present. 
2. The number of individuals. The second, of course, is really the 
correct count of the number of bacteria, but the former will give a count 
much closer to that obtained by the plate count, since the colonies upon 
the plate represent groups of bacteria rather than individuals, each 
group growing into a single colony only. Extensive tests have shown 
that there is a fair correspondence between the number of groups re- 
ported by experienced observers and the number of colonies that may 
grow in plates made from the same milk, although there are occasionally 
discrepancies of considerable extent. These discrepancies are caused 
by variations in judgment as to what constitutes a group, variations in 
the extent to which groups break up in the dilution waters when the 
smears are made, and the presence of dead bacteria or of bacteria which 
do not grow on the plates. Some experience is needed by the micros- 
copist in determining just what should be counted. In high-grade 
milks, an inexperienced person is apt to fail to recognize differences 
between bacteria and other minute objects. This results, as a rule, in 
an overcount by inexperienced men. In milk containing many readily 
recognizable bacteria in each field the inexperienced man is apt to over- 
look some of them, giving an undercount. These difficulties are over- 
come, however, by training and experience. 
Interpretation of Results. It must be recognized that the results 
obtained from the microscopic record give a closer approximation to the 
actual number of bacteria present in the milk than those obtained by 
the plate method, since the plate method will count as one, either a 
single bacterium or a group which may sometimes contain a hundred or 
even more individuals. Inasmuch, however, as the plate count has 
become a method of analysis that is well known and commonly applied, 
it becomes desirable to know as closely as possible what relations there 400 
MILK AND MILK PRODUCTS 
may be between the plate count and the microscopic count. Experi- 
ence has shown that the count of individual bacteria is ordinarily 1.5 
to 8 times as great as the plate count, the ratio between the two being 
largely dependent upon the size of the clumps of bacteria present. 
Where the bacteria are mostly isolated, the ratio of the two counts 
would be much closer than where there are present long chains of strep- 
tococci or masses of cocci, lifter one has had a little experience in 
counting clumps it is found that the number of groups shown by the 
microscope, agrees fairly well with the number of colonies shown by 
the plate count, though even here there are occasionally discrepancies, 
due among other things to the appearance in the microscope of kinds of 
bacteria which fail to grow in the culture media used in making plates. 
In all cases, however, the direct count of raw milk will give a much 
closer approximation to the actual numbers of bacteria than the plate 
count. In view of these facts it is difficult to interpret one count in 
terms of the other; but a few suggestions will give a fairly satisfactory 
idea as to how the two may be related. 
Grade A raw milk, which should have less than 100,000 bacteria 
per cubic centimeter, will not show more than three to four small clumps 
of bacteria for each 30 fields of the microscope where the diameter of 
the fields is .205 mm. Such milk also ought not to contain more than 
500,000 individual bacteria per cubic centimeter when counted by the 
microscope. For Grade A pasteurized milk (which should have less 
than 200,000 per cubic centimeter by the plate count before pasteuri- 
zation) the microscope should not show more than six to eight clumps 
per 30 microscopic fields, and not more than 1,000,000 individual bac- 
teria when counted with the microscope. 
Grade B milk, which is supposed not to have more than 1,000,000 
bacteria before pasteurization, when counted by the plating method, 
should not show more than 20 individual bacteria per field, where the 
diameter of the fields is .205 mm., and not more than three to four 
groups of bacteria per field. 
While the above relation between the plate count and the microscopic 
counts cannot be relied upon as having a very great amount of accuracy, 
it will serve to give a general idea of the ratio between the two under 
ordinary conditions, and may serve as a guide in the use of direct, 
microscopic method. 
The direct microscopic method is not as yet recommended by this 
committee as a method of estimating the numbers of bacteria that are 
present in samples of milk. For this purpose the plate method, which 
has long been in use and is fairly well understood, is still recommended as FROST'S MICROSCOPIC PLATE METHOD 
401 
the standard method to be employed. For the purpose of rapidly 
dividing raw milk into a series of grades, in such a way that the results 
can be obtained in the quickest possible time, the direct microscopic 
method seems to be extremely useful, and the results which are obtained 
will, in nearly all cases, agree with those obtained by the plate count, 
and probably in all cases will give a closer approximation to the fair 
grading'than the plate count can do. For these reasons the use of the 
direct microscopic method is extremely valuable at the dairy end of the 
milk route, where the farmer wishes to know the kind of milk he is pro- 
ducing, or the purchaser at the shipping station wishes to know the kind 
that he is receiving from the farmer. It is of less value at the city end 
of the milk route, especially if pasteurization of the milk has been intro- 
duced anywhere along the route. It is especially useful in the data it 
gives concerning the kinds of bacteria present in milk, since it sometimes 
enables the farmer quickly to pick out from his herds such cows as are 
discharging large numbers of streptococci, thus giving a very efficient 
means of protecting the milk supply from this type of organisms that 
are to-day recognized as suspicious and decidedly undesirable. 
Goodrich (1914) finds a marked correlation between the microscopic 
and plate counts. He regards the factor of 20,000 which is used to 
reduce the microscopic count to terms of the plate count as satisfactory. 
Breed and Brew (1917) claim that their microscopic method is as satis- 
factory for controlling city milk supplies as is the agar plate method. 
Breed and Stocking (1917) found that the plate counts are characterized 
by greater regularity in the hands of laboratory assistants carrying out 
routine analyses of milk, than the microscopic method of Breed and 
Brew. Inexperienced workers are said to secure great errors in the use 
of the microscopic method. About the same conclusion was reached by 
Conn (1915). He stated that considerable experience was needed by 
the analyst to distinguish between bacteria and dirt particles. 
The Breed method has been demonstrated by the work of different 
investigators to be fairly accurate with milks which have large numbers 
of bacteria. It may scarcely be conceded that where the bacteria are 
so few that many of the fields of a Breed smear contain no bacteria, the 
count is very accurate. In this connection the Frost microscopic plate 
method or Allen's microscopic method may be found to be more satis- 
factory. Brew has mentioned this last fact as a disadvantage of the 
microscopic method. To secure the greatest possible accuracy a large 
number of fields should be counted. 
Frost's Microscopic Plate Method. This differs from the Breed 
Method of securing a microscopic count in that little plates are made 402 
MILK AND MILK PRODUCTS 
instead of smears; otherwise the method is somewhat the same. In 
general, some of the same objections which are applied to Breed's method 
may be applied to the Frost method. The method is outlined by Frost 
in the following words: 
Preparation of Glass Slides. The little plates arc made on the 
ordinary microscopic glass slides (2.5 by 7.5 cm.). These arc carefully 
cleaned and arc then ruled with a grease pencil so that a square surface, 
over the center of the slide, is surrounded by a grease line. The included 
area is 4 sq. cm. The ruling is best done by having a wooden or card- 
board frame for the slide and some arrangement, as brads, to hold the 
straight-edge in place. These ruled slides ere then sterilized by passing 
them through a gas flame, and then they arc placed on a warm box 
(45° C., 113° F.) provided with an overhanging top to protect them from 
dust. 
Preparation of the Plates. A few tubes of ordinary nutrient agar 
are then melted and placed in a water bath at 45° C. to cool. Some 
sterile plugged test tubes arc also put in this water bath to warm up (one 
for each sample of milk to be tested). The milk sample to be tested is 
then thoroughly shaken and 1 c.c. is transferred to one of the warm 
sterile test tubes. To this is added an equal amount (1 c.c.) of the 
melted agar. The milk and agar are thoroughly shaken, care being 
taken to prevent the agar from cooling below 40° C. It is best to warm 
it up frequently by putting it into the 45° C. bath. 
One-tenth (0.1 c.c.) of the milk and agar mixture is then transferred 
to a warm glass slide and spread as quickly and evenly as possible over 
the ruled surface. This may be readily done with the help of a pipette 
while the slide is on the warm plate. The agar is allowed to set firmly 
by placing it on a level surface under cover for a few minutes. This 
makes a little plate culture containing 0.05 c.c. of milk. 
When the milk being examined is supposed to contain more than a 
million bacteria per cubic centimeter, it should be diluted with sterile 
water or sterile milk before it is mixed with the agar. The dilution 
may be 1 : 10. This would make the final dilution as much as 
1 : 200. 
Incubation. This is accomplished by placing the little plates in 
an incubator in a specially prepared cabinet, or moist chamber with a 
layer of water in the bottom to maintain a saturated atmosphere. A 
Petri dish may be used for a few slides. The incubation temperature 
is 37° C. for a varying length of time. Five or six hours should be 
allowed. For samples which contain many bacteria such as market 
milk which has been kept in a warm place three or four hours will be FROST'S MICROSCOPIC PLATE METHOD 
403 
sufficient. For pasteurized milk from ten to twelve hours may be 
necessary. 
Drying the Plates. When the colonies are sufficiently large the 
plates are dried. This should be done carefully. Too rapid drying 
will crack the Rims. They will dry well in the incubator or over a 
steam radiator. 
Staining. This is accomplished in the following manner: 
1. Fix in the flame. 
2. Put in a 10 per cent solution of acetic acid in 95 per cent alcohol. 
3. Stain with Loeffler's methylene blue (1:4) for three minutes. 
4. Decolorize in 95 per cent alcohol for a few seconds or until the 
background is a pale blue and the colonies stand out prominently. 
5. Dry without washing. 
Counting of Colonies. For the purpose of counting the colonies, 
the slides may be examined with the low-power dry lens. If the struc- 
ture of the colonies is to be studied it is always best to use a mounting 
medium and the cover glass. Cedar oil serves quite well and in that 
case the cover glass may be removed for use with the oil immersion 
but Canada balsam may be used in the usual way. A microscopic 
examination of these plates reveals colonies of considerable size stained 
a dark blue in a light blue field. The staining process should be so 
regulated that the colonies will be stained a dark blue in a light blue 
field. At least twenty fields should be counted on each plate and the 
plate should be gone over carefully in the selection of these twenty 
fields. 
Calculation. The number of bacteria per cubic centimeter may be 
determined by multiplying the number of colonies in a microscopic 
field by the number of times the area of this field is contained in 400 
sq. mm. or the area of the little plates times the dilution used. The 
area of the microscopic field for any definite combination of lenses and 
tube length is a constant and may be determined by the formula: 
Area=/?. The radius may be determined by a stage micrometer. 
The same tube length must be used that was used in the standardization 
of the microscope. When the microscope factor is known this formula 
may be used: 
Number of colonies counted T, . . c ... ,. - ... 
= ;-j- = Reciprocal of dilution of milk. 
Number of fields counted 
Microscope factor = number of bacteria per cubic centimeter in the 
milk. Example: 404 
MILK AND MILK PRODUCTS 
600 2,400,000 1onnnnv . • . 
-- X 20 X 200 = ~ = 120,000 bacteria per cubic centimeter, where 
A J 
600 = number of colonies counted; 
20 = number of fields counted; 
20 = dilution-1-20; 
200 = microscope factor. 
Frost (1915, 1917) has secured a satisfactory agreement between his 
method and the standard plate method. In his second communication, 
this author reports some results secured with his method during a prac- 
tical test on the Boston milk supply. The table which is presented in 
this book shows that Frost's microscopic method gives results which 
are different than those obtained on the standard agar plate. 
Allen's Microscopic Method for Counting Bacteria in Milk. Allen 
(1918) has described his method as based on the fact that a water sus- 
pension of aluminum hydroxide readily collects bacteria in milk. This 
may then be thrown down in one end of a centrifuge tube leaving the 
normal milk constituents suspended. Allen has described his tech- 
nique as follows: Preparation of Hydroxide Suspension. Mix equal 
parts of N/20 A1K(SO4)2 - I2H2O and N/20 sodium hydroxide. After 
precipitation is complete, wash the precipitate thoroughly by decanta- 
tion using scrupulously distilled water, keeping in mind that any dirt 
in the wash water will be taken up by the aluminum hydroxide and will 
appear in the film under the microscope. Make up to one-half the 
original volume and continue to dilute until a proper amount of alu- 
minum hydroxide is obtained after centrifuging. Procedure. 1. 
Add to the sample of milk to be analyzed enough of the washed alu- 
minum hydroxide precipitate suspension so that it becomes 20 per cent 
of the mixture. Shake thoroughly for several minutes. 
2. Add 2.5 c.c. of the above mixture to a centrifuge tube holding 
this amount when stoppered. (Use plain centrifuge tube open at both 
ends for stoppers.) 
3. Centrifuge for fifteen minutes at 5000 r.p.m. in a centrifuge of 
10-in. diameter or give equivalent centrifugalization. 
4. Pull stopper at cream end of the tube and remove the cream with 
a needle allowing the milk to run out. Then carefully pull the stopper 
at the other end of the tube and transfer the plug of aluminum hydroxide 
to a clean glass slide laying over glass or cardboard ruled off in square 
centimeters. By use of a needle or loop grind up the precipitate on the ALLEN'S MICROSCOPIC METHOD 
405 
slide and spread it evenly over an area of 2 sq. cm. using a loopful of 
sterile water if necessary for uniform spreading. 
5. Place in the incubator until nearly dry, then finish drying at room 
temperature. 
6. Heat slightly above the Bunsen flame. 
7. Place in a tray of toluene and agitate the tray several minutes 
to remove butter fat, then take the slides out of the tray and allow the 
toluene to evaporate from them. 
8. Place the slides in 95 per cent alcohol to remove all traces of 
toluene. 
9. Stain in 25 per cent saturated aqueous solution of methylene 
blue until the film reaches a proper depth. 
10. Dry in the warm air above a Bunsen flame and examine under 
the microscope, using an oil immersion lens of which the number of 
fields per square centimeter has been determined. 
11. Count 20 representative fields on the square centimeter and 
obtain the average number of bacteria per field. 
12. The number of bacteria per cubic centimeter of milk is obtained 
by multiplying the average number of bacteria per field by the number 
of microscopic fields in a square centimeter. Usually there are a little 
over 3000 fields per square centimeter when the ordinary 1.8 oil immer- 
sion lens is used but using 3000 as an even number to multiply by is 
satisfactory in counting. 
Allen also gives the following precautions: Care must be exercised 
not to use a suspension of aluminum hydroxide too strong to give too 
thick a smear. The thickness of the plug of aluminum hydroxide after 
centrifugalization should be about 1 mm. No fat or toluene should 
appear in the field under the microscope. Some samples of pasteurized 
milk show fine particles of curd due to heating. These may be removed 
by filtering through a small amount of cotton. The film should be 
uniformly stained. Rubber stoppers should be used with the centrifuge 
tubes as cork stoppers will not hold the liquid in the centrifuge. If toe 
deep a stained film is secured, it may be decolorized sufficiently by dip- 
ping in alcohol. 
Interpretation of Results. " The interpretation of the bacteriologi- 
cal analysis of market milk must depend upon the history of the milk. 
It is, therefore, difficult to give any general interpretation. The fol- 
lowing are a few significant conclusions: 
1. " Where the analysis can be made immediately after the milking 
the number of bacteria enables conclusions to be drawn as to the cleanli- 
ness and care in the dairy and the thoroughness in the cleaning and 406 
MILK AND MILK PRODUCTS 
sterilizing of the milk vessels, or sometimes the presence of cows with 
infected udders. With properly cleaned and sterilized milk vessels 
and proper care in the farm and dairy the numbers of bacteria should 
not exceed 10,000, and may easily be brought down to 5000. Numbers 
beyond these in milk analyzed immediately after the milking may be 
regarded as an indication of unclean dairy methods, dirty and unsterile 
milking vessels, or to infected udders. Apart from infected udders the 
factors in dairying that most noticeably increase the bacteria count 
are unsterile milk vessels, unsterile strainers, unclean udders, and failure 
to cool the milk promptly. 
2. " If the milk is properly cooled with ice the numbers should not 
materially increase in five to seven hours. Communities within five to 
seven hours of their dairies should, if perfect conditions prevail, be able 
to obtain milk with nearly as low a count as above indicated. Hence, 
in such communities bacterial counts above these numbers should not 
be found in properly guarded milk. A count of 50,000 in such a com- 
munity is an indication either of unsatisfactory dairy conditions or 
of failure to properly cool the milk during transportation. Night's 
milk, if properly cooled, can also easily be brought within these limits 
if analyzed the next morning. A count of over 50,000 for a community 
close to the dairies must be regarded as unsatisfactory, and the number 
should approach the 10,000 mark for high-grade milk. In hot summer 
weather the difficulties of keeping low counts are greater, but even 
then they need not surpass 50,000 if the milk is properly cooled. 
3. 11 Where milk must be a longer time in transportation from the 
dairy there will inevitably be an increase in bacteria, depending on the 
length of time and the temperature. Experience has shown, however, 
that even in these conditions the excessively high numbers that have 
frequently been found in city milk are in reality due to diseased udders, 
to dirty dairy conditions, to dirty and unsterile milk utensils, or culpable 
neglect of cooling. Moreover, such high bacterial counts at the ship- 
ping station are frequently traceable to a few dirty dairies whose milk 
with an abnormally high count contaminates the rest of the supply. 
Dirty shipping cans and warm temperatures in shipping are respon- 
sible for most of the high bacterial counts in city milk. Where the milk 
from healthy cows reaches the city within twenty-four hours, however, 
the number should not be over 100,000 in winter or 200,000 in summer, 
and numbers in excess of this may be regarded as due either to improper 
dairy conditions, dirty milk vessels, insufficient cooling or, perhaps, to 
diseased udders. In larger cities where much of the milk is forty-eight 
hours in reaching the city, higher numbers may naturally be expected; MICROSCOPIC VS. PLATE COUNT 
407 
but even under these conditions there is no good reason why the number 
of bacteria should reach 1,000,000; and it may mostly be brought down 
to below 200,000. In such cities, therefore, milk with more than 1,000,- 
000 bacteria must be regarded as improperly guarded either at the dairy 
or on its transit. 
4. 11 For a Grade A milk higher demands should be made than for 
the ordinary grade. The standard set by the Milk Commission for 
Grade A, viz.; of 200,000 for milk to be subsequently pasteurized or 
for 100,000 to be used raw, is stated by that Commission to be an 
extreme limit for the most unfavorable conditions. Cities situated 
near the supplying dairies should demand a much higher standard, 
which should not allow over 10,000 in bacterial content in Grade A milk 
in communities favorably situated. 
5. " For communities situated where ice is not available it may be 
necessary to accept a milk with a higher bacterial content; but as 
rapidly as possible the standard should be made to approach the limits 
as given above." 
Relation of the Microscopic to the Plate Count. The relation of 
Breed's microscopic to the standard plate count has received some study. 
Brew (1914), after a rather extensive study of the question pointed out 
some interesting facts in his summary. He stated that " the relative 
differences between the two counts are greater where the bacteria are 
few in number." In such a case, there is probably an error in the 
microscopic count since it is difficult to see how it is well adapted to 
milks with few bacteria. Brew finds a greater difference when the indi- 
vidual bacteria on the smear are counted than when only groups of 
bacteria are counted. This is said to be due to the fact that a colony on 
a standard plate has developed either from a single bacterium or a 
group of bacteria. Goodrich (1914) reported a marked correlation 
between the two counts. He thinks that, for accurate work, more than 
one slide should be prepared. Brew again studied this relation with the 
aid of Dotterer (1917) and found that the plate counts were higher than 
the microscopic counts on milks with a small number of bacteria. Brew 
has made an attempt to explain this by " unrecognized contamination " 
on the standard agar plate which would increase this count and by over- 
looking bacteria under the microscope. This seems a rather feeble 
attempt to bolster up the Breed microscopic count in one of its apparent 
deficiencies. More reasonable does it seem that " bacteria where they 
are very few in number, even though well stained and conspicuous may 
not occur in the microscopic fields examined." This is probably one 
of the major objections to Breed's microscopic method. Unlike Allen's 408 
MILK AND MILK PRODUCTS 
microscopic method, no concentration of the bacteria in the sample is 
made. Breed and Brew (1917) compared again, the relation of the two 
counts when studying the application of the microscopic method to the 
control of bacteria in market milk. No general agreement was observed. 
Frost's method seems to have had little application under commercial 
conditions. Frost (1916, 1917) has carried some studies himself and 
reported a smaller variation with his " little plate " method than was 
observed with the standard plate. 
The Allen microscopic method may overcome some of the disad- 
vantages of the Breed method. Allen has devised a procedure which is 
said to be applicable to all grades of milk. With the Breed smear gross 
errors are involved on milks with low counts. In his original contribu- 
tion Allen (1916) has shown that on milk with a count of 1000 and 
slightly over, he was able to check the standard plate count very closely. 
Ayers' Milk Tube Method. This is a very satisfactory method for 
studying the types of bacteria in milk. The milk is plated on agar 
plates which are incubated at 37° C. for six days. After counting the 
plates, each colony should be picked from the plates and transferred 
to a sterile tube of litmus milk. After fourteen days' incubation these 
tubes are examined and as a result of the reactions shown, the bacteria 
picked from the plates are divided into five groups: the acid-forming, 
acid-forming and coagulating, inert, alkali-forming, and peptonizing. 
Ayers has used this method in studying the flora of different milk 
products. 
Leucocytes in Milk 
These cells in milk were first given much attention by Stokes and 
Wegefarth (1897). It was noticed that they had much resemblance to 
the white corpuscles and from this it was concluded that they repre- 
sented " pus " from the cow's udder. Breed (1914) has given a good 
summary of the literature besides reporting the results of his own inves- 
tigations. He used the direct microscopical method for counting the 
cells first suggested by Prescott and Breed (1911) and later applied to 
the counting of bacteria in milk by Breed (1911). No relationship, if 
any exists, was established between the number of cells discharged and 
bacterial infections of the udders. Breed (1913) reports a milk having 
the enormous number of 54,300,000 cells per cubic centimeter. This 
milk when partaken of was found to have a normal taste and the imbiber 
suffered no evil after effects. 
Stokes' Method for Leucocytes in Milk. Centrifugal sediments 
from 10 c.c. of milk are stained and examined under the one-twelfth oil SAVAGE'S METHOD FOR LEUCOCYTES 
409 
immersion objective. The presence of cells in such a field was regarded 
by Stokes as justification for excluding an animal from a herd. 
Reed's Method for Leucocytes in Milk. 1. Fill 10 c.c. centrifuge 
tubes with milk and heat for ten minutes at 70-75° C. 
2. Centrifuge the tubes at high speed for ten minutes. Remove the 
upper layers of cream and milk with a pipette and refill the tubes with 
distilled water. Centrifuge again for three or four minutes. 
3. Draw off all except j c.c. of liquid in the point of the centrifuge 
tube. Wipe out the upper part of the tube with a bit of absorbent 
cotton fastened to the end of a glass rod. Mix thoroughly the remaining 
liquid and sediment. 
4. Transfer a drop of this mixture to a clean Thoma-Zeiss blood- 
counting cell and place the cover glass over it. Count the cells under 
a one-sixth objective. If the number of leucocytes is low, the entire 
area of the cell should be counted, using a mechanical stage to move the 
slide. If their number is large, five or six small squares may be counted 
and averaged. The average number per small square multiplied by 
200,000 will give the number of leucocytes per cubic centimeter in the 
original milk. 
Savage's Method for Leucocytes in Milk. (Savage, 1914.) The 
ordinary Thoma-Zeiss blood-counting chamber is employed. Direct 
counting of the cells is impossible owing to the opacity caused by the 
large amount of fat. One c.c. of the milk is accurately transferred 
to a centrifugal tube (about 15 c.c. capacity) of the usual pattern, 
and freshly filtered Toisson's solution is poured in to almost fill the 
tube. The two fluids are well mixed and then centrifugalized for 
ten minutes. The cream is well broken up by a clean glass rod, to 
disentangle leucocytes carried to the surface, and the mixture centrifu- 
galized for an additional five minutes. All the fluid is then removed 
down to the 1 c.c. mark, great care being taken not to disturb the deposit. 
This can be conveniently and readily done by means of a fine glass tube 
connected to an exhaust pump. Theoretically, all the cellular elements 
present in the original 1 c.c. of milk are now present in the 1 c.c. of fluid. 
The deposit is thoroughly well mixed (with a wire), and disturbed 
through the 1 c.c. A sufficient quantity is placed on the ruled squares 
of the Thoma-Zeiss apparatus, and the cover glass put on. The number 
of cells is counted in a number of different fields of vision, moving reg- 
ularly from one field of vision to another. The diameter of the field of 
vision is ascertained before counting by drawing out the microscope tube 
until an exact number of sides of the squares spans a diameter of the 
field of vision. 410 
MILK AND MILK PRODUCTS 
The number of cellular elements per cubic millimeter of milk = 
w^ere avcra8e number per field of vision, d = the number 
of squares which just spans the diameter, d is determined once for all 
by marking the ;nicroscope draw tube so that only 20 fields have to be 
counted, and the figures substituted in the formula. 
Doane-Buckley Quantitative Method for Estimating Leucocytes in 
Milk. With this method 10 c.c. of milk are centrifuged for four min- 
utes in graduated sedimentation tubes, at an approximate speed of 
2000 r.p.m. The cream is lifted out with a cotton swab, care being taken 
to get as much as possible of the fat. It is then centrifuged one minute 
more and the cream again removed with a cotton swab. Any fat re- 
maining in the milk interferes seriously with the counting, as, if there 
are more than a few globules they form a layer on the top of the liqiuid 
in the counting chamber, and, as the leucocytes settle to the bottom of 
the chamber, it is difficult to see through the fat. It is only with cows 
giving milk difficult of separation where this trouble is experienced,'and 
with such animals considerable care is necessary in removing all the 
cream gathered at the top of the sedimentation tube. The method of 
removing the fat with cotton is the best one that has occurred to us, and 
it is the only part of the process that does not operate with entire satis- 
faction in every instance. 
Following the removal of the cream, after the second centrifuging, the 
bottom of the tube will contain a portion of the sediment which is easily 
seen. This sediment may, in extreme cases of cows suffering from 
garget amount to as much as 1 c.c. Ordinarily it will be considerably 
less than j c.c. The amount varies considerably with the number of 
leucocytes, but not absolutely. The milk above this sediment is 
removed with a small siphon, which can be easily arranged with bent 
glass tubes drawn to a fine point and supplied with a small rubber end 
pinch cock. In using the siphon it is better to keep the point near the 
surface of the milk in the tube in order not to agitate the precipitated 
leucocytes and draw a number of them off with the milk. The milk in 
the tube may be siphoned within an eighth of an inch of the sediment 
in the tube. This will usually be below the |-c.c. mark. Two drops of 
saturated alcoholic solution of methylene blue are then added, thor- 
oughly mixed with the sediment by shaking, and then set in boiling water 
two or three minutes to assist the leucocytes in taking the color. The 
contents of the tube can be boiled by holding it directly in the flame, but 
it has no advantage over the use of the water bath, and it is very likely 
to break the glass. After heating, some water is added to the tube to DOANE-BUCKLEY METHOD 
411 
render the color less dense. Ordinarily filling the tube to the 1 c.c. 
mark will be sufficient, and this quantity gives an easy factor for cal- 
culating the final results. 
In putting this liquid containing the leucocytes into the blood 
counter considerable care is necessary, owing to the tendency of the 
leucocytes to sink to the bottom. At this place a' capillary tube is used, 
and the cover glass was held in one hand ready to cover the chamber as 
soon as the drop was transferred. to the counting counter. After 
placing the glass cover over the chamber, about a minute is allowed 
the leucocytes to settle to the bottom of the chamber. There are very 
few foreign bodies likely to be mistaken in counting for leucocytes. 
Ordinarily the polynuclear leucocytes predominate and the stained 
nuclei with the unstained surrounding cell show up very distinctly. A 
few small leucocytes with large nuclei may be found and these may be 
confounded with yeast cells until the worker becomes familiar with the 
distinction. 
As regards counting we have taken a standard with a cubic centi- 
meter as a basis quantity of milk, though we are, of course, aware that 
the corpuscles in the blood are enumerated with a cubic millimeter basis. 
We adopted the centimeter largely for two reasons. In counting bac- 
teria in the milk the cubic centimeter is always the basis employed. 
Simply because the leucocytes were derived from the blood seemed 
to be no reason why the same basis for counting should be employed as 
was used with the blood, while to the ordinary bacteriological worker to 
whom this work will fall, if ever adopted to any extent, the cubic cen- 
timeter standard would be a little more easily comprehended because 
more frequently used. The blood counter holds To c. mm. and 1/10,000 
c.c. If 10 c.c. of milk are used and the 1 c.c. of fluid is in the tube after 
siphoning, and the coloring matter and the water used to dilute has been 
added, then the resulting number of leucocytes in the counting multi- 
plied by 1000 will be the total number of leucocytes per cubic centi- 
meter in the milk. If a total of 75 leucocytes was counted in the 
chamber there would be 75,000 leucocytes per cubic centimeter in the 
milk. • 
In the actual counting under the microscope a square millimeter 
of the counting chamber will be found to be ruled oft into 400 smaller 
equal squares. This facilitates an accurate and rapid count. Where 
the number of leucocytes is not great the entire field can be counted 
in a short time. Where there is a great number of leucocytes a few 
squares or sets of squares in different parts of the ruled surface will give 
approximately the number. 412 
MILK AND MILK PRODUCTS 
There are occasionally a few variations desirable from these rules, 
but it may be well to state that the details have been pretty carefully 
and thoroughly worked over and compared, and it is seldom that short 
cuts can be made if correct results are desired. The time and speed of 
centrifuging are placed as low as possible for accurate work. When 
there is | c.c. or more of sediment, it is necessary to use more of the 
methylene blue for staining, as there will be too great a number of leu- 
cocytes to make a satisfactory count in the counting chamber, it is better 
to add water until there are 2 c.c., or sometimes even more in the sedi- 
mentation tube. 
This method of counting, while long in explaining is in reality short 
and simple in application. Moreover, it is based on accurate measure- 
ments in every detail, and the results are correspondingly reliable. 
(From Report of Committees of the Laboratory Section, American 
Public Health Assn. Am. J. Pub. Hyg., 6 (1910). 
♦ 
Pathogenic Bacteria in Milk 
Bacillus tuberculosis. The tubercle bacillus is recognized as one 
of the most important pathogens in relation to the milk question. While 
it may gain entrance from a tubercular person handling the milk, it is 
generally admitted that a tubercular cow is usually the source. Human 
tuberculosis is probably transmitted directly from human to human 
but may be transmitted by bovines, especially those with udder infec- 
tions. Griffith (1913) reports the discharge of virulent tubercle bacilli 
in the milk of a heifer which had been vaccinated when four days old 
with human tubercle bacilli. Other experiments by Smit (1908), 
Coquot (1908), Hessler (1909), indicate that there is slight possibility 
of the tubercle bacilli being discharged into milk unless there are open 
lesions. Smit found few tubercle bacilli in milk from tuberculous 
animals with sound udders but where open tuberculosis existed the 
bacilli could gain entrance to the milk from all channels which com- 
municate with the exterior. This subject has been well summed up by 
Schroeder (1907). 
Delepine's Method for Determining Tubercle Bacilli in Milk (from 
Savage, 1914). Collect two tubes of milk containing 40 c.c. and cen- 
trifugalize for fifteen minutes at 3000 r.p.m. Decant or draw off the 
cream and milk by means of a pipette leaving about 2 c.c. of milk and 
residue in each tube. Examine microscopically and inject into guinea 
pigs. TUBERCLE BACILLI IN MILK 
413 
Campbell's Method for the Detection of Tubercle Bacilli in Milk. 
Ten c.c. of a thoroughly mixed sample were placed in each of three 
centrifuge tubes by means of a sterile pipette. These should be cen- 
trifuged' for thirty minutes at about 1200 r.p.m. The tubes are then 
removed from the centrifuge and the cream removed by means of 
a large sterile platinum loop. Three c.c. of sterile water should be 
added to this cream to bring the volume up for inoculation. The 
milk remaining in the centrifuge should be drawn down to 1 c.c. These 
1 c.c. portions should be transferred to a sterile test tube. Slides may 
be made from these samples. Three c.c. of the cream emulsion and 
3 c.c. of the milk emulsion should be injected subcutaneously into guinea 
pigs. The pigs should be observed closely for from six to ten weeks and, 
if not dead before, should be autopsied. 
Anderson's Method. Mix 50 c.c. of the milk with 50 c.c. of sterile 
water and centrifuge for one hour at 2000 r.p.m. Inject 4 c.c. of the 
sediment into a guinea pig. If the guinea pig dies, a careful autopsy 
should be made. If death does not occur by the end of two months, 
test with tuberculin, chloroform and perform a careful autopsy. All 
suspicious lesions should be cultured and cultural and morphological 
studies made. 
Besson's Method for Isolation of Bacterium tuberculosis from Milk. 
Allow the fresh milk to stand for twenty-four hours and examine the 
deposit. Centrifuge and use the precipitate for making a microscopical 
examination. Coagulate 200 c.c. of the milk with a little citric acid and 
filter. Dissolve the precipitate on the filter in a solution of sodium 
phosphate (Na2HPO44-12HoO) and pour the liquid into a large test 
tube. Add a few cubic centimeters of ether and, after shaking for 
about ten minutes, pour off the ether which will carry the fat with it. 
Centrifuge the aqueous fluid and examine microscopically the sediment. 
Beattie and Lewis (1913) have isolated acid fast bacilli from milk 
which were totally different from tubercle bacilli. They emphasize 
the futility of using microscopic methods instead of animal inoculation. 
The prevalence of tubercle bacilli in market milk has received the 
attention of investigators in practically all countries. There is an 
element of error in comparing closely data from the different investi- 
gations since a different number of samples were used. This may ex- 
plain the high incidence of tubercle bacilli which has been reported by 
some investigators. Table XLIII gives the data which have been se- 
cured by some of the investigators on market milk. 
Microspira Cholerae. Basenai (1895) reported that there was no 
germicidal action of milk on Microspira choleras and that the organisms 414 
MILK AND MILK PRODUCTS 
could retain their vitality in milk for thirty-eight days. Some of the 
cells even retained their vitality after the milk had curdled. These 
statements arc interesting when compared to the results secured by 
Laser (1891) in butter. 
Table XLIII 
INCIDENCE OF B. TUBERCULOSIS IN MARKET MILK 
(Quoted from Parker 1917) 
Date. 
Place. 
Investigator. 
Samples 
Examined. 
Number 
Positive. 
Percentage 
Positive. 
1899 
England 
Macfayden. . . 
77 
17 
22 1 
1904 
Germany 
Muller 
1596 
97 
6.2 
1904 
Germany 
Beatty 
272 
27 
10.0 
1898 
Liverpool 
Delepine 
12 
22 
17.6 
1897 
Liverpool 
Hope 
228 
12 
5.2 
1900 
London 
Klein 
100 
7 
7.0 
1893 
Petrograd 
Scharbekow.. . 
80 
4 
5.0 
1900 
Kiew 
Pawlowsky. . . 
51 
1 
2.0 
1900 
Krakow 
Buiwid 
60 
2 
3 3 
1900 
Naples 
Marconi 
14 
7 
50.0 
1898 
Berlin 
Petri 
64 
9 
14 0 
1900 
Berlin 
Beik 
56 
17 
. 30 3 
1898 
Schev. Gueund.. 
Ott 
27 
27 
11 1 
1898 
Konigsburg 
Jaeger 
100 
7 
7.0 
1908 
Leipsic 
Eber 
210 
22 
10.5 
1905 
Rotterdam 
Smit 
567 
14 
2.7 
1906 
Rotterdam 
Smit 
1584 
45 
2.8 
1908 
Washington 
Anderson 
223 
15 
6.7 
1909 
Louisville 
Field 
119 
46 
29.5 
1909 
New York 
Hess 
105 
17 
16.2 
1909 
Philadelphia.... 
Campbell 
130 
18 
13.8 
1910 
Chicago 
Tonney 
144 
. 15 
10.5 
1910 
Bochester 
Goler 
237 
30 
12.6 
Bacillus Diphtherias. Diphtheria is a typical milk-borne disease. 
Marshall (1907) has reported the isolation of the organisms from milk. 
Trask (1912) has given a good resume of such epidemics. 
Pasteurization 
Pasteurization is the heating of a food substance for a time below the 
boiling-point. It is now almost exclusively applied to the heating of 
milk in order to make it a safer food. 
It is an old idea having been used by Scheele (McIntosh, 1901), in 
1782, to preserve vinegar. In 1831, Appert preserved various animal THEORY OF PASTEURIZATION 
415 
and vegetable products by heating in the closed containers. Pasteur 
(1879) applied heat to bottled beer to make it keep. In recognition of 
Pasteur's researches, which revealed the causes of deterioration in fer- 
mented liquors and the means of preserving them, the term " pas- 
teurization is now used. Soxhlet (1886) proposed pasteurization in 
the home for baby feeding. There has been much confusion between the 
terms " pasteurized " and " sterilized " as applied to milk. Rosenau, 
Fig. 68.-Complete Milk Plant. (New York Milk Committee.) 
(a) Milk Clarifier; (b) Pasteurizing and Holding Plants; (c) Milk Cooler; (d) Storage Tank 
for Cold Milk; (e) Bottle Filling and Capping Machine. 
to avoid this has suggested that each bottle indicate the time and tem- 
perature of heating. 
Theory of Pasteurization. This involves the use of moist heat as a 
disinfectant. As discussed in the chapter on sterilization and disin- 
fection, bacteria die according to the monomolecular law and this 
indicates that the killing is a time process. The bacteria do not die 
at once as soon as the heat is applied. The same law must obtain in 
the killing of bacteria by pasteurization. From this it would seem 416 
MILK AND MILK PRODUCTS 
that the " continuous " process where heat is applied over a longer 
time would be more satisfactory. 
Gable (1915) has summed the advantages which might be raised 
against pasteurization as follows: In the light of recent knowledge some 
of these are now untenable. 
OBJECTIONS 
1. Increased cost. 
a. By additional apparatus required. 
b. By fuel required for heating. 
c. By ice or ammonia required for cooling. 
d. By increased labor of handling. 
2. Conceals inferiority. 
a. By encouraging carelessness. 
b. Discouraging proper care. 
3. Interferes with cream rising and whipping. 
4. Modifies taste. 
5. Less wholesome. 
a. Acid less as the lactic acid bacteria are destroyed. 
b. Spores forms of bacteria are not destroyed. 
c. Injurious by-products of bacteria are not destroyed. 
d. Milk is chemically changed. 
e. Is less easily digested. 
f. Produces scurvy and rachitis. 
ADVANTAGES 
1. Economic. 
a. Makes milk keep sweet longer. 
b. By saving milk which otherwise would spoil. 
c. By saving bills for sickness and milk-borne epidemics. 
2. More wholesome. 
a. Pathogenic bacteria are destroyed. 
b. Infant mortality less. 
c. Bacteria are decreased in numbers. 
The methods of pasteurization are two in number, the " flash " and 
" continuous " processes. In the continuous process the milk is heated 
for thirty minutes at 60° C.; the flash process requires a higher temper- 
ature (80° C. to 90° C.) for a shorter time (1-5 minutes), according PASTEURIZATION 
417 
to the theory of disinfection and since disinfection is a time process the 
continuous process allows a greater reduction of bacteria. 
Effect of Pasteurization on Bacteria. This has been given much 
study in order to refute the argument against pasteurization that 
pasteurized milk putrifies rather than sours. Ayers and Johnson (1913) 
Kaw 
Milk 
/62.8'C. 
(145° F.) 
71.1°C. 
(160° F.) 
Milk Pasteurized for 30 Minutes at 
93.3'0.' 
(200'F.) 
rrTTm 
76.7° C. 
(170° F.) 
82.2° C. 
(180° F.) 
87.8'C. 
(190° F.) 
Acid. 
Coagulating 
Group 
ALKALI AND PEPTONIZING GROUPS STILL MORE REDUCED. THE TOTAL ACID GROUPS ARE INCREASED IN SIZE, 
WHILE THE ACID COAGULATING GROUP REMAINS ABOUT THE SAME, THEY ONLY COAGULATE VERY SLOWLY 
THE TOTAL ACID GROUP REMAINS ABOUT THE SAME. THE ACID COAGULATING GROUP ONLY COAGULATE VERY SLOWLY. 
THE PEPTONIZING GROUP BEGINS TO INCREASE. ALKALI FORMING ORGANISMS ONLY FOUND OCCASIONALLY. 
LARGEST GROUP COMPOSED OF ACID ORGANISMS, ABOUT ONE THIRD OF WHICH COAGULATE MILK. 
THE ALKALI AND PEPTONIZING GROUPS ARE GREATLY REDUCED BY PASTEURIZATION. 
Acid 
Group 
NO ACID COAGULATING ORGANISMS, OR ONLY A TRACE. FEW ACID PRODUCING ORGANISMS. 
OCCASIONALLY A FEW ALKALI FORMERS. THE PEPTONI ZING GROUP IS THE LARGEST, 
LARGEST GROUP COMPOSED OF INERT ORGANISMS 
ABOUT THE SAME AS 82.2° C. 080°F.) 
ABOUT THE SAME AS 82.2° C. (18O°F.) 
Inert 
Group 
Alkali 
Group 
Peptonizing 
Group 
Fig. 69.-The Hypothetical Relation of the Bacterial Groups in Raw and Pasteur- 
ized Milk. (After Ayers and Johnson.) 
studied the bacteria in pasteurized milk by means of their milk-tube 
method. They used the holder process with a temperature of 62.8° C. 
for thirty minutes in most of their experiments. Their results are 
graphically summarized in Fig. 69 from which it will be seen that pas- 
teurization increased the percentage of acid-forming bacteria in the milk. 
Higher temperatures of pasteurization caused a different relation to 418 
MILK AND MILK PRODUCTS 
exist between the groups of bacteria in milk. In an earlier paper (1910), 
these same authors found that pasteurized milk soured about the same as 
a clean, raw milk. Weigmann et al. (1916) found that, in milk which 
had been heated from 60° C. to 63° C. for thirty minutes, lactic acid 
bacteria were present in a much larger proportion to remaining organ- 
Acid Coagulating 
Group Percentage 
|Peptonizing| 
Raw Mill? 
j 62.8°0.(145°F) 
71.1'0.(160° F.) 
76.7°C.(170° F) 
82.2'C.(180'F) 
87.8'C.(190'F. I 
93.3'0.(200 ^' 
Fig. 70.-The Hypothetical Relation of Bacteria in Raw and Pasteurized Milk. 
(After Ayers and Johnson, 1913.) 
Milk Pasteurized 30 Minutes 
isms than in raw milk. The same was also found in milk which had 
been heated for ten to twenty minutes. Souring, however, was much 
delayed over that in raw milk, which the authors attribute to attenuation 
of the lactic acid organisms. With regard to the resistance of Bacillus 
colon to heat, Ayers and Johnson (1915) state that this organism would PASTEURIZATION 
419 
not be expected, from their results, to survive 65.6° C. for thirty min- 
utes. Erratic results were secured and it is possible that some strains 
of Bacillus colon may be found which will survive pasteurization under 
the above conditions. Gage and Stoughton (1906) found that in 55 
per cent of thermal death-point determinations made with B. coli, the 
cultures withstood 80° C. for five minutes. Russell and Hastings (1902) 
report the characteristics of a micrococcus from milk which withstood a 
temperature of 76° C. for ten minutes. Different individual resistance, 
however, was found among the cells. Ford and Pryor (1914) claim that 
milk heated to any temperature between 65° C. and 100° C. and kept 
between 22° and 37° C. will be decomposed by spore-forming bac- 
teria and consequently may be dangerous. They recommend the 
boiling of milk and storage on ice since heated milk is liable to 
decompose. 
Pathogenic Bacteria. The effect of the pasteurization process on 
pathogens has been limited almost entirely to Bacillus tuberculosis. 
Since this organism is especially important in relation to milk hygiene, 
many attempts have been made to determine its thermal death point in 
milk. Rosenau has prepared table XLIV, which reviews concisely 
some of the work which has been done. 
The work of Smith (1899) and of Russell and Hastings (1904) has 
been especially convincing. The former of these investigators found 
that when tubercle bacilli were suspended in milk they were destroyed 
in from fifteen to twenty minutes at 60° C.; the greater portion being 
killed in 5-10 minutes. The latter tried short periods of pasteurization 
and found that a temperature of 160° F. (71.1° C.) or above for one 
minute destroyed the virulence of bovine tubercle bacilli so that guinea 
pigs were not killed when inoculated with from 2 to 5 mgs. Others have 
confirmed this work and these temperatures are now accepted in the con- 
tinuous process. The milkman pasteurizing milk is between two fires. 
He must avoid a cooked taste in the milk which interests the housewife 
and must kill all pathogenic bacteria which are the chief interest of the 
sanitarian. 
On the relation of pasteurization to other pathogenic bacteria in 
milk, our knowledge is very meager. Hesse (1894) stated that raw 
cows' milk was not a good medium for cholera spirilli and that they 
died in twelve hours at room temperature and more quickly at higher 
temperatures. The acidity and other bacteria in the milk killed them. 
Fig. 69 prepared by North and taken from the report of the Commission 
on Milk Standards gives some data concerning B. typhi, streptococci and 
Bad. diphtheria. 420 
MILK AND MILK PRODUCT^ 
Table XLIV 
SHOWING THE THERMAL DEATH POINT OF THE TUBERCLE BACILLUS 
AS FOUND BY VARIOUS INVESTIGATORS 
(After Rosenau) 
Investigator 
Killed at 
Not Killed at 
Martin, 1882 
80° C. 
May, 1883 
By cooking 
Boiling, 5 minutes 
Sormani, 1884 
Schill and Fisher, 1884. . . 
90° for 10 minutes. 
100°. 
Voelsch, 1887. . ' 
100° boiling twice. 
Yersin, 1888 
60°, 10 minutes, no spores. 
60°, 10 minutes, with spores. 
68°, 20 minutes. 
70°, 5 minutes (enfeebles). 
60°, 5 minutes (sometimes 
enfeebles). 
80°, sometimes kills. 
85°, always kills. 
60°, 20 minutes 
60°, 5 minutes (attenuates). 
70°, 1 minute (kills). 
60°, 6 hours 
95°, momentary 
55°, 4 hours 
Bitter, 1890 
Bang, 1881 
Bonhoff, 1892 
50°, 60 minutes. 
Gancher and Ledeux- 
Lebard, 1892 
Forster, 1892 
55°, 3 hours. 
60°, 45 minutes. 
80°, momentary. 
60°, 1 minute. 
DeMan, 1893 
Schroeder, 1894 
60°, 1 hour 
60°, 15 minutes. 
Woodhead, 1895 
50°, 15 hours. 
60°, 8 hours. 
60°, 45 minutes 
90°, results contradic- 
tory. 
60°, 10 minutes. 
Marshall, 1899 
70°, 45 minutes. 
70°, 21 minutes. 
68°, 28 minutes 
Th Smith, 1899 
60°, 15 to 20 minutes 
70°, 10 minutes. 
Morgenroth, 1900 
55°, 3 hours 
100°, momentary 
Kobrak, 1900 
50°, 4 hours. 
Beck, 1900 
100°, 3 hours 
100°. 
Galtier, 1900 
80°, 30 minutes. 
85°, 6 minutes. 
Russell and Hastings, 1900 
Herr, 1901 
60°, 20 minutes. 
65°, 15 minutes 
80°, 5 seconds. 
Hesse, 1901 
60°, 20 minutes. 
65°, 15 minutes. 
Levy and Bruns, 4901.. . . 
Barthel and Stenstrbm, 
1901 
70°, 15 minutes. 
60°, 15 minutes. 
Bang, 1902 
Tjden, 1903 
85°, 1 to 2 minutes. 
Rullmann, 1903 
65°, 30 minutes 
Barthel and Stenstrom, 
1904 
80°, 1 minute (uncoagulated) 
71°, 1 minute. 
80°, 1 minute (coagu- 
lated). 
76°, 20 minutes. 
Russell and Hastings, 1904 
Zelenski, 1906 
Rosenau, 1907 
60°, 20 minutes. PASTEURIZATION 
421 
Fat 
Sugar 
.Casein 
Salts 
Temperature Fahrenheit 
Taste 
• Albumen 
Enzymes 
-Cream Line 
Tuberculosis 
Typhoid. 
Streptococci 
Diphtheria 
Fig. 71.-Indicating the Relation between the Time and Temperature and Other 
Factors Involved in the Pasteurization of Milk. (After North.) 
Time in Minutes ' 422 
MILK AND MILK PRODUCTS 
Pathogenic Streptococci. Much evidence has accumulated to show 
that septic sore throat may be caused by streptococci which originate 
in milk from cows suffering from mastitis. Cows suffering from garget 
are regarded as of special importance in this connection. Capps and 
Downs (1914) found a dairy farm where mastitis was present in the 
cows and where the milkers had sore throat. The milk from this dairy 
was delivered to a dairy company which did not pasteurize its milk and 
consequently an extensive epidemic of septic sore throat resulted. 
Krumwiede and Valentine (1915) during the investigation of an epidemic 
regarded the infection as of human rather than of bovine origin. They 
suggest that " in tracing the source of an epidemic the effort should be 
toward finding cases of sore throat among those engaged in producing 
the milk, not mastitis in the cow alone. If human streptococci are 
found in mastitis, they are most likely secondary agents in an already 
existing inflammation due to bovine strains. The streptococci in dif- 
ferent epidemics differ culturally and those similar culturally differ in 
their immunity reactions. Cultural similarity of strains from man and 
cattle is insufficient to prove their identity. Cultural identity in every 
detail or immunological identity is essential." Rosenow and Moon 
(1915) studied an epidemic of septic sore throat and, after tracing it to 
milk, isolated virulent streptococci from the milk. These showed 
selective preference for joints, muscles, gall bladder, etc., and resembled 
certain rheumatic strains morphologically and culturally. These 
investigators regard virulent streptococci in milk as possible sources of 
those which cause rheumatism and other chronic infections. Rosenow 
and Hess (1917) also traced another epidemic of this disease to the milk 
from three cows. Winslow and Hubbard (1915) reported a contact 
epidemic of this infection. Smaller epidemics have been studied by 
Overman (1914), Henika and Thompson (1917) and others. Smillie 
(1917) has stated that the streptococci of septic sore throat resemble 
those of scarlet fever and that discharges from such patients may infect 
the milk and cause septic sore throat. Ayres and Johnson (1914) 
studied the possibility of streptococci surviving pasteurization. Out 
of 139 cultures isolated from cow feces, the mouth of the cow and milk, 
64.03 per cent of them survived a pasteurization temperature of 140° F. 
Thirty-three and seven hundredths per cent survived at 145° F. At 
165° F. none were able to survive. These data might indicate that the 
streptococci which were pathogenic for man could under certain con- 
ditions pass through the pasteurization process. 
Allen (1916, 1917) has shown that raw milk as compared with pas- 
teurized milk exerts a powerful suppressing influence on the multipli- BACTERIOLOGY OF BUTTER 
423 
cation of certain bacteria. The pasteurized milk was found to be more 
favorable to the attack of the gas forming colon bacillis and B. aerogenes. 
Allen emphasizes the point that pasteurized milk, although it is safer 
for human consumption, should be handled with much care since heating 
has decreased its resistance to many detrimental changes. Ayers and 
Johnson after a study of this question reported that the bacterial 
increase in clean raw milk and pasteurized milk was about the same 
when both were stored under the same conditions. 
Bacteriology of Butter 
The bacteriology of butter may be considered from two viewpoints- 
the use of bacteria to bring about desired changes and the dissemination 
disease-producing bacteria. 
Bacteria in Cream and Cream Ripening. According to present 
practice cream is pasteurized and ripened before being made into butter. 
The pasteurization process removes all of the extraneous bacteria and 
thus prevents spontaneous changes which are liable to produce unfav- 
orable flavors and tastes. After pasteurization, the cream is inoculated 
with a culture known to produce a desired flavor and allowed to ripen. 
Certain distinct improvements were introduced into the butter industry 
by ripening of the cream. (1) A better yield of butter is secured from 
sour cream. (2) The flavor of the butter is kept constant from batch 
to batch. (3) Sour cream churns more easily than sweet cream. Peiser 
(1916) has stated that the greater proportion of bacteria in ripened cream 
are removed during churning by the buttermilk. Butter contains about 
one-tenth as many as are in the cream. 
Flavor of Butter. This is dependent upon a large number of factors 
and opinion is still divided with regard to which is the most important. 
That bacteria are important in changing the flavor of butter after it 
has been made is improbable. Conn has attempted to make a distinc- 
tion between " flavor " and " aroma " but such a division is probably 
quite artificial. 
Rancidity is a term which covers a multitude of abnormalities in 
the taste of butter. Guthrie (1917) in his interesting paper has given a 
review of the literature. In his experiments, he attempted to determine 
whether rancidity was due to chemical cow enzyme or biological changes. 
He found that none of these factors were important and believes that 
rancidity as defined by butter judges is rarely found. No marked 
change in the iodine number was caused by high temperatures, light or 
air. 424 
MILK AND MILK PRODUCTS 
The undesirable flavors in butter may be due to absorbed odors or 
to products formed in the butter after manufacture. Dyer (1916) 
showed that the undesirable flavors developing in butter held in cold 
storage at a temperature of 0° C. were not due to an oxidation of the fat 
itself. Gas analyses indicated that the quantity of buttermilk left in 
Bacteria, Hundreds of Thousands per Gram. 
Fig. 72.-Diagram Showing Decrease in Bacterial Content of Butter Stored at Dif- 
ferent Temperatures. (After Mohler, Washburn and Rogers, 1909.) 
Months in Storage 
the butter has a relation to the quantity of carbon dioxide formed. 
Rancidity is probably the algebraic sum of a number of factors. 
Milk in the fresh condition is a very suitable medium for bacteria. 
Northrup (1911) studied the longevity of B. typhi in sour milk. Before 
these organisms were destroyed Bacterium lactis acidi had to produce TUBERCLE BACILLI IN BUTTER 
425 
80° acid in milk and 28° acid in whey. Bacillus bulgaricus had to pro- 
duce 208° acid in milk and 60° acid in whey. She quotes the work of 
other investigators as Bassenge, Behla and others that lactic acid is 
toxic for Bacillus typhosus. Krumwiede and Noble (1915) report that 
the typhoid bacillus is killed in sour cream by acids and that the destruc- 
tion is proportional to the amount of acid and number of bacilli. Wash- 
burn (1908), in artificially infected milk found that B. typhi suffered no 
diminution in numbers up to twenty days. They had practically dis- 
appeared after forty-three days. Potter (1910) concluded that Bacillus 
bulgaricus and Bacterium lactis acidi exerted no appreciable repressing 
effect on Bacillus typhi and that these organisms may not be depended 
upon to make milk, which has been contaminated, safe. 
Table XLV 
VIABILITY OF TYPHOID BACILLI IN SOUR CREAM 
(Krumwiede and Noble) 
Days 
0 
2 
7 
8 
9 
10 
11 
Reaction * 
Number of typhoid 
bacilli 
1.0% 
392,000 
2.2% 
65,000,000 
5% 
300,000,000 
113,000,000 
181,000 
10% 
400 
* Number of cubic centimeters of N alkali needed to neutralize 100 c.c. of cream. 
Molds in Butter. Thom and Shaw (1915) found that mold in 
butter usually took three forms: first, orange yellow spots produced 
by Oidium lactis; secondly, dirty green spots produced by cladosporium 
alternaria, thirdly, green spots which are caused by penicillium. A 
salt content of 2.5 per cent prevented the development of the above 
fungi and, therefore, their presence indicates a low salting. The pres- 
ence of much curd was stated to allow a more vigorous growth of mold. 
Hastings (1916) has recommended that butter tubs and liners be placed 
in boiling water heated to 150° to kill the mold spores. 
Tubercle Bacilli in Butter. While the literature on the spread of 
this disease by butter is not extensive, there are several important 
investigations on the question. Rosenau, Frost and Bryant (1914) 
examined twenty-five samples of Boston market butter and found tuber- 
cle bacilli in two of them, B. coli in six and streptococci in fourteen. 
Hill (1911, 1913) has reported the spread of typhoid fever and diph- 
theria by butter. M. Mohler, Washburn and Rogers (1909) found that, 
contrary to prevailing opinion, tubercle bacilli were not devitalized by 426 
MILK AND MILK PRODUCTS 
cold storage; salt was reported to have no effect on these organisms 
in butter. Salted butter was found to retain its virulence for six 
months. Schroeder and Cotton (1908) demonstrated the presence of 
tubercle bacilli in butter which had been kept for one hundred and sixty 
days. These data indicate that some butters may contain tubercle 
bacilli even after comparative long storage periods. Again, the value of 
pasteurization of dairy products is emphasized. 
Savage's Method for Tubercle Bacilli in Butter. To detect tubercle 
bacilli in butter the inoculation method is the only satisfactory one. 
The butter is placed in centrifugal tubes which are stood in warm water 
at 42° C. until the butter is completely melted. The material is cen- 
trifugalized when liquid, and the sediment inoculated into guinea pigs 
as described under milk. It is difficult to keep the butter liquid during 
the centrifugalization. 
Bacillus typhosus in Butter. Washburn (1908) studied the lon- 
gevity of B. typhi in butter which was artificially infected. In this 
experiment, the organisms were found 150 days after the preparation of 
the butter. Boyd (1917) reported an epidemic of typhoid fever from 
butter. He regards the pasteurization of the products before ripening 
as a wise procedure to prevent disease and undesirable fermentations. 
Washburn's data indicate that butter may contain virulent typhoid 
bacilli for 151 days. Probably the conditions under which the data 
are obtained greatly influence the results. 
Bergey's Method for Typhoid Bacilli in Butter. Transfer 5 gms. of 
butter by means of a sterile scalpel to a test tube containing ordinary 
nutrient bouillon. After incubation for several days, streak plates of 
Endo's medium, Drigalski-Conradi agar and Loeffler's malachite green 
agar. Incubate these plates for from 24-48 hours and transfer the 
suspicious colonies to agar slants and a melted tube of dextrose agar. 
After incubation only those cultures should be retained for further 
study which show no fermentation in dextrose agar. Morphological 
studies should then be made together with cultural studies in lactose, 
sarbite, raffinose, dextrose, saccharose, dulcite, adonite and inulin 
broths. Observation has shown that Bacillus typhosus produces a 
slight acidity in lactose broth and a more definite acidity and coagulation 
in the sorbite medium. 
Determination of Bacteria in Butter, Lohnis Method. For the 
sterile weighing of butter, cheese and similar substances, put a number 
of filter papers, 9 cm. and 7 cm. in diameter, into two Petri dishes 
respectively and sterilize in the air oven. By means of sterilized forceps, 
place one of the larger papers on the scale pan ami then one of the TUBERCLE BACILLI IN BUTTER 
427 
Table XLVI 
INCIDENCE OF TUBERCLE BACILLI TN MARKET BUTTER 
(After Briscoe and MacNeal, 1911) 
Author. 
Date. 
Place. 
Samples । Samples 
Per 
Cent 
Posi- 
tive. 
Remarks. 
Ex- 
amined. 
Posi- 
tive. 
Brusaferro 
1890 
Turin 
9 
1 
11.1 
Both 
1894 
Zurich 
20 
2 
10.0 
Microscopic method. 
Obermiiller 
1895 
Berlin 
13 
8 
61.0 
Schuchardt 
1896 
Marburg 
42 
0 
0.0 
Obermiiller 
1897 
Berlin. 
14 
14 
100 0 
16 tested, 2 lost. 
Groning 
1897 
Hamburg . . 
17 
8 
47 
Himesch 
1897 
Wien 
? 
0 
0 
Rabinowitch.. . . 
1897 
Berlin 
30 
0 
0 
Rabinowitch.. . . 
1897 
Philadelphia. . 
50 
0 
0 
Petri 
1897 
Berlin 
102 
33 
32.3 
Hormon and 
Morgenroth. . 
1897 
Berlin 
10 
3 
30.0 
Rabinowitch.. . . 
1899 
Berlin 
15 
2 
13.3 
First series. 
Rabinowitch.. . . 
1899 
Berlin 
? 
? 
87.2 
Second series. 
Rabinowitch.. . . 
1899 
Berlin 
15 
15 
100.0 
Third series. 
Rabinowitch.. . . 
1899 
Berlin 
19 
0 
0 
Fourth series. 
Obermiiller 
1899 
Berlin 
10 
4 
40.0 
Korn 
1899 
Freiburg 
17 
4 
23.5 
Ascher 
1899 
Konigsberg. . . 
27 
2 
7.4 ■ 
Jager 
1899 
Konigsberg. . . 
3 
1 
33.3 
Coggi 
1899 
Milan 
100 
12 
12 
Weissenfield. . . . 
1899 
Bonn 
32 
3 
9.4 
Grassberger 
1899 
Wien 
10.0 
0 
0 
Herbert 
1899 
Tubingen 
43 
0 
0 
Herbert 
1899 
Wurttemberg . 
58 
0 
0 
Herbert 
1899 
Berlin 
20 
0 
0 
Pseudo tuberculosis 8 per 
Herbert 
1899 
Munchen 
5 
0 
0 
Pseudo tuberculosis 4 per. 
Abenhausen.... 
1900 
Marburg 
39 
0 
0 
Hellstrom 
1900 
Helsingfors. . . 
8 
1 
12.5 
12 samples, 4 lost 
Bomhoff ...» 
1900 
Marburg 
28 
0 
o 
Pawlowsky 
1900 
Kiew 
23 
1 
4 3 
Tobler 
1901 
Zurich 
12 
2 
16 7 
Lorenz 
1901 
Dorpat 
30 
0 
0 
Markl 
1901 
Wien 
43 
0 
0 
Herr and 
Beninde 
1901 
Breslau 
52 
6 
11 1 
Two were doubtful. 
Aujeszky 
1902 
Budapest 
17 
3 
17 6 
Thu 
1902 
Christiania. . . 
16 
0 
0 
Teichert 
1904 
Rosen 
40 
12 
30 0 
Reitz 
1906 
Stuttgart 
94 
8 
8 5 
Butter from 88 dairies. 
Eber 
1908 
Leipsic 
150 
18 
12 0 
Briscoe and 
MacNeal 
1911 
Urbana, Ill. . . 
6 
2 
33.2 
Eber 
1912 
15 6 
Rosenau et al.. . 
1914 
Bcfeton 
21 
2 
9 4 
Marchiotti 
1917 
25 
24.0 
52 per cent of the samples 
contained acid-fast ba- 
• 
cilli. 428 
MILK AND MILK PRODUCTS 
smaller papers on top of it. After taring, with sterilized knife or spoon, 
place the substance to be weighed on the upper filter paper. Then 
transfer the material along with the small filter paper, to the first dilu- 
tion flask. Make the dilutions according to the quantity of butter to 
be examined and pour plates. The water used for dilutions should be 
previously warmed to 40° C. so that the butter (1 gm. in the first tubes 
or flasks) may be readily emulsified. 
Determination of Bacteria in Butter (Schneider's Method). To 
examine butter directly for the number of bacteria and other contamina- 
tions, place 1 gm. of the butter into 10 c.c. of ether and shake until all 
of the butter fat is dissolved. Pour the solution into a special cen- 
trifuge tube and centrifuge for five minutes. Wash the contents of the 
1 c.c. end into 10 c.c. of ether and again shake and centrifugalize. Pour 
off the ether and add 2 c.c. of a 2 per cent solution of sodium hydroxide 
and shake until the casein is dissolved. The sodium hydroxide emul- 
sifies the small amount of fat which is present. Examine the emulsion 
for bacteria and body cells by means of the haemocytometer. 
Conn's Method for Analysis of Butter. Weigh out upon accurate 
chemical scales 5 gms. of freshly made butter. Place this in a sterile 
mortar, with 9/5 c.c. of sterile water. Rub the water and the butter 
together thoroughly, so as to distribute the bacteria as uniformly as 
possible through the water. This mixing should be continued for some 
time, for, at best, many of the bacteria will remain clinging to the 
fat. Dilute this mixture to 10,000 times and make a series of agar or 
litmus gelatin plates. Incubate and count as usual. 
Cheese 
Cheese may be classified into two general types, rennin curd and acid 
curd. In the former type, the enzyme rennin is added to the milk which 
forms calcium paracaseinate from the calcium caseinate. The acid curd 
cheeses are made from curd secured by the natural or artificial souring of 
milk. 
Cheese Ripening. In this process develops the characteristics 
which, in part, separate one type of cheese from another. Many theo- 
ries have been held in the past with regard to this process. Duclaux 
(1880), Freudenreich (1897), Babcock and Russell (1898), Gorini (1904) 
and others, have studied the problem and given different theories. The 
following factors were at times considered the important ones: 
1. Enzymes which originated from the cow. 
2. Enzymes of bacterial origin. 
3. Pepsin which is added with rennin. BACTERIOLOGY OF CHEESE 
429 
Bacteriology of Cheddar Cheese. Harding and Prucha(1908) studied 
more than 300 pure cultures of bacteria from Cheddar cheese by means 
of the group number on the classification card of the Society of Amer- 
ican Bacteriologists. These finally reduced to 33 groups. Ten of these 
groups disappeared at once; 9 others were found in but single cheeses; 
the remaining 14 groups were indicated as the most important members 
of the cheese flora. Bacterium lactis acidi included 4 of these 14 groups 
and was the only species always found and it practically included over 
99 per cent of the total bacterial content. 
Hastings, Evans and Hart (1912) conducted a similar study. They 
state that the Bacterium lactis acidi groups is an important one and 
sum up the role of these organisms as follows: 
1. They favor the curdling process. 
2. They favor the expulsion of the whey. 
3. They permit of the fusing of the curd particles. 
4. They activate the pepsin in the rennin extract. 
5. They have a protective action against the putrefactive bacteria. 
They state that the development of B. lactis acidi is followed by the 
Bacillus bulgaricus group. 
These same authors in a later paper (Evans, Hastings and Hart, 
1914) found the following four groups and assumed that they must 
function in the ripening process: 
1. Bacterium lactis acidi. 
2. B. casei. 
3. Streptococcus. 
4. Micrococcus. 
When the Bacterium lactis acidi group was used alone as starters, 
in pasteurized milk cheese, no cheddar flavor was obtained. Com- 
binations of the Streptococcus and B. lactis acidi groups when added 
to pasteurized milk improved the character of the cheese. Hart, 
Hastings, Flint and Evans (1914) isolated coccus forms of bacteria 
from cheddar cheese and cultured them in sterile milk. Large amounts 
of volatile acids were found on analysis of the culture. These acids 
were produced from either citric acid lactose or protein because the 
milk contained no fat. One strain of streptococcus formed large 
amounts of alcohols and esters which contribute to the flavor of the 
cheese. Suzuki, Hastings and Hart (1910) isolated and identified vola- 
tile acids during the ripening of cheddar cheese. Alcohol was formed 
probably from lactose fermentation. The agencies operative in the 
production of volatile acids and esters are not defined. 
Harding and Prucha (1908) analyzed different cheeses and demon- 430 
MILK AND MILK PRODUCTS 
strated that the number of bacteria decreases rather rapidly. Fresh 
cheeses were found to contain large numbers of bacteria. These 
decreased so that at the end of thirty or forty days there were less than 
10,000,000 bacteria per gram. These authors state that a cheddar 
cheese commercially ripe usually contains some millions of living bac- 
Commercial score 
Bacteria content 
Millions of Bactaria per Gram of Cheese 
Days 
Fig. 73.-Bacteria Content and Commercial Score of Cheddar Cheese. (After 
Harding and Prucha, 1903.) 
teria per gram. Fig. 73 shows the number of bacteria that were found 
in a cheese at various ages. 
The relation of cheese to the spread of disease is an interesting ques- 
tion which has received some attention from investigators. Tuber- 
culosis has received the greater part of the attention. Mohler, Wash- BACTERIA IN CHEESE 
431 
burn and Doane (1909) have mentioned and reviewed some of the 
work which was carried on previous to their publication. Hermann 
and Morgenroth (1898) upon examination of 15 samples of cottage 
cheese found 3 of which contained tubercle bacilli. Rabinowitch (1907) 
reported 3 positive samples out of 5 of the same product. Three sam- 
ples of soft cheese purchased from the Berne markets by Harrison 
(1900) were found to produce tuberculosis when injected into guinea 
pigs. Heim (1889) and Galtier (1887) infected milk before making 
cheese. Heim reported tubercle bacilli in the cheese for fourteen days 
but none after four weeks. Galtier found tubercle bacilli in cheese 
which was two months and ten days old. He believes that such milk 
products may disseminate the disease among human beings, Mohler, 
Washburn and Doane (1911) prepared a cheese from infected milk and, 
by inoculation into guinea pigs, produced generalized tuberculosis from 
samples 220 days old. Injections of emulsions 260 days old caused 
slight lesions. Schroeder and Brett (1918) carried out an extensive 
piece of work which has greatly enriched our knowledge. They pur- 
chased 256 samples of cheese from the Washington market and sub- 
jected them to examinations for the presence of tubercle bacilli by means 
of guinea-pig inoculation. Nineteen or 7.42 per cent contained tubercle 
bacilli. Cheddar cheese was examined to the extent of 59 specimens; 
no tubercle bacilli were found. None were found in 32 specimens of 
Neufchatel cheese. Eighteen out of 131 samples of cream cheese were 
infected. They regard the danger of eating ripened cheese as very 
slight since the bacilli would die during the ripening process. According 
to Mohler's work, this would depend on the length of the ripening period. 
Rowland (1895) inoculated cheese and butter with M. choleras and B. 
typhi. After a few days no living organisms could be found, which is 
regarded as reassuring by this author. He used cheddar, Dutch and 
American cheeses and both fresh and salt butter. 
Cheese Poisoning. This type of food poisoning is said to be rather 
common. Vaughn named the poisonous substance in cheese tyrotoxi- 
con which he regarded as a ptomaine. Newman (1902) and Lepierre 
(1894) confirmed his work. Spica (1910) recently isolated a poisonous 
substance from cheese. Levin (1917) studied this subject and found 
by means of agglutination reactions that a member of the colon group 
was involved in a case of cheese poisoning, which he investigated. No 
tyrotoxicon could be found. 
Determination of the Number of Bacteria in Cheese. (Harding and 
Prucha's Method.) Remove a square inch of the rind by means of a 
sterile knife. Draw out a plug about 4 ins. long with a carefully flamed 432 
MILK AND MILK PRODUCTS 
cheese tryer. Cut a number of thin slices from different parts of this 
plug and weigh out 1 gm. of these slices on a flamed copper foil using 
an analytical balance. This sample should be triturated in a flamed 
mortar with 10 gms. of sterile granulated sugar or finely ground ster- 
ilized quartz. Care should be taken to have the room free from dust 
and the instruments which come in contact with the cheese, sterile. 
Dilute this freshly ground cheese to 30 c.c. with sterile water. An 
aliquot portion should be transferred to a water blank. Plate out in 
litmus whey gelatin in dilutions of from 1000 to 1,000,000 or there- 
abouts. Incubate for ten days at room temperature. 
Lohnis' Method. One gm. of cheese taken under aseptic conditions 
and weighed according to the method described in another place is 
ground in a sterile mortar with 10 c.c. of sterile tap water, and some 
sterile quartz sand or glass powder added if necessary. The mixture is 
put into a dry, sterile 1000 c.c. flask, which is then filled to the mark with 
sterile water. After continuous shaking for several minutes, further 
dilutions with 9 c.c. of water arc made in test tubes. Usually dilutions 
of 1 to 10,000 to 1 to 100,000 are sufficient. This is plated out in plain 
agar and gelatin. Cultures for anaerobic incubation should also be 
made. 
Harrison's Method. Remove a plug of cheese with a sterile cork 
barer and transfer to a sterile mortar. Triturate this with sterile water 
of bouillon. This is injected to a 500 gm. guinea pig. A hole is made 
in the skin with a large needle. Through this by means of a pipette the 
cheese emulsion is blown in. Animals should be kept under observa- 
tion and immediately after death should be examined. Smears should 
be made from the diseased organs and stained from the Ziel Nielson 
method. 
Determination of Tubercle Bacilli in Cheese. (Mohler's et al. 
Method.) Rub portions of the cheese in a mortar with physiological 
salt solution. After grinding strain the liquid through a layer of absorb- 
ent cotton and the equivalent of 2 gms. of cheese should be injected 
beneath the skin of each guinea pig. Also feed portions of the cheese 
to other pigs and keep for observations. 
Fermented Milks 
These are milks in which the lactose has been changed to lactic 
acid, alcohol or carbon dioxide by the action of fungi. An extensive 
literature of the subject, together with a discussion has been prepared by 
Rogers (1916). CONDENSED MICK 
433 
Condensed Milk 
Milk has been condensed for a long time. In 1835, Newton took out 
a patent in England. Later, in 1849, Harsford added lactose to con- 
densed milk. 
The nomenclature of concentrated milks is not set. Condensed milk 
often refers to concentrated milks to which a carbohydrate, sucrose or 
lactose has been added. The term evaporated milk is often restricted 
to the plain concentrated milk to which no carbohydrates have been 
added. Again, these may be referred to as condensed sweetened milk 
and condensed unsweetened milks. The following definition for sweet- 
ened condensed milk has been accepted by the Association of American 
Dairy, Food and Drug Officials, August 7, 1916, and by the Association 
of Official Agricultural Chemists, November 22, 1916. " Sweetened 
condensed milk, sweetened evaporated milk, sweetened concentrated 
milk is the product resulting from the evaporation of a considerable 
portion of the water from the whole fresh, clean, lacteal secretion ob- 
tained by the complete milking of one or more healthy cows, properly 
fed and kept, excluding that obtained within fifteen days before and 
ten days after calving, to which sugar (sucrose) has been added. It 
contains not less than 28.0 per cent of total milk solids and not less than 
8.0 per cent of milk fat." (U. S. Agriculture, Food Inspection Decision 
158). 
Condensed milk is not sterile. The data from many investigations 
are on record in support of this. There seems to be no more bacteria 
or body cells in condensed milk than in raw market milk. Some of 
the processes of condensation remove these. The centrifugal sepa- 
rators are, doubtless, responsible for much of this reduction. The bac- 
teria which do pass through the condensation processes lie dormant on 
account of insufficient moisture and too concentrated solution. Some 
are present in the spore stage. This is probably true in those cases 
where members of the Bacillus subtilis group such as Bacillus subtilis 
and Bacillus mesentericus have been isolated. Thayer (1912) exam- 
ined condensed milk which spoiled from twelve to twenty-four hours 
after condensing and found Bacillus subtilis. To obviate this difficulty, 
this author recommended a sterilization temperature of 125° C. for 
fifteen or twenty minutes. Hammar (1915) isolated an organism which 
merely coagulated the canned milk. The name Bacillus coagulans was 
given to the organism. The coagulum was firm and had a sweetish 
taste. There was no indication of putrefaction. Andrews (1913) found 434 
MILK AND MILK PRODUCTS 
that there were not enough pus cells in the milk which he examined to 
cause suspicion. The following sums up his opinion: 
The presence, in reasonable number of the bacteria commonly found in fresh 
milk-Bacillus coli, streptococci, a few staphylococci, and B. enteritidis sporo- 
genes-is comparatively unobjectionable. A large proportion of the bacteria of 
milk seem to be destroyed in the process of condensation. It was found that con- 
densed milk was almost a differential medium for staphylococci. From his work 
it would seem that if a few Staphylococcus pyogenes aureus were present in the 
milk after condensation many more might be found when the can was opened. 
Efficient pasteurization before condensation should greatly reduce the pos- 
sibility of the staphylococci getting into the final product. 
Park et al. (1915) found that evaporated milk contained fewer 
bacteria than condensed milk. This may have been due io the higher 
temperature at which it is prepared. Kossowicz (1908) found that 
condensed milk was not bacteria free. He found Bacillus fluorescens 
liquefaciens, Bacillus prodigiosus and Bacillus sinapwagus to be present. 
Savage (1914) reports several investigations in England, the data from 
which do not differ materially from those secured in this country. The 
very interesting experiment of Delepine with regard to the effect of the 
condensation processes on Bacterium tuberculosis is mentioned. The 
tubercle bacillus, even though heavily seeded into milks, was killed 
and could not be demonstrated in the final condensed milk. 
Delepine (1915) has studied the effect of preserving and drying milk 
on the virulence of the tubercle bacillus. In the manufacture of sweet- 
ened milk, the mixed raw milk had a bacterial content of 38,000,000 
which was reduced by the treatment to less than fifty in one case. The 
finished sweetened product failed to produce lesions when inoculated 
into and fed to rabbits and guinea pigs. 
Ice Cream 
The term is a general one and covers many types of similar sub- 
stances. Those interested in the history of this food should look up 
the paper by Washburn (1910). 
The bacteriology of ice cream has, of late, received much attention 
since it became necessary to establish some bacterial standards. Wiley 
(1912) and his co-workers studied the ice cream manufactured in the 
District of Columbia and found rather large numbers of bacteria. 
Many of them contained streptococci. Esten and Mason (1915) 
studied the effect of storage on the number and kinds of bacteria. The ICE CREAM BACTERIA 
435 
cream was frozen in the usual type of freezer and stored in quart bricks. 
The method of analysis is given in another place. They found that 
there was little change in the number of bacteria as shown by litmus 
lactose gelatin plates when cream was kept frozen for a month. 
Ayers and Johnson (1915) undertook an investigation on ice cream 
with the following objects: 
1. To determine the number of bacteria in commercial ice cream 
during the summer and winter seasons. 
2. To determine what groups of bacteria are found in commercial 
ice cream. 
3. To determine the relative value of different methods for the deter- 
mination of Bacillus coll in ice cream. 
They found that the average number of bacteria in ice cream is 
lower during the winter months. A summary of their results is given 
in the following table: 
Item. 
Summer Series 
(94 Samples). 
Winter Series 
(91 Samples). 
Average number of bacteria per cubic centimeter. 
Maximum number 
Minimum number 
37,859,807 
510,000,000 
120,000 
10,388,222 
114,000,000 
13,000 
Studies by means of the milk-tube method showed that the bacterial 
groups bore about the same relation to each other in the average summer 
and winter samples. During the summer months the averages based on 
71 samples were as follows: 
Acid coagulating 49.82 
Acid forming 20.72 
Inert group 13.98 
Alkali forming groups 1.86 
Peptonizing group 13.62 
The averages for the winter samples are: 
Acid coagulating group 30.84 
Acid forming group ... 38.03 
Inert group 4.81 
Alkali forming group 5.42 
Peptonizing group 20.90 
The peptonizing group is of most significance since it is these bac- 
teria which cause poisoning from ice cream. 436 
MILK AND MILK PRODUCTS 
Ayers and Johnson (1917) in a later paper report the results from a 
study of some phases of ice-cream analysis. It was found that bacteria 
are evenly distributed through a can of ice cream and that storage does 
not tend to cause an uneven distribution. These were factors which 
had been suspected of causing difficulties in the bacterial examination 
of ice cream. This was later confirmed by Heinemann and Gardan 
(1917). As freezing progressed clusters of bacteria were broken up and 
this tended to increase the count. No marked difference was found in 
the counts on samples from different parts of the same can. 
Hammar and Goss (1917) analyzed ice cream and water sherbets. 
They reported very few organisms in the water sherbets compared with 
the ice cream. Ice creams, other than vanilla, were found to contain 
from 130,000 to 40,850,000 bacteria per cubic centimeter. In general 
there was no increase in the number of bacteria during storage and gen- 
erally there was a reduction. With some samples increases were re- 
ported. Table XLVII contains a few analyses reported by Hammar 
and Goss. 
Table XLVII 
SHOWING THE EFFECT OF STORAGE ON THE NUMBER OF BACTERIA 
IN ICE CREAM 
(Hammer and Goss, 1917) 
Mix 
Freeze 
1 day old.. 
2 " . . 
3 " 
236,000 
735,000 
360,000 
310,000 
260,000 
32,800,000 
30,850,000 
7,750,000 
4,450,000 
2,435,000 
1,150,000 
120,000 
146,000 
137,000 
216,000 
172,500,000 
271,000,000 
157,000,000 
128,000,000 
52,000,000 
110,000,000 
170,000,000 
194,000,000 
216,000,000 
102,000,000 
120,000,000 
140,000,000 
70,000,000 
71,000,000 
4 " 
152,000 
300,000 
139,000 
156,000 
5 " 
310,000 
34,000,000 
39,000,000 
41,000,000 
6 " 
7 " 
31,000,000 
54,000,000 
8 " 
61,000,000 
9 " 
■ 
36,000,000 
15,000,000 
10 " .. 
11 " 
12 " .. 
No special difficulties appear in the bacterial analysis of ice cream. 
Secure a brick of the cream or a pint or quart container if it is not avail- 
able in brick form. As soon as it has reached the laboratory unwrap ICE CREAM BACTERIA 
437 
and remove samples from the interior of the mass of ice cream. This 
may be done by scraping off the exterior with a sterile spatula and by 
means of a sterile scoop transfer portions to sterile flasks. Heat these 
on a water bath at 40° C. with occasional stirring until melted. Dilute 
with sterile pipettes and plate out using the standard media. 
Bacillus Typhosus in Ice Cream. Several severe epidemics of 
typhoid fever have been caused by ice cream. Mitchell (1915) found 
that these bacteria could live for 12 to 39 days in ice cream and that 
ice cream could thus become an important epidemiological factor in the 
spread of the disease. Cumming (1917) reported an epidemic of 
typhoid fever caused by a carrier who participated in the preparation 
of the cream. Lumsden (1917) has given the characteristics of epi- 
demics caused by ice cream. The difficulty of securing accurate data in 
such epidemics is mentioned. Waterman (1917) has reported a small 
outbreak of typhoid fever which probably came from ice cream; the 
typhoid history of the people involved is rather striking. Bolton (1918) 
reported experiments on freezing B. typhi in cream. At the end of a 
month the number of bacteria is reported to have been reduced to about 
one-twentieth of the original number. At the end of about 45 days 
after freezing in brine, some of the bacilli were found to be alive. As 
the author points out, ice cream may be a potent factor in the dis- 
semination of typhoid fever. 
Determination of the Number of Bacteria in Ice Cream (Esten 
and Mason). Samples are taken with sterilized knives, the outer layer 
being cut off before the sample is taken out. Allow the cream to melt 
at room temperature in the same manner as milk samples. Plate out 
on litmus lactose gelatin; incubate for 7 days at 21° C. and count. 
Make four plates from each sample and report the results as an average 
of the four plates. By using litmus lactose gelatin the number of acid- 
forming bacteria and the number of liquefying bacteria may be deter- 
mined. 
Savage's Method for Bacterial Analysis of Ice Cream. The ice 
cream should be collected in a sterile vessel, e.g., a wide-mouthed sterile 
bottle with glass stopper-and packed in ice if it cannot be examined 
at once. 
To examine, melt the ice cream by placing for fifteen to twenty min- 
utes in the 22° C. incubator then treat as a milk sample. 
The degree of dilution and the methods of examination are similar 
to those used in the examination of milk. 
Determination of Streptococci in Ice Cream (Wiley et al.). The 
method for the detection of streptococci in ice cream has been used by 438 
MILK AND MILK PRODUCTS 
Wiley and his co-workers as follows: The melted sample should be 
centrifuged for half an hour in a Stewart lactocrite with a speed of 
approximately 3000 r.p.m. This apparatus, which consists of a flat 
aluminum pan holding twenty tubes of 1 c.c. capacity and stoppered 
at the outer end with a specially constructed rubber plug, causes the 
sediment not only to be thrown to the end of the tube but drives it 
against the rubber plug with such a force that it is almost quantitatively 
adherent to the plug. Accordingly, if one removes the rubber stopper 
and by rubbing on a glass slide and over an area of known surface 
attaches the sediment, one can obtain, on staining and examining the 
film microscopically, an approximation of the number of organisms and 
leucocytes in 1 c.c. of the liquid. Because of the debris in the ice 
cream, which ordinarily renders the usual method of centrifuging milk 
and cream samples quite impracticable, the above method was resorted 
to so far as the detection of the presence of streptococci was concerned, 
it was found eminently satisfactory. 
Davis (1914), in connection with investigations of septic sore throat 
decided that all of the streptococci concerned were of the hemolytic 
variety. These were found to remain alive in ice cream for eighteen 
days with little diminution in numbers. He concludes that ice cream 
is a suitable medium for the growth and dissemination of dangerous 
streptococci. 
The Food and Drugs Act of the United States Department of 
Agriculture attempts to promote the production of pure clean foods. 
With certain foods, as with ice cream, a satisfactory method of labora- 
tory control has not been worked out, and to this end more attention 
must be given to the conditions of manufacture. The Commission on 
Milk Standards has proposed the following score card by which the ice 
cream plants may be rated. It is then assumed that there is some direct 
relation to the quality of ice cream and the conditions under which it is 
produced. ICE CREAM 
439 
Perfect. 
Allow. 
Location 
15 
15 
30 
40 
Above ground 5 
Free from contaminating surroundings (no score if bad) ... 3 
Protected from street dust 3 
Not connected with any other room 2 
No other business in same establishment 2 
Construction 
Well lighted (natural) ,. 2 
Well ventilated 1 
Thoroughly screened 2 
Water-closet does not open directly into establishment. .. 2 
Separate room for washing utensils 2 
Floor: Smooth, water-tight, well drained 4 
Walls and ceiling: Smooth and tight 2 
Equipment 
Steam at all times 5 
Hot water at all times (no credit unless running hot water). 3 
Sterilizer for utensils 3 
Connections for sterilizing apparatus 2 
Pasteurizer: 
Holding machine 4 
Automatic recording device 1 
Refrigeration: Mechanical (proper icebox, 1) 2 
Freezer: Type, connections, etc 2 
Sanitary piping 2 
Wash basins and towels ample 1 
Utensils: 
Condition 1 
Ample for the service 2 
Racks for 1 
Employees: 
Health certificates for 1 
Clean suits provided 1 
Methods *. 
Freedom from flies 2 
Protection of material: 
Before manufacture 3 
During manufacture 3 
After manufacture 3 
Utensils and apparatus sterilized (washed in hot water, 1) 3 
Cleanliness: 
Floors 3 
Windows 1 
Apparatus / 3 
Walls and ceiling 1 
Utensils 3 
Employees 3 
Character of materials used: 
Milk and cream, Grade A (Grade B, 4; Grade C, 1). .. 6 
Condensed milk, eggs, etc 2 
Thickeners, none used 1 
Artificial coloring, none 1 
Degree of refrigeration of final product 2 
Total 
100 
SCORE CARD FOR ICE-CREAM MANUFACTURING PLANTS 440 
MILK AND MILK PRODUCTS 
BIBLIOGRAPHY. 
Abenhausen. 1900. Investigation on the Presence of Tubercle Bacilli in 
Marburg Butter and Margarine. Dissertation, Marburg, 1900. 
Allen, P. W. 1916. Comparison of the Rate of Multiplication of Bacteria 
in Raw Milk with the Rate in Pasteurized Milk. Jour, of Infectious Dis- 
eases, 19 (1916), 721-728. 
Allen, P. W. 1917. Comparisons of the Rate of Gas Production by Certain 
Bacteria in Raw and Pasteurized Milk. Jour. Infectious Diseases, 21 
(1917), 219-225. 
Andrews, F. W. 1913. The Cytology and Bacteriology of Condensed Milk. 
Jour. Path, and Bact., 18, 169-178. Exp. Sta. Rec., 1913. 
Anon. 1912. Tubercle Bacilli and Uncooked Food. Vt. Bd. Health Bull. 12, 
58-60. 
Ascher, N. 1899. Untersuchungen von Butter und Milch auf TuberkeL 
bacillen. Zeit. f. Hyg., 32, 329. 
Ayers, S. H. and Johnson, W. T. 1910. The Bacteriology of Commercially 
Pasteurized and Raw Market Milk. U. S. Dept, of Ag., Bureau of 
Animal Industry, Bull. 126. 
Ayers, S. H. and Johnson, W. T. 1913. A Study of the Bacteria which Sur- 
vive Pasteurization. U. S. Dept, of Ag., Bureau of Animal Industry, Bull. 
161. 
Ayers, S. H. and Johnson, W. T. 1915. A Bacteriological Study of Retail 
Ice Cream. U. S. Dept, of Agr. Bull. 303. 
Ayers, S. H. and Johnson, W. T. 1915. Ability of Colon Bacilli to Survive 
Pasteurization. Jour. Agr. Research, 3, 401-410. 
Ayers, S. H. and Johnson, W. T. 1917. The Determination of Bacteria in 
Ice Cream. U. S. Dept. Agr. Bull. 563. 
Ayers, S. H., Cook, L. B. and Clemmer, P. B. 1918. The Four Essential 
Factors in the Production of Milk of Low Bacterial Content. U. S. Depart- 
ment of Agriculture Bulletin 642. 
Babcock, S. M. and Russell, H. L. 1897. Unorganized Ferments of Milk; 
a New Factor in the Ripening of Cheese. Wise. Ag. Exp. Sta. 14th Annual 
Report, 1897, 161-193. 
Baer, A. C. 1916. Ice Cream Making. Agr. Exp. Station, Univ, of Wis. 
Bull. 262. 
Balazs, A. 1913. About the Detection of Boiled and Raw Milk. Abst. in 
Chern. Ztg., 37, 554. 
Bang, B. 1891. Experimentelle Untersuchungen liber Tuberculose Milch. 
Deut Zeit. f. Thiermed, 17, 1. 
Basenar, F. 1895. The Behavior of Cholera Bacilli in Raw Milk. Arch. 
Hyg., 23 (1895), 170-183. BIBLIOGRAPHY 
441 
Beattie, J. M. and Lewis, F. C. 1913. Acid Fast Bacilli in Milk. Jour. 
Path, and Bact., 18, 122-123, Abstract. 
Bergey, D. H. 1911. The Isolation of Bacillus Typhosus from Butter. Jour. 
Med. Res., 25 (1911-12), 231-33. 
Bolten, J. 1918. Effect of Freezing on the Organisms of Typhoid Fever and 
Diphtheria. Public Health Reports, 33, 163-166. 
Bosworth, A. W. 1913. The Action of Rennin on Casein. N. Y. Agr. Exp. 
Station Tech. Bull. 31. 
Boyce. 1900. Results Quoted from Aunett Lancet, 1900, p. 160. 
Boyd, M. F. 1917. Butter as a Vehicle of Infection in Typhoid. Jour. Amer. 
Med. Assn., 69, 2030-2032. 
Brainerd, W. K. Clean and Sanitary Milk. Virginia Ag. Exp. Sta. Bull. 185. 
Breed, R. S. 1911. The Determination of the Number of Bacteria in Milk 
by Direct Microscopical Examination. Cent. Bakt. Abt. II, 30, 337-340. 
Breed, R. S. 1913. The Significance of Body Cells in Milk. Proceedings, 
6th Ann. Convention International Milk Dealers' Assn., 1913, 25-32. 
Breed, R. S. 1914. Cells in Milk Derived from the Udders. N. Y. Agr. Exp. 
Station Bull. 380. 
Breed, R. S. and Brew, J. D. 1917. The Control of Bacteria in Market Milk 
by Direct Microscopic Examination. New York Agr. Exp. Station 
Bull. 443. 
Brew, J. D. 1915. Milk Quality as Determined by Present Dairy Score 
Cards. N. Y. Agr. Exp. Sta. Bull. 398. 
Brew, J. D. and Dotterrer, W. D. 1917. The Number of Bacteria in Milk. 
New York Agr. Exp. Station (Geneva) Bull. 439. 
Briscoe, C. F. and MacNeal, W. J. 1911. Tuberculosis of Farm Animals. 
Illinois Ag. Exp. Sta. Bull. 149, 317-431. 
Browne, Wm, W. 1915. A Comparison of the Acid Production of the Bacillus 
Coli Group Isolated from Different Sources. Am. J. Pub. Health, 5 (1915), 
39-48. 
Bruck, C. Experimentelle Beitrage zur Frage der Typhusverbreitung durch 
Butter. Cent. Bakt. Abt. I. Referate, 34, 778. 
Brussaferro. 1890. Experiments on the Inoculation of Commercial Butter. 
Gironali di Med. Veter. Prat. Torino. Cited by Reitz. 
Campbell, H. C. 1909. Tubercle Bacilli in Market Milk in Philadelphia. 
26th Ann. Rept. Bureau Animal Industry, 163-177. 
Campbell, H. C. 1916. Comparison of the Bacterial Count of Milk with 
the Sediment or Dirt Test. U. S. Department of Agriculture Bulletin 361. 
Capps, J. A. and Davis, D. J. 1914. The Relationship of Septic Sore Throat 
to Infected Milk. Jour. Inf. Dis., 15, 130-134. 
Colwell, R. C. 1917. The Influence of Gargety and High-count Cows on 
the Number of Bacteria in Milk. Abstracts Bact., 1, 48-9. 
Combs, W. B. and Eckles, C. H. 1917. The Relation of Oidium Lactis and 
Penicillium to the Keeping Qualities of Butter. Jour. Dairy Science, 1, 
347-355. 442 
MILK AND MILK PRODUCTS 
Commission on Milk Standards, Third Report of (1917), appointed by the 
New York Milk Committee. Reprint 386. Public Health Reports, 32, 
271-296. See also 27, No. 19, May 10, 1912. 
Committee Report. 1915. Standard Methods of Bacteriological Analysis of 
Milk. Am. J. Pub. Health, 5, 1315-25. 
Conn, H. W. 1915. Standards for Determining the Purity of Milk. Reprint 
No. 295. Public Health Reports, 30, No. 33. 
Conn, H. W. 1916. Reliability of the Bacteriological Analyses of Milk. Milk 
Dealer, 6, 50-45. 
Conn, H. W. and Esten, W. M. 1902. The Comparative Growth of Different 
Species of Bacteria in Normal Milk. Ann. Report Storrs Ag. Exp. Sta., 
14, 13-80. 
Conn, H. W. and Esten, W. M. 1904. The Effect of Different Temperatures 
in Determining the Species of Bacteria which Grow in Milk. Ann. Rept. 
Storrs Agr. Exp. Sta., 16, 27-88. 
Conn, H. W. and Stocking, W. A. 1903. Aseptic Milk. Ann. Rept. Storrs 
Ag. Exp. Sta., 15, 52-62. 
Conn, H. W. and Stocking, W. A. 1903. Strained and Unstrained Milk 
Preserved at 70° and 50° F. Ann. Rept. Storrs Ag. Exp. Sta., 15, 38-51. 
Coquot, A. 1908. The Passage of Tubercle Bacilli into Milk. Bull. Soc. 
Cent. Med. Vet., 85, 146-152. 
Cumming, J. G. 1917. An Epidemic Resulting from the Contamination of 
Ice Cream by a Typhoid Carrier. Jour. Am. Med. Assn., 68, 1163-1165. 
Dauson, C. F. 1898. Vitality and Retention of Virulence by Certain Path- 
ogenic Bacteria in Milk and its Products. U. S. Dept. Agr., Bureau Animal 
Industry. Ann. Rept., 1898, 224-228. 
Davis, D. J. 1914. The Growth and Viability of Streptococci of Bovine 
and Human Origin in Milk Products. Jour. Inf. Dis., 15, 378-388. 
Delepine, S. 1915. Effects of Preserving Processes upon the Virulence of 
Tubercle Bacilli in Milk. Reviewed in the American Jour. Pub. Health, 
5, 379-380. 
Doane, C. F. and Lawson, H. W. 1908. Varieties of Cheese. U. S. Dept, of 
Agr. Bureau of Animal Industry Bulletin 105. 
Du Claux, E. 1894. Le Lait, Paris. 
Dyer, D. C. 1916. Progressive Oxidation of Cold-storage Butter. Jour. 
Ag. Res., 6, 927-952. 
Eber, A. 1908. Investigations on the Tubercle Bacilli Content of Milk and 
Milk Products Sold in Liepsic. Zeit. Fleisch u. Milchhyg., 18, 309-319. 
Eber, A. 1912. Investigations on the Presence of Tubercle Bacilli in Milk 
and Milk Products. Zeit. Fleisch u. Milchhyg., 22, 243-349. 
Ernst, H. C. 1890. How Far May a Cow Become Tuberculous before her 
Milk Becomes Dangerous as an Article of Food. Amer. Jour. Med. Sciences, 
88, 439. 
Esten, W. M. 1909. Bacterium Lactis Acidi and its Sources. Storrs Ag. 
Exp. Sta. Bull. 59. BIBLIOGRAPHY 
443 
Esten, W. M. and Mason, C. J. 1907-9. Sources of Bacteria in Milk. Ann. 
Rept. Storrs Ag. Exp. Sta. 1907-9, 65-107. 
Evans, Alice C. 1916. Bacteria of Milk Freshly Drawn from Normal 
Udders. Jour. Inf. Dis., 18, 437-476. 
Evans, Alice Hastings, E. G. and Hart, E. B. 1914. Bacteria Concerned 
in the Production of the Characteristic Flavor in Cheese of the Cheddar 
Type. Jour. Agricultural Research, 2, 167-192. 
Ford, W. W. and Pryor, J. C. 1914. Observations upon the Bacteria found 
in Milk Heated to Various Temperatures. Bull. Johns Hopkins Hosp., 25, 
270-276. 
Fremy. 1839. Quoted from another author. Ann. de pharm., 31, 188. 
Freudenreich, E. 1897. Ueber die Erreger der Reifung der Emmenthaler 
Kase. Landw. Jahrb. Schweiz. Jahrg., 11, 85-101. 
Freudenreich, E. 1902. Milchsauerefermente und Kasereifung. Landw. 
Schweiz. Jahrb., 16, 91-104. 
Freudenreich, E. and Thoni, J. 1905. Sur Faction de differents lactiques 
sur la maturation du fromage. Rev. Generale du Lait., 4, 169-181; 200- 
209; 225-232; 247-259. Also see Vol. 2, 241-247; 271-280. 
Frost, W. D. 1915. A Microscopic Test for Pasteurized Milk. Jour. Amer. 
Med. Assn., 64, 821-822. 
Frost, W. D. 1916. Confirmation Tests for B. Coli in Routine Water Exam- 
ination. Am. Jour. Pub. Health, 6, 1916, 585. 
Frost, W. D. 1916. Comparison of a Rapid Method of Counting Bacteria 
in Milk with the Standard Plate Method. Journal Infect. Dis., 19, 273-287. 
Frost, W. H. 1916. Relationship of Milk Supplies to Typhoid Fever. Public 
Health Reports, 31, 3291-3302. 
Frost, W. D. 1917. Counting the Living Bacteria in Milk-A Practical Test. 
Jour, of Bact., 2, 567-583. 
Frost, W. D. and Ravenel, M. 1911. A Microscopic Test for Heated Milk. 
Proc. Am. Assn. Med. Milk Commissions, 1911, p. 127. 
Gage, S. M. and Stoughton, Grace V. 1906. A Study of the Laws Governing 
the Resistance of Bacillus Coli to Heat. Technology Quarterly, 19, 41-45. 
Galtier, B. 1887. Dangers de Futilization des produites tel que le petit- 
lait et le fromage, oblenus avec le lait de vaches tuberculeuses. Comp. 
Rend. Acad. Sci., 104, 1333-1335. 
Gebhardt, F. 1890. Experimentelle Untersuchungen uber den Einfluss der 
Verdiinnung auf die Wirksamkeit des Tuberkelosen Giftes. Virch. Arch., 
109, 127. 
Goodrich, G. W. 1914. Comparison of the Plating and Microscopic Methods 
in the Bacteriological Examination of Milk. Jour. Inf. Dis., 14, 512-519. 
Gorini, C. 1904. Sur la presence de bacteries productrices d'acidite et de 
presure dans Ies fromages en maturation. Rev. Generate du Lait, 3, 505- 
510. 
Grassburger, R. 1899. The Intraperitoneal Injection of Market Butter 
and the Resulting Changes. Miinchener Med. Wochenschr., 46, 341-344. 444 
MILK AND MILK PRODUCTS 
Groning. Cited in Kolle und Wassermann. Handbuch der Pathogenen 
Mikroorganisms, 2, 140. 
Guthrie, E. S. 1917. Concerning Rancidity of Butter. Jour. Dairy Science, 
1, 218-233. 
Hammar, B. W. 1915. Bacteriological Studies on the Coagulation of Con- 
densed Milk. Iowa Ag. Exp. Sta. Res. Bull. 19. 
Hammar, B. W. 1912. Bacteria and Ice Cream. Iowa Agr. Exp. Sta. Ames. 
Bull. 134. 
Hammar, B. W. and Goss, E. F. 1917. Bacteria in Ice Cream. II. Iowa 
Agric. Exp. Sta. Bull. 174. 
Hammarsten. Quoted from another author. Maly's Jahresbericht, 1872, 
p. 118; 1874, p. 135; 1877, p. 158. 
Harding, H. A. 1916. Present Status of the Score Card of Milk Quality. 
Amer. J. Pub. Health, 6 (1916), 562. 
Harding, H. A. 1917. Bacterial Count as an Index of Cleanliness in Milk. 
Abstracts of Bacteriology, 1 (1917), 53-54. 
Harding, H. A. and Prucha, M. J. 1908. The Bacterial Flora of Cheddar 
Cheese. New York Agr. Exp. Station Tech. Bull. 8. 
Harding, H. A., Breed, R. S., Stocking, Jr., W. A. and Hastings, E. E. 1917. 
What is Meant by Milk Quality. University of Illinois Agr. Exp. Sta. 
Circular No. 205. 
Harding, H. A., Ruehle, G. L., Wilson, J. K. and Smith, G. A. 1913. The 
Effect of Certain Dairy Operations upon the Germ Content of Milk. 
N. Y. (Geneva) Agr. Exp. Sta. Bull. 365. (Contains a number of refer- 
ences on this subject.) 
Harmann and Morgauroth. 1898. Weitere Mittheilungen fiber Tuber- 
kelbacillen-Befunde in Butter und Kase. Hyg. Rund., .8, 1081-1084. 
Harrison, F. C. 1896. Bacterial Contamination of Milk. Ann. Rept. 
Ontario Ag. Coll., 22, 105-114. 
Harrison, F. C. 1902. The Duration of the Life of the Tubercle Bacillus in 
Cheese. U.S.Dept. Agr., Bureau Animal Industry 19th Ann Rept., 217-233. 
Hart, E. B., Hastings, E. G., Flint, E. M. and Evans, Alice C. 1914. 
Relation of the Action of Certain Bacteria to the Ripening of Cheese of 
the Cheddar Type. Jour. Agr. Res., 2, 193-216. 
Hastings, E. G. 1916. Cause and Prevention of Mold on Butter. Proc. 
Wis. Buttermakers' Assn., 16, 145-152. 
Hastings, E. G., Evans, A. C. and Hart, E. B. 1912. Studies on the Factors 
Concerned in the Ripening of Cheddar Cheese. Wisconsin Agr. Exp. Sta- 
tion. Research Bulletin 25. 
Hastings, E. G., Evans, A. C. and Hart, E. B. 1912. The Bacteriology of 
Cheddar Cheese. U. S. Dept, of Agriculture. Bureau of Animal Industry 
Bull. 150. 
Heim. 1889. Ueber das Verhalten der Krankheitserreger der Cholera, des 
Unterleibtyphus und der Tuberculose in Milch, Butter, Molken, und Kase. 
Arb. a. d. kaiserl. Gesundh., 5, 294-311. BIBLIOGRAPHY 
445 
Heinemann, P. G. and Gordan, J. E. 1917. Creamery and Milk Plant, 5 
44-52. 
Hellstrom, F. E. 1900. The Estimation of Tubercle Bacilli in Butter and a 
Few Investigations on Pathogenic Bacteria in Butter from Pasteurized 
and Unpasteurized Cream. Cent. Bakt. Abt. I. Ref., 28, 542-555. 
Henike, G. W. and Thompson, I. F. 1917. Septic Sore Throat: Epidem- 
iological study of a milk-borne epidemic at Galesville, Wis., due to the use 
of infected milk. Jour. Am. Med. Assn., 68, 1307. 
Herbert. Investigation on the Presence of Tubercle Bacilli in Butter. Cited 
by Reitz, A. (1906). 
Herr, F. and Beninde, M. 1901. Investigation on the Presence of Tubercle 
Bacilli in Butter. Zeit. Hyg., 38, 152-181. 
Hess, A. H. 1908. The Incidence of Tubercle Bacilli in New York City Milk. 
Jour. Am. Med. Assn., 52, 1011-1016. 
Hesse, W. 1894. Die Beziehung zwischen Kuhmilch und Cholera Baketerien. 
Zeit. f. Hyg., 17, 238. 
Hessler, G. 1909. Ein Beitrag zur Frage der Infektiositat der Milch tuber- 
kuldser Kiihe. Inaug. Dissert. Univ. Bern., 1909. 
Hill, H. W. 1911. Typhoid Fever from Butter. Third Biennial Rept. 
Minn. State Bd. Health, p. 203. 
Hirschberger, K. 1889. Experimentelle Beitrage zur Infektiositat der 
Milch tuberculdser Kiihe. Deut. Arch. Klin. Med., 44, 400. 
Horman and Morgenroth. 1898. The Presence of Bacteria in Butter. 
Hygienische Rundschau, 8, 217-230. 
Horman and Morgenroth. 1898. Further Contribution on the Presence of 
Tubercle Bacilli in Butter and Cheese. Hyg. Rund., 8, 1081-1084. 
Ice Cream, Report of Hearing on, before Dr. C. A. Alsberg, Chief of the Bureau 
of Chemistry, U. S. Dept, of Ag., February 10, 1914 and March 7, 1914. 
Subject: The Use of Colloids as Stabilizers in Ice Cream, the Butter Fat 
Standards for Ice Cream and the Bacteriology of Ice Cream, with Special 
Reference to the Cincinnati Ice Cream Cases. Published by the National 
Association of Ice Cream Manufacturers, June, 1914. 
Jensen, Orla. 1916. The Use of Amyl Alcohol in Gerber's Method for the 
Determination of Fat in Milk. Maelkeritid, 29, 104-106. 
Jordan, E. O. 1917. The Bacterial Examination of Foods. American Food 
Journal, 12, 251-252. Jour. Am. Med. Assn., 68, April 14, 1917. 
Joshi, L. L. 1913. Observations on the Bacteriological and Chemical Exam- 
ination of the Milk Supply of Bombay. (Calcutta, 1913, 20 pp.) 
Joshi, L. L. 1915. Bacteriological Examination of the Bombay Milk Supply. 
Jour. Dairying (India), 3, 5-36. 
Kinyoun, J. J. 1914. A Bacteriological Index for Dirt in Milk. U. S. Naval 
Medical Bulletin 8, 435-442. 
Klein, A. 1900. Zur Kenntniss der Verbreitung des Bacillus Tuberculosis 
und Pseudotuberculosis in der Milch Sowie der Biologie des Bacillus Tuber- 
culosis. Cent. Bakt. Abt. I, 28, 111. 446 
MILK AND MILK PRODUCTS 
Korn, O. 1899. Tubercle Bacilli in Butter. Arch. Hyg., 36, 57-65. 
Kossowicz, A. 1908. Bacterial Content of Condensed Milk. Z. landw. 
Vers. Wes. Oesterr., 11, 719-24. 
Krumwiede, C. and Noble, W. C. 1915. The Viability of the Typhoid 
Bacillus in Sour Cream. Collected Studies from the Bureau of Labora- 
tories. City of New York, Vol. 8, 311. 
Krumwiede, C. and Valentine, E. 1915. A Bacteriological Study of an 
Epidemic of Septic Sore Throat. Jour. Med. Res., 33, 231-238. 
Lapierre, C. 1894. Chimie animale-analyse d'un fromage avarie extrac- 
tion d'une ptomaine nouvelle. Comp. Rend. Acad. Sci., 118, 476-482. 
Laser, H. The Behavior of Bacteria of Typhoid Fever, Tuberculosis and 
Cholera in Butter. Zeit. Hyg., 10, 513-520. 
Levin, W. 1917. Cheese Poisoning. A toxicogenic bacillus isolated from 
cheese. Jour. Lab. Clinical Medecin, 2, 3-26. 
Lumsden, L. L. 1917. The Determination of Ice Cream as a Factor in the 
Spread of Typhoid Fever Infection. Am. Jour. Pub. Health, 7, 1005-1009. 
Luxwolda, W. B. 1911. The Growth of Some Milk Bacteria at Different 
Temperatures. Cent. Bakt. Abt. II, 31, 129-175. 
Marchisotti, A. C. 1917. The Presence of the Tubercle Bacillus in Butter. 
Bev. Facult. Agron, y. Vet. La Plata, 2, Series 2, 12. 279-298. 
Markl. 1901. On the Presence of Tubercle Bacilli in Vienna Market Butter 
and Margarine. Cent. Bakt. Abt. I. Ref., 29, 954. 
Marshall, W. E. 1907. Note on the Occurrence of Diphtheria Bacilli in 
Milk. Jour. Hyg., 7, 32-34. 
Mason, C. J. 1915. The Bacteria in Ice Cream. Storrs (Conn.) Agr. Exp. 
Sta. Bull. 83. 
Melvin, A. D. 1912. The Score-card System of Dairy Inspection. U. S. 
Dept. Agr. Bur. of Anim. Ind. Cir. 199. 
Mohler, J. R.. Washburn, H. J. and Doane, C. F. 1909. The Viability of 
Tubercle Bacilli in Cheese. 26th Ann. Rent. Bureau Animal Industry, 
187-191. 
Mohler, J. R., Washburn, H. J. and Rogers, L. A. 1909. The Viability 
of Tubercle Bacilli in Butter. U. S. Department of Agriculture, Bureau of 
Animal Industry, Annual Report for 1909, 179-185. 
Morganroth. 1899. On the Presence of Tubercle Bacilli in Margarine. 
Hyg. Rundschau, 9, 1121. 
North. 1913. Discussion following Article by Pease. (1913). 
North, C. E. 1916. Bacterial Testing versus Dairy Inspection. Am. J. 
Pub. Health, 6 (1916), 569. 
North, C. E. 1917. A Survey of Dairy Score Cards. Amer. Jour. Pub. 
Health, 7, 25-39. 
Northrup, Z. 1911. The Influence of the Products of Lactic Organisms upon 
Bacillus Typhosus. Mich. Ag. Exp. Sta. Tech. Bull. 9. 
Obermuller. 1899. Tubercle Bacilli in Market Butter. Hyg. Rund., 9, 
57-79; 7, 712-714, 1897; 5, 877, 1895. BIBLIOGRAPHY 
447 
Overton, F. 1914. An Epidemic of Septic Sore Throat at Easthampton 
(N. Y.) and its Control. Health News, N. Y. State Department of Health, 
9, 379-371. 
Overton, F., Krumwiede, C. and Jaques, A. D. 1914. An Epidemic of 
Septic Sore Throat in Rockville Center, N. Y. Health News, N. Y. State 
Department of Health, 9, 230-233. 
Park, W. H., et al. 1915. A Sanitary Study of Condensed Milk. N. Y. 
Med. Jour., 102, 1073-1083. 
Park, W. H. 1918. " What Constitutes Good Milk." Jour. Am. Med. 
Assn., 70, 720. 
Park and Williams. 1917. Pathogenic Microorganisms. Lea & Febiger. 
Parker, H. N. 1917. City Milk Supplies. McGraw-Hill Book Co., New 
York City. 
Pawlowsky. 1900. The Presence of Tubercle Bacilli in Market Butter. 
Comp. Rend. 10 Intern. Cong. Hyg. et Demog., 187-189. 
Pease, H. D. 1913. The Sanitary Significance of Bacteria in Milk. 6th 
Ann. Convention, International Milk Dealers' Association Proceedings, 
1913, 105-116. 
Pease, H. D. 1916. Some Observations on Causes of High Bacterial Counts 
in Market Milk. Amer. Jour. Pub. Health, 6, 563-568. 
Peiser, L. 1916. Bacteriology of Cream Ripening. Milk Dealer, 5, 26-27. 
Pennington, M. E. et al. Bacterial and Enzyme Changes in Milk and Cream 
at 0° C. Jour. Biol. Chern., 16, 331-368. 
Pennington, M. E. 1908. Bacterial Growth and Chemical Changes in 
Milk Kept at Low Temperature. Jour. Biol. Chern., 4, 353. 
Petri, R. F. 1898. Zum Nachweis der Tuberkelbacillen im Butter und Milch. 
Arb. Gesundsheitsamt, 14, 1. 
Prescott, S. C. and Breed, R. S. 1911. The Determination of the Number 
of Body Cells in Milk bya Direct Method. Jour. Inf. Dis., 7, 632-40. 
Prucha, M. J., Harding, H. A. and Weeter, H. M. 1915. Utensils as a 
Source of Bacterial Contamination of Milk. Science, 42, 353. 
Prucha, M. J., Weeter, H. M. and Chambers, W. H. 1918. Germ Content 
of Milk. II. As influenced by the utensils. Univ, of Ill. Ag. Exp. 
Sta. Bull. 204. 
Prucha, M. J., Weeter, H. M. and Chambers, W. H. 1918. Germ Life in 
Cow Feces. Abstracts of Bacteriology, 2, 6. 
Rabinowitch, L. 1897. On the Question of Tubercle Bacilli in Market 
Butter. Zeit. Hyg., 26, 90. Exp. Sta. Record, 9, 689. 
Rabinowitch, L. 1899. Tubercle Bacilli in Butter. Deut. Med. Wochen- 
schr., 25, 5. 
Rabinowitsch, M. 1907. Verbreitung der Tuberkelbacillen durch Milch 
und Fleisch. In. G. Cornet's Die Tuberkulose. 2d Edit. Pt. I, 123 
Vienna, 1907. 
Ravenel, M. P., Hastings, E. G. and Hammar, W. B. 1910. The Bacterial 
Flora of Milk held at Low Temperatures. Jour. Inf. Dis., 7, 38-46. 448 
MILK AND MILK PRODUCTS 
Reed, H. S. 1914. Manual of Bacteriology. Ginn & Co. 
Reed, H. S. and Reynolds, R. R. 1916. Some Effects of Temperature upon 
the Growth and Activity of Bacteria in Milk. Virginia Ag. Exp. Sta. 
Tech. Bull. 10. 
Reitz, A. 1906. Bacteriological Examination of Butter. Cent. Bakt. Abt 
IL, 16, 193-212. 
Rogers, L. A. 1916. Fermented Milks. U. S. Dept. Ag. Bull. 319. 
Rogers, L. A., Clark, W. M. and Evans, A. 1914. The Characteristics of 
Bacteria of the Colon Type found in Bovine Feces. Jour. Inf. Dis., 15, 
100-123. 
Rogers, L. A. and Dahlberg, A. 0. 1914. The Origin of Some Streptococci 
Found in Milk. Jour. Agr. Res., 1, 491. 
Rogers, Clark and Evans. Colon Bacteria and Streptococci and their 
Significance in Milk. Am. J. Pub. Health, 6 (1916), 381. 
Rosenar, M. J. 1912. The Number of Bacteria in Milk and the Value of 
Bacterial Counts. In milk and its relation to the public health. Hyg. 
Lab. Bull. 56, 427-453. 
Rosenar, M. J., Frost, W. D. and Bryant, R. 1914. A Study of Market 
Butter of Boston. Jour. Med. Res., 30, 69. 
Rosenow, E. C. and Hess, C. L. 1917. An Epidemic of Septic Sore Throat 
Due to Milk. Jour. Am. Med. Assn., 68, 1305. 
Rosenow, E. C. and Moon, V. H. 1915. On an Epidemic of Sore Throat 
and the Virulence of Streptococci Isolated from the Milk. Jour. Inf. Dis., 
17, 69-71. 
Rowland, S. D. 1895. Cheese and Butter as Possible Carriers of Typhoid 
and Cholera. Brit. Med. Jour., 1895, No. 1799. 
Ruehle, G. L. A. and Kulp, W. L. 1915. Germ Content of Stable Air and 
its Effect upon the Germ Content of Milk. N. Y. (Geneva) Agr. Exp. 
Sta. Bull. 409. 
Russell, H. L. 1893-4. The Source of Bacterial Infection and the Relation 
of the Same to the Keeping Quality of Milk. Ann. Rept. Wis. Ag. Exp. 
Sta., 11, 150-165. 
Russell, H. L. and Hastings, E. G. 1902. A Micrococcus, the Thermal 
Death Point of which is 76° C. Cent. f. Bakt. Abt. IL, 8, 339-342. 
Russell, H. L. and Hastings, E. G. 1904. Effect of Short Periods of Ex- 
posure to Heat on Tubercle Bacilli in Milk. 21st Ann. Rept. Wis. Ag. 
Exp. Sta., 178-192. 
Savage, W. G. 1906. Streptococci and Leucocytes in Milk. Jour. Hygiene, 
6, 123-138. 
Savage, W. G. 1909-10. Report upon the Bacterial Measurement of Milk 
Pollution. Ann. Rept. Local Govt. Bd. (Gt. Brit.), 39, 474-503. 
Savage, W. G. 1914. The Bacteriological Examination of Food and Water. 
Cambridge Univ. Press, p. 112. 
Schrieder, M. C. 1914. Dirt Sediment Testing-a Factor in Obtaining 
Clean Milk. Amer. Jour. Pub. Health, 4, 50-64. BIBLIOGRAPHY 
449 
Schroeder, E. C. 1907. Milk and its Products as Carriers of Tuberculous 
Infection. 24th Ann. Rept. Bureau of Animal Industry, 183-199. (Good 
Bibliography appended.) 
Schroeder, E. C. and Brett, G. W. 1918. Public Health Studies Con- 
cerning Cheese. Jour. Am. Vet. Assn., 52, 674-685. 
Schroeder, E. C. and Cotton, W. E. 1908. Tubercle Bacilli in Butter. 
Their Occurrence, Vitality and Significance. U. S. Dept. Agric., Bureau 
of Animal Industry Circular 127. 
Schuchardt. 1896. An Investigation of the Presence of Tubercle Bacilli 
in Butter. Dissertation, Marburg. 
Sherman, J. M. 1915. Studies on the Production of Sanitary Milk. Penn. 
Ag. Exp. Sta. Rept., 1915, 299-305. 
Sherman, J. M. and Reynolds, F. P. 1915. Bacteriological Methods for 
Determining the Quality of Milk. I. The reaction of agar for the bac- 
terial analysis of milk. Penn. Ag. Exp. Sta., 1915, 294-299. 
Sherman, J. M. 1916. The Advantages of a Carbohydrate Medium in the 
Routine Bacterial Examination of Milk. Jour. Bact. I., 481-492. 
Slack, F. H. 1917. Some Observations on the Bacterial Examination of 
Milk. Amer. Jour. Pub. Health. 7, 690-697. 
Smillie, W. G. 1917. Studies of the Beta Hemolytic Streptococcus (Smith 
and Brown). Jour. Inf. Dis., 20, 45-48. 
Smit, H. J. 1908. Ueber das Vorkommen von Tuberkelbacillen in der Milch 
und den Lymphdriisen des Rindes. Inaug. Diss. Univ. Bern., 1908. 
Smith, Th. 1899. The Thermal Death Point of Tubercle Bacilli in Milk and 
Some Other Fluids. Jour. Med. Res., 4, 233. 
. 1916. Standard Methods of Bacteriological Milk Analysis. 
Provisional report of the laboratory section of the American Public Health 
Association. 
Stein, G. 1884. Experimentelle Beitrage zur Infektion der Milch Perl- 
siichtiger Kiihe. Inaug. Dissert., Berlin. 
Stocking, W. A. 1906. Quality of Milk Affected by Common Dairy Prac- 
tices. Storrs. Ag. Exp. Sta. Bull. 42. 
Stokes, W. R. and Wegeforth, A. The Microscopic Examination of Milk. 
Med. News, 71 (1897), 15-48. 
Suzuki, S. K., Hastings, E. G. and Hart, E. B. 1910. The Production of Vola- 
tile Acids and Esters in Cheddar Cheese and their Relation to the Develop- 
ment of the Flavor. Wisconsin Agr. Exp. Station Research Bull. 11. 
Sweet, E. A. 1917. Safe Milk, an Important Food Problem. Public Health 
Reports Supp. No. 31. 
Taylor, G. B. 1917. Experiments on Determinations of Cow Manure in 
Milk; Moisture Content and Solubility of Cow Manure. Journal of 
Dairy Science, 1, 303-312. 
Teichert. 1904. Bacteriological and Chemical Studies of the Butter in the 
Province of Posen with Special Reference to the Tubercle Bacillus. Cent. 
Bakt. Abt. II., 13, 560-561. 450 
MILK AND MILK PRODUCTS 
Thayer, L. A. 1912. A Bacterial Infection of Condensed Milk. Vermont 
Agricultural Experiment Station Bull. 170, 123-124. 
Thom, J. 1914. On the Hygienic Bacteriological Character of Bern Market 
Milk with Reference to the Presence of Tubercle Bacilli. Cent. Bakt. 
Orig. Abt. I., 74, 11-69. 
Thue, H. 1905. Investigations on the Presence of Tubercle Bacilli in Milk, 
Butter and Margarine in Christiania. Cent. Bakt. Abt. I., Ref., 36. 597. 
Tobler, M. 1901. Contribution on the Question of the Presence of Tubercle 
Bacilli and other Acid-fast Bacteria in Market Butter. Zeit. f. Hyg., 36, 
120-148. 
Trask, J. W. 1912. Milk as a Cause of Epidemics of Typhoid Fever. Scarlet 
Fever and Diphtheria. In milk and its relation to the public health. Bull. 
56. Hyg. Lab. U. S. Public Health Service. 
Van Slyke, L. L. and Bosworth, A. W. 1914. Studies Relating to the 
Chemistry of Milk and Casein. N. Y. Ag. Exp. Sta. Tech. Bull. 37. 
Van Slyke, L. L. and Bosworth, A. W. 1912. Composition and Properties 
of Some Casein and Paracasein Compounds and their Relations to Cheese. 
New York Agricultural Experiment Station, Tech. Bull. 26. 
Van Slyke, L. L. and Bosworth, A. W. 1916. Chemical Changes in the 
Souring of Milk. N. Y. Agr. Exp. Sta. Tech. Bull. 48. 
Vaughn, V. C. 1886. A Ptomaine from Poisonous Cheese. Zeit. Physiol. 
Chern., 10, 146. 
Washburn, H. J. 1908. The Viability of Typhoid Bacilli in Milk and Butter. 
25th Ann. Rept. Bureau Animal Industry, 297-300. 
Washburn, R. M. 1910. Principles and Practice of Ice Cream Making. 
Vermont Agr. Exp. Sta. Bull. 155. 
Washburn, R. M. and Dahlberg, A. C. 1917. The Influence of Salt on the 
Changes Taking Place in Butter. Journal of Dairy Science, 1, 114-126. 
Waterman, E. L. 1917. Typhoid Fever from Ice Cream. Public Health 
Bulletin of the Michigan State Board of Health, 5, 157-159. 
Weigmann, H., Wolff, A., Treusch, Marg., Steffen, M. 1916. The 
Behavior of Streptococcus Lacticus on Heating Milk at 60° to 73° C. 
(modern pasteurization). Cent. f. Bakt. Abt. IL, 45, 63-107. 
Weinzire. A Bacteriological Method for Determining Manural Pollution of 
Milk. Am. J. Pub. Health, 5 (1915), 862-66. 
Weissenfeld. 1899. Bacteria in Butter and Other Milk Products. Berl, 
klin. Wchschr., 36, 1053-1055. 
Wiley, H. W. 1912. Ice Cream. In milk and its relation to the public 
health, pp. 249-312. 
Winslow, C. E. A. and Hubbard, L. W. 1915. A Contact Outbreak of 
Septic Sore Throat in Westchester County (N. Y.). Health News, N. Y. 
State Dept. Health, 9, 266-270. CHAPTER XII 
EXAMINATION OF EGGS 
Eggs have always been an important factor in the diet. As the 
density of population increased, especially in urban centers, the problem 
of securing fresh eggs became more and more acute. This was ren- 
dered more intricate by the fact that the greatest part of the annual lay 
is secured during the months of March, April, May and June. This 
makes it necessary to hold over, either by cold storage or preservation, 
the abundance of these months against the want of the others. It has 
become of much importance to determine what factors are important 
in the keeping of this food product. 
A knowledge of the physiology of the hen which is concerned with the 
development of the egg is important in this connection. This has been 
well described by Benjamin (1915) from whose publication the following 
has been adapted. The oviduct of the hen is located in the rear part 
of the body cavity. The yolk is the first part to develop and this 
takes place in the ovary which may contain a large number of minute 
yolks. Each is contained in a sac, or follicle through which it secures 
its nourishment while developing. After development the yolk en- 
closed in its vitellin membrane escapes from the yolk sac find descends 
through the oviduct. If fertilization occurs, it takes place soon after 
the egg has entered the oviduct and before any albumen is deposited 
about it. About 40 per cent of the albumen is said to be laid down as 
the yolk passes through**the upper half of the oviduct. After passage 
through this albumen-forming region, it reaches the isthmus where the 
shell membranes are added with about 10 to 20 per cent more albumen. 
The uterus is then reached, where the remainder of the shell is added. 
During its passage through the vagina, just before its expulsion from 
the cloaca, it probably receives the outer gelatinous coating on the shell. 
The structure of the egg and shell is given in Figs. 75 and 76. 
The chemical composition of any food substance is important in 
determining the type of composition which may take place. This is 
especially true with a substance like eggs. The analysis quoted in 
Table XLVIII is taken from the paper of Benjamin (1915). 
451 452 
EXAMINATION OF EGGS 
Mar hi aa Duv<i del. 
1, ovary, with minute ovules; .2-3, yolk sacs; 4, suture line; 5, empty yolk sac; 7, funnel 
opening into oviduct; 8, yolk in oviduct; 9, albumen-secreting region; 10, albumen being secreted; 
11, yolk passing through oviduct; 12, germinal disk; 13, isthmus; 14, uterus; 15, large intestines; 
17, cloaca. 
On the right-hand side of the figure are shown, from the top downwrd: Complete egg; yolk 
of egg incubated for sixteen hours; completed egg in uterus-(1) isthmus, (2) glands of uterus, 
(3) complete egg, (4) vagina, (8) cloaca. STRUCTURE OF EGGS 
453 
Fig. 75.-Structure of an Egg. (After Benjamin, 1914.) 
A, outer shell; B, outer shell membrane; C, inner shell membrane; D, air cell; E, outer 
thin portion of albumen; F,G, inner dense portion of albumen; H, vitelline membrane; I, chalaza; 
J, thin film of white yolk inside of vitelline membrane: K, layers of yellow yolk separated by thin 
layers of white yolk; L, germinal disk; M, central part of yolk filled with white yolk; N, slender 
tube connecting center of yolk with region of germinal disk. 
A 
P 
Fig. 76.-Shell Membranes of an Egg Highly Magnified. (After Benjamin, 1914.) 
A, inner shell membrane. Note the fine cellular structure. The cells are bound together by 
many intertwining fibers. B, outer shell membrane. This is much coarser in structure than the 
inner membrane. The fibers are very transparent. 
Table XLVIII 
COMPOSITION OF HENS' EGGS 
Yolk, 
Percentage. 
White of Egg, 
Percentage. 
Whole Egg, 
Percentage. 
Water 
46-52 
80-88 
70-76 
Fat 
30-35 
Traces 
9-14 
Protein 
14-16 
10-13 
10-15 
Shell and shell membranes 
9-12 454 
EXAMINATION OF EGGS 
From Table XLVH1 it is seen that the whole egg usually contains 
over 10 per cent of fat. Nearly all of this fat is in the yolk, and obvi- 
ously the fowl must have a surplus of fat before the yolks can begin 
to develop. The eggs of ducks, geese, turkeys, guinea fowls, and other 
birds vary to some extent from the analysis noted above, but the dif- 
ferences are slight. 
The detailed analysis of hens' eggs which follows is taken from Simon's 
textbook of physiological chemistry: 
Analysis of shell (9-11 per cent of whole egg). 
Percentage. 
Calcium carbonate 90.00 
Magnesium carbonate Small amount 
Calcium and magnesium phosphate Small amounts 
Water 1.00 
Analysis of albumen (60.5 per cent of whole egg). 
Percentage. 
Water 80.00-86.68 
Solids 13.32-20.00 
Albumins 11.50-12.27 
Extractives 0.38-0.77 
Glucose 0.10-0.50 
Fats and soaps Traces 
Mineral salts 0.30-0.66 
Lecithins and cholesterin Traces 
The mineral ash of the albumen has been found by Poleck and 
Weber to consist of: 
Percentage. 
Sodium (Na20) 32.56-32.93 
Potassium (K2O) 27.66-28.45 
Calcium (CaO) 1.74-2.290 
Magnesium (MgO) • 1.60- 3.17 
Iron (Fe2O3) 0.44-0.55 
Chlorine (Cl) 23 84-28.56 
Phosphoric acid (P2O2) 3.16- 4.83 
Carbonic acid (CO2) 9.67-11.60 
Sulphuric acid (SO3) 1.32- 2.63 
Silicic acid (SiO2) 0.28- 0.49 
Fluorine (Fl) Traces 
Analysis of yolk (29 per cent of whole egg). GRADING EGGS 
455 
According to Gautier we have the following: 
Percentage. 
Water ' 47.19-51.49 
Solids 48.51-42.81 
Fats (olein, palmitin, and stearin) 21.30-22.84 
Vitellin and other albumins 15.63-15.76 
Lecithins 8.43-10.72 
Cholesterin 0.44- 1.75 
Cerebrin 0.30 
Mineral salts 3.33- 0.36 
Coloring matter 
Glucose 
0.553 
Poleck and Weber also give the following as the analysis of the min- 
eral salts of the yolk: 
Percentage. 
Sodium (Na2O) 5.12-6.57 
Potassium (K2O) 8.05- 8.93 
Calcium (CaO) 12.21-13.28 
Magnesium (MgO) 2.07- 2.11 
Iron (Fe2O3) 119- 1.45 
Phosphoric acid, free (P2O3) 5.72 
Phosphoric acid, combined 63.81-66.70 
Silicic acid (SiO2) ; 0.55- 1.40 
Chlorine Traces 
According to Simon, the shell is made up of an organic matrix of the 
nature of keratin. This matrix is largely impregnated with lime salts. 
The pigments of the shell are said to be derived from the common 
pigment of blood. The shell membranes are also composed largely of 
keratin, with a small amount of mineral salts, principally calcium phos- 
phate. The white of the egg is supposed to consist of compartments 
which are divided by thin membranes and which contain the liquid 
albumen. These membranes are continuous with the chalazae and the 
shell membranes. The yolk consists largely of spherical cells, most of 
which are filled with fat. . 
The bacteriology of eggs has received considerable attention and 
much interesting and useful data are available. As far as is known 
eggs have not been important in spreading pathogenic bacteria. 
Grading of Eggs. This is essentially a commercial problem. Under 
some conditions, the grading of eggs is very difficult. However, in 
practice they are graded by the candling process and observations with 456 
EXAMINATION OF EGGS 
regard to their internal condition and appearance, are made. Frazier 
(1916) and Stiles and Bates (1912) describe the different grades of 
eggs. 
Firsts and Extras. This class includes all freshly laid, sound, whole, 
clean shell, medium and good-sized eggs. The term " fresh " is used to 
indicate an egg with practically no age. Before the candle such an egg 
is characterized by a very small air cell at the large end, absence of 
defects, transparence and a slow-moving yolk. They appear perfectly 
homogeneous. Some fresh eggs are laid with defects as " liver spots " 
" meat spots," foreign material, etc. The appearance of the opened 
egg gives much information to the microbiologist. The yolk should 
stand up with a definite shape on account of the strong vitelline 
membrane about it. The albumen should be transparent and firm. 
The contents should have no odor and should be practically free from 
a large number of bacteria. 
Table XLIX 
COMMERCIAL SAMPLES OF LEAKING EGGS BROKEN IN THE 
CANDLING ROOM (Pennington) 
Sample 
No. 
Source. 
Date 
of Col- 
lection. 
Bacteria per 
Gram on Plain 
Agar Incubated. 
Gas- 
produc- 
ing bac- 
teria 
per 
Gram 
in Lac- 
tose 
Bile. 
Liquefy- 
ing or- 
ganism 
per 
Gram. 
Ammonial 
Nitrogen 
(Folin). 
Mois- 
ture. 
Wght. 
of 
Sam- 
ples, 
Lbs. 
20° C. 
37° C. 
Wet 
Basis. 
Dry 
Basis. 
4214 
F-l 
May 1 
4,300,000 
2,100,000 
10,000 
1,400,000 
.0017 
.0054 
69.74 
420 
4224 
F-l 
May 2 
3,800,000 
1,800,000 
100,000 
430,000 
.0016 
.0054 
70.35 
4243 
F-l 
May 3 
1,600,000 
950,000 
10,000 
650,000 
.0017 
.0062 
72.55 
4370 
F-l 
May 23 
25,000,000 
6,300,000 
100,000 
12,000,000 
.0020 
.0067 
70.09 
150 
Seconds. In this grade are classed all eggs not included under the 
first, except the third grade mentioned below. Stiles and Bates report 
the following divisions: 
(a) Undersized. If not for their small size these eggs would be 
classed as firsts. They conform to the standards mentioned above for 
a first-grade product. 
(6) Checks and Cracks. Eggs, the shells of which have become 
broken by careless handling but which have intact shell membranes. GRADING EGGS 
457 
(c) Leakers. Eggs whose shells and shell membranes are suffi- 
ciently broken to permit a portion of their contents to escape. 
(d) Dirties. Eggs, the shells of which have become soiled from un- 
clean nests, etc. 
(e) Weak Eggs. In this grade are placed all eggs in which the 
albumen has become weak or watery, due to high or varying tempera- 
ture. 
Leakers and dirties present the greatest problem to food bacteriol- 
ogists since they allow the ingress of bacteria. The leakers are either 
sold for local consumption or are broken for drying. Table XLIX pre- 
sents some data on commercial samples of leaking eggs secured by 
Pennington et al. (1914). 
Dirties appear during wet weather and sometimes during hot 
weather when moisture from the hen's body allows more dirt to adhere 
to the'shell. Many of these are used for local consumption or for 
drying since they do not keep well. In the examination of eggs with 
dirty shells Pennington (1914) secured widely divergent results. The 
minimum number of bacteria was 400 and the maximum number 
1,600,000 per gram at 20° C. The number of B. coli varied from 
10 to 10,000 in six samples. It has been reported by many 
investigators that dirty eggs do not keep well. Often these eggs 
are washed but this process reduces the keeping quality since it 
removes the delicate membrane or film of mucus on the surface 
of the shell. 
Spots. All eggs which show a spot before the candle are put into 
this class. These may be due to bacterial, meat, blood or mold spots. 
The term " spot-rot " is applied to a heated egg caused by decom- 
position of the dead germ. Molds often develop in eggs and the hyphae 
extend toward the air cell. To prevent this eggs must be stored in a 
dry place. Kossowicz found that old eggs were more susceptible to 
mold invasion than are fresh eggs; this is somewhat to be expected. 
Postolka (1916) confirmed this opinion. Marked growth of molds took 
place after either natural or artificial infection in the testacea but rarely 
in the yolk. Natural infection was caused by Penicillium glaucum and 
Cladosporium herbarum. Under experimental conditions almost any 
mold will attack and penetrate the egg. Postolka states that with 
even great infection with molds, no putrefaction takes place. Meat 
spots are caused by broken-down tissue of the hen's oviduct. In 
the candling process they appear as small floating bodies and 
are usually near the shell. Blood clots result from injuries to 458 
EXAMINATION OF EGGS 
the oviduct which causes blood to bo laid down with the albumen. 
Bloody eggs are not used by the average housewife and they cause 
a great loss. 
Rots. Different kinds of rots are known since bacteria develops 
in eggs in different ways. The fertile egg is much more liable to rot 
than is the sterile egg. Benjamin (1915) describes the following rots: 
White rots are common and often called watery rots, sour, or addled eggs. 
They represent the first stages in bacterial decomposition. Such eggs 
have an enlarged cell before the candle and a mixed interior. When 
opened they are usually a light yellow in color and watery. Mixed 
rots represent a more advanced stage of decomposition and are charac- 
terized by thin interiors; in the open condition they give off an odor 
of hydrogen sulphide and sourness. The yolk is rarely intact. Black 
rots are eggs in which the contents are very dark and may be easily 
shaken about the shell. When opened the odor is much like*that of 
hydrogen sulphide. In appearance the contents are mixed and very 
watery. 
Kuhl (1914) from the examination of large numbers of eggs con- 
cluded that for trade purposes the following would be a good classifica- 
tion of commercial eggs: fresh eggs, those upto eight or ten days old; 
eggs, those not over four weeks old; cooking eggs, any offered for sale 
which are not spoiled. 
Factors Influencing Bacterial Content of Eggs. A study of this side 
of the question involves two points, first the entrance of bacteria before 
the shell is put down and, secondly the contamination after laying, due 
to improper handling, etc. While the first point has been studied, the 
data are not convincing. Mauer (1911), in quoting the work of Per- 
not (1909) and Conradi states that the infection of the yolks even in 
the normal ovary is possible. Horowitz (1902), Zimmermann (1878), 
Abel (1895), Cao (1908), Draer (1895), McClintock (1894), Poppe 
(1910) and others, maintain that the oviduct is not sterile. Contrary 
to this are the data of Horowitz (1902) and Rettger (1913). This stage 
in our knowledge points out that more work is needed on this subject. 
Obviously, under abnormal conditions, the oviduct may be infected. 
This has been found to be true with Bacillus pullorum and the white 
diarrhoea of chicks. Bushnell and Mauer (1914) point out that there 
are factors which lower the vitality of the hen and render her unable to 
resist invading bacteria. Hadley and Caldwell (1916) think that the 
preponderance of yolk infections indicates that bacteria are present in 
the ovaries of the hen. BACTERIA IN EGGS 
459 
Permeability of the Shell. The shell about the egg may be regarded 
as a semi-permeability membrane and, therefore, not resistant to bac- 
teria. Wittich (1851) reported the infection of eggs by molds. Wilm 
(1895) succeeded in infecting eggs with cholera vibrios. When the eggs 
were covered with a broth culture, the organisms passed through the 
shell in from fifteen to sixteen hours, Golokow (1896), Piorowski (1895), 
Lange (1907), and Poppe (1910) demonstrated the same thing with 
other bacteria both pathogenic and non-pathogenic. Opposed to this 
work is that of Mauer (1911), Hoppert (1912), Sachs-Miike (1907), 
Zorkendorfer (1894) and Schlegel (1904). Kossowicz (1913) showed that 
B. proteus penetrated the shell very easily. The above indicates the 
ability of bacteria to penetrate the shells of eggs and this emphasizes 
the necessity of storing eggs under sanitary conditions. 
Bacteria in Egg White. Available data on this subject are con- 
flicting. Many of those who have found no bacteria in egg white have 
assumed that this compDnent of the egg possesses a bactericidal action. 
Wurtz (1890), Rettger and Sperry (1912), Scholl (1893), Horowitz 
(1902), Turro (1902), Laschtschenko (1909), and Riezicka (1912) pro- 
duce data indicating the same result. Sperry (1913) showed that cold 
storage eggs exhibited the same action. Bainbridge (1911), Poppe 
(1910), Hoppert (1912), Mauer (1911), Laschtschenko (1909) found no 
germicidal action. Mauer was especially interested in B. coli. Rettger 
and Sperry employed B. putrificus and B. edematis maligni as the best 
organisms. Egg white in test tubes was heavily inoculated with the 
test organisms and incubated under conditions favorable for the devel- 
opment of the organisms. In the yolk which was treated in the same 
manner evident putrefaction set in while in the egg white the bacteria 
were destroyed for microscopic examination failed to demonstrate the 
presence of the cells. Hadley and Caldwell (1916) in their excellent 
report on eggs reported data which confirm that of Rettger and Sperry. 
Bacteria in the Egg Yolk. Practically all of the investigators are 
agreed that the yolk contains the greater number of the bacteria. Had- 
ley and Caldwell found 8.7 per cent of 2520 eggs infected in the yolks. 
They quote from other authors: Mauer is said to have found 18.1 
per cent of the yolks to be infected. Bushnell and Mauer raised this 
to 23.7 per cent. Rettger's data is corrected to show a yolk infec- 
tion of 9.9 per cent covering examinations over a period of three 
years. 
Qualitative studies of the bacteria in eggs have been reported by 
several investigators. Rettger (1913) gives the following bacteria: 460 
EXAMINATION OF EGGS. 
Fresh Eggs. 
Number of Times Found. 
Staphylococcus, usually aurusu or albus 
74 
Subtilis growths usually B. mesentericus or B. ramosus 
60 
B. coli and closely related organisms 
43 
Proteus group 
30 
Streptococcus 
14 
Micrococcus (tetragenus, etc.) 
9 
Streptothrix 
6 
Diptheroid bacillus 
5 
Putrefactive anaerobes 
5 
B. fluorescens 
2 
Mold 
4 
B. mocosus 
3 
Mixed 
2 
257 
Hadley and Caldwell studied 40 different strains isolated from eggs. 
Among them were found 11 cocci, 28 rods, and 1 spirillum. No strep- 
tococci were reported. They observed no member of the hemorrhagic, 
septicemia, intestinal, proteus, colon, enteritidis, typhoid dysentery 
nor diphtheria groups. Bacterium pullorum was not observed in 
2520 eggs. No anaerobic bacteria were sought. 
That eggs in the fresh condition contain bacteria is still an open one. 
As pointed out by Hadley, it is difficult to explain the presence of bac- 
teria by contamination during plating. Hadley and Caldwell noticed 
different types of bacteria on the control plates than on the egg plates. 
The percentage of infected eggs found by different investigators is given 
below: 
Name. 
Examined. 
Per cent Infected. 
Rettger 
3510 
9.5 
Rettger (10 c.c. samples) 
647 
3.86 
Bushnell and Mauer 
2759 
23.70 
Hadley and Caldwell 
2520 
8.70 
Mauer 
600 
18.10 
Enzymes in Eggs. Rullman (1915) found catalase in eggs which 
were bacteria free. The amount had no relation to the age and was 
about equal in the yolks and whites. In putrid eggs the amount was 
so large that the sample had to be diluted. Pennington and Roberston 
(1912) studied this question using eggs of known history. They experi- CANDLING EGGS 
461 
mented to determine the presence of pepsin, trypsin, lipase, catalase, 
and reductase. Lipase content increases from a little in a fresh egg to a 
large amount in a stale egg. The catalase content of a fresh egg was 
found to be variable. 
Dried Eggs. The production of desiccated eggs has become a well- 
established industry. Most of our knowledge with regard to its con- 
trol rests on the work of Pennington (1916). The methods of manu- 
facture and their bacteriological control are carefully gone over. From 
their data the following facts were established. For breaking pur- 
poses reputable firms use all under-sized or over-sized, dirty, cracked or 
Fig. 77.-Types of Candling Devices. (After Benjamin.) 
The one at the left is made by arranging an ordinary oil lamp inside of a pasteboard box. The 
one at the right is a metal device which is placed on a lamp in place of the chimney. 
shrunken eggs. Such eggs should be candled, broken and dried under 
chilled conditions. The lowest count on flaky dried eggs was 65,000 
per gram and the highest count was 20,000,000 per gram. The average 
count for 48 samples was 3,600,000. The number of B. coli varied 
from 0 to 1,000,000. About three times as many bacteria were found 
in the dried product as in the liquid egg. Ross (1914) examined 248 
samples of dried eggs which had been stored at different temperatures. 
He gave the following conclusions to his work. " The desiccated egg 
loses a large percentage of the bacteria originally present if stored for a 
relatively short period. A more rapid diminution of bacteria results 
if the storage takes place at higher temperatures. This author states 462 
EXAMINATION OF EGGS 
PLATE 2.-CANDLING APPEARANCE OF MARKET EGGS 
1. Oviduct lines on shell. Note spiral lines of darker pigment. 
2. A fresh white egg. Note the small air cell, the indistinct yolk, the pink albumen, and the 
chalaza in the upper left-hand part. 
3. A fresh brown egg. Note the small air cell, the dark contents, and the very indistinct yolk. 
4. A light float. Note the heated yolk and the enlarged air cell. 
5. A heavy float. Note the yolk is very dark and low, the air cell is very large and loose, and 
the entire egg appears heated. 
6. A blind check. Note the five indistinct lines of breakage, invisible except by eandling. 
• 7. A check. Note that the shell is broken, but there is no leaking of contents. The air cell is 
enlarged. 
8. A meat spot. Note the distinct dark spot floating in the albumen, and the chalaza above 
and at the left of the meat spot. PLATE 2 CANDLING EGGS 
463 
that a product prepared even from spots and worse, might satisfy the 
ordinary bacterial tests if stored for a period of a few months. 
Frozen Eggs. Stiles and Bates (1912) prepared frozen eggs in the 
laboratory from second-grade eggs. Such a product had generally a 
bacterial content of 1,000,000 per gram. Frozen eggs prepared from 
spots, rots and blood rings gave a bacterial content of from 4,000,000 to 
1,000,000,000 per gram with a relatively high content of B. coli and 
streptococcus. Pennington (1916) reported a careful study of this sub- 
ject and pointed out many features in the sanitary control of the in- 
dustry. 
Candling of Eggs. The control of the commercial egg industry by 
chemical or bacterial analyses is very impracticable. Pennington (1909) 
has pointed out that each egg is a package by itself and an analysis of 
it would in no way indicate the condition of the lot. This has made 
it necessary for those engaged in food control to look for other methods 
for determining the fitness of eggs for consumption. Candling has 
developed to satisfy this need. Frazier (1917) states that this consists 
in ascertaining the character of an egg by allowing light to penetrate 
the contents. Different contrivances are used to do this. An electric 
light yields the most satisfactory results since it is constant in inten- 
sity and quality. Many cheap types of candling devices are used. 
Benjamin has described two common types. These are shown in 
Fig. 77. The accurate use of the candling apparatus demands some 
experience before reliable results may be obtained. The colored plates 
will be of much assistance. They are taken by permission from the 
bulletin by Professor Benjamin. Pennington et al. (1918) have recently 
published instructions for candling eggs. Those wishing a complete 
classified description of eggs before the candle and outside of the shell 
will be rewarded by reading this bulletin. 
Bacterial Examination of Eggs. Much discussion has passed back 
and forth over the methods of egg analysis. The early methods were 
probably faulty as Hadley has said and data from the earlier work must 
be accepted with some reservation. Hadley has discussed the methods 
of analysis and any one interested in that subject should consult his 
work. 
Stiles' Method. The eggs should be washed in a solution of bi- 
chloride of mercury (1/1000) or 5 per cent phenol for a few minutes 
after which they should be dried with sterile cotton and placed with 
the large end uppermost in a small beaker. The air space is then 
scorched with a gas flame for a few seconds. An opening should be 
made immediately into the cavity with sterile forceps, a sufficient 464 
EXAMINATION OF EGGS 
PLATE 3.-OPENED APPEARANCE OF MARKET EGGS . 
25. A fresh egg. Note the round, firm yolk, the colorless, dense albumen and the hard twisted 
chalazae. 
26. A light float. Note the heated and somewhat flattened yolk, the tinted and slightly 
watery albumen, and the weak vitellin membrane. 
27. A heavy float. The yolk is darkened by heat and very much flattened, the vitellin mem- 
brane is very weak, and the albumen is colored and very watery. 
28 A meat spot. This is similar in appearance to a piece of liver. The meat spot may be 
easily removed. PLATE 3 BACTERIAL ANALYSIS OF EGGS 
465 
amount of the shell being removed without rupturing the membrane 
below. When this is accomplished the latter should be broken with a 
hot platinum spatula and with a sterile pipette 0.5 c.c. of the white of 
the egg quickly removed and placed in the necessary Petri dishes for 
cultures. The remaining egg white is then decanted, leaving the un- 
broken yolk in the shell. With another sterile pipette, the yolk sac 
is ruptured and suitable portions of its contents removed for study. 
While this procedure guards against contamination, the breaking up of 
the respective layers of the egg when out of this shell is difficult and 
sometimes the inability to do so interferes seriously with the obtaining 
of quantitative results. With the eggs which have been in cold storage 
for considerable periods a separation of the whites and the yolks is not 
possible. 
Bushnell and Mauer's Method. The egg should be cleaned with 
brush and soap and immersed for ten minutes in a 1 to 500 solution of 
corrosive sublimate. It is transferred with sterile crucible forceps to a 
small conical graduate, acute pole uppermost. The corrosive subli- 
mate is removed and the egg dried by washing it first with alcohol 
and then with ether. The acute pole is scorched to kill spores, etc., 
that might remain. The egg is then immediately removed from the 
graduate by the operator's holding it by the blunt pole, turning the 
acute pole down. The hands of the operator should have been thor- 
oughly greased with vaseline to avoid contamination of the flasks by 
bacteria which might drop off the hands while handling the eggs. 
With sharp, stout forceps, which have been sterilized in the flame, a 
hole about | cm. in diameter is made into the acute pole. Holding the 
egg with the acute end down, and making the stab from below prevents 
contamination from above. The shell around the hole is flamed briskly 
and the egg is put with the acute pole upon the neck of a tall 300 c.c. 
Erlenmeyer flask containing 100 c.c. of sterile bouillon. The blunt 
end of the egg is now heated with a Bunsen flame, while a close watch is 
kept on the hole. The heating expands the air in the air space and 
this expels the contents of the egg. As soon as about half of the albu- 
men has run into the flask the heating is interrupted. The cotton plug 
is quickly removed from a sterile flask, the neck of the flask is flamed 
and the egg is transferred from the first to the second flask. Some- 
times it is necessary to invert the egg, as soon as the heating is discon- 
tinued, to prevent all of the albumin from running into the first flask. 
In this case it often happens that a little of the egg content runs down 
the outside of the shell, where it may become contaminated. To prevent 
such material from getting into the next flask, it is cemented to the 466 
EXAMINATION OF EGGS 
PLATE 4.-CANDLING APPEARANCE OF MARKET EGGS 
17. The live embryo after twelve hours' incubation. Note the slightly raised yolk, darkness by 
heat, and the embryo on the upper surface of the yolk. 
18. The live embryo after five days' incubation. Note the embryo and the blood vessels, and 
the dark mixed yolk. 
19. A live embryo after twelve days' incubation. Note the clouded contents, with few large 
blood vessels in view, and the enlarged air cell. 
20. A live embryo after eighteen days' incubation. Note the general dark appearance, the 
outline of the chick and the enlarged air cell. 
21. A dead embryo after five days' incubation. Note the ring or band of blood, the enlarged 
air cell, and the clouded appearance. 
22. An infertile egg after five days' incubation. Note the distinct yolk, the enlarged air cell 
with a dark boundary, and the general heated appearance. 
23. A bloody egg and blood clot. Note the bright red clot on the yolk and the general redness 
of the albumen. 
24. A soft-boiled egg. Note the slaty blue color. PLATE 4 BACTERIAL EXAMINATION OF EGGS 
467 
shell by being heated in the flame. The expulsion of the albumin into 
the second flask should be done slowly and watched closely. As soon 
as the yolk appears in the hole the heating is interrupted and the egg is 
tilted from one side to the other to allow the rest of the albumen to run 
out. In the same manner the yolk is expelled in two portions. The 
success of this method depends largely on the size of the hole. If this 
is too small, it is hard to separate the white from the yolk; if it is too 
large, it is difficult to expel the yolk in two separate portions. Some- 
times the yolk obstructs the hole before all of the albumen is obtained. 
If the yolk does not retract after cooling, the egg is inverted for a 
moment. Often the vitelline membrane will not rupture, and the yolk 
will come out in one piece. This can be prevented by puncturing the 
membrane with a sterile platinum needle. In this manner four flasks 
are obtained from each egg, two of them containing albumin and two 
of them containing yolk. The flasks are repeatedly shaken to mix the 
contents well. It is of advantage to have tall flasks because the con- 
tents can be mixed more easily without wetting the cotton plug. Two 
flasks, one with albumin and one with yolk, are incubated at 38° C. for 
forty-eight hours, and the other flasks are incubated at 20° C. for five 
days. After this period of development subcultures on agar slants are 
made to determine if growth has taken place. 
Rettger's Method for the Bacterial Examination of Eggs. The 
egg to be examined is placed small end up, in an egg cup or holder. 
The upper half of the egg is flamed with a Bunsen burner, the cup being 
turned constantly so that every part of the upper half of the shell is 
brought into brief contact with the flame. While the egg is held in 
one hand the upper end of the shell is removed with sterile scissors, 
leaving an opening about 1 in. in diameter. The white is poured out, 
care being taken to prevent it from running down the side of the shell. 
At this point the edge of the opening is flamed after which the entire 
yolk is poured out into a wide-mouthed flask, or better still, a large 
tube especially designed for this work by Rettger. Previous to intro- 
ducing the yolk, definite amounts of nutrient bouillon are placed in the 
flasks (25-50 c.c.) or tubes (25 c.c.) which are plugged with cotton and 
sterilized. The yolk and bouillon should be thoroughly mixed. In 
case the test tube is used this process is greatly facilitated by the pres- 
ence of a small glass rod about 1| in. in length. The tubes should then 
be placed in the 37° C. incubator and after seventy-two hours' incuba- 
tion agar streaks made from them. These should be incubated for 
from twenty-four to forty-eight hours. 468 
EXAMINATION OF EGGS 
PLATE 5.-OPENED APPEARANCE OF MARKET EGGS 
33. A live embryo after five hours' incubation. Note the indistinct circle around the embryo, 
the heated and flattened yolk, and the watery and flattened albumen. 
34. A live embryo after twelve hours' incubation. Note the distinct rings around the embryo, 
the heated and flattened yolk, and the watery and darkened albumen. 
35. A live embryo after five days' incubation. Note the embryo and blood vessels, the rup- 
tured vitellin membrane, and the watery nature of the albumen. 
36. A live embryo after twelve days' of incubation. Note the distinct form of the developing 
chick. PLATE 5 BACTERIAL ANALYSIS OF EGGS 
469 
Hadley and Caldwell's Method. The egg shell should be thor- 
oughly disinfected before any opening is made. This may be accom- 
plished by first washing the egg, if soiled, with soap and water and a 
wad of cotton, then immersing for ten minutes in 1 : 5000 mercuric 
chloride solution, containing either citric acid or ammonium chloride 
to increase the penetrating power. The egg may then be plunged 
into 95 per cent alcohol, removed, drained and ignited to dry the sur- 
face. These operations can be carried on in a suitably constructed 
wire rack in which the eggs can await examination, being meanwhile 
protected from air contamination. 
Next, as a final precaution, the egg should be well flamed at the end 
opposite the air space until a very thin layer (1-2 mm.) of the albumen 
lying close to the shell is coagulated. The amount of heating required 
can be ascertained by experience. After this, by means of sterile forceps, 
a hole about 2 cm. in diameter is made at the flamed end and the white 
poured into a tube or flask containing 25 c.c. of the desired medium. 
Next, the opening in the shell is enlarged to about 3 cm. The yolk is 
gently allowed to run out of the shell onto a circle of sterile filter paper 
(kept in a small pile under the bell jar). The paper is then so inclined 
that the yolk rolls about until the white is entirely removed by the paper, 
after which the yolk is rolled off the edge of the paper into a tube or 
flask containing at least 25 c.c. of culture medium, an amount some- 
what greater than the volume of the average yolk. Great care must, 
of course, bo taken not to rupture the yolk membrane before the yolk 
is poured into the tube. The yolk may then be broken by means of a 
sterile glass rod and mixed with the broth. 
The preparation is now ready for incubation which may be carried 
on for forty-eight hours at 37° C., followed by forty-eight hours at 20° C. 
The white may be mixed with the broth and grown in the same manner. 
At the end of the period of incubation the tubes should be again mixed 
by rotation to insure distribution of the bacteria, and a small amount 
on a straight needle transferred from each to ordinary tubes contain- 
ing broth. The broth tubes are incubated at 37° and at 20° C., as were 
the original tubes and examined for growth at the end of four days. In 
case growth appears plate cultures are made.* 
* In addition to the points of technique mentioned above, minor details can per- 
haps best be worked out by the individual investigator. For instance, cotton plugs 
in the large tubes are unwieldy. They may be replaced by close-fitting glass covers, 
coming well down over the tops of the egg tubes. The breaking of the yolk and the 
mixing with the broth may be accomplished by means of broken glass placed in 
the tubes with the broth, rather than by means of glass rods, a procedure which 
may favor contamination from the air. Wire cages or racks of various sizes and 470 
EXAMINATION OF EGGS 
PLATE 6.-OPENED APPEARANCE OF MARKET EGGS 
37. A live embryo after eighteen days' x>f incubation. Note the fully-shaped chick, with the 
embryo sac attached. 
38. A dead embryo after five days' of incubation. Note the ring or band of blood, the mixed 
yolk and albumen, and the signs of deterioration. 
39. An infertile egg after five days of incubation. Note the heated appearance of the yolk, 
the very watery contents, the slightly colored albumen, and the very weak vitellin membrane. 
40. A bloody egg and blood clot. The blood clot is adhering to the yolk. A small part of the 
albumen is bloody, but the greater part of the egg is perfectly normal. PLATE 6 BIBLIOGRAPHY 
471 
It should be borne in mind that the recommendations made above 
are suitable only for fresh eggs in which it is desired to examine the yolk 
and white separately. In old market eggs, or preserved eggs in which 
the yolk is weakened, and in decomposed eggs in which the yolk and 
white have become mixed, different methods would be required; and 
here, as in the bacteriological examination of other egg products, the 
technique must be evolved in accordance with the particular aim of the 
investigation. 
BIBLIOGRAPHY 
Abel, R. and Draer, A. 1895. Das Hiihnerei als Kulturmedium fiir Cholera 
Vibrionen. Zeit. f. Hyg., 19, 61. 
Artault, T. 1893. Recherches bacteriologiques, mycologiques, zoologiques 
et medicales sur 1'ceuf de poule. These Paris. Cent. Bakt., 1894, 16, 461. 
Artault, T. 1893. Le bacille pyocyanique dans un ceuf de poule. Compt. 
Rend. soc. biol., series 9, 5, 78. 
Artault, T. 1895. Tuberculose provoquee chez lapins par des injections de 
contenu d'ceufs de poule. Comp. rend. soc. biol. ser. 10, 2, 683. 
Bainbridge, F. A. 1911. The Action of Certain Bacteria on Proteins. 
Journal of Hygiene, 11, 341. 
Barthelmy, M. A. De 1'incubation des ceufs d'une poule atteinte du cholera 
des poules. Compt. Rend. Acad. Sciences, 96, 1322. 
Bechamp, A. and Eustache, G. 1877. Sur l'alteration des ceufs provoquee 
par des moississures venus de I'ext^rieur. Comp. Rend. Acad. Sci., 85, 
854. 
Beckwith, T. D. and Horton, G. D. 1914. Is Poor Hatching of Normal Eggs 
Due to the Presence of Microorganisms within the Egg? Science N. S., 
40, 240. 
Benjamin, E. D. 1914. The Interior Quality of Market Eggs. New York 
Agricultural Exp. Station Bull. 353. Cornell Univ., Ithaca. 
Bornaud, M. 1914. Recherches sur les parasites vegetaux des ceufs des 
poules. Cent. Bakt. Ref., 63, 43. 
Bushnell, L. D. and Mauer, 0. 1914. Some Factors Influencing the 
Bacterial Content and Keeping Quality of Eggs. Kansas Ag. Ex. Sta. 
Bull. 201. 
Cao, G. 1908. Sur la permeabilita delle uova ai microorganismi. Bull. 
Inst. Pasteur, 6, 472. 
shapes may be used for the immersion of the eggs in the disinfecting solution, in the 
alcohol and during ignition. 
Methods for the detection of anaerobic microorganisms probably do not present 
unusual difficulties. The method of Hesse, involving the use of a layer of sterile 
olive or paraffin oil over the surface of the mixture is probably the best. In this 
case samples may be withdrawn by means of a sterile pipette. 472 
EXAMINATION OF EGGS 
PLATE 7.-CANDLING APPEARANCE OF MARKET EGGS 
9. A home-preserved egg. Note the medium-sized air cell and the fresh appearance of the 
yolk. 
10. A No. 1 cold-storage egg. Note the enlarged air cell and the slightly darkened yolk. 
11. A No. 2 cold-storage egg. Note the very large and loose air cell, and the darkened yolk 
with a very distinct boundary. 
12. A stuch yolk. Note the spots where the yolk adheres, and the^enlarged air cell. 
13. Mold spot. Note the small mold spots on the shell membrane. 
14. White rot. Note the slimy and mixed appearance of the egg contents and the enlarged 
air cell. 
15. Mixed rot. Note the darkened and mixed contents and the broken air cell. 
16. Black rot. Note the very dark contents, and the large, loose air cell. PLATE 7 BIBLIOGRAPHY 
473 
Donitz. 1895. Ueber das Verhalten der Choleravibrionen Hiihnerei. Zeit. 
Hyg., 20, 31. 
Frazier, D. J. 1917. Candling Eggs. American Food Journal, 12, 39. 
Gayon, U. 1873. Sur l'alteration spontanee des ceufs. Comp. Rend. Acad. 
Sci., 76, 232. 
Gayon, U. 1875. Recherches sur l'alteration spontanee des ceufs. Ann. 
Sci. 1'Ecole Norm. Superieur, Series 2, 4, 205. 
Golokow. 1896. Ueber das Eindringen von Cholera Vibrionen im Hiihnerei. 
Baumgarten's Jahresbericht, 12, 583. 
Grigoriew, A. W. 1894. Vergleichende Studien fiber die Zersetzung des 
Hiihnereiweisses durch Vibrionen. Arch. Hyg., 21, 142. 
Hadley, P. B. and Caldwell, D. W. 1916. The Bacterial Infection of Eggs. 
Rhode Island Ag. Exp. Sta. Bull. 164. 
Hammerl, H. 1894. Ueber die im rohen Eiren durch das Wachstum von 
Cholera Vibrionen hervor gerufen Veranderung. Zeit. Hyg., 18, 153. 
Hoppert, M. J. Factors Relating to the Bacteria Content of Eggs. Thesis 
University of Wisconsin. 
Horowitz, A. 1902. Contribution a 1'etude des moyens de defense de 1'organ- 
isms centre 1'invasion microbienne; recherche sur 1'oviducte de la poule 
de le blanc d'ceuf. These Paris. Baumgarten's Jahresbericht, 19, 984. 
Hueppe, F. 1888. Ueber die Verwendung von Eiern zu Kulturwecken. 
Cent. Bakt., 2, 80. 
Kempner, W. 1894. Ueber Schwefel wasserstoffbildung der Choleravibrionen 
im Hiihnerei. Arch. Hyg., 21, 317. 
Kossowicz, A. 1913. Die Zersetzung und Haltbarmachung der Eier. Wies- 
baden. J. F. Bergman. 
Kuhl, H. 1914. The Relation between Spoiling and the Age of Eggs. Hyg. 
Rundschau, 24, 253-259. 
Lamson, G. H. 1909. Infection and Preservation of Eggs. Storrs Ag. Exp. 
Sta. Bull. 55. 
Lange, R. 1907. Ueber das Eindringen von Bakterien in das Hiihnerei 
durch die Eischale. Arch. Hyg., 62, 201. 
Laschtschenko, P. 1907. Ueber die keimtotende und entwicklungshemmende 
Wirkung von Hiihnereiweiss. Zeit. Hyg., 64, 419. 
Linnossier et Lemoine. 1910. Sur la toxicite normale des aliments albu- 
minoides frais. Influence de la conservation. Comp. Rend. soc. biol., 
68, 671. 
McClintock. 1894. Bacteria in Eggs. Modern Medicine and Bact. Rev., 
3, 144. 
Mauer, O. 1911. Bacteriological Studies on Eggs. Kansas Ag. Exp. Sta. 
Bull. 180. 
Menini, G. 1908. Richerche intorno alia penetrazione dei bacteria nelle 
uova di gallina. Lo. Sperimentale, 61, 711. 
Mosler, F. 1864. Mykologische Studien am Hiihnerei. Arch. Path. Anat. 
Physiol. Virchow., 29, 510. 474 
EXAMINATION OF EGGS 
PLATE 8.-OPENED APPEARANCE OF MARKET EGGS 
29. A home-preserved egg. Note the pink and watery albumen, and the flattened and slightly 
darkened yolk. 
30. A No. 1 cold-storage egg. Note the well-rounded yolk, and the slightly watery albumen 
with a slightly darkened tint. 
31. White rot. Note the mixed and very watery contents, and the light yellow color. 
32. Mixed rot. The contents are of various colors, mixed, and very watery. PLATE 8 BIBLIOGRAPHY 
475 
Pennington, M. E. 1909. A Chemical and Biological Study of Fresh Eggs. 
Jour. Biol. Chem., 7, 109. 
Pennington, M. E., et al. 1914. A Bacteriological and Chemical Study of 
Commercial Eggs in the Producing Districts of the Central West. U. S. 
Dept. Agric. Bureau Chem. Bull. 51. 
Pennington, M. E. and Robertson, H. C. 1912. A Study of the Enzymes 
of the Egg of the Common Fowl. Circular 104, U. S. Dept. Agric. Bureau 
of Chemistry. 
Pennington, M. E., et al. 1916. A Study of the Preparation of Frozen and 
Dried Eggs in the Producing Section. U. S. Dept. Agric. Bull. 224. 
Pennington, M. E., Jenkins, M. K. and Betts, H. M. P. 1918. How to 
Candle Eggs. U. S. Dept. Agriculture Bulletin 565. 
Piorkowski. 1895. Ueber die Einwanderung der Typhus Bacillen in das 
Hiihnerei. Arch. Hyg., 25, 145. 
Poppe, K. 1910. Zur Frage der Uebertragung von Krankheitserregern durch 
das Hiihnerei. Zugleich ein Beitrag zur Bakteriologie des normalen Eise. 
Arb. a. d. Kaiserl. Gesndhsmt., 34, 186. 
Postolka, A. 1916. The Growth of Molds in Eggs. Cent. Bakt. Abt. IL, 
46, 320-330. Chem. Abts., 11 (1917), 3215. 
Raebiger, W. 1900. Ueber die Rotfiirbung eines Hiihnereis durch B. pro- 
digiosus. Zeit. Fleisch u. Milchhyg., 11, 115. 
Rettger, L. F. and Sperry, J. A. 1912. The Antiseptic and Bactericidal 
Properties of Egg White. Jour. Med. Res., 26, 55. 
Rettger, L. F. 1913. The Bacteriology of the Hen's Egg with Special Ref- 
erence to its Freedom from Microbic Invasion. Cent. Bakt. Abt. IL, 
39, 611. Storrs. Ag. Exp. Sta. Bull. 75. 
Rettger, L. F. 1911. Bacilliary White Diarrhoea of Chicks. Storrs Ag. 
Exp. Sta. Bull. 60. 
Riezicka, V. 1912. Ueber die natiirliche Schutzkraft in Entwicklung be- 
griffener Hiihnerei. Arch. Hyg., 77, 369. 
Ross, L. S. 1914. Bacterial Content of Desiccated Eggs. Proc. Iowa Acad. 
Sci., 21, 33-49. 
Rullman, W. 1915. The Bacteria and Catalase Content of Hen Eggs. 
Cent. Bakt. Abt. IL, 45, 219-230. Chem. Abts., 11, 2509. 
Schenk. 1850. Ueber Pilzbildung in Hiihnereiern. Verhandl. Phys. Med. 
GesselL, 1, 73. 
Scholl, H. 1892. Untersuchungen uber Giftige Eiweisskorper bei Cholera 
Asiatica und einigen Faulnisprocessen. Arch. Hyg., 15, 172. 
Scholl, H. 1893. Bakteriologische und chemische Studien uber das Hiihnerei- 
weiss. Arch. Hyg., 17, 535. 
Schrank, J. 1888. Untersuchungen uber den im Hiihnerei die stinkende 
Faulniss hervorrufenden Bacillus. Wiener, med. Jahrb., 84, 303. 
Schranck, J. 1895. Bakteriologische Untersuchungen fauler Kalkeier. 
Zeit. Allg. Osterr-Apoth. Ver., 33, 295. 476 
EXAMINATION OF EGGS 
' Fig. 78.-Rate of Evaporation of Hens' Eggs. 
1 to 9. Eggs that have been held for one day, one week, two weeks, three weeks, four weeks, 
five weeks, six weeks, seven weeks, and eight weeks, respectively. The eggs were kept at a living- 
room temperature of about 70° F., in an open pasteboard carton. There was no breeze blowing 
over the eggs, and each egg illustrated is typical of several eggs that were examined at each stage; 
therefore this series represents the normal results of such holding. 
10 to 12. Eggs held for twelve weeks, one week, and one-half week, respectively, at a tem- 
perature of 40° F. This is a very good temperature at which to hold eggs for short periods. Even 
at twelve weeks of age (section 10), the egg is less evaporated than at four weeks of age, if held at a 
living-room temperature of 70° F. (section 5). Eggs may be held for two weeks at 40° F. without 
much change. 
13 and 14. Each of these eggs has been held for six weeks at a temperature of about 50° F. 
The porous shell of the second egg (14) has caused more rapid evaporation. The porosity of the 
shell may be easily distinguished in candling. 
15 and 16. These eggs have been held for five weeks and for two and one-half weeks, respec- 
tively, at a temperature of 50° F. It should be noted that the evaporation is more rapid than that 
which took place at 40° F., but much slower than that at 70° F. BIBLIOGRAPHY 
477 
Fig. 78. 478 
EXAMINATION OF EGGS 
Simon, C. E. 1904. A Text Book of Physiological Chemistry for Students of 
Medicine and Physicians, 455. 
Sperry, J. A. 1913. On Antiseptic and Bactericidal Properties of Egg White. 
Science N. S., 38, 413. 
Spring, A. Des champignons qui se developent dans les oeufs de poule. 
Bull. Acad. Roy. Sci. Let. St. Beaux-Arts Belg., 19, 555. 
Stiles, G. W. and Bates, C. 1912. A Bacteriological Study of Shelled, 
Frozen and Desiccated Eggs made under Laboratory Conditions at Wash- 
ington. U. S. Dept. Agric. Bur. Chern. Bull. 158. 
Tice, W. G. A Chemical and Bacteriological Study of Eggs. 35th Ann. Rept. 
New Jersey Bd. Health, 275. 
Turro, R. 1902. Zur Bakterienverdauung IV. Bakteriologische Wirkung 
des Hiihnerei. Cent. Bakt. Abt. I. Orig., 32, 107. 
Von Wittich. 1851. Ueber Pilzbildung im Hiihnerei. Zeit. Wiss. Zool., 
3, 213. 
Wiley, H. W., et al. 1908. A Preliminary Study of the Effects of Cold 
Storage on Eggs, Quail, and Chicken. U. S. Dept. Agric. Bureau of 
Chern. Bull. 117. 
Wilm. 1895. Ueber die Einwanderung von Choleravibrionen im Hiihnerei. 
Arch. Hyg., 23, 145. 
Wurtz, R. De Faction bactericide du blanc d'oeuf. Semaine Med., 3, 21. 
Zimmermann, 0. E. R. 1878. Ueber dir Mikroorganismen welche die Ver- 
derbnis der Eier veranlassen, 6. Ber. d. Natur. Gesell. Chemnitz, p. 3, 
Landwir. Jahersb., 755. 
Zorgendorfer. 1893. Ueber die im Hiihnerei vorkommenden Bakterien- 
arten nebst Vorschlagen zu rationellen Verfahren der Eiconservierung. 
Arch. Hyg., 16, 367.z CHAPTER XIII 
MEAT AND MEAT PRODUCTS 
Meat is interesting from the bacteriological viewpoint for several 
reasons. As the activities of man became more complex, it became 
necessary to keep meat for future consumption. In order to do this, 
he had to study the action of the bacteria. The meat industry is one 
in which a large amount of capital is invested. Since meat is very sus- 
ceptible to the attacks of bacteria and since it may be taken from dis- 
eased animals, it has become necessary for the Federal authorities to 
control the slaughter and disposal of meat. The Federal Meat Inspec- 
tion Service of the United States Department of Agriculture is a public 
health agency which is constantly protecting the consumers. Melvin 
(1906) has outlined the scope and application of the laws governing 
this product. The opinions regarding what constitutes diseased meat 
are by no means in agreement. Stiles (1917) has discussed this subject 
and pointed out that there are several definitions of the term " diseased " 
as applied to meat. Meat is essentially protein in composition and this 
chemical constitution determines the type of decomposition to which 
it is susceptible. 
Origin of Bacteria in Meat. The presence of bacteria in the organs 
and flesh of healthy animals is still a debated question. Many investi- 
gators have reported their presence but the health of the animal in 
these instances may be questioned. Maurel (1911) found a diplococcus 
in meat. Conradi (1909), using a special enrichment method found 
aerobic and anaerobic bacteria in muscles and organs. Bierotti and 
Machida (1910) secured similar results. Zwick and Weichel (1911) 
found bacteria frequently in the liver but not in the muscles with the 
exception of one case. Conradi (1909) in examining many organs 
found bacteria but concluded that they came from the intestines, being 
absorbed along with the food. Hoagland et al. (1917) in studying the 
changes in beef during storage above freezing, states as follows with 
regard to the presence of bacteria: 
Certain bacteria (chiefly micrococci) may be normally present in the carcass 
of healthy animals slaughtered for beef. These bacteria possess no pathological 
479 480 
MEAT AND MEAT PRODUCTS 
significance and do not appear to multiply in the cold-stored carcasses provided 
the cold-storage room is maintained at the proper temperature. 
Chopped Meats. These may contain many bacteria for a number 
of reasons: (1) The meat that is used for such purposes may be dirty 
and old and unfit for consumption in other ways. (2) The hashing 
process probably distributes bacteria throughout the meat This 
treatment increases the surface area of the meat, allowing a more vigor- 
ous development of bacteria. (3) Careless handling of the product may 
cause high counts in the same way as in other food. 
The bacteriology of chopped meat has been studied by a number of 
investigators. Zweifel (1911) regarded the count as of little sanitary 
significance. Marxer (1903) made a bacteriological study of Hamburger 
steak and concluded that 1,000,000 bacteria per gram or a large number 
of representatives of the protein group indicated a meat that was on the 
verge of decomposition. Weinzirl and Newton (1914), after developing 
a satisfactory method for enumerating bacteria in chopped meats studied 
forty-four samples of market Hamburger steak. To determine the 
degree of composition comparisons were made between the organoleptic, 
ammonia tests, and the bacterial content. From their data, they con- 
clude that the Marxer standard given above is too low since nearly all 
samples have to be condemned without regard to the evidence of putre- 
faction. There was a good correlation between the ammonia and organ- 
oleptic tests but not one between these and the bacterial content. 
These authors propose a standard of 10,000,000 bacteria per gram which, 
according to their data, would condemn about 50 per cent of the sam- 
ples. Le Fevre (1917), using the technique devised by Weinzirl and 
Newton, made a semi-qualitative study of the bacteriain Hamburger 
steak. The presence of liquefying bacteria was evident in nearly all 
of the samples. Acidifiers presented in the table do not agree with 
statements in the article. Anaerobic bacteria were found in some 
samples. Le Fevre believes that the Marxer standard is high enough 
and that the bacterial count should be relied upon for the control of 
retail supplies of chopped meat. 
Whether this may be done is doubtful. At least much more infor- 
mation should be accumulated before any " standard " is established. 
The kind of bacteria may be more important than the numbers. Wein- 
zirl and Newton (1915) undertook an investigation to determine the 
effect of cold storage below freezing on the bacterial content. After 
one year at -10° C. six of the ten samples fell within the 10,000,000 
standard. This compared favorably with the organoleptic tests. CANNED MEATS 
481 
Sausage. The bacteriology of sausage is essentially the same as 
that of Hamburger steak. One difference is the content of spices in 
sausage. While most of the work on bacteria in sausage has been con- 
cerned with meat poisoning, a little has been done which gives informa- 
tion with regard to the normal bacteria which may be present. V. I). 
Sloonton (1907) found few bacteria in sausage and in one instance the 
sausage was sterile. Drying and smoking greatly reduced the number 
of bacteria. Highly seasoned sausage was very detrimental to certain 
bacteria. Cary (1916) made a comprehensive study of the sausage with 
regard to the numbers of bacteria which were present and the factors 
influencing them. The sausages were purchased upon the market in 
Chicago. It is interesting to note that Cary regards the bacterial count 
of sausage as of little importance. Many factors such as precautions in 
manufacture, handling in shops, and presence of preservatives influence 
the count. 
The following bacteria which are of special importance were isolated 
from thirty-four samples: 
Bacillus co1 on 30 times 
Proteus vucgaris 11 times 
B. paracolon 9 times 
B. fecalis 8 times 
Yeast 8 times 
Streptococcus 5 times 
Staph, aureus 2 times 
The cultures of B. paracolon resembled B. paratyphosus morphologically 
and culturally but were not agglutinated by either paratyphoid or enter- 
itidis sera. The casings were regarded as unimportant in influencing 
the count of sausage, if they were properly prepared. 
Canned Meats. Some aspects of the canning problem have been 
taken up in another chapter. More attention may be given, however, to 
the canning of meats at this time. Practically all kinds of meats are 
canned. The procedure which is followed depends very much on the 
kind of meat. Canned meat possesses characteristics which are in sharp 
contrast with some of those of fresh meat. It is trimmed free from 
bone cartilage and contains a smaller amount of water than fresh meat. 
The cost, therefore, is not much more than that of fresh meat. 
Some investigations have been made on canned meats. Beveridge 
and Fawcus (1908) reported that when tins of meat of identical size and 
shape are heated, there is a difference in the length of time required 
for the interior of the cans to reach the same temperature. The lowest 482 
MEAT AND MEAT PRODUCTS 
temperature that could be used for sterilizing tinned meats was 120° 
for sixty minutes. These experiments were carried out with B. cadaveris 
sporogenes and B. putrificus coli. Fowler (1909) studied five samples of 
canned meat, three of which were blown. An organism like B. putri- 
ficus coli was isolated. The spores of the organism resisted boiling for 
two minutes. Beveridge (1909) found that the blackening of the inte- 
rior of the tins was due to products of bacterial action such as H2S, etc. 
McBryde (1907) reported a study of the spoilage of tinned corned 
beef. The sound cans were found to be sterile while the leakers revealed 
the presence of bacteria yeasts and molds. The bacteria were facul- 
tative with regard to oxygen. McBryde points out that while the bac- 
teria which he found were non-pathogenic, they may give rise to toxic 
substances which may serve to cause severe physiological disturbances 
in those who partake of the meat. 
Bushnell and Utt (1917) examined canned salmon purchased on the 
open market and found it sterile and relatively free from tin salts. 
Apparently all of the tins which they examined were normal. Swelled 
canned sardines have been investigated by Sadler (1918). B. vulgaris 
and members of the colon-aerogenes group were isolated. Apparently 
no effort was made to determine the presence of anaerobic bacteria. 
Obst (1918) found a bacillus possibly identical with B. W alfishrausch- 
brand in pure culture in 287 cans of swelled sardines. Koch's postulates 
were satisfied using this organism and sound cans of sardines. The 
organism was an anaerobic, gas-producing, non-spore forming bacillus. 
In a rather extended investigation on spoiled canned mackerel and cod, 
the author has isolated both aerobic and anaerobic organisms. These 
were put according to the usual factory methods and processed at the 
usual temperatures. 
BACTERIAL EXAMINATION OF MEAT AND MEAT PRODUCTS 
Hoagland's et al. Method. This is applicable to large pieces of meat 
and was used by these workers in studying changes in meats under cold- 
storage conditions. A slice or section from | in. thick is cut off (of the 
round) and from this a chunk measuring 4| by 8 ins. is taken for bacterial 
examination. This is immersed in boiling water for three minutes and 
next in mercuric chloride (0.5 per cent) for five minutes. It is then 
wrapped in sterile gauze which has been wrung out in the mercuric 
solution. This is done in order to sterilize the surface of the meat and 
to prevent the growth and possible penetration of bacteria from the 
outside. Beginning about 1 in. down from the outer surface take a MEAT EXAMINATION 
483 
series of cultures at intervals of an inch, proceeding from the outside 
toward the center. In taking the cultures, use a serus scalpel. Plugs 
of meat about 1 cm. square should be taken and cultures made in neutral 
beef broth and glucose agar. When clouding appears, the contents of 
these tubes should be plated out. 
Weinzirl and Newton's Method for the Examination of Hamburger 
Steak. Three different portions. of the sample to be analyzed are 
taken. The meat is triturated in a mortar with sand by means of a 
pestle. After grinding with sand for a time sterile salt solution is 
added and grinding continued. Finally the salt solution is increased 
to 100 c.c. and a thorough mixture is made. This is sampled and incu- 
bated at room temperature. Ordinary media may be used. 
Eyre's Method for Examination of Meat. The meat is minced by 
means of sterile instruments and added to plain broth. The flasks are 
incubated at 42° C. for thirty minutes. They are shaken from time to 
time. Then this is sampled. 
Examination of Sausage. Thoroughly sterilize the sausage by pass- 
ing through a flame. Cut open the casing with a sterile knife and 
remove 3 or 4 gms. of the interior. This should be added to a sterile 
tared weighing bottle and carefully weighed. Thoroughly emulsify in 
sterile ■water and put on shaking machine for twenty minutes. Plate 
out on plain agar, plain gelatin and litmus lactose agars. 
St. John's Method for Examination of Sausage. The skin is seared 
and removed; then small fragments are torn from the muscular tissue. 
These, weighing about 0.3-0.7 gm. are shaken for ten minutes, in 
sterile salt solution with ground glass. Plates were '^ade with neutral 
glycerol agar and plain agar. Three incubation temperatures were used 
37°, 20°, and 1.6°. (Effects of cold storage on eggs, quail and chickens. 
Wiley et al. Bull. 115 Bu. of Chem., 1908, p. 75.) 
Bacterial Examination of Shellfish. Oysters were regarded by 
sanitarians for some time as possible agents in the spread of typhoid 
fever and other intestinal infections. Not until 1894, however, were 
they definitely connected with this disease. Professor H. W. Conn, of 
Wesleyan University, in a masterly piece of epidemiological work, proved 
to the satisfaction of those interested in public health that an outbreak 
of typhoid fever among those who had attended a fraternity banquet 
was caused by oysters. The chain of evidence was so complete that the 
study of oyster grounds, in relation to possible sewage pollution, was 
greatly stimulated. Since this time many investigations have been 
carried out. Stiles (1911) has given a rather complete bibliography of 
the articles up to the date of his paper. Hindman and Goodrich (1917) 484 
MEAT AND MEAT PRODUCTS 
upon examination of Puget Sound oysters found a great variation in 
the bacterial content. They state that oysters intended for human 
consumption should be grown in water free from pollution and that 
after collection they should be handled with great care. 
The relation between polluted shellfish and disease is so close that 
the American Public Health Association appointed a committee to pre- 
pare a code of standard methods of shellfish examination. The report 
of this committee was published in 1912 and included the following rec- 
ommendations for the bacteriological examination of shellfish. 
Many laymen assume and argue that because oysters and other 
foods are cooked, they are safe for human consumption. In many cases 
cooking may render a food product sterile. So many experiments have 
been performed, however, along this line that a student of the subject 
does not accept such a sweeping statement. Stiles (1911) reported data 
which were secured in five different experiments to demonstrate the 
effect of heat for the destruction of bacteria in oysters. The shell- 
fish were exposed to live steam (98° and 99° C.) in a steam sterilizer for 
periods varying from two to thirty minutes. He found that by steaming 
contaminated oysters and clams in the shell, or cooking them after 
shucking for fifteen minutes at boiling temperature destroyed prac- 
tically all organisms of a questionable character, but since in practice 
shellfish are rarely cooked for this length of time, cooking cannot be 
depended upon to remove the danger. In this connection it is well to 
remember the work of Howell (1912), Roussel (1907), and Marchland 
(1909) on bread and of Sawyer (1914) on cooked spaghetti. 
Recommendations for Standard Methods for the Bacteriological 
Examination of Shellfish 
Oysters in the Shell. Selection of Sample. 
Twelve (12) oysters of the average size of the lot under examination, 
with deep bowls, short lips, and shells tightly closed, shall be picked out 
by hand and prepared for transportation to the laboratory. 
As complete a record of such data as is possible to obtain shall be 
made covering the following points: 
The exact location of the bed from which the sample has been 
selected. 
The depth of the water over the bed at time of collection. 
The state of the tide. 
The direction and velocity of the wind. EXAMINATION OF SHELL FISH 
485 
Other weather conditions. 
The day and hour of the removal of the stock from the water. 
The conditions under which the stock has been kept since removal 
from the water and prior to the taking of the sample. 
The day and hour of the taking of the sample. 
Transportation of the Sample. The oysters so selected shall be 
packed in suitable metal or pasteboard containers of such size and 
shape that a number of them can be enclosed in a shipping case capable of 
satisfactory refrigeration by means of ice. The important points in 
this connection are: 
A. The prevention of the mixing of the oyster liquor of different 
samples, and of the mixing of the ice water with the oysters. 
B. The icing of the samples, if they are not to arrive at the point of 
laboratory examination inside of thirty-six hours, or, if the outside tem- 
perature is above 50° F. 
It is not necessary to enclose the oysters in an absolutely tight con- 
tainer, providing the above conditions are maintained. 
Condition of Samples. Record shall be made of the general condi- 
tion of the oysters when received, especially whether the shells are open 
or closed; of the presence of abnormal odors; and of the temperature 
of the stock. 
Technical Procedure. The bacteriological examination shall be 
started as soon as possible after the receipt of the sample. 
The oysters shall be thoroughly cleaned with a stiff brush and clean 
running water and then dried. The edges of the shell shall be passed 
through the flame or burned w'ith alcohol. 
The opening of the shell shall be accomplished by either of the fol- 
lowing methods: 
A. By the use of a sterile oyster knife in the usual manner. 
B. By drilling through a flamed portion of the shell near the hinge 
with a sterile drill. The drill shall be sterilized, and the site of the oper- 
ation on the shell shall be flamed at least once during the drilling process. 
Bacterial Counts. Bacterial counts shall be made of a composite 
sample of each lot obtained by mixing the shell liquor of five oysters. 
Agar shall be used for the culture medium and in general the pro- 
cedure shall be in accordance with the method recommended for the 
examination of water by the Committee on Standard Methods of Water 
Analysis of the American Public Health Association. 
The water used for dilution purposes shall contain 1 per cent of 
sodium chloride, in order to approximate the natural salinity of oyster 
liquor. 486 
MEAT AND MEAT PRODUCTS 
The agar plates shall be incubated at 20° C. for three days and the 
colonies then counted. 
Determination of Bacteria of the Bacillus coli Group. The quanti- 
tative determination of the presence of B. coli shall be in accordance 
with the following procedure: 
Measured quantities (1.0; 0.1; 0.01 c.c., etc., or their equivalents 
in dilutions) of the shell water of each of five oysters selected from the 
dozen, shall be placed in fermentation tubes containing lactose peptone 
bile, prepared according to the method recommended by the Committee 
on Standard Methods of Water Analysis. These shall be incubated for 
three days at 37° C.; and the presence or absence of gas noted daily. 
For all ordinary purposes of routine work a development of from 10 to 
85 per cent of gas during this time period shall constitute a positive test 
indicating a presumption of the presence of at least one bacterium of 
the B. coli group in the quantity of the shell water tested. But no final 
B. coli rating based on these results shall be used for official approval or 
condemnation unless positive confirmatory tests for the presence or 
organisms of the B. coli group shall have been obtained from the tube of 
highest or next highest dilution from each oyster, showing the presence 
of gas. These confirmatory tests shall be begun immediately upon 
noting the formation of gas, and carried out in accordance with the 
procedure recommended by the Committee on Standard Methods of 
Water Analysis. 
Statement of Results. The results of the bacterial counts shall be 
expressed as Number of Bacteria per Cubic Centimeter. The results 
for the tests for B. coli shall be expressed either in the form of the fol- 
lowing arbitrary numerical system to be known as " The American 
Public Health Association Method of Rating Oysters for B. coli-, or in 
Estimated Number of Bacteria of the B. coli Group per Cubic Centi- 
meter of the Sample." 
The American Public Health Association Method of Rating Oysters 
for B. coli.* The following values shall be assigned to the presence of 
bacteria of the B. coli group in each of the five oysters examined, these 
figures being the reciprocals of the greatest dilutions in which the test 
for B. coli was positive: 
If present in 1.0 c.c. but not in 0.1 c.c., a value of 1. 
If present in 0.1 c.c. but not in 0.01 c.c., a value of 10. 
If present in 0.01 c.c. but not in 0.001 c.c., a value of 100, etc. 
* Where the term B. coli is used, it refers in all cases to bacteria of the B. coli 
group and not to the specific prototype. EXAMINATION OF SHELL FISH 
487 
The sum of these values for the five oysters gives the total value 
for the sample and this figure shall be taken as the " rating for B. coli." 
The results shall be expressed in the following tabular form: 
Oysters. 
1.0 c.c. 
0.1 c.c. 
0.01 c.c. 
Numerical Value. 
1 
+ 
+ 
0 
10 
2 
+ 
+ 
0 
10 
3 
+ 
0 
0 
1 
4 
+ 
0 
0 
1 
5 
+ 
0 
0 
1 
Total, or rai 
ing for B. coli.. 
23 
RESULTS OF TESTS FOR B. COLI IN DILUTIONS INDICATED 
+ = Presence of bacteria of the B. coli group in fermentation tube test with lactose bile. 
0= Failure to demonstrate presence of bacteria of the B. coli group. 
Estimated Number of B. coli per Cubic Centimeter. If the stand- 
ard B. coli rating above described is divided by 5 or, in general, if the 
rating is divided by the number of oysters tested, the result will be 
approximately the number of B. coli per cubic centimeter of shell water. 
Partly because it does not do this exactly, but also for simplicity 
and the avoidance of fractions, the method of stating results as an 
aibitrary rating is preferred by the committee. Practical experience 
with the method also has appeared to justify this preference. 
Illustrations of the Application of the Method of Rating Oysters 
for B. coli. Sometimes results similar to the following are obtained, 
that is, one or more oysters may show positive results in small quan- 
tities of shell water, while an equal number may show negative results 
in larger quantities. In this case the next lower numerical value shall 
be given to the positive results in the high dilutions, and such positive 
results shall be considered as being transferred to a lower dilution giving 
negative results in another oyster. This is done on the theory that 
inconsistent results, mathematically considered, may follow naturally 
from an unequal distribution of the bacteria in the shell water. This 
recession of the assigned values, however, shall not be carried beyond the 
point where the number of such recessions is greater than the number of 
instances where other oysters in the series failed to give positive B. coli 
results. 
As examples of the method of obtaining the rating for B. coli, the 
following illustrations are given. They represent results that may be 
met in practice. 488 
MEAT AND MEAT PRODUCTS 
CASE A-RESULTS OF B. COLI TESTS IN DILUTIONS INDICATED 
Oysters. 
1.0 c.c. 
0.1 c.c, 
0.01'c.c. 
Numerical Value. 
1 
+ 
+ 
0 
10 
2 
+ 
+ 
0 
10 
3 
+ 
+ 
0 
10 
4 
+ 
0 
0 
10 (not 1) 
5 
+ 
+ 
+ 
10 (not 100) 
50 = Rating 
CASE B.-RESULTS OF B. COLI TESTS IN DILUTIONS INDICATED 
Oysters. 
1.0 c.c. 
0.1 c.c. 
0.01 c.c. 
Numerical Value. 
1 
+ 
+ 
+ 
10 (not 100) 
2 
+ 
+ 
+ 
10 (not 100) 
3 
+ 
0 
0 
1 
4 
0 
0 . 
0 
1 (not 0) 
5 
0 
0 
0 
1 (not 0) 
23 = Rating 
CASE C.-RESULTS OF B. COLI TESTS IN DILUTIONS INDICATED 
Oysters. 
1.0 c.c. 
0.1 c.c. 
0.01 c.c. 
Numerical Value. 
1 
+ 
+ 
0 
10 
2 
+ 
+ 
0 
10 
3 
+ 
+ 
+ 
100 
4 
+ 
+ 
+ 
10 (not 100) 
5 
+ 
0 
0 
10 (not 1) 
140 = Rating 
Oysters Removed from the Shell (Opened or Shucked Stock). 
Except as hereinafter stated, all the procedures and requirements for 
the examination of opened oysters, i.e., shucked stock, shall be those 
specified for the examination of oysters in the shell. 
Selection and Preparation of Sample. The stock in the container 
from which the sample is to be taken shall be thoroughly mixed, and 
one or more wide-mouthed sterile jars of a total capacity of one quart 
shall be each half filled with the sample by means of a clean ladle or 
other instrument sterilized by flaming alcohol. The jar or jars shall be 
so sealed as to exclude all possibility of contamination from without. EXAMINATION OF SHELL FISH] 
489 
Transportation of Samples. When the time between the collection 
of the sample and its examination exceeds three hours, or, if the outside 
temperature is above 50° F., the sample shall be thoroughly refrigerated 
by means of ice placed around, but not in, the sample jars. 
Technical Procedure. The bacteriological examination shall be 
begun as soon as possible after taking the sample. The sample shall be 
thoroughly shaken at least twenty-five times immediately before 
opening. 
Bacterial Counts. The procedure specified for oysters in the shell 
be followed: 
Determination of Bacteria of the Bacillus coli Group. The pro- 
cedure specified for oysters in the shell shall be followed, but attention is 
called to the fact that higher dilutions than 1/100 c.c. are usually 
required. Triplicate fermentation tubes shall be inoculated from each 
dilution of the sample. 
Statement of Results. The results of the bacteriological examina- 
tion of the opened oysters, or shucked stock, shall be expressed in the 
same way as that specified for oysters in the shell, except that in the 
calculation of the B. coli rating the values for the results of the positive 
fermentation tests after confirmation shall be recorded for each of the 
inoculations of each dilution. In order that the rating from these 
triplicate tests may be compared with that obtained from testing five 
oysters in the shell, the sum of the values for the triplicate tests shall be 
multiplied by 5/3. If, instead, the sum is divided by 3, the result will 
give approximately the number of B. coli per cubic centimeter. 
Clams and other Shell Fish 
The methods for examining clams and shell fish, other than oysters, 
shall be those given above. Certain modifications are necessary in 
the method of handling the samples and the opening of the shells, etc. 
Clams are more likely to lose water during transportation than 
oysters. It is, therefore, necessary to take greater precautions to sep- 
arate different samples of clams from each other than in the case of 
oysters. 
In opening soft clams it has been found that if two incisions are 
made through the mantle the shell water may be poured out without 
opening the shell. 
Hard clams are more difficult to open, but if the shell be struck over 
the dorsal muscle with a small hammer an opening will be formed per- 
mitting the insertion of the knife to cut the muscle. 490 
MEAT AND MEAT PRODUCTS 
Sometimes clams and other shell fish contain too little liquor to make 
all of the tests above described. This is always the case when the 
shells are very small. Under these conditions the water from two or 
more shellfish shall be taken together and tested and considered 
as one. 
The English practice for the examination of oysters is somewhat 
different than that used in America. Savage (1914) has outlined the 
method which was used by Houston for the Royal Commission on 
Sewage Disposal, as follows: 
1. The outside of the oyster shells are well scrubbed with soap and 
water, and cleaned as thoroughly as possible in running tap-water, 
finally with sterile water. 
2. The hands of the investigator are thoroughly cleaned, washed in 
1 in 1000 corrosive sublimate solution, and finally with sterile water. 
3. The oysters are opened by a sterile knife held in position by a 
sterile cloth, and with the concave shell underneath. Great care must 
be taken to avoid any loss of the liquor. The liquor in the shell is 
poured into a 1000 c.c. cylinder, and the oyster and oyster liquor are 
added after the oyster has been cut into small pieces by sterile scissors. 
4. Ten oysters are treated as above in each experiment. 
5. The volume of oysters plus oyster liquor is read off, and usually 
varies between 80 and 120 c.c. For qualitative work 100 c.c. may, 
therefore, be taken as a fair average of the total shell contents of ten 
oysters. 
Sterile water is then poured into the cylinder up to the 1000 c.c. 
mark and the whole well stirred with a sterile rod. Each 100 c.c. of 
this liquor may be considered to contain the bacteria in one oyster. 
The Precipitin Test for the Detection of Foreign Protein in Meats. 
The food microbiologist may be called upon at any time to determine 
the presence or absence of a suspected protein in certain foods-horse or 
deer meat in sausage. The chemical methods for doing this are very 
indirect if not impossible. The bacteriological methods are very sen- 
sitive. 
Meat Extract. About 30 gms. of the meat under examination should 
be covered with 100 c.c. of sterile physiological salt solution. This is 
then allowed to stand over night in a refrigerator after which it should 
be filtered clear by any of the usual methods. Hiss and Zinsser (1914) 
advise that this should be adjusted to a neutral reaction and diluted 
with sterile water until only a slight even turbidity is formed by the 
addition of concentrated nitric acid. The less the amount of fat in the 
above protein solution, the easier will it filter. Before injection, the BIBLIOGRAPHY 
491 
protein solutions should be sterilized by passage through a stone filter 
in order not to cause an infection in the animal. 
Anti-protein Sera. These should be active in fairly high dilution 
and each serum must be specific for its protein. As many anti-sera 
will have to be made as kinds of meat suspected. Usually, however, 
the problem is simpler and involves the question of horse meat, in 
sausage, etc. Schneider (1915) gives the following for this examina- 
tion: 
Tube 1, 2 c.c. unknown extract (1-300)+0.1 c.c anti-horse serum. 
Tube 2, 2 c.c. unknown extract (1-300)+0.1 c.c. normal rabbit serum. 
Tube 3, 2 c.c. horse-flesh extract (1-300) +0.1 c.c. of anti-horse serum. 
Tube 4, 2 c.c. pork extract (1-300)+0.1 c.c. of anti-horse serum. 
Tube 5, 2 c.c. beef extract (1-300)+0.1 c.c. of anti-horse serum. 
Tube 6, 2 c.c. saline solution+0.1 c.c. of anti-horse serum. 
Schneider advises stratifying the immune sera in the tube. They 
should be kept at room temperature. If " tubes 1 and 3 show a clouding 
within five minutes and if a definite precipitate forms within thirty 
minutes, the other tubes remaining perfectly clear, the extract is prob- 
ably one of horse flesh or the flesh of some other single-toed animal." 
BIBLIOGRAPHY 
Bates, C. 1916. The Handling of Shucked Oysters. Amer. Jour. Public 
Health, 6, 987. 
Bates, C. 1916. Sanitary Control of Shellfish Industry. Amer. Jour. Pub. 
Health, 6, 453. 
Bates, C. and Round, L. 1916. A Comparison of Bacteriological Methods for 
the Examination of Oysters. Amer. Jour. Pub. Health, 6, 841. 
Beveridge, W. W. O. and Fawcus, H. B. 1908. Experiments on Canning 
Meat. London. Great Britain War Office, 57-72. 
Beveridge, W. W. O. 1908. Report on the Nature and Causes of the Black- 
ening of the Interior of Tins. Third report of the committee on physio- 
ological effects of food, training and clothing on the soldier. London. 
Great Britain War Office, 1908. Jour. Royal Army Medical Corps, 
13, 326-332. 
Bierotti and Machilda. 1910. Miinchener med. Wochenschr., 57, 636. 
Bushnell, L. D. and Utt, C. A. A. 1917. The Examination of Canned 
Salmon for Bacteria and Tin. Kansas State Board of Health, 13, 36-38. 
Jour. Ind. Eng. Chern., 12, 678. 
Cary, W. E. 1916. The Bacterial Examination of Sausages and its Sanitary 
Significance. Amer. Jour. Pub. Health 6, 124-135. 
Conn, H. W. 1894. 17th Annual Report of the Connecticut State Board of 
Health. 492 
MEAT AND MEAT PRODUCTS 
Conradi. 1909. A New Bacteriological Method for Meat Analysis. Zeit. 
Fleisch. u. Milchhygiene, 19, 341-345. See also Munch. med. Wochen- 
schr., 56, 1318-1320. 
Fowler, C. E. P. 1909. Bacteriological Report. Great Britain War Office, 
13, 323-325. 
Hindman, E. F. and Goodrich, F. J. 1917. A Study of Puget Sound Oysters. 
Amer. Food Jour., 12, 611-614. 
Hoagland, F., McBryde, C. N. and Powick, W. C. 1917. Changes in Fresh 
Beef During Cold Storage above Freezing. U. S. Department of Agricul- 
ture Bulletin 433. 
Howell, K. 1912. The Bacterial Contamination of Bread. Am. J. Pub. 
Health, 2, 321-324. 
Johnstone, J. 1909. Routine Methods of Shellfish Examination with 
Reference to Sewage Pollution. Jour. Hyg., 9, 412. 
Kossowicz, A. and Nassau, R. 1916. Contribution to Bacteriology and 
Technology of the Preservation of Meat. Wiener Tierartz. Wochenschr., 
3, 81-102. Chern. Zentr., 1916, II, 69. 
LeFevre, E. 1917. A Bacteriological Study of Hamburger Steak. Amer. 
Food Journal, 12, 140-142. 
MacManus, R. D. 1917. The Manufacture of Meat Food Products. Amer. 
Food Journal, 12, 559-563. 
McBryde, C. N. 1907. A Study of the Methods of Canning Meats with 
Reference to the Proper Disposal of Defective Cans. 24th Ann. Rep. 
Bureau of Animal Husbandry, U. S. Dept. Ag., 279-296. 
Marchland, H. 1909. The Cooking of Bread. Meun. Franc., 25, 208-210. 
Exp. Sta. Rec., 22 (1910), 64. 
Marxer. 1903. Beitrage zur Frage des Bakteriengehalts und der Halt- 
barkeit des Fleisches. Quoted from LeFevre, 1917. 
Maurel, E. 1911. The Occurrence of Microorganisms in the Interior of 
Meat, Potatoes, and Sausage. Comp. Rend. Soc. Biol., 70, 241-244. 
Exp. Sta. Rec., 25 (1911), 265. 
Melvin, A. D. 1906. The Federal Meat Inspection Service. 23d Ann. 
Report Bureau of Animal Industry, 65-100. 
Pennington, M. E. 1911. The Comparative Rate of Decomposition of 
Drawn and Undrawn Market Poultry. U. S. Dept. Ag. Bu. Animal 
Industry. Circular 70. 
Round, L. 1916. Comparative Bacteriological Examination of Oysters. 
Amer. Jour. Pub. Health, 6, 686. 
Roussel, J. 1907. The Survival of Pathogenic Bacteria in Bread after Baking. 
Rev. Intend. Mil., 20, 122-131. Chern. Abts., 2 (1908), 1168. 
Sadler, W. 1918. The Bacteriology of Swelled Canned Sardines. Amer. 
Jour. Pub. Health, 8, 216-220. 
Sawyer, W. A. 1914. Ninety-three Persons Infected by a Typhoid Carrier 
at a Public Dinner. Jour. Amer. Med. Assn., 63, 1537-1542. 
Smith, G. H. 1913. Size of the Sample Necessary for the Accurate Deter- BIBLIOGRAPHY 
493 
mination of the Sanitary Quality of Shell Oysters. Amer. Jour. Pub. 
Health, 3, 705. 
Stiles, G. W. 1911. Shellfish Contamination from Sewage Polluted Waters 
and from Other Sources. U. S. Department of Agriculture, Bureau of 
Chemistry Bull. 136. 
Stiles, C. W. 1917. What is Diseased Meat? Amer. Food. Jour., 12, 193- 
194. Jour. Amer. Med. Assn., 68, March 3, 1917. 
Weinzirl, J. and Newton, E. B. 1914. Bacteriological Analysis of Ham- 
burger Steak with Reference to Sanitary Conditions. Amer. Jour. Public 
Health, 4, 413-417. 
Weinzirl, J. and Newton, E. B. 1915. The Fate of Bacteria in Frozen Meat 
Meat Held in Cold Storage and its Bearing on a Bacteriological Standard 
for Condemnation. Amer. Jour. Public Health, 5, 833-835. 
Zwick and Weichel. The Presence of Bacteria in the Flesh of Normal 
Slaughtered Animals and the Technique of the Bacteriological Meat 
Examination in Forced Slaughter. Arb. kaiserlich. Gesundh., 3, 327-337. CHAPTER XIV 
FOOD PRESERVATION 
The relation of bacteria to foods may be considered from several 
viewpoints. With regard to the changes which they induce in foods 
they may be divided into two general classes-the desirable and the 
undesirable. These may be divided somewhat after the following 
manner: 
I. Desirable microorganisms: 
A. Bacteria 
(a) Fermented milks 
(b) Cheese .and butter 
(c) Pickles, sauerkraut, etc. 
(d) Ensilage 
(e) Industries, leather, retting, etc. 
B. Yeasts 
(a) Leavening agents 
(6) Wine and beer 
C. Molds 
(c) Cheese 
II. Undesirable Microorganisms: 
A. Those which cause spoilage 
(a) Fermentation, putrefaction 
B. Those which cause food poisoning 
The question of food spoilage may be discussed from a number of 
viewpoints. Some have taken up each special food such as milk, meat, 
etc., and discussed the types of change, organisms, etc., which are 
involved. More recently, however, Rahn (1913) has pointed out the 
possibility of discussing the large groups of food substances which 
undergo decomposition and not mentioning each particular food. For 
this purpose, he has -devised the following groups, which serve very well 
as a basis for a consideration of food spoilage: 
494 ASEPSIS 
495 
Group I. Proteins (Acids and carbohydrates in traces). 
Example: Meat, fish and eggs. 
Group II. Carbohydrates (Acids and proteins in traces). 
Example: Starch, sugar, honey. 
Group III. Proteins+Carbohydrates (Acid in traces). 
Example: Milk, cereals, flour, greens (cabbage), vege- 
tables. 
Group IV. Acids (Proteins and carbohydrates in traces). 
Example: Vinegar. 
Group V. Acid+Proteins (Carbohydrates in traces). 
Example: Hard cheese, sauerkraut. 
Group VI. Acid fl-Carbohydrate (Protein in traces). 
Example: Must. 
Group VII. Acid + Protein fl-Carbohydrate. 
Example: Sour milk, butter, fruit. 
Group VIII. Acids, carbohydrates and proteins in traces. 
Example: Water. 
As Rahn points out the type of decomposition in each group is quite 
sharply defined. The decomposition of foods in Group I is a typical 
putrefaction (Faulnis). The foods under Group II contain too little 
water to permit of microbial cleavage. Acid fermentations are the 
types of change for Group III. 
Food Preservation 
Asepsis. According to Rahn's system the following groups of food 
substances may be preserved by asepsis. 
Group I. Meat, fish, eggs. 
Group III. Milk, vegetables, nuts. 
Group VII. Fruits. 
By asepsis is meant the prevention of contamination of food sub- 
stances by undesirable microorganisms during their preparation. The 
following factors influence this method of food preservation. 
I. Conditions of production. 
II. Amount of moisture. 
III. Physical structure. 
• IV. Chemical composition. 
V. Temperature. 
Few foods are produced absolutely free from microorganisms. If 
such were the case, they would keep indefinitely. Since this is prac- 
tically impossible, it has become necessary to reduce to a minimum 496 
FOOD PRESERVATION 
unnecessary contamination during production. In this connection, it 
should be remembered that many foods contain bacteria in the tissue. 
This has been shown in other places in this book. Hoagland, McBryde 
and Powick (1917) found a micrococcus to be normally present within 
the tissues of fresh beef. Bacteria have also been found in tissues from 
other sources. 
The relation of environmental conditions during production to 
infection of foods needs no extended discussion. Many cases of food 
poisoning from meats have been traced back to infection during slaugh- 
ter. Other factors tend to render the aseptic production of meat im- 
practicable and impossible. Certified milk is the nearest approach to 
the production of aseptic milk. Water glass is used for preservation of 
eggs and this is probably a good example of aseptic preservation of foods. 
The humidity of the atmosphere in which foods are kept, is an im- 
portant determining factor. Water is necessary to carry food into the 
cell and remove the waste products from the cells of microorganisms. 
Spores will not germinate if there is insufficient moisture present. 
The physical structure of the food may determine the type of 
decomposition. Nature has provided many foods with efficient bar- 
riers against the attack of microorganisms. Unless these are broken in 
some way, the microorganisms are not able to accomplish any damage. 
Fruits, such as apples, cherries, pears, etc., are provided with a thick 
cellulose skin which is very resistant to the attack of microorganisms. 
Few of them possess a cellulase with which to attack this barrier. Eggs 
are protected by a shell which is covered with a gelatinous layer. If 
this is removed by washing, the egg will spoil much more quickly. 
In general, the conditions under which a food is produced greatly 
influence any method that may be adopted to preserve it. 
Spices. Condiments have probably been a little over-emphasized 
as food preservatives. Bitting (1909), in studying the preserving 
action of spices in ketchup found that cinnamon and cloves were the 
strongest antiseptics. He boiled 20 gms. of the whole spice in 200 c.c. 
of water for forty-five minutes. This was then filtered and from 0.1 
to 5 c.c. of the filtrate added to tomato bouillon. Mustard, paprika 
and cayenne pepper checked growth, but 5 c.c. of the highest strength 
did not inhibit growth. Bachmann (1916), using different technique, 
secured somewhat the same results. Using the oils, she found that 
cinnamic aldehyde was most effective in preventing the growth of all 
organisms studied. Eugenol and oil oi allspice had a distinct preserv- 
ative action. Molds were found to be more sensitive than the bac- 
teria or yeasts. FERMENTATION 
497 
The investigations carried out at the University of Wisconsin have 
given us much information on the use of spices as preservatives (Hoff- 
mann and Evans, 1911, Bachmann, 1916). Cinnamic aldehyde and 
eugenol were found to be especially important in preserving. Both of 
these are found in cinnamon and cloves and probably accounts for 
the preserving action of these condiments. In the later paper by Bach- 
mann a new method was used by which the plain and spice agar could 
be used in the same Petri dish. The results are quite similar to those 
presented in a former paper. Pepper and nutmeg were not able to 
retard growth of microorganisms. Cloves and allspice in large amounts 
were effective. In a later paper Bachmann (1918) reported that bac- 
teria, molds and yeasts show a marked sensitiveness to different brands 
of spice. The growth of microorganisms on a spiced medium may be 
taken as a criterion of the preservative value of the spice. 
Garlic was found to possess a prophylactic action by Minchin (1917) 
in diseases of diphtheria, typhus and typhoid. It would possibly 
possess the same action in food preservation. 
Fermentation. This process of preservation is limited to a certain 
few foods. The acids, principally lactic, are relied upon to suppress 
the growth of putrefactive bacteria. Brown (1916), in studying the 
brine pickle fermentation reports that many bacteria may enter the 
tank, but only those which can tolerate 12 to 20 per cent of salt are con- 
cerned in the normal fermentation. He found that the acidity of new 
brine is practically zero and gradually increases to 50 .per cent N/10 or 
above. Lactic and acetic acids predominate with a ratio of about 2:1. 
When the scum yeasts reduced the acid from the surface downward, the 
pickles were liable to decomposition. 
From the fact that lactic acid is so important in the keeping of 
pickles it may be wondered whether the salt is really necessary. Rahn 
(1913) has answered this: 
This question must doubtless be answered with yes. If no salt, or only 
little salt would be added the keeping quality of brine pickles would be like 
that of the dill pickles, or even less, since the spices of the dill pickles have a 
certain, though very small, disinfecting value. Salt retards all life activities 
of bacteria; it makes the fermentation go slower and it makes the acid destruc- 
tion by the scum yeast slower, too. The more salt the longer will the pickle 
keep. 
The fermentations involved in the preservation of sauerkraut are 
little different from those of pickles. According to Round (1916) 
bacteria are the important organisms concerned in the fermentation 498 
FOOD PRESERVATION 
and when air is allowed to come in contact with the brine or kraut, 
there is a vigorous growth of scum yeasts. Vats which showed an 
abnormal fermentation contained a different species of bacteria from 
the normal which got into the brine before the acid was high enough 
to prevent their growth. In a later paper (1917) Round states that 
the respiration of living tissue is important in gas production. Bacillus 
brassica: (Wehmer) and Bacillus cucumeris fermenlatte (Henneberg) 
represented the types which produced the greatest amounts of acid. 
Smoking. Smoke has been used for a long time to preserve foods, 
especially meats. It probably acts by closing the pores of the meat and 
thus preventing the entrance of bacteria. The smoke contains different 
volatile products which are deleterious to the organisms causing putre- 
faction. The creosote compounds are also objectionable to certain 
insects which might pass their larval stage on it. 
Smoke also imparts a flavor to meats which is desired. This is 
especially true with meats which have passed through a pickle. Resin- 
inous woods are never used since these impart an undesirable flavor to 
the meat. Green hickory is advised for smoking under domestic con- 
ditions or corn cobs may serve as fuel. Under commercial conditions, 
piles of hickory logs are burned under the meat (hams). Toward the 
end of the smoking period, hickory sawdust is thrown on to give the hams 
a brown color (MacManus, 1917). 
McBrydc (1911) has studied an economically important infection of 
hams known as ham souring. In advanced cases, the ham will possess 
a distinctly putrefactive odor while in the early stages the odor may 
be indefinite and a little " off." McBryde isolated an anaerobic bacillus 
(B. putrefacies) with which he satisfied the postulates of Koch. Since 
it is an anaerobic bacillus it develops in the deeper portions of the ham. 
The portions nearest the bone seem to be attacked first. 
Concentrated Solutions. The cell membrane should be regarded 
as a semi-permeable membrane and the passage of liquids through it 
is governed by the laws of osmosis and osmotic pressure. This is 
dependent upon the size of the molecule and type of substance in 
solution. The osmotic pressure is proportional to the number of 
molecules in solution. In salt and sugar solutions then, of equal con- 
centration by weight, the salt will exert a greater destructive action than 
the sugar which is present in a fewer number of molecules. 
Salt as a Preservative. Much more data are available in regard 
to the use of salt than carbohydrates. In this case it is used to prevent 
the growth of certain organisms and favor the growth of others. In 
early times salt served as currency and was too expensive to be used PRESERVATION BY SALTING 
499 
in food preservation. As methods for its manufacture were developed 
it became too abundant for currency and gradually was used for preser- 
vation of foods. Different reasons have been ascribed for the keeping 
power of salt. 
I. Exerted a poisonous action. 
II. Made the moisture unavailable for the microorganisms. 
III. Destroyed cells by plasmolysis. 
Rapin and Weigman (1913) found that salt contained bacteria. Salt 
which was kept in barrels had a high number of bacteria in the upper 
layers. Spore-forming varieties were found which would attack milk 
fat when this was salt used dairying. 
Peterson (1900) reported spine interesting data from which the 
change of species of microorganism with salt concentration may be 
noted. In low concentrations the flora is more heterogeneous. Between 
12 and 15 per cent concentration the rods are killed. Wild yeasts 
develop in 25 per cent of salt. These data were secured from studies on 
fish. (See Table 48.) 
Table XLVIII 
INFLUENCE OF SALT UPON THE BACTERIAL FLORA AND DECOM- 
POSITION OF FISH 
(Peterson, 1900) 
The figure indicates the day after salting when bacteria or chemical change was 
first noticed. 
Per 
Cent. 
Per 
Cent. 
Per 
Cent. 
Per 
Cent. 
Per 
Cent. 
Per 
Cent. 
Per 
Cent. 
Per 
Cent. 
Salt concentration 
5 
8 
10 
12 
15 
18 
20 
23 
Rods, appear 
2 
14 
23 
52 
Rods, plenty 
12 
29 
50 
75 
Cocci, appear 
2 
2 
2 
2 
14 
15 
50 
75 
Cocci, plenty 
2 
2 
6 
6 
34 
15 
Yeast, appear 
6 
14 
15 
50 
75 
Yeast, plenty 
14 
15 
Odor 
6 
8 
8 
12 
34 
75 
Alkaline reaction 
4 
6 
10 
12 
34 
69 
Butyric acid 
12 
23 
23 
12 
34 
75 
75 
Peptones 
10 
30 
58 
75 
75 
Ammonia 
7 
6 
10 
12 
h2s 
6 
14 
28 
58 
Indol 
16 
29 
68 
75 
Phenol 
55 
75 
The blank spaces indicate that change was not observed in seventy-five days, 500 
FOOD PRESERVATION 
De Freytag (1890) studied the effect of concentrated salt solutions 
on pathogenic bacteria. The brine used for preserving meat had the 
following effect on bacteria: 
In Salt-Concentrated-the Following Died: 
(After de Freytag) 
• 
B. anthracis After hours 
B. anthracis spores Not in 6 months 
B. typhosus After 5 months 
B. of hog erysipelas After 2 months 
Vibrio cholera; After 6-8 hours 
Strep, erysipelatis Not in 2 months 
Staph, pyogenes After 5 months 
B. tuberculosis Not in 3 months 
B. diphtheria; Not in 3 weeks 
(After Stadler) 
B. coli Not in 6 weeks 
B. enteriditis After 4| weeks 
B. morbificans bovis After 3 weeks 
B. proteus .' Not in 3 weeks 
B. typhosus Not in 6 weeks 
B. diphtherice ; Not in 4| weeks 
Staph, pyogenes Not in 6 weeks 
B. I act. aerogenes Not in 6 weeks 
B. pestis Not in 16 weeks 
Stadler (1899) found that Bad. coli commune, Bac. morbificans 
bovis, Bac. enteritidis, Bac. {proteus) vulgaris and Bacillus botulinus 
were inhibited in salt concentrations of 7-10 per cent. Fettick (1908) 
studied the relation of salt to butter preservation. His conclusions 
were drawn from counts. They were about the same on salted and 
unsalted butter. Inoculating pure cultured into sterile butter, several 
molds Bacillus fluorescens liquefaciens, and lactic acid bacteria grew at 
3 per cent concentration. Bacillus colon, Bacillus subtilis, Bacillus 
aerogens and some torulae multiplied at 6 per cent concentration. From 
these data, Fettick concludes that a concentration of about 3 per cent 
of salt should be used in butter. This will allow the lactic acid bacteria 
to grow but prevent the others. In connection with butter the data of 
Washburn and Dahlberg (1917) is interesting. They conclude that 
salt hastened the deterioration of the butter. They found little relation PRESERVATION BY SALTING 
501 
between the bacteria acidity and score. Rahn, Brown and Smith 
(1909) found that salted butter kept better than unsalted. Micro- 
organisms were found in slated butter which multiplied slowly at -6° C. 
Thom and Shaw (1915) found that 2.5 to 3 per cent of salt was necessary 
to keep down mold growth in butter. 
Giltner and Baker (1915) in studying the effect of salt on the flora 
in butter have given some information which might be considered in 
this place. They found that 12 per cent of salt does not retard growth 
in all cases and that some bacteria are able to withstand 20 per cent of 
salt. Streptococci were found to be sensitive to salt while staphylo- 
cocci and micrococci were not. The greater part of the yeasts and 
torulae in butter cannot withstand as much salt as the cocci. Eight per 
cent of salt was believed to limit the physiological activities of most 
bacteria. Continued cultivation of some organisms on salt agar in- 
creased the salt tolerance. 
Weichel (1910) studied the effect of salt on the bacteria which are 
concerned in food poisoning. He found that by inoculating broth con- 
taining various amounts of salt with B. enteritidis, B. paratyphi " B," 
B. aertryke, that the cultures lived for between 33-95 days. Extending 
this study to meat inoculated before pickling, he found that inoculated 
meat showed a complete reduction in bacteria in 12 to 19 per cent of 
salt only after 75 days. Ptomaines formed by bacteria were not de- 
stroyed by salting. In this connection Serkowski and Tomczak (1911) 
stated that 15 to 20 per cent of salt was required to be of prophylactic 
value with respect to bacteria which cause food poisoning. 
Lewandowsky (1904) observed a differential action of certain salts. 
A 25 per cent solution of sodium chloride was about as efficient as a 
30 per cent solution of potassium nitrate. The chlorides of potassium 
and sodium possessed about the same efficiency in solutions of equal 
molecular concentration. 
Bitting (1909) in studying spoilage of tomato ketchup tried the effect 
of salts. Increasing amounts were put into 100 c.c. portions of bouillon 
which were later inoculated with molds and yeasts. A 5-gm. solution 
had no effect on molds. A 30-gm. solution allowed no development 
of the mold. The yeast did not develop in a 15-gm. solution. These 
data are interesting in connection with the work of Wehmer and Petters- 
son (1900). Wehmer isolated a yeast from herring brine which was 
inhibited by 15 per cent of salt. Pettersson confirmed this during a 
study of fish brine. 
Thom (1914) inoculated Czapek's solution containing different 
amounts of salt with different cultures of penicillin. The results are 502 
FOOD PRESERVATION 
shown in Table XLIX. From this, as Thom has pointed out, it will be 
seen that the lower percentages of salt delayed growth but did not 
prevent it completely. At 15 per cent salt concentration the molds are 
inhibited. 
Table XLIX 
SHOWING THE EFFECTS OF DIFFERENT CONCENTRATIONS OF SALT 
ON THE DEVELOPMENT OF MOLDS 
Check. 
With 5 Per 
Cent Sodium 
Chloride. 
With 10 Per 
Cent Sodium 
Chloride. 
With 15 Per 
Cent Sodium 
Chloride. 
7 days. 
34 days. 
7 days. 
34 days. 
7 days. 
34 days. 
7 days. 
34 days. 
P. camembcrti 
0.8 
1 .0 
0.7 
1.0 
0.3 
0.6 
0.2 
0.8 
P. italicum 
0.8 
1.0 
0.3 
0.0 
0.4 
0.0 
0.1 
P. expansum 
0.9 
7.0 
0.8 
1.0 
0.5 
0.8 
0.1 
0.4 
P. roqueforti 
0.6 
1.0 
0.4 
0.7 
0 3 
0.6 
0.1 
P. commune 
0.8 
1.0 
0.7 
1.0 
0.1 
0.7 
0.1 
0.8 
P. chrysogenum 
0.8 
1.0 
0.9 
1.0 
0.4 
0.6 
0.3 
0.8 
P. viridicatum 
0.8 
1.0 
0.9 
1.0 
0.4 
0.8 
P. cit. glabrum 
0.9 
1.0 
0.8 
1.0 
0.3 
0.6 
0.2 
P. cyclopium 
0.8 
1.0 
0.6 
1.0 
0.4 
1.0 
0.2 
0.9 
P. solitum 
0.9 
1.0 
0.9 
1.0 
0.5 
0.9 
0.2 
0.8 
P. piscarium 
0.9 
1.0 
0.9 
1.0 
0.4 
1.0 
0.2 
0.9 
P. lanosum 
0.7 
1.0 
0.7 
1.0 
0 3 
0.6 
0.2 
0.8 
Tn recording observations 8 the normal colony on this medium as represented by the check cul- 
ture at its typical stage of development is recorded as 1.0. Less vigorous growths are recorded in 
tenths upon the observer's judgment. Germination of spores without further development of a 
colony is recorded arbitrarily as 0.1. (After Thom, 1914.) 
Karaffa-Karbutt (1912) found salt to possess weak bactericidal 
power. Saprophytic bacteria required a higher concentration than 
pathogenic bacteria. Eight to 9 per cent inhibited the colon groups 
while the pyogenic bacteria required 10 to 12 per cent. Many yeasts 
grew in a 25 per cent solution. Concentrated salt solutions kill vege- 
tative bacteria in two to three months but not spores. 
The preparation of cod which has been described by Bitting (1911) 
furnishes a good illustration of the use of salt for preserving meat. He 
reports that 25,000 long tons of salt are used in a year at Gloucester, 
Mass., alone. As soon as the fish are caught, they are cleaned, which 
consists in removing the head, viscera and backbone. They are then 
piled up in the hold and sprinkled with salt. About 1.5 bu. of salt 
may be used per 100 lbs. of fish. The water is drawn out of the fish and 
this brine runs to the bottom of the hold from which it is pumped out. PRESERVATION BY PICKLING 
503 
The fishermen often attempt to reduce the amount of water taken from 
the fish by using lesser amounts of salt so that the fish will weigh more 
in port. Souring may result. After the fish are on shore they may be 
further dried by means of salt. The amount of salt taken up by fish 
is given in the following table from Bitting. 
AMOUNT OF SALT TAKEN UP BY FISH PUT UP IN DIFFERENT FORMS 
(After Bitting, 1911) 
Salt, Per Cent. 
Moisture, Per Cent. 
Fish cakes 
18.9 
47.6 
Slack salted 
14.5 
37.2 
Export 
19.6 
25.6 
Shredded 
20.6 
46.2 
Reddening of Cod Fish. Bitting (1911) has discussed the bibliog- 
raphy of this subject. His own work on this question yielded a coccus 
and bacillus as the possible causes. He has reported the complete 
cultural characters of these organisms. Indications point to the 
infection of the fish during its preparation. Undoubtedly the organism 
is able to resist the action of salt. 
Pickling. This might be discussed under another heading. Pickles 
are usually combinations of sugar, salt and often molasses. They are 
usually used in such combinations and concentrations that they inhibit 
bacterial life but do not blot it out entirely. Salt acts as a dehydrating 
agent. One result of this is a hardening of the tissue. Sugar does not 
do this, and is, therefore, often used with salt. It imparts a sweet 
flavor to the meat. Hoagland (1908) regards the action of saltpeter 
in influencing the color of meat as due to the formation of no hemoglobin. 
Saltpeter is reduced within the meat to nitrites which, in turn, are 
changed to nitric oxide. This unites with the hemoglobin to give the 
characteristic red color. Saltpeter, which is used in many pickling 
solutions, probably exerts little effect on bacteria. 
Sugar. Carbohydrates lack some of the preserving action of the 
salts. Bitting (1909) also tried the effect of sugar in preserving tomato 
bouillon. No effect of sugar was noticed until the amount of sugar 
had reached 25 gms. per 100 c.c. of bouillon. With the molds studied, 
sirups were prepared up to 200 gms. of sugar per 100 c.c. of tomato 
bouillon. The growth became slower up to 170 gms. Above 170 gms. 
it required two months to get growth. Yeasts were not able to survive 
80-gm. solutions of sugar. 504 
FOOD PRESERVATION 
Drying. All forms of life demand moisture. Especially is this the 
case with the lower forms whose body structures are made up of about 
90 per cent water. Nature uses this method for preserving the cereals 
against attack of microorganisms. Crackers, if kept dry, may be pre- 
served indefinitely. Various meats are not susceptible to the attack 
of bacteria in the dry condition. 
According to Rahn's classification the following groups may be pre- 
served by drying: 
Group I. Protein foods. 
Group II. Carbohydrate foods. 
Group III. Proteins+carbohydrates. 
Group VII. Acid + protein + carbohydrates. 
Some of the food products coming under these groups do not lend 
themselves conveniently to evaporation or drying. Beattie and Gould 
(1917) gave an excellent treatise on the methods and apparatus used in 
the commercial application of drying to food preservation. 
Cold Storage. All microorganisms are very susceptible to changes 
in temperature. The great majority have their range of optimum tem- 
perature between 20° C. and 40° C. In any discussion of cold storage 
the psychrophilic bacteria are concerned. About all that may be said 
with regard to freezing is that it inhibits or delays bacterial development 
and exerts a selective action. Much data has been recorded in the lit- 
erature to prove this. Pictet and Joung (1884) soon after pure cultures 
were made possible, subjected many bacteria to low temperatures 
(-130° C.). Koch (1884) demonstrated that the cholera vibrio could 
resist -10° C. for ten hours. MacFayden (1900), using liquid air tem- 
peratures of -190° C., found that bacteria were resistant. Paul and 
Prall (1907) used the garnet method to study the effects of low tem- 
peratures on bacteria and the rate of death. For 100 days the number 
of bacteria remained constant at liquid air temperatures. Those which 
were kept at room temperature and. icebox temperature decreased 
rapidly. Sedgwick, Hamilton and Funk (1917) have shown that 
some bacteria remain alive longer in solutions at low temperatures 
which, at higher temperatures, are decidedly toxic. They assume 
" that the mechanical protection given bacteria from shearing ice crys- 
tals by solutes is important." Ruata (1918) has secured results in this 
connection which indicate that cold if applied long enough will destroy 
bacteria. He worked with dry cold at temperatures of -4° C. and 
-12° C. Tubes were withdrawn from the freezing environment and 
incubated on successive days. On the fourth day the number of col- 
onies which developed had decreased to from 3 to 49 while, on the second COLD STORAGE 
505 
day, thousands of colonies had appeared. After the fourth day none 
developed. This author believes that other investigators on this sub- 
ject have not allowed a sufficiently long contact with the cold. It is 
stated that even one cell on the interior could start a culture. 
Some difference is made with regard to whether the food is kept 
above or below freezing. Below freezing, growth and multiplication 
are probably impossible on account of lack of moisture. Above freezing, 
the moisture is available and multiplication takes place even though it 
is very slow. Just above freezing, it is possible for the putrefaction 
bacteria to grow. These may cause the formation of toxic substances. 
Cold storage is relied upon to save many foods. Some of these are 
produced in greater abundance in one season or are eaten many miles 
from the source of production. Refrigeration has made this possible. 
Mutton preserved by cold storage during shipment from Australia is 
now eaten in England. Beef from Argentina is shipped to many parts 
of the world. Burrows (1914) described a quarter of beef which had 
been kept frozen for eighteen years. Microscopic examination of the 
fibers showed a normal appearance. The meat was consumed with no 
signs of digestive disturbance. Burrows stated that the meat had 
been kept in a chamber in and out of which other beef was passing 
and regarded this as a factor in the preservation of eighteen-year beef. 
The bacteriological features concerned with cold storage have been 
studied by the Bureau of Chemistry of the U. S. Department of Agri- 
culture. Milk stored at freezing temperatures was found to contain 
many millions of bacteria at the end of a month. The chemical char- 
acteristics of the milk also underwent a detectable change. (Penning- 
ton, 1908). 
Wiley (1908) and his colleagues have secured much data with regard 
to the cold storage of eggs. They found that the flavor of the eggs 
began to deteriorate quickly. Certain constituents during continued 
cold storage were found to develop rosette crystals in the yolks. Eggs 
which were stored for a year decreased about 10 per cent in weight. 
With regard to poultry, these investigators found that undrawn poultry 
kept better than the drawn. It was stated that cleaning of the fowl 
should be done carefully or there was danger of serious infection of the 
muscle tissue. 
Pennington has studied the changes which take place in poultry 
during cold storage. Visible changes were found. The cold-stored 
poultry were found to contain appreciable numbers of bacteria in the 
tissue while the tissues of fresh fowls were sterile. In a paper published 
in 1917, Pennington reports the results of chemical, bacteriological 506 
FOOD PRESERVATION 
and histological studies made on poultry stored between temperatures 
of -9 and -13° C. below freezing. These birds were frozen hard. 
There was no loss in nutritive value but a change in flavor was detected 
after nine months. Chemical changes which were observed were 
thought to be due to enzymic rather than bacterial causes. The aerobic 
bacteria decreased in the muscle during storage as the period lengthened. 
Above freezing, different lots were stored at 29.3° C. at from 7.2 «to 
12.8° C. and at 0° C. Bacteria in muscles and skin increased at all 
three temperatures but the increase was smaller as the temperature 
approached 0° C. The amino and basic nitrogen increased at the 
expense of protein nitrogen. 
Pressure. Hite, Giddings and Weakley (1914) studied the effect 
of pressure on bacteria in canned fruits and vegetables. The bacteria 
which caused spoilage of sweet, ripe fruits could be destroyed by pres- 
sure. One hundred thousand pounds for ten minutes stopped the fer- 
mentation- of grape juice. Apple juice which was subjected to 60,000 
to 80,000 lbs. for thirty minutes remained sweet. These authors kept 
apple juice which had been subjected to 90,000 to 120,000 lbs. for five 
years. Inconsistent results, however, were secured with blackberries 
and raspberries. Tomatoes also gave such results. The work indi- 
cates that more study is necessary to overcome some of the difficulties 
of the process in its application to food preservation. Bridgman (1914) 
found that egg white became somewhat stiffened when it was exposed 
to 75,000 lbs. per square inch for thirty minutes at 20° C. Larson, 
Hartzell and Diehl (1918) found that a direct pressure of 6000 atmos- 
pheres would kill non-spore forming bacteria when applied for fourteen 
hours. It required 12,000 atmospheres for the same length of time to 
kill the spore formers. This has much significance in the sterilization 
of foods by this method since the spore formers cause much of the trouble 
in spoilage. In an atmosphere of CO2 50 atmospheres for 1.5 hours 
killed non-spore formers. 
Canning. Appert in 1802 first showed that foods could be pre- 
served if they were sterilized and kept in hermetically tight containers. 
This discovery stimulated the use of the process and it spread rapidly 
to various parts of the world. Bitting (1917) has shown the extent of 
the canning industry. For the year 1914, 103,765,923 cases of canned 
food products were put up. These were valued at 8258,798,036. He 
states that the United States is not only the largest producer but also 
the largest consumer of canned. foods. The future of this industry 
seems very bright and each day sees a new type of food preserved by 
this method. CANNING 
507 
Process of Canning. The following pages on this topic are taken 
from the various publications of Bitting (1917, 1915, 1912): 
Raw Materials. These must be of first-class quality in order to 
secure a high-grade canned product. The canning factory should be 
located near the point of production in order to have the produce 
delivered in fresh and unharmed condition. Corn and peas lose their 
fresh appearance soon after harvesting. Beans become tough and 
stringy. The same may be said with regard to the canned fish industry. 
A recent type of fish cannery is constructed on a boat which is floated 
to the fishing grounds. 
Preparation. The procedure in this case depends solely on the 
product for obviously, the operations will depend on the food to be 
canned. As the produce is brought to the cannery it is roughly graded 
according to size, quality or maturity. These are handled separately. 
Some fruits need no more preparation than picking out foreign matter 
and effective material. Others need peeling, pitting, coring and sizing. 
Certain vegetables, on the other hand, require more preparation than 
the fruits. Peas must be threshed from the vines, corn cut from the 
cob, and string beans snipped and stringed. Most of this work is done 
by machinery. 
Grading. This is done to secure a uniformity of the product. 
Bitting regards this as having been carried to excess when it is realized 
that there are ten or twelve grades of peas, fifteen to eighteen grades of 
peaches and ten grades of cherries. Grading for size is purely mechan- 
ical. The produce, when possible, is sifted through perforated cylinders, 
screens, vibrating rolls, etc. 
Washing. This is a thorough process in industrial canning. The 
packing of peas requires nearly 1 gal. of water for each can. 
Blanching. This is carried out for several reasons, depending upon 
the product. The object is not to whiten as the term would indicate. 
It may be to remove the sticky or gelatinous coating from beans, peas, 
etc., or to make more flexible and of uniform color as with cherries. 
Filling the Cans. Whether this is done by hand or machinery 
depends upon the product. Material like peas, squash, corn, beans, and 
hominy are put into the cans by machinery while such fruits as pears, 
peaches, must be put into the cans by hand in order to secure uniform 
filling of the cans. 
Siruping and Brining. Most canned foods have a sirup or brine 
added. The lowest grade products are packed in water. This brine 
may be either salt, sugar or a mixture of both depending upon the 
product. 508 
FOOD PRESERVATION 
Green Corn 
Storage Shed 
| Wagon~j 
Dump 
Fact ory & Hr sking Roo tu 
E31 ~ Tiean Corn [Conveyor | | 
Husking Machines 
... J>«8ecti ig. Floor. 
Distributin Conveyor Ot er 
1 
Scale I 
Platform 
Office 
Track 
Track 
Track 
Fig. 79.-Plan of Modern Corn Packing Plant. 
(Courtesy of the Sprague Canning Machine Co.) 
Warehouse 
Cob 
Bin 
^C"b ('onvevur I 
C itters 
Cooling Tank 
Engine 
Room 
Cook 
Room 
Boiler 
Room CANNING 
509 
Exhausting. The can is heated until hot in order to drive out as 
much air as possible. This is not done with produce which is canned 
hot or which receives a hot brine. 
Closing the Cans. This is now done mostly by machinery whether 
the cans are soldered or crimped. 
Processing. This is the final treatment given the cans and is done 
soon after the cans are capped. This may be done either below or 
above the boiling-point. The desired temperature is maintained either 
by hot water or an atmosphere of steam. After the " process " the 
cans must be cooled else the products will be over cooked. This is 
probably one of the most important steps in the canning procedure. 
Much loss is often caused by errors at this point. It is often necessary 
to make the " process " a subject of careful study because the length 
must often vary even from day to day during the packing of the same 
goods. 
Furthermore, it is known that the process is dependent upon the 
character of the contents of the cans. Acid products like strawberries 
require a lower process than a product like corn or beans. The thermal 
death point of microorganisms is lowered by the presence of acid. Bit- 
ting and Bitting (1917) have studied the heat penetration of cans and 
found a great variation with the character of the food. The tempera- 
ture of a No. 3 can filled with water raised from 85° F. to 210° F. in four 
minutes. Sweet potatoes gave different results and were decidedly 
more sluggish in their change of temperature. 
The Container. In commerce tin cans are almost exclusively used 
although paper cans have a promising outlook. Many shapes and 
varieties are now used. Bitting makes the following statement with 
regard to this: 
First, the cans were made to utilize a standard sheet of metal with the min- 
imum of waste by the method of can making in vogue at the time. This resulted 
in cans of arbitrary volume, bearing no definite relation to standards of volume, 
like the pint, quart or gallon and unfortunately they did not bear a close rela- 
tionship to the quantity that would be consumed at a single meal by an average 
family. A size of package was started, however, which has persisted because 
of a fixture in trade and the expense in changing machinery, cans, shipping 
cases, etc. Secondly, was the introduction of sizes to fit a given weight of a 
certain product. This has been particularly true of meat products, as 4, 8, 12, 
and 16-ounce cans. The sizes that will hold these weights of a ground meat 
will not hold the same quantity if cereal be added or if large cuts or pieces be 
used in place of the finely ground product. Thirdly, is the attempt to make 
cans that will hold a quantity of a given article to retail at a popular price, like 510 
FOOD PRESERVATION 
10 cents. This applies to soup, beans, etc. Table L gives the sizes and vol 
umes of the standard cans. 
Table L 
SHOWING THE SIZES AND CAPACITIES OF STANDARD CANS 
(After Bitting) 
Number. 
Diameter in Inches. 
Height in Inches. 
Capacity in Ounces. 
1 
2H 
4 
12 
2 
3 A 
4A 
22.2 
21 
4A 
42 
32.6 
3 
41 
4 7 
36.4 
10 
6A 
7 
116.1 
Under domestic conditions, glass cans have been much used. Great 
care, however, has to be used to secure a tight seal, which is often ren- 
dered difficult on account of poor rubber gaskets. 
Swells. According to Bigelow (1914) a " swell " is a can which 
has undergone decomposition by microorganisms accompanied by 
generation of gas, which first releases the vacuum and then causes 
pressure in the can. This decomposition is often of putrefactive nature 
and may be rapid or slow, according to the organisms and temperature. 
They are caused by different factors. Insufficient sterilization or leaky 
cans are evident causes. One leaky can may infect other cans in a 
similar condition. 
Baker (1912) has given some attention to the composition of gases 
occurring in commercial canned foods. Some of the analyses given 
below are taken from his paper: 
Can of red raspberries, eighteen months old: 
Per Cent. 
Carbon dioxide 8.40 
Oxygen 00 
Hydrogen 65.50 
Nitrogen 26.10 
Can of strawberries, eighteen months old: 
Per Cent. 
Carbon dioxide 12.60 
Oxygen 00 
Hydrogen 72.40 
Nitrogen 15.00 EXAMINATION OF CANNED FOODS 
511 
Baker states that the analyses of probably 100 samples of gas from 
sound cans has never shown any oxygen. He attributes this to the 
oxidation of tin and iron salts, and combination with nascent hydrogen. 
Springers. Bigelow (1914) defines a springer asa " can whose ends 
are more or less bulged, owing to pressure from hydrogen generated as a 
result of the chemical action of the contents on the metal of the con- 
tainer or because the can was overfilled or insufficiently exhausted." 
Cans with bulged ends may be caused by expansion due to a rise in 
temperature. These will soon return to normal shape when the tem- 
perature is lowered. Baker (1912) states that the gases in the head 
spaces of springers are never more than three; carbon dioxide, nitrogen 
and hydrogen. Often hydrogen is absent and oxygen is rarely found. 
Flat Sour. This is a general term and may be used to cover many 
abnormalities of canned goods. Flat sours contain no gas and since 
there is nothing to indicate any abnormal condition when looking at a 
can, their discovery is delayed until the can is opened. 
Examination of Canned Foods 
The examination of canned foods is accompanied with danger unless 
the examiner has had experience in interpreting his data, both chem- 
ical and bacteriological. Bigelow (1917) has pointed out this side of 
the question very strikingly. The methods which are used are quite 
similar to those used in other branches of bacteriology. The objects 
of the examination may be varied. Quite often it is to determine the 
sterility of certain foods in order to check up the process of manufacture; 
or the quality of raw materials may be the object. The examination 
of canned foods presents the same difficulty that Pennington has men- 
tioned with regard to the examination of eggs: the examination of one 
can or one egg may not yield information with regard to other cans or 
eggs. Each must be regarded as a sealed package and when once 
opened may be brought back to the original condition with a little 
difficulty. 
The bacteriological examination of canned foods has been fully 
discussed by Bitting and Bitting (1917). Some of the discussion below 
has been taken from that paper. 
The first step in the examination is to observe the appearance of the 
can. Rusty cans suggest the possibility of perforations. The cans 
should be without dents and with slightly concave or flat ends. In 
certain- cases bulged ends may be due to springers, which will go down 512 
FOOD PRESERVATION 
a little after the can is cooled. Bitting points out that incubation at 
37° C. and at from 50 to 55° C. for fifteen days should precede the bac- 
terial examination. 
The second step, is the opening of the can, which must be carried 
out under very carefully controlled conditions. The can should be 
thoroughly sterilized about the place for opening either by flaming or 
covering with alcohol and allowing it to burn off. The can may be 
opened by cutting around the top with a sterile can opener or a hole 
put through the top by a sterile awl. The contents for examination 
may then be removed by means of a sterile loop or a sterile pipette. 
When the hole is punched into the can a flame should be played on the 
spot so that no bacteria will be sucked into the can with the air. The 
can should be entirely opened, later as soon as possible, in order to deter- 
mine the appearance of the contents. 
The third step, is concerned with the actual microscopical examina- 
tion. This should be direct as well as cultural. The direct methods 
of examination have been sufficiently discussed elsewhere in this book. 
The cultural methods require special media such as corn, agar, tomato 
bouillon, etc., depending upon the substances which are being exam- 
ined. Such media create for the organisms which are searched for, an 
environment as much as possible like the normal. Both aerobic and 
anaerobic methods of culture should be used. It is often advisable to 
incubate the cultures in a vacuum, to come more nearly to the conditions 
inside of the can. 
Organisms in Canned Foods. These have not received very extended 
study. Much of our knowledge must come from analogy and there 
is some danger in reasoning thus. In relatively few instances has time 
been taken to identify the various varieties of microorganisms. Bur- 
gess (1912) isolated and identified about 12 organisms from " flat sour " 
corn. Among others, he found B. coli, B. acidi lactici, Ps. syncyanea, 
B. megatherium and B. vulgatus. The first work on the bacteriology of 
canned foods in this country was done by Russell (1895). He exam- 
ined swelled canned peas. He isolated two organisms, one of which 
was a copious gas producer. Prescott (and Underwood, 1897, 1898, 
1900), gave this subject quite a little attention. They did much to 
establish the close relationship of bacteria to the spoilage of canned 
foods. The importance of the canned food industry should stimulate 
the examination of these foods. 
The author has found spore-forming facultative anaerobes to be 
common. In a few cases vegetative cells have been isolated. In one 
case mushrooms packed in brine of the following composition-5 lbs. EXAMINATION OF CANNED GOODS 
513 
salt, 1 lb. citric acid, 12| gals, of water, developed swelled cans. Bac- 
teria with the following group numbers were isolated. 
Bacillus 111.2232033 
Bacillus 111.2232032 
Bacillus 111.2222022 
Bacillus 222.1112031 
Bacillus 222.1212032 
The growth of such organisms in the finished products probably 
indicates insufficient sterilization. The gas-forming bacteria as indi- 
cated by the above group numbers are members of the colon group. 
These, undoubtedly, got onto the mushrooms during growth, since 
they are usually grown on heavily manured areas. 
Examination of Cans for Leaks. Several ingenious methods have 
been proposed. 
1. One which is widely used is to tap the end of the can. A sound 
can with a vacuum will give a characteristic ring while one which is a 
leaker with no vacuum will give a dead sound. The examination of a 
few cans of both types will show the difference. 
2. The suspected cans may also be examined for leaks by plunging 
them into hot water. If leaks are present, bubbles will be given off. 
3. The Meade tester may be used. This subjects the suspected can 
under water to a vacuum. If the can is a leaker, a stream of bubbles 
will be given off from it. 
Storage of Food in Tin Cans. It has become almost an instinct for 
people to immediately take food out of a tin can after it has been opened. 
When the housewife is asked why she does this, various reasons are 
given. One which has often been given is that " ptomaine " poisoning 
will result. Bigelow (1918) has given some definite information on this 
subject. He regards the storage of food in the tin cans after part has 
been removed as no more dangerous than storage in containers made of 
other material, since it is becoming more and more known that food 
" poisoning " is really an infection. Bigelow determined the tin 
content in progressive samples from the same tin of food and it is inter- 
esting to note that after fermentation had set in (pineapple) larger 
amounts of tin were dissolved. Bigelow considers that the only reasons 
which make it advisable to remove food from opened cans are appear- 
ance and economy of space secured by putting the food into a smaller 
container, 514 
FOOD PRESERVATION 
Tomato Products 
The canning of tomato products is often beset with many pitfalls 
for the canner. The chemical constitution of the tomato renders it 
liable to attack from microorganisms. The following table is prepared 
from some data reported by Bigelow and Fitzgerald (1914). 
Table LI 
COMPOSITION OF TOMATO PULP 
(Bigelow and Fitzgerald) 
Sample 
No. 
Description of Sample. 
Specific Gravity. 
Total 
Solids. 
Per Cent. 
Salt. Free 
Solids.. 
Per Cent. 
Insoluble 
Solids. 
Per Cent. 
Salt Free 
Soluble 
Solids. 
Per Cent. 
68° F. 
68 
203° F. 
203 
893 
701 
Peeled tomatoes 
concentrated.. . 
Trimming stock 
concentrated.. . 
not 
not 
4.47 
4.53 
4.42 
4.43 
0.56 
0.50 
3.86 
3.90 
1048 
Whole tomatoes.. 
5.57 
5.53 
1.12 
4.41 
1051 
Whole tomatoes.. 
6.55 
6.43 
0.75 
5.68 
' 
Sample 
No. 
Description of Sample. 
Sugar. 
Per Cent. 
Acid as 
Citric. 
Per Cent. 
Salt. 
Per Cent. 
Undeter- 
mined. 
Per Cent. 
Salt Per 
Cent of 
Total 
Solids. 
Insoluble 
Solids. 
Per Cent 
of Total 
Solids. 
893 
701 
Peeled tomatoes 
concentrated.. . 
Trimming stock 
concentrated.. . 
not 
not 
2.26 
2.36 
0.40 
0.30 
0.05 
0.10 
1.20 
1.04 
1.2 
2.4 
12.50 
11.00 
1048 
Whole tomatoes.. 
.... 
3.55 
0.35 
0.04 
0.51 
0.7 
20.10 
1051 
Whole tomatoes.. 
.... 
3.96 
0 37 
0.12 
1.35 
1.80 
11.50 
Ketchup. This is one of the most important tomato products 
and one which has received much attention with regard to the enforce- 
ment of the Food and Drugs Act. It is made in the following way: 
Clean, ripe tomatoes are thoroughly washed and pulped. The skins, 
seeds, etc., are removed after which the pulp is concentrated. To this 
are then added the condiments to taste. The ketchup is then sieved 
and bottled. After the bottles are sealed, they may be processed to 
insure sterility. 
Much trouble has been experienced in the control of this industry 
from the standpoint of the Food and Drugs Act. Undoubtedly, in Yearbook U. S. Dept, of Agriculture, 1911. 
PLATE 9 
Fig. 1.-Spores and Fragments of Filaments of Mold from Decaying Sweet Pepper 
(X150) 
The same or an allied species is one cause of " dry rot " in tomatoes. 
Fig. 2.-Yeasts and Spherical Bacteria 
from Decaying Tomatoes (X500). 
The oval bodies are the yeasts, some in budding 
stage; the bacteria appear as small spheres, 
or pairs of spheres. 
Fig. 3.-Rod-shaped Bacteria from 
Tomato Pulp, Common in Bad 
Ketchups (X500). 
f o face page 51b. EXAMINATION OF CANNED PRODUCTS 
515 
former times, many unscrupulous manufacturers used spoiled fruit, 
and by-products from the tomato canning industry for making ketchup. 
Satisfactory methods for separating good and bad ketchups have been 
hard to find. In general, a bad or low-grade ketchup is one which has 
been made from decayed or partly decayed fruit. The methods which 
are now used are microscopical and center around determining the num- 
bers of bacteria, yeasts and molds. Bitting (1909) used superficial 
methods for studying spoilage of tomato ketchup. He found that 
ketchup prepared from whole ripe stock had a low content of microor- 
ganisms and in that prepared from decayed fruit large numbers of micro- 
organisms were present. 
Howard (1911) proposed a method for the examination of ketchup 
which has been in use since then probably because no one has proposed 
a better one to replace it. This method is based on examining the 
tomato product for yeasts, molds and bacteria, and it is thus assumed 
that these microorganisms indicate that decayed fruit was used or that 
the method of manufacture is faulty. This latter possibility was found 
to be the case in an instance quoted by Howard (1917). A manufac- 
turer was using ripe fresh tomatoes and still secured high counts in the 
final product. 
Howard's Method for the Microanalysis of Tomato Products 
and Interpretation of the Results 
Apparatus Required. The outfit used is as follows: 
A good compound microscope giving magnifications of approxi- 
mately 90, 180, and 500 diameters. This is accomplished by the use 
of a 16 mm. (two-thirds of an inch) objective and an 8 mm. (one-third 
of an inch) objective, together with a medium (X6 compensating) and 
also a high-power ocular (X18 compensating). A Thoma-Zeiss blood- 
counting cell,* a 50-c.c. graduated cylinder, and ordinary slides and 
cover glasses complete the apparatus required. It is impractical to 
use objectives of a higher power than those mentioned, because of their 
short working distance, which makes their use with the counting cell 
inpossible. 
Estimation of Molds. A drop of the product to be examined is 
placed on a microscope slide and a cover glass is placed over it and 
* This is a cell named after the designer of the form of rulings used, and consists 
of a slide with a disk ruled in mm. squares, so arranged that when the cover is 
in place the film of liquid under examination is mm. deep. They were originally 
intended for counting corpuscles in the blood and are obtainable from practically 
all manufacturers of microscopic accessories. 516 
FOOD PRESERVATION 
pressed down till a film of the product about 0.1 mm. thick is obtained. 
After some experience this can be done fairly well. A film much thicker 
than this is too dense to be examined successfully, while a much thinner 
film necessitates pressing the liquids out, which gives a very uneven- 
appearing preparation. When a satisfactory mount has been obtained, 
it is placed under the microscope and examined. The power used is 
about 90 diameters, and such that the area of substance actually exam- 
ined in each field of view is approximately 1.5 sq. mm. 
A field is examined for the presence or absence of mold filaments, 
the result noted, and the slide moved so as to bring an entirely new 
field into view. This is repeated till approximately 50 fields have 
been examined, and the percentage of fields showing molds present 
is then calculated. Our experience has demonstrated that for home- 
made ketchups this is practically zero, and with some manufactured 
ketchup it is as low as from 2 to 5 per cent, while for carelessly made 
products it may be 100 per cent; that is, every field would show the 
presence of mold. Investigations under factory conditions clearly 
indicate that with only reasonable care the proportion of fields having 
molds can be kept below 25 per cent. A specimen in which 60 per cent 
of the fields have molds is in more than twice as bad a condition as one 
containing 30 per cent. 
After the percentage reaches 30 to 40 per cent it will be found that 
some of the fields frequently have more than one filament or clump of 
mold, and the number of such fragments might be counted, but in this 
laboratory this usually is not done. A Thoma-Zeiss counting cell with 
a center disk of 0.75 in. instead of 0.25 in., as usually furnished, would 
give a regular depth of liquid and would be more exact than the method 
described, but this must be specially manufactured, not being listed 
in any of the catalogues of microscopic supplies, and the method as given 
is sufficiently accurate for the purpose. When the number of fragments 
of mold per cubic centimeter is estimated, it has been found to range 
from virtually zero to over 20,000. There is no excuse for a manufac- 
turer allowing such conditions to prevail that his ketchup shows more 
than 2000 per cubic centimeter, while some manufacturers by careful 
handling hold it down to 150. 
Estimation of Yeasts and Spores. Though the spores referred to 
are those coming from molds and correspond to seeds in more highly 
developed plants, it is frequently very difficult to differentiate some of 
them with certainty from some yeasts without making cultures, which is 
obviously impossible in a product that has been sterilized by heat. 
For this reason the yeasts and spores have been reported together, and Yearbook U. S. Dept, of Agricultuie, 1911. 
PLATE 10 
Fig. 1.-Normal Tomato Ketchup, Showing Cells and Granular Contents 
(X200). 
Fig. 2-Mold Filament from Ketchup Made from Partially Decayed Stock 
(X150). 
To face page 516. EXAMINATION OF TOMATO PRODUCTS 
517 
if there seemed to be a larger percentage of the latter, mention was made 
of that fact. 
To make a count 10 c.c. of the product is thoroughly mixed with 
20 c.c. of water, and, after being allowed to rest for a moment to permit 
the very coarsest particles to settle out, a small drop is placed on the 
central disk of the Thoma-Zeiss counting cell and then covered with 
a glass. Care must be exercised to have the slide perfectly clean, so 
that, when the cover glass is put in place, a series of Newton's rings * 
results from the perfect contact of the glass surfaces; and furthermore, 
the drop should be of such size as not to overrun the moat around the 
central disk and creep in underneath the cover glass, thus interfering 
with the contact. 
With the magnification of 180, it has been the practice in this 
laboratory to count the number of yeasts and spores on one-half of 
the ruled squares on the disk. With the dilution used this calculates 
back to a volume equal to one-sixtieth of a cubic millimeter in the 
original sample, and reports are made on that basis rather than on 
the number in a cubic centimeter, because the former number is more 
readily grasped by the mind and affords a simpler notation. To 
obtain the numbers per cubic centimeter the count made is simply 
multiplied by 60,000. 
It has been found in practice that the number of yeasts and spores 
varies, for one-sixtieth of a cubic millimeter, from practically none in 
homemade and first-class commercial ketchups up to 100 or 200, and 
in one sample the number was as high as 1200. Laboratory experiments 
show that, when the number of yeasts in raw pulp reaches from 30 to 35 
in one-sixtieth of a cubic millimeter the spoilage may frequently be 
detectable by an expert by odor or taste, and from experiments made 
under proper factory conditions, it seems perfectly feasible to keep the 
number in commercial ketchups below 25. 
Estimation of Bacteria. The bacteria are estimated from the same 
mounted sample as that used for the yeasts and spores. A power of 
about 500, obtained by using a high-power ocular, is employed in this 
case, and because of the greater number present a smaller area is counted 
over. Usually the number in several areas, each consisting of five of the 
small-sized squares, is counted and the number of organisms per cubic 
centimeter is calculated by multiplying the average number in these 
areas by 2,400,000. Thus far it has proved impracticable to count the 
micrococci present, as they are likely to be confused with other bodies 
* These are rainbow-colored rings produced at the point of contact when polished 
plates of glass are pressed against each other. 518 
FOOD PRESERVATION 
frequently present in such products, such as particles of clay, etc. A 
comparison of this method with the ordinary cultural methods on 
samples in which the organisms had not been killed has almost invari- 
ably shown that the one used gives too low instead of too high results. 
In some cases it was found to give not more than one-third of the entire 
number present. The estimates of the laboratory on this point may, 
therefore, be considered very conservative. 
As regards the limits which may be expected in the examination of 
ketchups for bacteria, it might be stated that some manufactured 
samples as well as good, clean products made by household methods, 
have been examined and the count found to be so low when estimated 
by this method that the numbers present were reported as negligible. 
In other words, it was found that for the areas counted over the number 
of bacteria averaged less than one-that is, less than 2,400,000 per cubic 
centimeter. It is unusual, however, for the final number per cubic 
centimeter to be less than from 2,000,000 to 10,000,000 organisms. 
Contrasted with this number as a minimum, it has been found that the 
number has occasionally exceeded 300,000,000 per cubic centimeter. 
Such a number as this would indicate extremely bad conditions and 
carelessness in handling, as the studies of factory conditions have shown 
that there is little excuse for the number ever exceeding 25,000,000 per 
cubic centimeter. While experiments have also shown that although 
the effect produced by the bacteria on the product varies with different 
species, it is true that their presence can frequently be detected in the 
raw pulp by odor or taste when the number exceeds 25,000,000 per cubic 
centimeter and sometinkes when the count is as low as 10,000,000. 
To one who has not been initiated into the mysteries of the micro- 
scope the presence of such a number of bacteria in a food product 
seems inexcusable. It must be remembered in this connection that the 
most of these are probably nonpathogenic forms, and many occur 
naturally on the skins of the fruits. It does not seem just to set a 
standard so high as to virtually prohibit the manufacture of the product 
under commercial conditions; rather the idea is to set a limit that the 
manufacturer can attain if due care is exercised and which will insure 
a cleanly product. It is, however, perfectly possible to make a cleanly, 
wholesome product commercially even though the number of bacteria 
exceed that in the homemade article. 
The allowable limits for the bacterial content of tomato pulp vary 
according to the concentration. The number, however, should be 
low enough so that when the amount of concentrating necessary for 
its conversion into ketchup has been accomplished the final product TOMATO PRODUCTS 
519 
will still be within permissible limits (25,000,000 per cubic centimeter). 
Thus, for a pulp which must be concentrated one-half the bacterial 
counts should not exceed about half the limits stated above for the 
ketchup itself, i.e., it should not be more than 12,500,000 per cubic 
centimeter. The same general rule should also apply to the content of 
molds and of yeasts. 
To insure a sound product, free from decay or any filthy material, 
many factors must be carefully watched, for not infrequently over- 
sight in one particular has been found to have undone the good effects 
of the care exercised in all other ways. Thus it is possible for the 
washing of the fruit to be ideal and the sorting out or removing of 
the decayed portions beyond criticism, and yet a delay in making up 
the pulp into the final product may allow an amount of decomposi- 
tion to occur which offsets the care previously exercised. It has been 
a matter of surprise to some manufacturers to find with what rapidity 
some of these organisms increase. In one factory where this point 
was tested, the bacterial content in a batch of tomato trimming juice 
was found to be about 7,000,000 per cubic centimeter when taken 
from the peeling tables, and after standing at room temperature for 
five hours it had increased to 84,000,000. This was a twelvefold 
increase in a length of time which was less than half the working day 
for some of the factories visited. At the end of five days the number 
had increased to nearly 3,000,000,000 per cubic centimeter. Thus it 
is seen that delay in manufacture is very liable to result disastrously. 
Such facts as these serve to emphasize the great importance of 
absolute cleanliness in every detail about factories of this kind. Dirty 
floors and ceilings and apparatus left with residues of tomato product 
clinging to them are most fruitful sources for the contamination of 
new batches of the product. To clean such an establishment properly 
it is almost imperative that machinery and woodwork be washed by 
means of live steam used lavishly at frequent intervals. To leave 
buckets, tables, conveyors, or any other part of the equipment or floors 
overnight without cleansing them, as was the practice in some fac- 
tories, is reprehensible and tends to contaminate the product and 
lead to spoilage and loss. 
There are some objections to this method. Prescott, Burrage and 
Philbrick (1917) have stated that the method is grossly inadequate. 
They pointed out the following facts which render the method open to 
criticism. 1. Low magnification for examining the unstained mounts. 
2. No account of coccus forms is taken. 3. Personal equation is given 
too much importance. 4. Directions arc too indefinite. Bitting and 520 
FOOD PRESERVATION 
Bitting (1915) have also stated that the method is unsatisfactory. 
Indefinite directions are especially mentioned. Vincent (1918) has 
maintained the above objections. Bitting and Bitting have pointed 
out the practical impossibility of concentrating tomato pulp to the 
consistence of tomato paste and having it pass the standard of 25,000,- 
000. The inadequacies of the procedure are soon apparent to anyone 
who attempted to use the method. The difficulty of distinguishing 
between particles of organic matter and bacteria makes the results very 
uncertain. These criticisms caused a restatement of the procedure. 
More definite details have been given by Howard (1917) as follows: 
Howard's Revised Method for the Microanalysis of Tomato 
Products 
Apparatus. («) Compound Microscope. Equipped with apochro- 
matic objectives and compensating oculars, giving magnifications of 
approximately 90, 180 and 500 diameters. These magnifications can 
be obtained by the use of 16 and 8 mm. Zeiss apochromatic objectives 
with X6 and X18 Zeiss compensating oculars, or their equivalents, 
such as the Spencer 16 and 8 mm. apochromatic objectives with Spencer 
X10 and X20 compensating oculars, the draw tube of the microscope 
being adjusted as directed below. 
(&) Thoma-Zeiss Blood-counting Cell.* 
(c) Howard Mounting Cell. Constructed like a blood-counting cell 
but'with the inner disk (which need not be ruled) about 19 mm. in 
diameter. 
Molds (Tentative). Clean the Howard cell so that the Newton's 
rings are produced between the slide and the cover glass. Remove the 
cover and place by means of a knife blade or scalpel, a small drop of the 
sample of the central disk; spread the drop evenly over the disk and 
cover with the cover glass so as to give an even spread to the material. 
It is of the utmost importance that the drop be mixed thoroughly and 
spread evenly; otherwise the insoluble matter and the molds are most 
abundant at the center of the drop. Squeezing out of the more liquid 
portions around the margin must be avoided. In a satisfactory mount, 
Newton's rings should be apparent when finally mounted and none of 
the liquid should be drawn across the moat and under the cover glass. 
* Comment by the authors. In using these cells the plane parallel cover glasses 
furnished with them should be used instead of the ordinary microscope cover glasses. 
Since the latter are subject to curvatures that introduce errors in the thickness of 
the mounts. YEASTS AND SPORES 
521 
Place the slide under the microscope and examine with a magnifi- 
cation of about 90 diameters and with such adjustment that each field 
of view represents approximately 1.5 sq. mm. of area on the mount.* 
This area is of vital importance and may be obtained by adjusting 
the draw tube to the proper length as determined by actual measure- 
ment of the field, a 16 mm. Zeiss apochromatic objective with a Zeiss 
X6 compensating ocular or a Spencer 16 mm. apochromatic objective 
with a Spencer X10 ocular, or their equivalents, being used to obtain 
the proper magnification. 
Observe each field as to the presence or absence of mold filaments 
and note the result as positive or negative. Examine at least 50 fields, 
prepared from two or more mounts. No field should be considered pos- 
itive unless the aggregate length of the filaments present exceeds approx- 
imately one-sixth of the diameter of the field. Calculate the propor- 
tion of positive fields from the results of the examination of all the 
observed fields and report as percentage of fields containing mold 
filaments. 
Yeasts and Spores (Tentative). Fill a graduated cylinder with 
water to the 20 c.c. mark, and then add the sample till the level of the 
mixture reaches the 30 cc.. mark. Close the graduate, or pour the 
contents into an Erlenmeyer flask, and shake the mixture vigorously 
fifteen to twenty seconds. To facilitate thorough mixing the mixture 
should not fill more than three-fourths of the container in which the 
shaking is performed. For tomato sauce or pastes or products running 
very high in the number of organisms, or of heavy consistency, 80 c.c. 
of water should be used with 10 c.c. or 10 gms. of the sample. In the 
case of exceptionally thick or dry pastes it may be necessary to make 
an even greater dilution. 
Pour the mixture into a beaker. Thoroughly clean the Thoma-Zeiss 
counting cell so as to give good Newton's rings. Stir thoroughly the 
contents of the beaker with a scalpel or knife blade, and then, after 
allowing to stand three to five seconds, remove a small drop and place 
upon the central disk of the Thoma-Zeiss counting cell and cover imme- 
diately with the cover glass, observing the same precautions in mounting 
the sample as given above. Allow the slide to stand not less than 
ten minutes before beginning to make the count. Make the count 
* Comment by the authors. In order to have an area of 1.5 sq. mm. the diameter 
of the microscopic field should be 1.382 mm. This is determined by using a stage 
micrometer and adjusting the length of the microscope draw tube. Obviously after 
the proper draw tube length has been secured the adjustment should be noted and 
always used in making the mold counts. 522 
FOOD PRESERVATION 
with a magnification of about 180 to obtain which the following com- 
binations, or their equivalents, should be employed: 8 mm. Zeiss 
apochromatic objective with X6 Zeiss compensating ocular, or an 8 mm. 
Spencer apochromatic objective with X10 Spencer compensating ocular 
with draw tube not extended. 
Count the number of yeasts and spores * on one-half of the ruled 
squares on the disk (this amounts to counting the number in eight of the 
blocks, each of which contains twenty-five of the small ruled squares). 
The total number thus' obtained equals the number of organisms in 
1/60 c.mm. if a dilution of one part of the sample with two parts of 
water is used. If a dilution of one part of the sample with eight parts 
of water is used the number must be multiplied by three. In making 
the counts the analyst should avoid counting an organism twice when 
it rests on a boundary line between two adjacent squares. 
Bacteria (Tentative). Estimate the bacteria from the mounted 
sample used above, but allow the sample to stand not less than fifteen 
minutes after mounting before counting.! Employ a magnification of 
about 500, which may be obtained by the use of an 8 mm. Zeiss apochro- 
matic objective with an X18 Zeiss compensating ocular with draw tube 
not extended, or an 8 mm. Spencer apochromatic objective with an X20 
Spencer compensating ocular with a tube length of 190, or their equiva- 
lents. Count and record the number of bacteria in a small area consist- 
ing of five of the small-sized squares. Move the slide to another por- 
tion of the field and count the number on another similar area. Count 
five such areas, preferably one from near each corner of the ruled portion 
of the slide and one from near the center. Determine the average num- 
ber of bacteria per area and multiply by 2,400,000, which gives the num- 
ber of bacteria per cubic centimeter. If a dilution of one part of the 
sample with eight parts of water instead of one part of the sample with 
two parts of water is used in making up the sample, then the total count 
obtained as above must be multiplied by 7,200,000. Omit the micro- 
cocci type of bacteria in making the count. 
Explanation of Calculations. Fig. 80 has been prepared to make 
somewhat clearer the explanation of the areas denoting the yeast and 
spore and bacterial counts. The light lines in the figure show the 
arrangement of rulings on the entire slide. The squares (A, A, etc.), 
and rectangles (B, B, etc.) designated in the figure by the heavy lines 
indicate the portions used for the yeast and spore and for the bacterial 
* Comment by authors. The organisms counted as " yeasts and spores " are 
the yeast cells, and yeast and mold spores, not bacteria spores. 
t The estimation of yeast, molds and bacteria are made from the same mount. CALCULATIONS 
523 
counts, respectively. The eight large squares, A, A, etc., are the squares 
used for yeast and spore counts. Each of these squares has twenty-five 
of the small squares. The sum of the organisms counted in the eight 
squares marked A, A, etc., is the number in 1/60 c.mm. if a dilution 
of one part of product to two parts of water is used. 
Yeast and Spore Count. The ruled square on the slide is 1 mm. on 
each side and the cell is 1/10 mm. deep. The volume of the ruled part 
is, therefore 1/10 c.mm. The ruled area is divided into sixteen large 
squares and the number of organisms is counted in eight of these, which 
is equivalent to 1/2 of 1/10 c.mm., or 1/20 c.mm. If a dilution of one 
Fig. 80.-Diagram of Thoma Rulings. (After Howard, 1917.) 
One millimeter divided by lines into twenty spaces in each direction each space equalling mm. 
To facilitate counting, every fifth space is subdivided by a line through the middle. 
part of the product to two parts of water is used 1/3 of 1/20 c.mm., or 
1/60 c.mm. as representing the actual amount of original stock in 
which organisms are counted, is obtained. 
Bacterial Count. The rectangles, B, B, etc., each including five of 
the smallest squares, represent the areas used in making the bacterial 
count. Similar rectangles of equal area might be selected, the object 
being to count five such areas well distributed over the ruled portion 
of the slide. The average number of bacteria counted on five rectangles, 
such as B, B, etc., multiplied by 2.4 million, equals the number of 524 
FOOD PRESERVATION 
bacteria per cubic centimeter. Tn calculating t he bacteria, it is observed 
that there are 400(20X20) small squares on the slide. The number of 
bacteria in rectangles (B, B, etc.), each containing five of these small 
squares, are counted and an average made. This average represents 
the bacteria in 1/80 of the total ruled area. Since the cell is 1/10 mm. 
deep, the volume represented by the organisms counted is 1/80X1/10 
or 1/800 c.mm. With the usual dilution of one part of product to two 
parts of water the actual volume in which the number of organisms is 
determined is 1/3 of 1/800 cm. or 1/2400 c.mm. or 1/2,400,000 c.c. 
Howard (1917) made a study of the relation between the number of 
yeasts and spores, mold counts, and bacteria (rods) and the amount of 
rot in the tomatoes. With regard to yeasts and spores a count of 
twenty was represented by 1 per cent decay. From this point the rate 
of increase was lower. A count of thirty-five yeasts and spores indicated, 
according to Howard about 4 per cent of rot. Fig. 81 devised by How- 
ard shows the " Zone of Possible Yeast and Spore Counts." From this 
chart it is possible to determine the relation between a count and the 
per cent of rot which it represents. 
The relation between the number of bacteria and the percentage of 
rot is shown in Fig. 82. Howard thinks that below 15,000,000 little is 
indicated with regard to the amount of rot. Above this " and up to 
20 per cent of rot, the ratio of increase as shown on this chart is about 
20,000,000 for each per cent of rot." Howard states that a low bac- 
terial count does not always indicate a sound stock but a high number 
of bacteria always indicates bad stock. 
Fig. 83, devised by Howard, shows the relation between the mold 
count and the percentage of rot. From this chart it is possible to 
calculate the percentage of decay from a mold count. The following is 
quoted from Howard. 11 Attention should be called to the fact that 
beyond 20 per cent of rot the chart is plotted on the basis of the assump- 
tion that 100 per cent rot would give a mold count of 100 per cent of the 
fields. As a matter of fact, the mold count reaches this maximum of 
100 per cent of the fields with less than this amount of rot. In the 
study of the relation between the rot and count under factory conditions, 
20 per cent of rot was the highest on which the full data was secured. 
From 0 to | per cent of rot the mold count rises rapidly. Beyond 
| per cent the rate of rise gradually decreases, until after 20 per cent of 
rot the rate of increase is slow. On the whole the zone was higher on 
factory than on laboratory samples. The chart shows that a count of 
60 per cent molds represents a rot content of not less than about 4 per 
cent. It is interesting that the mold count of twenty-five, which was YEAST AND SPORES 
525 
Per cent of Rot 
Fig. 81.-Percentage of Weight of Rot, and Yeast and Spore Count. 
The line showing the upper limit of " Zone of Possible Yeast and Spore Count " beyond 12 per cent of rot is based on a sample having 100 
per cent of rot by weight. 98 fields with molds, 103 yeast and mold spores per Jo c.c. and 860 million bacteria per cubic centimeter. 
ZONE OF . 
I POSSIBLE YEAST AND SPORE | 
COUNTS 1 
1 1 ! 1 F 
• Laboratory Samples 
x Factory Samples 
Toast and Spore Count per Veocmm. 526 
FOOD PRESERVATION 
-J 1 4 1- 
• Laboratory Samples 
x factory Samples | 
Bacteria-Millions per cc. 
j ZONE OF 1 
[ POSSIBLE BACTERIA] 
| COUNTS 
Fig. 82.-Percentage by Weight of Rot and Bacterial Count. 
(After Howard and Stevenson, 1917.) 
The line showing the upper limit of " Zone of Possible Bacteria Counts " beyond 20 per cent 
of rot is based upon the count of a sample having 100 per cent of rot, 100 fields with molds, 22 yeast 
and spores per c.c. and 960 million bacteria per cubic centimeter. 
Per cent of Kot MOLDS 
527 
Per cent of Rot by Weight, 
Fig. 83.-Relation Between Possible Mold Count and Percentage of Rot by Weight. (Factory Sample.) 
(After Howard, 1917.) 
. ZONE OF । 
I POSSIBLE MOLD | 
COUNTS 
Mold Counts 528 
FOOD PRESERVATION 
suggested as a factory-working basis in 1911, represents at least 0.8 to 
0.9 per cent of rot. There may be more rot than that present, but, on 
the basis of the data at hand, it is highly improbable that it represents 
less than that amount. From the chart it is possible to calculate the 
approximate minimum percentage of decay represented by a given mold 
count. Thus, for instance, a mold count of forty enters the ' Zone of 
Permissible Mold Counts ' at a point representing 2.2 per cent of 
decay. Therefore, a count of forty may be obtained in samples having 
any amount of rot between 2.2 and 100 per cent." 
Vincent's Method for the Examination of Tomato Paste. Vincent 
(1918) has attempted to overcome the errors of the Howard method by 
adapting the Breed smear to tomato products. The method is described 
as follows: " The catsup is diluted with two parts of sterile water, since 
this dilution has proven satisfactory in counting most specimens when 
the Zeiss counting chamber is used. With a sterile pipette calibrated 
to deliver 0.01 c.c. the diluted catsup is deposited on a second glass slide 
and evenly spread over an area of 1 sq. cm. by means of a sterile needle. 
After drying, the slide is immersed in 95 per cent alcohol for one minute 
to fix the smear, dried in air, stained with Loeffler's methylene blue 
for two minutes, washed in water, dried and examined with the 1/12 in. 
oil immersion lens." The rest of the procedure is identical with that 
outlined under Breed's microscopic method for counting bacteria in 
milk. The microscope is standardized to bring the diameter of the field 
to 0.205 mm. Thirty fields are counted and the average for one field 
is multiplied by three on account of the dilution of the catsup. This 
result is then multiplied by 300,000, which represents the number of 
bacteria per cubic centimeter. 
Vincent compared this microscopic count with the count secured 
with the Zeiss blood counter. The latter count was always much 
greater than the former. Vincent believes that the cocci should 
be counted since they probably take as important a part in the spoilage 
of tomatoes as the rods, 
Food Poisoning 
In the past, this term has covered a multitude of physiological 
disturbances in the alimentary tract. For some, it has been a very 
easy matter in the past to diagnose an ailment as food poisoning 
or ptomaine poisoning when other possibilities have been excluded. 
The term " ptomaine poisoning " was used by many for a time, but 
this is gradually being discarded for terms with more meaning. More PTOMAINES 
529 
recently the conception that foods may cause infection has crept in 
and this is being confirmed. Specific bacteria are now being recognized 
as the principal factors in many cases. In others, the toxins produced 
by bacteria are ingested and these produce intoxications. 
Certain characteristics of food infection or poisoning have pre- 
vented rapid advances in our knowledge. The occurrence of a small 
number of cases in a family is investigated with some difficulty because 
these individuals often are engaged in quite different activities. Much 
easier is it to study the problem when the individuals are members of 
units whose activities are much alike. Consequently some of the most 
valuable data with regard to food poisoning has been collected from 
prisons, barracks and similar groups. Probably, in the smaller units, 
cases of food poisoning have been overlooked and attributed to other 
causes. Then, in the past, many cases of food poisoning have been 
reported, the study of which included no bacteriological or chemical 
examination. 
The causes of food poisoning are varied because there is little agree- 
ment with regard to a definition for the term " poison," which is a 
difficult term to define. However, for convenience, the following 
causes of food poisoning may be mentioned: 
I. Ingestion of foods containing pathogenic bacteria. 
II. Ingestion of foods containing poisonous products of bacterial 
katabolism-toxins. 
III. Ingestion of foods which are naturally poisonous, such as mush- 
rooms. Certain alkaloids are present. 
IV. Ingestion of foods containing poisonous metals. 
Ptomaines. These are toxic amines and, as indicated in the chapter 
on intestinal bacteria, they are the products of bacterial action. They 
are basic in character and have been called " animal alkaloids." Most 
of them do not contain oxygen; the following are typical ptomaines: 
(CH3)NH2 methylamin 
(CH3)2NH dimethylamin 
(CH3)3N trimet hylamin 
NH2-CH2-CH2-CH2-CH2-NH2 putrescine 
NH2-CH2-CH2-CH2-CH2-CH2-NH2 cadaverine 
Not all of the ptomaines are poisonous. The more elementary amino 
compounds are probably not poisonous. The ptomaines do not hold the 
position in food poisoning as formerly. Their importance in the past 
probably originated in their isolation from bacterial cultures and the 530 
FOOD PRESERVATION 
lack of more definite bodies to which to attribute the effects of disease 
caused them to be emphasized in this connection. Little data was 
necessary to prove that they were not important in bacterial toxemia. 
In relation to food poisoning, their significance is negligible. It has 
been very easy in the past for diagnosticians to attribute the various 
alimentary disturbances to the presence of ptomaines. This was 
especially true if the patient had been eating of canned foods. It is 
unfortunate that such a close relation between ptomaine poisoning and 
canned foods has been established. Probably no food is less liable 
to be of any importance in this connection than canned foods. A food 
which has putrefied to the point where ptomaines are present will be 
excluded by the organoleptic tests; it is also reasonable to assume that 
the ptomaines themselves arc soon destroyed by bacterial action. 
Vaughan and Novy have given extended attention to the ptomaines and 
it may be due almost entirely to their investigations that ptomaines 
have held such an important position in food poisoning. They pro- 
posed a rather elaborate nomenclature for the different types of poison- 
ings. Some of them are as follows: 
Milk poisoning galactotoxismus 
Cheese poisoning tyrotoxismus 
Fish poisoning ichthyotoxismus 
Meat poisoning kreatoxismus 
Food poisoning bromototoxismus 
Relation of Carriers to Food Infections. Common infections of foods 
by carriers are, dysentery, typhoid fever, paratyphoid fever and strep- 
tococcus sore throat. Data are being accumulated very rapidly to 
enlighten this question. 
The question is often asked, how long may a person continue to 
distribute the bacilli in feces and urine? In answer to this question 
there are several cases which might be mentioned. Bolduan and 
Noble (1912) report a dairyman who had typhoid fever at fifteen years of 
age. He was a carrier for forty-six years showing B. typhi in his stools. 
Martz (1917) reported a carrier of fifty-five years' standing. The case 
of a woman who was apparently a carrier for fifty-four years is men- 
tioned in an Editorial of the Jour. Am. Med. Assn., Vol. 52 (1909), 388. 
The handling of carriers is not a settled question. Kendall (1916) 
pointed out the danger of carriers handling foods. Several severe 
epidemics of typhoid are known to have been caused by food infected 
from a carrier. The ideal situation will only be reached after all handlers PARATYPHOID FEVER 
531 
of foods in public places are examined for various diseases which arc 
transmissible through foods. 
In an effort to control and keep in touch with " carriers " the Cal- 
ifornia State Board of Health causes the following agreement to be 
entered into between the " carrier " and the department. (Cumming, 
1917). 
I have this day been informed that my excreta contain typhoid 
bacilli and that, unless unusual precautions arc taken, persons will con- 
tract the infection from me. Realizing this danger I agree to observe 
the precautions stated below, and request that I be permitted to remain 
in free communication with other persons. 
1. I will take no part in the preparation or handling of food which 
will be consumed by persons outside of my immediate family, and I 
will not participate in the management of a boarding house, restaurant, 
food store, or in any other occupation involving the preparation or 
handling of food. 
2. I will not dispose of my excretions in a toilet to which flies have 
access without first exposing such excretions to either a 5 per cent 
dilution of liquor formaldehyde or 5 per cent phenol (carbolic acid). 
3. I will notify the local health officer of any cases of typhoid among 
persons with whom I come in contact. 
4. I will inform that local health officer of any contemplated change 
of residence so that he can notify the State Board of Health and 
obtain its approval. 
5. I will submit specimens for examination when requested by the 
State Board of Health. 
6. I will fill out the following report blank when submitted to me 
semiannually, and return the same to the California State Board of 
Health: 
" I have, during the last six months, complied to the best of my 
knowledge with the five separate agreements entered into between 
myself and the California State Board of Health. Precautions involved 
in these separate agreements are for the purpose of preventing typhoid 
infection." 
That " carriers " may be found among the animals is suggested by 
much recent data. The work of Mitchell and Bloomer (1914) seems to 
indicate that chickens may not become carriers although the authors 
recommend further study. 
Paratyphoid Fever. This infection is produced by a bacillus closely 
related to B. typhi but possessing certain distinct differences. Many 
cases of food poisoning have been traced to the paratyphi bacilli. Bain- 532 
FOOD PRESERVATION 
bridge (1912) has attempted to make a distinction between paratyphoid 
fever and meat poisoning. He bases his argument on the inadequacies 
of agglutination reactions for separating members of the group and states 
that precipitation or complement fixation reactions are necessary to 
distinguish between the bacteria, to which food poisoning has been 
attributed. The symptoms of food or meat poisoning are quite differ- 
ent and Bainbridge is probably correct in making this distinction. The 
true paratyphoid fever resembles typhoid in many of its characteristics 
but the mortality from it and the mildness of the disease are distinct 
differences. 
Jordan (1917) has pointed out the occurrence of at least two clinical 
types of paratyphoid infection, one having almost the identical picture 
of the true infection with B. typhi and the other the commoner gastro- 
intestinal type. 
Bongartz (1910) studied samples of meat from both condemned 
animals and apparently normal animals. Many of these were found 
to contain paratyphoid bacilli. He suggests the education of the public 
to the eating of cooked meat which, to a certain extent, would reduce the 
danger of diseased meat or foods. Hoffenreich (1914), on the other 
hand, examined 249 samples of meat from typical abbatoirs and found 
no indications of the presence of bacteria of the paratyphoid-Gaertner 
group. He concludes that their distribution is not as general as has 
been supposed. Bernhardt (1914) has found Bacillus voldagsen and 
B. typhi suis (Glasser) in a case of meat poisoning which resulted in 
death. He advises that a polyvalent paratyphoid serum be used for 
diagnosis. 
Fowler (1909) has described an epidemic of paratyphoid fever 
resulting from eating goose at a Christmas dinner. He stated that the 
goose may have been infected during slaughter; bacteria may have 
invaded the flesh and excreted a toxin which would account for the 
acute onset of the cases. Bernstein and Fish (1916) described an 
epidemic of sixty cases and four deaths resulting from paratyphoid 
infection from a pie. This was confirmed by clinical and laboratory 
investigation. Such epidemics point out that restaurants (Kendall, 
1916) may be important factors in the dissemination of infection. 
Muller (1915) reported that several cases of fish poisoning were caused 
by bacteria of the paratyphoid-enteriditis group. Seele (1913) in dis- 
cussing food poisoning in the German army during the years 1912 and 
1913 stated that meat and sausage were the foods which caused 
most trouble. The following organisms were identified: B. coli 
communis, B. proteus, B. enteritidis and B. paratyphosus. From BOTULISM 
533 
the above epidemics it is apparent that foods may distribute para- 
typhoid bacilli. 
Bacillus enteritidis. Gaertner (1888) first showed the relation of 
this organism to food poisoning. The infection came from the meat of a 
cow which was abnormal before it was slaughtered. The bacillus was 
isolated from the diseased meat and also from the spleen of a fatal case. 
Later Durham (1898) and De Nobele (1899) also isolated bacilli like 
B. enteritidis from abnormal meat. Van Ermengen (1892) reported the 
study of an extensive epidemic of food poisoning which was believed 
to have originated from eating the meat of diseased calves. Bacteria 
like B. enteritidis were isolated. Other investigations have been 
reported by Holst (1895), Poels and Dhont (1894), Jacobitz and Kayser 
(1910) and others, 
Botulism 
Botulism or " sausage poisoning " has been known for a long time. 
The earliest report was by Kerner, who described a series of cases among 
which were a number of deaths. Dickson, in America (1915), has given 
the subject quite a little study. Most of the outbreaks in this country 
have been in California. This is probably due to their being alert to 
the possibility of botulism which, in other cases, has been diagnosed as 
ptomaine poisoning. 
The organism was discovered by Ermengen (1899) in a ham which 
had been responsible for a severe attack of meat poisoning. It was 
found in muscle in the spore stage. He also isolated this organism 
from the spleens of the fatal cases. A further study gave more evi- 
dence that the organism was distinctly pathogenic. 
The organism has the following characteristics: It is gram positive, 
anaerobic and a spore former. The temperature range is between 18 
and 30° C. The vegetative cells are easily destroyed by heat but the 
spores are quite resistant. Although Van Ermengen stated that they 
could be destroyed by heating at 85° C. for fifteen minutes, Dickson 
(1915) has reported a greater resistance. The organism does not grow 
above 30° C. and, therefore, the toxin is the important factor in this 
type of food poisoning. 
Toxin of Bacillus botulinus. The toxin of Bacillus botulinus is 
soluble and produces a strict toxemia. It is thermolabile and very 
toxic not only when administered by injection but when administered 
by the mouth. This separates it very closely from the toxins of B. 
tetani and B. diphtherice. It was formerly thought that the toxin for- 534 
FOOD PRESERVATION 
mation occurred only on certain food substances, as meat, but Dickson 
has shown that it may take place in such foods as canned beans and 
peas. 
The symptoms have been stated by Dickson (1915). 
The symptomology of botulism has been summarized by Van Ermengen as a 
neuroparalytic symptom complex with disturbances of secretion and symmet- 
rical partial or complete motor paralyses which apparently have their seat in 
lesions of the central nervous system. The first symptoms do not occur earlier 
than from twelve to twenty-four hours after ingestion of the infected food, 
although they may appear much later. In contrast to the usual types of food 
poisoning there is apt to be little evidence of gastro-intestinal disturbance, and 
obstinate constipation is more frequent than is diarrhoea. There is usually 
a decrease, but there may be an increase in the secretion of saliva and of mucus 
in the mouth and in the throat, when mucus is present it is extremely viscid. 
There is early disturbance of the external and internal muscles of the eyes which 
manifests itself by blepharoptosis mydriasis, disturbances of accommodation, 
diplopia and strabismus. General muscular weakness is common. There 
may be dysphasia or aphonia and there is usually great difficulty in swallowing. 
The absence of sensory disturbances is characteristic. 
The symptoms gradually increase in severity and when death occurs it is 
usually from respiratory or cardiac failure, as in bulbar paralysis. When 
recovery takes place the/e is usually a long and tedious convalescence with a 
slow return of muscular strength. The condition so closely resembles that 
seen in gelsemium or hyoscyamin poisoning, polyomyelitis, cerebral syphilis 
and bulbar paralysis that isolated cases may readily escape recognition, but 
when a series of cases is seen and especially if all have partaken of a common 
article of food there is usually little difficulty in establishing a diagnosis. 
Botulism may be diagnosed microscopically by detecting the Bacil- 
lus botulinus in the suspected food. The bouillon cultures should be 
tested for toxin and inoculated food fed to laboratory animals. In 
the cultural examination of the food, careful anaerobic technique should 
be used. 
Bacillus botulinus has caused severe poisonings in modern times. 
Wilbur and Ophuls (1914) reported the investigation of an epidemic 
which was caused by home-canned string beans. Dickson (1915) has 
mentioned some other epidemics. He (1917) called attention also to 
the fact that vegetables put up by the cold-pack method might cause 
the disease. This was later denied, to a certain extent, by the U. S. 
Department of Agriculture. Fischer (1906) studied an epidemic of 
botulism resulting from the eating of bean salad. The symptoms were 
characteristic and appeared between twenty-four and forty-eight hours. 
The beans were canned and when opened were not cooked since they BOTULISM 
535 
seemed to bo soft. Had they been thoroughly cooked it is improbable 
that any unusual results would have been noticed. Another epidemic 
of botulism from canned string beans has been reported by Landmann 
(1904). In this epidemic the value of boiling canned food is again 
emphasized. Some of the beans were boiled by accident and when 
eaten produced no evil results. Those who partook of the uncooked 
beans suffered the usual symptoms. The presence of B. botulinus was 
indicated by a bacteriological examination.* 
Diagonosis of Food Poisoning. Extract some of the suspected food 
with physiological salt solution. Inject boiled and unboiled decimal 
dilutions of this extract into guinea pigs. Feed the suspected food to 
experimental animals. If they will not eat it, sprinkle other foods with a 
physiological salt solution extract of the suspected food. 
BIBLIOGRAPHY 
Bachmann, F. M. 1916. A Study of the Effect of Spices on the Growth of 
Certain Organisms. Abstracts of Bacteriology, 1, 109. 
Bachmann, F. M. 1916. Inhibiting Action of Certain Spices. Jour. Ind. 
Chem., 8, 620-623. 
Bachmann, F. M. 1918. The Use of Different Microorganisms to Determine 
the Preservative Value of Different Brands of Spices. Jour. Ind. Eng. 
Chern., 10, 121-123. 
Bacon, R. F. and Dunbar, B. P. Changes Taking Place During Spoilage of 
Tomatoes with Methods for Detecting Spoilage in Tomato Products. 
U. S. Department of Agriculture, Bureau Chemistry Circular 78. 
Bainbridge, F. A. 1912. The Milroy Lectures on Paratyphoid Fever and 
Meat Poisoning. Lancet, 1912-1, 705, and lectures in two succeeding 
numbers. 
Baker, II. A. 1912. "Springers" in Canned Foods, Causes and Prevention. 
8th Intern. Cong. Applied Chem., 18, 39-41. 
Baker, H. A. 1912. The Disappearance of Oxygen in Canned Food Con- 
tainers. 8th Intern. Cong. Applied Chem., 18, 45-49. 
Beattie, J. H. and Gould, H. P. 1917. Commercial Evaporation and Dry- 
ing of Fruits. U. S. Dept. Agriculture, Farmer's Bulletin 903. 
Bernhardt, G. 1912. In Regard to the Causes of Meat Poisoning. Para- 
typhoid B. bacilli Voldagsen Type as a Cause of Meat Poisoning in Man. 
Zeit. f. Hyg., 73, 64-78. 
Bernstein, H. S. and Fish, E. S. 1916. Food Poisoning by the Bacillus 
paratyphosus B. An epidemic due to the organism isolated from a pie. 
Jour. Amer. Med. Assn., 66, 167-171. 
* Those desiring an extended discussion of botulism should read Monograph 
No. 8 (1918) of the Rockefeller Institute for Medical Research by Dickson. 536 
FOOD PRESERVATION 
Bigelow, W. D. 1914. Swells and Springers. Research Laboratory, Na- 
tional Canners' Assn., Washington, D. C., Bull. 2. 
Bigelow, W. 1). 1917. The Chemist in the Canned Food Industry. Jour. 
Ind. Eng. Chem., 9, 187-189. 
Bigelow, W. D. 1918. Problems of Canning Operations. Amer. Jour. Pub. 
Health, 8, 212-215. 
Bigelow, W. D. and Fitzgerald, F. F. 1914. Tomato Pulp. Research 
Laboratory, National Canners' Assn., Washington, D. C., Bull. 3. 
Bitting, A. W. 1909. Experiments on the Spoilage of Tomato Ketchup. 
U. S. Dept. Agriculture, Bureau of Chem. Bull. 119. 
Bitting, A. W. 1909. The Canning of Peas. U. S. Dept. Agriculture, Bureau 
of Chemistry Bull. 125. 
Bitting, A. W. 1911. Preparation of Cod and Other Salt Fish for the Market. 
U. S. Dept. Agriculture, Bureau of Chemistry Bull. 123. 
Bitting, A. W. 1915. Methods Followed in the Commercial Canning of 
Foods. U. S. Dept. Agriculture, Bull. 196. See also Bull. 151. 
Bitting, A. W. 1917. Canned Foods. Modern Processes of Canning in the 
United States, General System of Grading and Description of Product 
Available for Export. U. S. Dept. Commerce. Bureau of Foreign and 
Domestic Commerce. Miscellaneous Series No. 54. 
Bitting, A. W. and Bitting, K. G. 1915. Ketchup. Murphey-Bevins Co., 
Lafayette, Indiana. 
Bitting, A. W. and Bitting, K. G. 1917. Bacteriological Examination of 
Canned' Foods. Research Laboratory, National Canners' Laboratory 
Bull. 14. 
Bolduan, C. F. 1909. Bacterial Food Poisoning by A. Dieudonne. E. B. 
Treat & Co. 
Bolduan, C. and Noble, W. C. 1912. A Typhoid Bacillus Carrier of Forty- 
six Years' Standing and a Large Epidemic from this Source. Jour. Amer. 
Assn., 58, 7. 
Bongartz, C. 1910. Do Paratyphus-like Bacteria Occur in Meat under 
Normal Conditions and if so is Bacteriological Meat Inspection Necessary? 
Inaug. Dissert. Univ. Berne. 
Bridgman, P. W. 1914. The Coagulation of Albumin by Pressure. Jour. 
Biol. Chem., 19, 511-512. 
Brown, C. W. 1916. Notes on Brine Pickle Fermentation. Abstracts of 
Bacteriology, 1, 104-105. 
Burgess, P. S. 1912. A Bacteriological Study of " Flat Soured " Canned 
Corn and Pumpkin Together with Length of Time Required for Complete 
Sterilization. The Canner and Dried Fruit Packer, about 1912. 
Cathcart, E. P. 1906. The Bacterial Flora of " Blown " Tins of Preserved 
Foods. Jour. Hyg., 6, 248-250. 
Courmont and Rochoux. 1910. Dogs as Typhoid Carriers. Amer. Jour. 
Med, Sciences, 140, 899. BIBLIOGRAPHY 
537 
Cumming, J. G. 1917. An Epidemic Resulting from Contamination of Ice 
Cream by a Typhoid Carrier. Jour. Amer. Med. Assn., 68, 1163-1165. 
De Noble. 1899. Quoted from Bolduan, 1916. 
Dickson, E. C. 1915. Botulism, an Experimental Study. Jour. Amer. 
Med. Assn., 65, 492-496. 
Durham, H. E. 1898. On an Epidemic of Gastro-enteritis Associated with 
the Presence of a Variety of the Bacillus enteritidis (Gaertner). Brit. 
Med. Jour., 1898-1. 
Durham, H. E. 1899. Infection by Unsound Meat. Brit. Med. Jour., 
1899-1, 1216. 
Fettick, 0. 1908. Quantitative und Qualitative Untersuchungen uber die 
Bakterien, Hefen, und Pilze der Butter und iiber den Einfluss des Koch- 
salzgehaltes auf dieselben. Cent. Bakt. Abt. IL, 22, 32. 
Fischer, A. 1906. Ueber eine Massenerkrankung an Botulismus infolge 
Genusses " verdorbener." Zeit. f. klin. Med., 59, 58-77. 
Fowler, G. E. P. 1909. Outbreak of Food Poisoning Following a Christmas 
Dinner. Jour. Roy. Army Medical Corps, 13, 271-274. 
Freytag, C. L. de. 1890. Ueber die Einwirkung konzentrierter Kochsalz- 
losungen auf Bakterien. Arch. Hyg., 11, 60. 
Giltner, W. and Baker, J. D. 1915. Effect of Salt on Butter Flora. Mich. 
Sta. Rept., 1915, 209. 
Gould, R. G. 1917. Commercial Dehydration. Amer. Food Journal, 
373-375. 
Hilliard, C. M. 1918. The Fish Canning Industry. Amer. Jour. Pub. 
Health, 8, 202-204. 
Hite, B. H., Giddings, N. J. and Weakly, C. E. 1914. The Effect of Pres- 
sure on Certain Microorganisms Encountered in Preserving Fruits and 
Vegetables. West Virginia Ag. Exp. Sta. Bulletin 146. 
Hoffenreich, E. 1914. Investigations of the Presence of Bacteria in Places 
where Meat is Slaughtered and Sold with Special Reference to the Para- 
typhoid-Gaertner Group. Tierarz. Zentrl., 37, 337-346. 
Hoffman, C. and Evans, A. 1911. The Use of Spices as Preservatives. 
Jour. Ind. Eng. Chem., 3, 835-838. 
Howard, B. J. 1911. Decomposition and its Microscopical Detection in 
Some Food Products. Year Book, U. S. Dept. Agriculture, 1911, 297-308. 
Howard, B. J. 1911. Tomato Ketchup under the Microscope. U. S. Dept. 
Agriculture, Bureau of Chemistry Circular 68. 
Howard, B. J. and Stevenson, C. H. 1917. Microscopical Studies on 
Tomato Products. U. S. Dept. Agriculture Bull. 581. 
Howard, B. J. and Stevenson, C. H. 1917. The Sanitary Control of Tomato 
Canning Factories. U. S. Department of Agriculture Bull. 569. 
Jacobitz and Kayser, H. 1910. Bacterial Food Poisoning. Cent. Bakt. 
Abt., 1. Orig., 53, 377-378. 
Jordan, E. O. 1917. Food Poisoning. The University of Chicago Press. 
Jordan, E. O. 1918. Food-borne Infections. Science, 47, 80-86. 538 
FOOD PRESERVATION 
Karaffa-Korbutt, K. 1912. The Influence of Common Salt on the Life 
and Growth of Microorganisms. Zeit. Hyg., 71, 162-170. 
Landmann, G. 1904. Ueber die Ursache der Darmstadter Bohnenvergiftung. 
Hyg. Rund., 14, 449-452. 
Larson, W. P., Hartzell, T. B. and Diehl, H. S. 1918. The Effect of High 
Pressures on Bacteria. Journal of Infectious Diseases, 22, 271-279. 
Lewandowsky, F. 1904. Ueber das Wachstum von Bakterien in Lablo- 
sungen von hoher Konzent ration. Arch. Hyg., 49, 47. 
MacFayden, A. 1900. On the Influence of the Temperature of Liquid Air 
on Bacteria. Lancet, 1900-1., 849, 1130. 
Martz, Hans. 1917. A Typhoid Carrier for Fifty-five Years. Zeit. Hyg., 
80, No. 3. Quoted from Amer. Jour. Pub. Health, 7, 431. 
Minchin, W. C. 1917. The Germicidal and Therapeutic Action of Garlic. 
Med. Press and Circular 103, 493-495. 
Mitchell, O. W. II. and Bloomer, G. T. 1914. Experimental Study of the 
Chicken as a Possible Typhoid Carrier. Jour. Med. Res., 31, 247-250. 
Muller, M. 1914. The Value and Purpose of Animal Experimentation in 
Meat Examination. Zeit. Hyg., 16, 115-138. 
Muller, R. 1914. Fish Poisoning by Bacteria of the Paratyphoid-enter- 
iditis Group. Munch, med. Wochenschr., 61, 471-473. 
Nelson. 1910. The Pungent Constituent of Pepper. Jour. Ind. Eng. Chern., 
2, 419. 
Pennington, M. E. 1911. The Comparative Rate of Decomposition in 
Drawn and Undrawn Market Poultry. U. S. Dept. Agriculture, Bureau 
of Chemistry, Circular 70. 
Pennington, M. E., et al. 1917. The Influence of Temperature above 
Freezing on the Changes in Composition, Bacterial Content and His- 
tological Structure of the Flesh of the Common Fowl. Jour. Biol. Chern., 
29,31,32. 
Pernot, E. F. Experiments on the Canning of Fruits. Oregon Ag. Exp. 
Sta. Bull. 81. 
Pettersson, A. 1900. Experimentelle Untersuchungen fiber das Conser- 
vieren von Fisch und Fleisch mit Salzen. Arch. Hyg., 37, 171. 
Pfule. E. 1904. Beitrag zur bakteriologischen Untersuchung der Fleisch- 
konserven. Zeit. Hyg., 48, 121. 
Poels and Dhont. 1894. Quoted from Bolduan, 1916. 
Prescott, S. C. 1900. The Bacteriology of Canned Foods. Science, 11, 442. 
Prescott, S. C. and Underwood, L. 1897. Microorganisms and Sterilizing 
Processes in the Canning Industry. Tech. Quarterly. 
Prescott, S. C. and Underwood, L. 1898. Contributions to our Knowledge 
of the Microorganisms and Sterilizing Processes of Canning Industries. 
Tech. Quarterly. 
Prescott, S. C., Burrage, S., and Philbrick, B. G. 1917. Some Sources 
of Error in the Microscopical Examination of Certain Food Products. 
Abstracts of Bacteriology, 1, 51. BIBLIOGRAPHY 
539 
Rahn, 0. 1913. Bacteriology of Foods on a Physiological Basis. Cent. 
Bakt. Abt. IL, 37, 492-497. 
Rahn, 0., Brown, C. W. and Smith, L. M. 1909. Keeping Qualities of 
Butter II, and III. Mich. Ag. Exp. Sta. Tech. Bull. 2. 
Rapin. The Microbic Flora of Salt as a Cause of Butter and Cheese Defects. 
Milk Zeitung, 20, 433. 
Round, L. A. 1916. Normal Fermentation of Sauerkraut. Absts. Bact., 1, 
108. 
Round, L. A. 1917. The Bacteriology of Sauerkraut, a Further Study. 
Abstracts of Bacteriology, 1, 50. 
Ruata, A. Q. 1918. Action of Cold on Microorganisms. Annali d'lgiene 
Rome, 28; Jour. Amer. Med. Assoc., 70, 1125 (1918); Amer. Jour. Pub. 
Health, 8 (1918), 399. 
Sadler, W. 1918. The Bacteriology of Swelled Canned Sardines. Amer. 
Jour. Pub. Health, 8, 216-220. 
Sedgwick, W. T., Hamilton, H. W. and Funk, F. J. 1917. Experimental 
Studies on the Effects of Various Solutions upon the Viability of Bacteria 
at Low Temperatures. Abstracts of Bact., 1, 49. 
Seele, W. 1913. Cases of Food Poisoning Noted in the German Army 
During the Last Two Years. Inaug. Dissert. Univ. Berlin. Hyg. Rund., 
24, 531, 532. 
Serkowski, S. and Tomczek, P. 1911. The Influence of Common Salt on 
the Bacteria which Cause Meat Poisoning. Zeit. Untersuch. Nahr. 
Genussmitel, 21, 211-216. 
Stadler, E. 1899. Ueber die Einwirkung von Kochsalz auf Bakterien die 
bei den sog. Fleischvergiftungen eine Rolle spielen. Arch. Hyg., 35, 
40-82. 
Thom, C. 1914. The Salt Factor in the Mold Ripened Cheeses. Storrs 
Agric. Exp. Sta. Bull. 79, 387-394. 
Thom, C. and Shaw, R. H. 1915. Moldiness in Butter. Jour. Ag. Research, 
3, 301-310. 
Weichel, A. 1910. The Effect of Salt upon Bacteria of the Ptomaine Group. 
Arb. kaiserl. Gesundh., 34, 247-265. Chern. Abstracts, 4, 3261 
Weigmann, H. 1913. Germ Content of Salt. Jahresbericht. Vers. Stat. 
Molkw. Landw. Kammer. Schleswig-Holstein, 1913, 9, 10. 
Wilbur, R. L. and Ophuls, W. 1914. Botulism. A report of food poisoning 
apparently due to eating of canned string beans with a report of a fatal case. 
Arch. Intern. Med., 589. CHAPTER XV 
EPIDEMIOLOGY 
No attempt will be made here to present a complete discussion of 
the subject, but merely a few remarks about epidemics and the method 
of investigating them. Boudreau (1914) has defined an epidemic as an 
unusual number of cases of a communicable disease, arising within a 
short period, in a limited area, and traceable to a common source or 
sources. The food bacteriologist may often find it necessary to inves- 
tigate the cause of an epidemic either to protect an innocent article of 
food or to check the spread of infection. It is by no means an easy task 
in some epidemics to pick out the real cause; in others, by using a logical 
procedure, the epidemiologist may select with great accuracy the focus 
of the infection and see the reward of his efforts by the immediate 
decrease of the number of cases. He must be on his guard for any and 
all information which may help him towards his goal. 
Different methods have been used in the past by epidemiologists. 
It used to be customary for him to use indirect methods in securing his 
data. If called to a community where there was an unusual amount of 
disease, he would go equipped with sample bottles for the collection of 
specimens of milk and water and would spend much time in the early 
part of his investigation in inspecting water supplies, methods of sewage 
treatment, etc. The mode of attack is now quite different and marks 
one of the distinct advances which preventive medicine has made. 
The present practice in investigating epidemics has been well stated by 
Hill (1912). A certain amount of information is always necessary with 
regard to the characteristics of the disease and some of the different 
methods of spreading. Certain characteristics have been worked out for 
epidemics caused by food, water, carriers, etc. These are by no means 
constant but serve in a general way along with such other general data 
such as, population of the community, endemicity of the disease in the 
community, general appearance, etc. If this preliminary, general 
study is well made the epidemiologist may save much time when he 
comes to analyze his other data because suggested causes of the infec- 
tion may be quickly eliminated. 
540 WATER-BORNE EPIDEMICS 
541 
Milk-borne Epidemics. The character of this article and the type 
of consumers gives a milk-borne epidemic of infection rather marked 
characteristics. Newman (1904) has mentioned the following: 
(a) There is a special incidence of the disease upon the track of the 
implicated milk supply. It is localized to such area. 
(b) Better class of houses and persons suffer most. 
(c) Milk drinkers are chiefly affected and they suffer most who are 
large consumers of raw milk. 
(d) Women and children suffer most and frequently adults suffer 
proportionately more than children. 
(e) Incubation periods are shortened. 
(f) There is a sudden onset and rapid decline. 
(f/) Multiple cases in one house occur simultaneously. 
(A) Clinically the attacks of the disease are often mild. Contact 
infectivity is reduced and the mortality rate is lower than usual. 
The above characteristics as outlined by Newman (1904) for milk- 
borne epidemics also fit other food epidemics. Sawyer (1914, 1915) 
reported investigations of two typhoid epidemics, one caused by water 
and the other caused by food. Fig. 84 shows the incidence of cases for 
both epidemics. These curves show very well the types which are 
secured for food and water-borne epidemics of typhoid fever. 
Water-borne Epidemics. Such epidemics of disease have been 
common in the past and many such epidemics are described in the liter- 
ature. The important characteristics of such epidemics may be de- 
scribed as follows: 
(a) Water-borne epidemics may be preceded by a period of dysen- 
tery. 
(b) There is usually a slow onset of the epidemic and a rapid decline. 
(c) The cases are evenly distributed over the city if the city is served 
by a municipal supply. 
(d) The larger water-borne epidemics have occurred in the spring. 
Epidemics caused by other foods will show, in general, other char- 
acteristics. If the typhoid fever is caused by oysters, it will be con- 
fined to that part of the year when oysters are usually eaten. Further- 
more, the great majority of the cases will be primary cases-those who 
have partaken of the oysters. To detect carriers may require much 
inferential evidence and the elimination of other suggested causes. 
After this has been done, the indication that a carrier is concerned must 
be supported by bacteriological examinations of the urine and feces of 
the suspected individual. 542 
EPIDEMIOLOGY 
Number of Cases 
Fig. 84.-Showing the General Characteristics of a Food-borne and Water-borne Epidemic of Typhoid 
Fever. (After Sawyer, 1915.) 
WATER-BORNE TYPHOID FEVER EPIDEMIC 
AT HEALDSBURG, SONOMA CO. 
CONTRASTED WITH 
FOOD-BORNE TYPHOID FEVER EPIDEMIC 
AT HANFORD, KINGS CO. 
Investigations by the Hygienic Laboratory of the 
California State Board of Health 
Cases 
Fatal Cases 
HEALDSBURG EPIDEMIC 
HANFORD EPIDEMIC 
Kumber of Cases 
Number of Cases WATER-BORNE TYPHOID FEVER 
543 
Fig. 85.-Spot Map of Typhoid Fever Epidemic of Water-borne Origin. (After Bartow.) 
ROCK island arsenal 
I l> I 1 J J II 11-11 U-I 1 -Ll u * -" 
DISTRIBUTION TYPHOID PEYER " ROCK ISLAND ILL. 
FEBURARR /9 * APRIL- 24 ML 
DAVENPORT 544 
EPIDEMIOLOGY 
Determination of the Origin of an Epidemic. In the majority of 
epidemics, the foci may be discovered with a fair degree of accuracy. 
Hill (1912) has given the detailed procedure and part of his paper is 
reproduced below. On account of the standing of this authority in this 
field, the author feels justified in quoting rather extensively from his 
work. In the last analysis, the epidemiologist must be a many-sided 
man; he must be somewhat of a bacteriologist, psychologist, and 
detective. He must have the intuition to separate the essential from 
the non-essential data. Then, somewhat like the lawyer, he should have 
studied reported epidemics in order to determine how others have been 
investigated. 
" To illustrate the general principles, let us suppose notification 
be received that a typhoid fever outbreak exists in a far-off community. 
The public health detective packs his grip and goes. He knows no 
details; he has never heard of this particular community before; he 
has not even any general information about the character of the country; 
he enters the community with no preconceived ideas. But he does know 
how typhoid fever originates and how it spreads. Water, milk, food, 
flies, and fingers are the routes-typhoid cases or typhoid carriers the 
source. His duties are to find both; and to find them, not as a scien- 
tific amusement, or as a matter of record; not to furnish food for spec- 
ulation-above all not to make a show of doing something-but to 
stop the outbreak; and then to advise measures to prevent recurrence. 
" The public health detective on entering the community affected 
by typhoid fever does not first examine the water supply, the milk sup- 
ply, the sewage disposal system, the markets, the back alleys, the 
dairies or anything else. He goes directly to the bedsides of the patients. 
Of course he must obtain the names and addresses of the patients 
from someone-from the local health officer, if he has them; from the 
attending physicians, if the health officer has no list; from the lay citi- 
zens themselves, if no one else is immediately available. The more 
complete the list, the faster he can work, because then he is not com- 
pelled to hunt up the cases personally. But if there be no list, he 
begins making one himself. His intention is to see just as many patients 
as he can, for each furnishes evidence and he wants it all. But he knows 
that it is not always necessary at this stage to see absolutely all the 
patients, so long as he sees the majority. 
" Reaching the patient's * bedside, his investigation begins. Auto- 
* If the patient is a child or delirious or not strong enough for an interview, or 
speaks only some foreign tongue, the relatives, friends or associates must supply the 
information. ORIGIN OF EPIDEMICS 
545 
matically, almost mechanically, he decides whether or not the patient 
has typhoid fever or not. Satisfied on that point, his first question is 
not, ' Tell me all the different water supplies you have used, or all the 
sources of milk you have used.' The first question is, ' When did you 
first show the earliest symptoms of the disease?' Why? Because 
this date once fixed, the date at which infection entered the patient's 
mouth is fixed also, i.e., a date between one and three weeks previous 
to the date of earliest symptoms.* Remember that at this stage the 
detective may not have even an inkling as to which of the usual factors, 
water, milk, food, flies or fingers, is involved. Still less can he guess 
which particular water supply, milk supply, etc., of the many possible 
ones, may be the guilty one. But the answer to this question reduces 
possible routes to those used by this patient-not at anytime-but during 
a specific period, i.e., from one to three weeks preceding his date of 
earliest symptoms. 
" Not yet, however, are the milk and water questions offered. The 
second question is, ' Where were you during that period? ' Why? 
Because if the patient were not in the community during that period, 
he could not have contracted his infection within it, and does not 
belong to the outbreak under examination at all but to some other. 
He is, in brief, an ' imported case ' and while, of course, he is to be 
supervised lest he spread his infection to others, he cannot help to 
locate the source of the main outbreak-unless perchance he be himself 
that source, i.e., the introducer to the community of the original infection. 
If he be an imported case he is noted for further reference and the 
detective goes to another patient. If not, the questions continue. 
But not yet is water or milk or flies mentioned. The third question is 
' Were you associated during your period of infection with any then 
known typhoid case? ' Why? Because such association, especially if 
intimate, makes it more than probable that the case under examination 
received his infection from the preceding case, rather than from any 
general route and that he is, therefore, a ' secondary ' case. If he had 
such associations, this is noted for further reference and the investi- 
gator passes on to another bedside. If not, the questions continue 
and now, at last, take up milk, water, food, etc., but, of course, only so 
far as to determine those used by the patient during his infection 
period. 
" Then the investigator passes to the next patient. What has he 
learned so far? Nothing much yet. But he has narrowed the possible 
* The occasional exceptions do not affect the validity .of this statement as a 
practical working rule. 546 
EPIDEMIOLOGY 
routes of infection to certain water supplies, certain milk supplies, 
certain food supplies, etc., i.e., those used by the first patient during a 
certain period, and he has done this in thirty minutes-in scarcely the 
time it takes for the old-style investigator to get his bottles ready to 
collect his first water sample. 
11 At the bedside of the second patient, the same inquiries in the 
same order arc made. If this second patient be an imported case, or a 
secondary case, he also is merely noted for future reference. If he be 
a primary, however, the origins of his drinking water, milk, food, etc., 
during his infection period are also ascertained. Perhaps he coincides 
with the first patient in every detail of alimentary supplies, in history 
and associations. If so, nothing much has been added to the detective's 
knowledge. But more than likely, dissimilarities have developed. 
Since the responsible water supply, milk supply, etc., must be one of 
those water supplies, milk supplies, etc., used in common by primary cases 
all those not common to both of these primary cases may be dropped 
from consideration (except in rare instances of multiple routes). Thus, 
if both have used the same water, water from that origin remains as a 
possibility. But if the water supplies have been different, water is 
eliminated from the question entirely. If the milk supplies are identical, 
milk remains as a possible route of infection; if not, milk is eliminated 
from the question entirely. 
" In brief, provided the information obtained be reliable, and it is a 
part of the public health detective's training to distinguish at a glance 
truth from falsehood, the honestly mistaken, or forgetful, or stupid 
replies from the reliable ones-and above all never to believe anything 
(to the extent of recording it) unless it is checked, confirmed and estab- 
lished as a fact, the modern investigator has in one hour narrowed his 
investigation to a point which the old-style investigator often would 
not reach for weeks. 
" And so from patient to patient the inquiry proceeds. In the course 
of the day the investigator has seen perhaps thirty patients. The 
tabulation (probably already made in his own mind) shows, say, three 
imported cases, five secondaries, two uncertain or indefinite. The 
remaining primary cases show in common, say, one water supply only, 
the milk, etc., varying; or one milk supply only, the water, etc., vary- 
ing; or no connection except attendance at some one social function. 
" Going straight to the route thus indicated, the public health 
detective quickly confirms the indications of his results. He knows 
that the route indicated must be the guilty one for only that route 
can account for all the cases. He concentrates on that route until ORIGIN OF EPIDEMICS 
547 
the evidence is complete-when and how that route became infected, 
when and by what sub-routes the infection was distributed, why it 
infected the patients found and not others, etc. 
"In this illustration I have assumed complete ignorance on the 
part of the epidemiologist as to everything connected with the com- 
munity he is investigating, except what he finds by cross-examining 
the patients. As a matter of fact, every epidemiologist, however much 
a stranger to the particular community he enters, begins to learn about 
it from the moment he enters it. 
" Thus almost unconsciously he notes the size of the town and com- 
pares it with the number of cases reported as existing; if it is summer 
time he almost automatically notes the presence or absence of open 
toilets in the back yards, of manure piles and of garbage cans-all 
bearing upon fly infection. If it is winter time or the community be 
well sewered, he does not even consider flies. If the cases are grouped 
in one quarter of the town, while the public water supply extends all 
over it, he tentatively eliminates the water supply, before he asks a 
question. If good surface drainage and a sandy soil exist, or driven 
wells arc chiefly in vogue, he tentatively eliminates well water-even 
before he registers at the hotel. 
" This is not and cannot be a complete synopsis of all the combina- 
tions of circumstances which the epidemiologist meets. It is intended 
to illustrate his methods and to show why they are incredibly rapid 
and incredibly accurate-how they eliminate speculation and guarantee 
a correct solution-which means of course the achievement of the 
great end, the finding of proper measures for suppression. 
" As soon as the route is indicated, he must go to that route, and 
establish beyond peradventure that it was in truth responsible. A 
water supply cannot convey typhoid if typhoid fever discharges have 
not entered it. There is no object in attributing an outbreak to fly 
infection from toilets into which typhoid feces have not been dis- 
charged at such a time as to account for the cases. A milk supply, 
not handled at some point by an infected person, nor adulterated at 
some time with infected extraneous matter cannot convey typhoid. 
Whatever his results, they cannot be true unless they are consistent- 
they should not be accepted unless they are provable-and proved. 
" If the public health detective is familiar with the community 
where the outbreak occurs, including its water supplies, its milk sup- 
plies, the sociological relationships of its people, etc., etc., he can often 
tentatively determine the cause of the outbreak by a mere inspection 
of the names and addresses of primary cases, especially if plotted on 548 
EPIDEMIOLOGY 
a map of the community, taking into account also the time of year, 
and other general points. But such deductions, while often wonderfully 
reliable, can never be as conclusive and satisfactory as are the results 
of an investigation by even a total stranger, if the investigation be con- 
ducted as above described." 
The data received from each patient should be recorded on a case 
card. Many different cards have been described in the literature. The 
one given below has been used by the Illinois State Water Survey. 
Others have been described by Hill (1912), Boudreau (1914), Ferguson 
(191 ) and Hansen (1914). 
Suggested Form for Securing Information Relative to Typhoid Fever Cases by 
the Local Health Authorities. 
Record No 
TYPHOID FEVER RECORD 
1. Name of Patient 
2. Residence, Street and Number 
3. Age 
4. Sex 
5. Give Occupation and Place of Business 
6. Name of Physician 
7. Date when Patient Took to Bed 
8. Date of First Symptoms 
9. Date of Physician's First Visit 
10. Date of Leaving Bed on Recovery 
11. Date of Relapse 
12. Date of Death 
13. Was Widal Test Made? Date .Positive or Negative 
14. Was Spleen Enlarged and Were Rose Spots Present? 
15. Was Patient Absent from City During the Two Months Previous to 
Illness? If so, state where he visited and between 
what dates 
16. If Patient Changed Residence During Two Months Previous to Illness, 
Give Former Address and Date of Change 
17. Character of Residence (private house, boarding house, apartment, hotel, 
etc.) . 
18. State Source of Drinking Water. At Home, at Place of Business, Else- 
where CASE CARD 
549 
19. State Source of Milk Supply and Name of Dealer. Was Milk Habitually 
Drunk? Was it Occasionally Used, as in Tea or Coffee or in Ice Cream? 
20. Were Raw Oysters, Lettuce or Celery Eaten Raw within Three Weeks before 
Illness? 
21. State from Whom Bread was Obtained 
22. Did Patient Fish or Bathe in Neighboring Streams? 
If so, State at what Point or Points 
23. Did Patient use any Public Baths or Swimming Pools? 
If so, give Dates 
24. Give Number of Persons Living in Same House or Apartment 
Also Give Number in Whole Building in Case of Hotel, Apartment House, 
etc 
25. Give Names of Other Cases in Same Building and Dates when they Went 
to Bed 
26. Give Names and Approximate Dates for Cases among Business Associates 
27. Give Names and Approximate Dates for Cases among Friends (Include 
Schoolmates) 
28. Did Patient Attend any Social Gatherings where Food was Served within 
Two Months before Illness? If so, Gives Dates 
29. Describe Sanitary Condition of Premises. Note Especially Outdoor 
Privies, Cesspools, Wells and Refuse Piles 
30. Is House Well Screened? 
31. Had Patient a Separate Room? 
32. Had Patient a Trained Nurse or was Nursing done by Member of the Family 
or Household? 
33. Where Stools and Urine Disinfected? If so, Describe How this was Carried 
Out and Note Length of Time Continued 
Remarks. To Include any Facts, Statements or Comments not Included 
in the Foregoing that May Throw Light on the Source and Mode of Infec- 
tion 
Information Given by 
Information Obtained by 
Date of Obtaining Information 550 
EPIDEMIOLOGY 
APPENDIX I. FORMS FOR TYPHOID INVESTIGATIONS 
Suggested Form for Physicians' Report on Typhoid Fever 
Typhoid Fever 
Name 
Street and Number 
Sex 
Age 
Place of Business 
Occupation. 
Physician 
Date of Taking to Bed 
Date of First Symptons 
Date of Physician's First Visit 
Was Widal Test Made? Positive or Negative 
Was Patient Out of Town within Thirty Days? 
Between what Dates 
Are There Other Cases in House? 
If So, How Many 
State Source of Drinking Water 
Give Name of Milk Dealer. 
Remarks 
The preparation of a spot map is very often an aid in studying epi- 
demics. If the cases are about evenly distributed over a city it is a 
fair indication that the water supply is to blame. If the cases are 
grouped in several localities, it may indicate that a milk supply or a 
carrier is to blame. Fig. 84 shows such a map which was prepared 
by Bartow (1912) for a water-borne epidemic. 
BIBLIOGRAPHY 
Bartow, E. Discussion to Paper by Hill (1912). 
Boudreau, F. G. 1914. The Mode of Procedure in the Study of Epidemics. 
Ohio State Board of Health Bull. 4, 1277-1286. 
Ferguson, H. F. 1916. Epidemic of Typhoid Fever Caused by Polluted 
Water Supply at Old Salem Chautauqua. Illinois State Water Survey 
Bulletin No. 13, 272-286. 
Hansen, P. 1914. A Study of Typhoid Fever in Rockford, Illinois, in the 
Late Summer and Fall of 1913. Illinois State Water Survey Bull No 11 
384-430. BIBLIOGRAPHY 
551 
Hill, II. W. 1912. The Detailed Procedure to be Followed in an Epidem- 
iological Determination of the Origin of a Typhoid Outbreak. Proceedings 
Illinois Water Supply Assn, 1912, 96-105. 
Jordan, E. 0. and Irons, E. E. 1912. The Rockford (Ill.) Typhoid Epi- 
demic. Jour. Inf. Diseases, 11, 21-43. 
Sawyer, W. A. 1914. Ninety-three Persons Infected by Typhoid Carrier 
at a Public Dinner. Jour. Amer. Med. Assn., 63, 1537-1542. 
Sawyer, W. A. 1915. A Water-borne Typhoid Fever Epidemic. California 
State Board of Health, 10, 303-311. APPENDIX 
Indicator Solutions. The following table is taken from Hawk's 
Physiological Chemistry and will be found useful in bacterial work. 
The amounts of the indicator solutions and their strengths to be used in 
the determination of hydrogen ion concentrations in 10 c.c. portions 
of unknown solution are indicated below: 
INDICATOR SOLUTIONS 
Drops. 
Preparation of Solution. 
1. Alizarin yellow R (p-nitro- 
benzine-azo-salicylic acid) 
10-5 
0.1 gm. in 1000 c.c. water. 
2. Azolitmin (litmus) 
Aqueous solution. 
Alcoholic solution. 
3. Cochineal 
4. 2.5-dinitro-hydrochinone.. 
5-2 
1 gm. to 1000 c.c. alcohol. 
5. Mauvein 
8-1 
0.5 gm. to 1000 c.c. 
6. Methyl orange 
5-3 
0.1 recrystallized salt to 1000 c.c. water. 
7. Methyl red 
4-2 
Saturated solution in 50 per cent alcohol. 
8. Methyl violet 
8-1 
0.5 gm. in 1000 c.c. water. 
9. Neutral red 
20-10 
0.1 gm. in 500 c.c. alcohol and 500 c.c. 
10. p-nitrophenol 
20-3 
water. 
0.4 gm. to 60 c.c. alcohol, 940 c.c. water. 
11. Phenolphthalein 
20-3 
0.5 gm. to 500 c.c. water, 500 c.c. alcohol. 
12. Rosolic acid 
15-6 
0.4 gm. in 400 c.c. alcohol, 600 c.c. water. 
13. Thymolphthalein 
10-3 
0.4 gm. to 500 c.c. alcohol, 500 c.c. water. 
14. Tropaeolin 0 
10-5 
0.1 gm. to 1000 c.c. water. 
15. Tropaeolin 00 
5-3 
Of recrystallized salt, 0.1 gm to 1000 c.c. 
16. Tropaeolin 000 
10-4 
water. 
0.1 gm. to 1000 c.c. water. 
In an extensive study of the application of H-ion determination to 
bacteriological work, Clark and Lubs (1917) have pointed out the 
error in determining reaction by titration. They used the electro- 
metric method and by means of it prepared a series of new indicators 
553 554 
APPENDIX 
which will be valuable in future bacteriological work. They are given 
in Table LII. 
Table LII 
LIST OF INDICATORS, PREPARED BY CLARK AND LUBS, 1918 
Chemical Name. 
Common Name. 
Concen- 
tration, 
Per Cent. 
Color Change. 
Range 
Ph 
Thymol sulphon phthalein (acid range) 
Thymol blue 
0.04 
Red-yellow 
1.2-2.8 
Tetra bromo phenol sulphon phthalein 
Ortho carboxy benzine azo di methyl 
Brom phenol blue 
0.04 
Yellow-blue 
3.0-46 
aniline 
Methyl red 
0.02 
Red-yellow 
4 4 6 0 
Ortho carboxy benzine azo di propyl 
aniline 
Propyl red 
0.02 
Red-yellow 
4 8 6 4 
Di bromo ortho cresol sulphon phtha- 
lein 
Brom cresol purple 
0.04 
Y ellow-purple 
5.2-6 8 
Di bromo thymol sulphon phthalein. . 
Brom thymol blue. 
0.04 
Yellow-blue 
6.0-7 6 
Phenol sulphon phthalein 
Ortho cresol sulphon phthalein 
Thymol sulphon phthalein (see above). 
Phenol red 
0.02 
Y ellow-red 
6.8-8 4 
7 2 8 8 
Cresol red 
0.02 
Yellow-red 
Thymol blue 
0.04 
Yellow-blue 
8.0-9.6 
Ortho cresol phthalein 
Cresol phthalein. . . 
0.02 
Colorless-red 
8.2-9.8 
Preparation of Standard Reagents 
A standard solution contains a known amount of reacting substance 
and is to be distinguished from a normal solution. A normal solution 
contains the hydrogen equivalent of the active constituent in grams per 
liter. This is that amount which brings into the reaction 1.008 gms. of 
hydrogen, 8 gms. of oxygen, etc., or the equivalent. This definition has 
so many distinct advantages that it is now generally accepted. 
The following are some of the standard solutions used in bacteriology: 
N Sodium Hydroxide. Weigh out 40.06 or better about 45 gms. of 
sodium hydroxide and dissolve in 1 liter of boiled cooled distilled water. 
Standardize against normal hydrochloric acid. It is best to allow the 
two solutions to stand at the same temperature for several hours or 
overnight. The titration should be repeated several times. Calcula- 
tion is as follows: 
If 50 c.c.NaOH = 52.6 c.c. NHC1 
the NaOH is too strong by 2.6 c.c. per every 50 c.c. 
mnn . 2.6X1000 _ 
50 : 2.6=1000 : x x = - = 50.c.c. 
50 APPENDIX 
555 
Table II. 
A 
1 
2 
3 
- 
5 
6 
- 
7 
- 
8 
9 
10 
11 
12 
13 
14 
True Acidity =CH+ 
1 
10-1 
10-2 
IO-' 
10-' 
10 
10-• 
IO-' 
10-» 
10-» 
10-w 
10-11 
10-" 
10-is 
IO-1* 
True Alkalinity = COH~ 
10 
lO-n 
10-*2 
10-" 
lO-io 
io-» 
10-' 
10 -♦ 
io-« 
10-5 
10-' 
IO-' 
10-' 
io-* 
1 1 
Dimethylamidoazobenzine 
Cherry- 
red 
-F- 
Flesh- 
Colored 
Golden- 
Yellow 
4- 
4- 
4- 
4- 
4- 
Methyl Orange 
Rose- 
red 
-F- 
-1- 
-t- 
Orange- 
red 
Yellow 
4- 
4- 
4- 
4- 
Sodium-alizarinsulphonate 
Greenish 
Yellow 
-b 
-F- 
-F- 
-F- 
-1- 
Red 
4- 
4- 
4- 
4- 
4- 
Rosolic Acid 
-F- 
Yellow 
-F- 
-F- 
-1- 
Rose 
Red 
4- 
4- 
4- 
Guiac Tincture 
-F- 
Color- 
less 
-F- 
-F- 
Greenish 
Yellow 
4- 
4- 
4- 
4- 
Phenolphthalein 
Color- 
less 
-F- 
-4- 
-1- 
Red 
4- 
4- 
4- 
4- 
Thymolphthalein 
Color- 
less 
-F- 
-F- 
-F- 
Blue 
4- 
4- 
4- 
Tropaolin 0 
Yellow 
-F- 
-F- 
-F- 
-F- 
Orange 
Yellow 
4- 
4- 
Trinitrobenzine 
Color- 
less 
-F- 
-F- 
-F- 
Orange 
4- 
Benzopurpurin 
Yellow 
-F- 
-1- 
-4- 
Red 
Methyl Red 
-F- 
Red 
-I- 
Pink 
Yellow 
4- 
•fc- 
Congo Red 
-*■ 
Blue 
Violet 
Reddish- 
Violet 
Orange 
Orange- 
Red 
4- 
4- 
Litmus 
-F- 
Red 
Red 
Red 
Rod- 
Violet 
Violet 
Blue 
4- 
4- 
Cochineal 
-F- 
-F- 
Yellow 
Yellow- 
Pink 
Lilac 
4- 
4- 
Water Suppb'Vss^iadon^ Wtana' Ilf' 1910 93The significance of the term alkalini'y >" water analysis and the determination of alkalinity by means of indicators. Proceedings Illinois 556 
APPENDIX 
This amount of water should be added, after which the solution will 
be exactly normal. It should be checked, however, against the N HC1. 
From this the N/20 NaOH may be prepared by diluting 50 c.c. of the 
N NaOH to a liter. 
N Potassium Hydroxide. Dissolve 56 gms. of KOH in 1000 c.c. of 
distilled water and standardize againstN HClas was done with N NaOH. 
N Sodium Carbonate. Ignite the bicarbonate NaHCO3 to redness 
and dissolve 53 gms. in a liter of water. Standardize against normal 
acid and dilute accordingly with distilled water. 
N Sulphuric Acid. Follow the procedure which has been given else- 
where for N HC1. This may be standardized against N NaOH or grav- 
imetrically by precipitating with BaOH. 
N Hydrochloric Acid. This should contain 36.46 gms. of HC1 in 
a liter of water. The pure concentrated HC1 may be diluted to a 
specific gravity of 1.020. According to the table on page 559, it will be 
seen that this solution is too strong, having about 4.13 gms. of HC1 in a 
liter. To be made exactly normal, it should be titrated against pure 
Na2COa. This is obtained by heating the NaHCO3 being careful not 
to allow it to fuse. This is then put into a weighing bottle from which 
it is weighed into Erlenmeyer flasks for titrating. Dissolve in boiled 
distilled water and titrate with the HC1 solution using methyl orange as 
the indicator. Calculations are made as follows, assuming for example 
that 1.9864 gms. of Na2CO3 neutralized 37.28 c.c. of the HC1 solution. 
If our solution had been exactly normal 1 liter would have neu- 
tralized 
Na2CO3 106.00 __ , ,T 
--- = --- =53 gms. of Na2CO3 
53 : 1000 = 1.9864 : x 
x = 37.48. 
37.28 c.c. of the HC1 solution were required and it is evident that 
this solution is too strong and that for each 37.28 c.c. of the solution, 
37.48 -37.28 = .20 c.c. of water must be added. The amount of water 
to be added to each liter of the HC1 solution to make it exactly normal 
is computed as follows: 
37.28 : .20 = 1000 : x 
x = amount of distilled water to be added. 
N Nitric Acid. See preparation of N HC1. 
N Oxalic Acid. Dissolve 47 gms. of pure dry oxalic acid in 1000 c.c. 
of distilled water. Standardize against N Na2CO3. APPENDIX 
557 
N/10 Potassium Permanganate. Dissolve 3.163 gms in a liter of 
water and standardize against ferrous ammonium sulphate, oxalic acid 
or pure iron. 
1 c.c. = .0056 gm. Fe 
.0072 gm. FeO 
.008 gm. Fe2O3. 
1 c.c. = . 0008 gm. O. 
.0063 gm. H2C4O4.2H2O. 
N 10 Iodine. Dissolve 12.685 or 12.8 gms. of pure iodine and 18 
gms. of potassium iodide in a liter of distilled water. Standardize 
against N/10 Na2S20s. 
1 c.c. = .0158 gm. Na2S2O3, 
= . 00495 gm. AS2O3, 
= .00575 gm. AS2O5. 
N/10 Sodium Thiosulphate. Dissolve 24.9 gm. of the crystals in 
1000 c.c. of water and standardize against N/10 iodine. 
N/10 Sodium Chloride. Dissolve 5.9 of the salt in 1000 c.c. of water. 
Standardize gravimetrically by precipitating as AgCl. 
1 c.c. = .0058 gm. NaCl, 
= . 0035 gm. Cl, 
= .0108 gm. Ag. 
N/10 Silver Nitrate. Dissolve 16.966 gms. of AgNOs in a liter of 
water and standardize by precipitating as AgCl. The preparation of 
standard silver nitrate for water analysis is outlined in another place. 
Preparation of Antiformin. Dissolve 720 gms. of chloride of lime 
and 260 gms. of sodium carbonate in 2500 c.c. of water. Allow to stand 
and filter. By means of N/10 sodium thiosulphate determine the 
amount of available chlorine and adjust so that it will contain about 
5.6 per cent. Add 7.5 per cent of sodium hydroxide, filter and store for 
future use. 
Azolitmin Solution. Add 1 per cent of Kahlbaum's azolitmin to 
distilled water and boil for five minutes. Adjust the reaction, if 
necessary, with NaOH. It should give a distinct blue plate when it is 
diluted in the Petri dish with the medium. 
Benedict's Reagent for the Quantitative Estimation of Dextrose. 
Copper sulphate (crystallized) 18.00 gms. 
Sodium carbonate (anhydrous) 100.00 gms. 
Sodium citrate 200.00 gms. 
Potassium thiocyanate 125.00 gms. 
Potassium ferrocyanide (5 per cent solution). 5.00 c.c. 
Distilled water to make 1 liter. 558 
APPENDIX 
Dissolve the carbonate, citrate and thiocyanate in 800 c.c. of water 
and filter if necessary. Dissolve exactly 18 gins, of copper sulphate in 
100 c.c. of distilled water. Add this to the first solution with constant 
stirring after which add the 5 c.c. of potassium ferrocyanide and dilute 
to a liter. 
Diluting Fluid for Leucocytes. Prepare a 0.5 per cent solution of 
acetic acid and tinge slightly with gentian violet. 
Hayem's Diluting Fluid for Erythrocytes. 
Sodium sulphate (Na2SO4) 5 gms. 
Mercuric chloride (HgCU) 0.5 gm. 
Sodium chloride (NaCl) 1.0 gms. 
Distilled water 200 c.c. 
Litmus or Azolitmin Solution. The standard solution of litmus 
shall be a 2 per cent solution of reagent litmus in water. Crush the 
litmus in a mortar with a little water and, after diluting to the mark, 
boil for five minutes. Distribute in flasks or tubes and sterilize. This 
solution generally needs no correction but may be adjusted writh dilute 
NaOH or HC1 if necessary. 
Millon's Reagent. Dissolve 30 c.c. of mercury in 570 c.c. of con- 
centrated nitric acid and dilute with two parts of water. 
Molisch Reagent. Dissolve 15 gms. of a-naphthol in 95 per cent 
alcohol. 
Nylander's Reagent. Boil 4 gms. of potassium hydrogen tartrate 
and 2 gms. of bismuth subnitrate in 100 c.c. of 10 per cent KOH. When 
cool filter. 
Phenylhydrazine Mixture. Thoroughly mix two parts of sodium 
acetate and one part of phenylhydrazine hydrochloride.. 
Seliwanoff's Reagent. Dissolve .05 gm. resorcinol in 100 c.c. of 
dilute HC1. 
Toisson's Diluting Fluid for Erythrocytes. 
Sodium sulphate (Na2SO4) 8 gms. 
Sodium chloride (NaCl) 1 gm. 
Glycerol 30 c.c. 
Distilled water 160 c.c. 
Color with gentian violet. 
Uffelmann's Reagent. Add ferric chloride to phenol until a bluish- 
violet color results. APPENDIX 
559 
SPECIFIC GRAVITIES OF HYDROCHLORIC, NITRIC, AND SULPHURIC 
ACIDS 
(After G. Lunge) 
Specific 
Gravity 
x 15° 
at 
(Vacuo). 
Per Cent by Weight. 
Specific 
Gravity 
x 15° 
at 4^- 
(Vacuo). 
Per Cent by Weight. 
HC1. 
HNOj. 
H2SO4. 
HC1. 
HNO3 
H2SO4. 
1.000 
0.16 
0.10 
0.09 
1.225 
36.03 
30.48 
1.005 
1.15 
1.00 
0.83 
1.230 
36.78 
31.11 
1.010 
2.14 
1.90 
1.57 
1.235 
37.53 
31.70 
1.015 
3.12 
2.80 
2.30 
1.240 
38.29 
32.28 
1.020 
4.13 
3.70 
3.03 
1.245 
39.05 
32.86 
1.025 
5.15 
4.60 
3.76 
1.250 
39.82 
33.43 
1.030 
6.15 
5.50 
4.49 
1 255 
. . . 
40.58 
34.00 
1.035 
7.15 
6.38 
5.23 
1.260 
41.34 
34.57 
1.040 
8.16 
7.26 
5.96 
1.265 
42.10 
35.14 
1.045 
9.16 
8.13 
6.67 
1.270 
42.87 
35.71 
1.050 
10.17 
8.99 
7.37 
1.275 
43.64 
36.29 
1.055 
11.18 
9.84 
8.07 
1.280 
44.41 
36.87 
1.060 
12.19 
10.68 
8.77 
1.285 
45.18 
37.45 
1.065 
13.19 
11.51 
9.47 
1.290. 
45.95 
38.03 
1.070 
14.17 
12.33 
10.19 
1.295 
46.72 
38.61 
1 075 
15.16 
13.15 
10.90 
1.300 
47.49 
39.19 
1 080 
16.15 
13.95 
11.60 
1.305 
48.26 
39.77 
1.085 
17.13 
14 74 
12.30 
1.310 
49.07 
40.35 
1.090 
18.11 
15.43 
12.99 
1.315 
49.89 
40.93 
1.095 
19.06 
16.32 
13.67 
1.320 
50.71 
41.50 
1.100 
20.01 
17.11 
14.35 
1.325 
51.53 
42.08 
1 105 
20.97 
17.89 
15.03 
1.330 
52.37 
42.66 
1.110 
21 92 
18.67 
15.71 
1.335 
53.22 
43.20 
1 115 
22.86 
19.45 
16.36 
1.340 
54.07 
43.74 
1.120 
23.82 
20.23 
17.01 
1.345 
54.93 
44.28 
1.125 
24.78 
21.00 
17.66 
1.350 
55.79 
44.82 
1.130 
25.75 
21.77 
18.31 
1.355 
56.66 
45.35 
1 135 
26.70 
22.54 
18.96 
1.360 
57.57 
45.88 
1.140 
27.66 
23.31 
19.61 
1.365 
58.48 
46.41 
1.145 
28.61 
24.08 
20.26 
1.370 
59.39 
46.94 
1.150 
29.57 
24.84 
20.91 
1.375 
60.30 
47.47 
1.155 
30.55 
25.60 
21.55 
1.380 
61.27 
48.00 
1.160 
31 52 
26.36 
22.19 
1.385 
62.24 
48.53 
1.165 
32.49 
27.12 
22.83 
1.390 
63 23 
49.06 
1.170 
33.46 
27.88 
23.47 
1.395 
64.25 
49.59 
1.175 
34.42 
28.63 
24.12 
1.400 
65.30 
50.11 
1.180 
35.39 
29.38 
24.76 
1.405 
66.40 
50.63 
1.185 
36.31 
30.13 
25.40 
1.410 
67.50 
51.15 
1.190 
37 23 
30.88 
26.04 
1 .415 
68.63 
51.66 
1 195 
38.16 
31.62 
26.68 
1.420 
69.80 
52.15 
1.200 
39.11 
32.36 
27.32 
1 .425 
70.98 
52.63 
1.205 
33.09 
27.95 
1.430 
72.17 
53.11 
1.210 
33.82 
28.58 
1.435 
73.39 
53.59 
1.215 
34.55 
29.21 
1 .440 
74.68 
54.07 
1.220 
35.28 
29.84 
1.445 
75.98 
54.55 
From Reed's Manual of Bacteriology, Ginn & Co. 560 
APPENDIX 
SPECIFIC GRAVITIES OF HYDROCHLORIC, NITRIC, AND SULPHURIC 
ACI DS.-Con tinued 
Specific 
Gravity 
. 15° 
at . 
(Vacuo). 
Per Cent by Weight. 
Gravity 
x 15° 
at 
(Vacuo). 
Per Cent by Weigth. 
HC1. 
HNOs. 
H2SO4 
HCl. 
HNOi. 
H2SO4. 
1.450 
1.455 
1.460 
1.465 
1.470 
1.475 
1.480 
1.485 
1.490 
1.495 
1.500 
1.505 
1.510 
1.515 
1.520 
1.525 
1.530 
1.535 
1.540 
1.545 
1.550 
1.555 
1.560 
1.565 
1.570 
1.575 
1.580 
1.585 
1.590 
1.595 
1.600 
1.605 
1.610 
1.615 
1.620 
1.625 
1.630 
1.635 
1.640 
1.645 
1.650 
1.655 
1.660 
1.665 
77.28 
78.60 
79.98 
81.42 
82.90 
84.45 
86.05 
87.70 
89.60 
91.60 
94.09 
96.39 
98.10 
99.07 
99.67 
55.03 
55.50 
55.97 
56.43 
56.90 
57.35 
57.83 
58.28 
58.74 
59.22 
59.70 
60.18 
60.65 
61.12 
61.59 
62.06 
62.53 
63.00 
63.43 
63.85 
64.26 
64.67 
65.08 
65.49 
65.90 
66.30 
66.71 
67.13 
67.59 
68.05 
68.51 
68.97 
69.43 
69.89 
70.32 
70.74 
71.16 
71.57 
71.99 
72.40 
72.82 
73.23 
73.64 
74.07 
1.670 
1.675 
1.680 
1.685 
1.690 
1.695 
1.700 
1.705 
1.710 
1.715 
1.720 
1.725 
1.730 
1.735 
1.740 
1.745 
1.750 
1.755 
1.760 
1.765 
1.770 
1.775 
1.780 
1.785 
1.790 
1.795 
1.800 
1.805 
1.810 
1.815 
1.820 
1.825 
1.830 
1.835 
1.840 
1.8405 
1.8410 
1.8415 
1.8410 
1.8405 
1.8400 
1.8395 
1.8390 
1.8385 
74.51 
74.97 
75.42 
75.86 
76.30 
. 76.73 
77.17 
77.60 
78.04 
78.48 
78.92 
79.36 
79.80 
80.24 
80.68 
81.12 
81.56 
82.00 
82.44 
82.88 
83.32 
83.90 
84.50 
85.10 
85.70 
86.30 
86.90 
87.60 
88.30 
89.05 
90.05 
91.00 
92.10 
93.43 
95.60 
95.95 
97.00 
97.70 
98.20 
98.70 
99.20 
99.45 
99.70 
99.95 APPENDIX 
561 
ALCOHOL TABLE MODIFIED FROM WINDISCH 
Specific 
Gravity of 
Distillate. 
Per Cent of 
Alcohol by 
Weight. 
Per Cent of 
Alcohol by 
Volume. 
Specific 
Gravity of 
Distillate. 
Per Cent of 
Alcohol by 
Weight. 
Per Cent of 
Alcohol by 
Volume. 
1.0000 
0.00 
0.00 
0.9908 
5.20 
6.55 
0.9998 
0.11 
0.13 
6 
5.32 
6.71 
6 
0.21 
0.27 
4 
5.45 
6.86 
4 
3.32 
0.40 
2 
5.57 
7.02 
2 
0.42 
0.53 
0 
5.70 
7.18 
0 
0.53 
0.67 
0.9898 
5.83 
7.33 
0.9988 
0.64 
0.80 
6 
5.95 
7.50 
6 
0.74 
0.93 
4 
6.08 
7.66 
4 
0.85 
1.07 
2 
6.21 
7.82 
2 
0.96 
1.20 
0 
6.34 
7.99 
0 
1.06 
1.34 
0.9888 
6.47 
8.15 
0.9978 
1.17 
1.48 
6 
6.59 
8.31 
6 
1.28 
1.61 
4 
6.73 
8.48 
4 
1.39 
1.75 
2 
6.88 
8.64 
2 
1.50 
1.88 
0 
6.99 
8.81 
0 
1.60 
2.02 
0.9878 
7.12 
8.98 
0.9968 
1.71 
2.16 
6 
7.26 
9.15 
6 
1.82 
2.30 
4 
7.39 
9.32 
4 
1.93 
2.44 
2 
7.53 
9.48 
2 
2.04 
2.58 
0 
7.66 
9.66 
0 
2.16 
2.72 
0.9868 
7.80 
9.83 
0.9958 
2.27 
2.86 
6 
7.94 
10.00 
6 
2.38 
3 00 
4 
8.07 
10.17 
4 
2.49 
3.14 
2 
8.21 
10.35 
2 
2.60 
3.28 
0 
8.35 
10.52 
0 
2.72 
3.42 
0.9858 
8.49 
10.70 
0.9948 
2.82 
3.56 
6 
8.63 
10.88 
6 
2.94 
3.71 
4 
8.77 
11.05 
4 
3.06 
3.85 
2 
8.91 
11.23 
2 
3.17 
4.00 
0 
9.06 
11.41 
0 
3.29 
4.14 
0.9848 
9.20 
11.59 
0.9938 
3.40 
4.29 
6 
9.34 
11.77 
6 
3.52 
4.43 
4 
9.49 
11.95 
4 
3.64 
4.58 
2 
9.63 
12.14 
2 
3.75 
4.73 
0 
9.79 
12.32 
0 
3.87 
4.88 
0.9838 
9.92 
12.50 
0.9928 
3.99 
5.03 
6 
10.07 
12.69 
6 
4.11 
5.18 
4 
10.22 
12.88 
4 
4.23 
5.33 
2 
10.36 
13.06 
2 
4.35 
5.48 
0 
10.52 
13.25 
0 
4.47 
5.53 
0.9828 
10.66 
13.44 
0.9918 
4.59 
5.78 
6 
10.81 
13.63 
6 
4.71 
5.93 
4 
10.96 
13.82 
4 
4.83 
6.09 
2 
11.12 
14.01 
2 
4.95 
6.24 
0 
11.27 
14.20 
0 
5.08 
6.40 562 
APPENDIX 
Specific 
Gravity of 
Distillate. 
Per Cent of 
Alcohol by 
Weight. 
Per Cent of 
Alcohol by 
Volume. 
Specific 
Gravity of 
Distillate. 
Per Cent of 
Alcohol by 
Weight. 
Per Cent of 
Alcohol by 
Volume. 
0.9818 
11.42 
14.39 
0.9718 
19.30 
24.32 
6 
11.57 
14.58 
6 
19.45 
24/51 
4 
11.72 
14.77 
4 
19.60 
24.70 
2 
11.88 
14.97 
2 
19.76 
24.89 
0 
12.03 
15.16 
0 
19.91 
25.08 
0.9808 
12.19 
15.36 
0.9708 
20.06 
25.27 
6 
12.34 
15.55 
6 
20.21 
25.47 
4 
12.50 
15.75 
4 
20.36 
25.66 
2 
12.65 
15.95 
2 
20.51 
25.84 
0 
12.81 
16.14 
0 
20.66 
26.03 
0.9798 
12.97 
16.34 
0.9698 
20.81 
26.22 
6 
13.13 
16.54 
6 
20.96 
26.41 
4 
13.28 
16.74 
4 
21.10 
26.59 
2 
13.44 
16.94 
2 
21.25 
26.78 
0 
13.60 
17.14 
0 
21.40 
26.96 
0.9788 
13.76 
17.34 
0.9688 
21 ..54 
27.14 
6 
13.92 
17.54 
6 
21.69 
27.33 
4 
14.08 
17.74 
4 
21.83 
27.51 
2 
14.23 
17.94 
2 
21.97 
27.69 
0 
14.29 
18.14 
0 
22.12 
27.87 
0.9778 
14.55 
18.34 
0.9678 
22.26 
28.05 
6 
14.71 
18.54 
6 
22.40 
28.23 
4 
14.87 
18.74 
4 
22.54 
28.41 
2 
15.03 
18.94 
2 
22.68 
28.59 
0 
15.19 
19.14 
0 
22.82 
28.76 
0.9768 
15.35 
19.34 
0.9668 
22.96 
28.94 
6 
15.51 
19.55 
6 
23.10 
29.11 
4 
15.67 
19.75 
4 
23.24 
29.29 
2 
15.83 
19.95 
2 
23.38 
29.46 
0 
15.99 
20.15 
0 
23.52 
29.64 
0.9758 
16.15 
20 35 
0.9658 
23.65 
29.81 
6 
16.31 
20.55 
6 
23.79 
29.98 
4 
16.47 
20.75 
4 
23.93 
30.15 . 
2 
16.63 
20.96 
2 
24.06 
30 32 
0 
16.79 
21.16 
0 
24.19 
30.49 
0.9748 
16.95 
21.36 
0.9648 
24.33 
30.66 
6 
17.11 
21.56 
6 
24.46 
30.82 
4 
17.27 
21.76 
4 
24.59 
30.99 
2 
17.42 
21.96 
2 
24.73 
31.16 
0 
17.58 
22.16 
0 
24.85 
31.32 
0.9738 
17.74 
22.35 
0.9638 
24.99 
31.49 
6 
17.90 
22.55 
6 
25.12 
31.65 
4 
18.05 
22.75 
4 
25.25 
31.81 
2 
18.21 
22.95 
2 
25.37 
31.98 
0 
18.37 
23.14 
0 
25.50 
32.14 
0.9728 
18.52 
23.34 
0.9628 
25.63 
32.30 
6 
18.68 
23.54 
6 
25.76 
32.46 
4 
18.84 
23.73 
4 
25.88 
32.62 
2 
18.99 
23.93 
2 
26.01 
32.78 
0 
19.14 
24.12 
0 
26.13 
32.93 
ALCOHOL TABLE MODIFIED FROM WINDISCH-Continued APPENDIX 
563 
INTERNATIONAL ATOMIC WEIGHTS* 
0 = 16 
Name. 
Symbol. 
Atomic 
Weight. 
Name. 
Symbol 
Atomic 
Weight. 
Aluminium 
Al 
27.1 
Molybdenum 
Mo 
96.0 
Antimony. . ., 
Sb 
120.2 
Neodymium 
Nd 
144.3 
Argon. 
A 
39.88 
Neon 
Ne 
20.2 
Arsenic 
As 
74.96 
Nickel 
Ni 
58.68 
Barium 
Ba 
137.37 
Niton 
Nt 
222.4 
Bismuth 
Bi 
208.0 
Nitrogen 
N 
14.01 
Boron 
B 
11.0 
Osmium 
Os 
190.9 
Bromine 
Br 
79.92 
Oxygen 
O 
16.00 
Cadmium 
Cd 
112.40 
Palladium 
Pd 
106.7 
Caesium 
Cs 
132.81 
Phosphorus 
P 
31.04 
Calcium 
Ca 
40.07 
Platinum 
Pt 
195.2 
Carbon 
C 
12.00 
Potassium 
K 
39.10 
Cerium 
Ce 
140.25 
Praseodymium.... 
Pr 
140.6 
Chlorine 
Cl 
35.46 
Radium 
Ra 
226.4 
Chromium 
. Cr 
52.0 
Rhodium 
Rh 
102.9 
Cobalt 
Co 
58.97 
Rubidium 
Rb 
85.45 
Columbium 
Cb 
93.5 
Ruthenium 
Ru 
101.7 
Copper 
Cu 
63.57 
Samarium 
Sm 
150.4 
Dysprosium 
Dy 
162.5 
Scandium 
Sc 
44.1 
Erbium 
Er 
167.7 
Selenium 
Se 
79.2 
Europium 
Eu 
152.0 
Silicon 
Si 
28.3 
Fluorine 
F 
19.0 
Silver 
Ag 
107.88 
Gadolinium 
Gd 
157.3 
[ Sodium 
Na 
23.00 
Gallium 
Ga 
69.9 
1 Strontium 
Sr 
87.63 
Germanium 
Ge 
72.5 
Sulphur 
S 
32.07 
Glucinum 
G1 
9.1 
Tantalum 
Ta 
181.5 
Gold 
Au 
197.2 
Tellurium 
Te 
127.5 
Helium 
He 
3.99 
Terbium 
Tb 
159.2 
Holmium 
Ho 
163.5 
Thallium 
T1 
204.0 
Hydrogen 
H 
1.008 
Thorium 
Th 
232.4 
Indium 
In 
114.8 
Thulium 
Tm 
168.5 
Iodine 
I 
126.92 
Tin 
Sn 
119.0 
Iridium 
Ir 
193.1 
Titanium 
Ti 
48.1 
Iron 
Fe 
55.84 
Tungsten 
W 
184.0 
Krypton 
Kr 
82.92 
Uranium 
U 
238.5 
Lanthanum 
La 
139.0 
Vanadium 
V 
51.0 
Lead 
Pb 
207.10 
Xenon 
Xe 
130.2 
Lithium 
Li 
6.94 
Ytterbium 
1 b 
172.0 
Lutecium 
Lu 
174.0 
(Neoytterbium) 
Magnesium 
Mg 
24.32 
Ytterium 
Yt 
89.0 
Manganese 
Mn 
54.93 
Zinc 
Zn 
65.37 
Mercury 
Hg 
200.9 
Zirconium 
Zr 
90.6 
- 
- 
* Compiled by the International Committee on Atomic Weights consisting of F. W. Clarke, 
W. Ostwald, T. E. Thorpe, and G. Urbain. 564 
APPENDIX 
SOLUBILITY OF OXYGEN IN WATER 
(Winkler, Berichte, 22, 1772.) (1889) 
Tempera- 
ture. 
Oxygen. 
Temperature. 
Oxygen. 
Temperature. 
Oxygen. 
0 
14.70 
9 
11.58 
18 
9.56 
1 
14.28 
10 
11.31 
19 
9.37 
2 
13.88 
11 
11.05 
20 
9.19 
3 
13.50 
12 
10.80 
21 
9.01 
4 
13.14 
13 
10.57 
22 
8.84 
5 
12.80 
14 
10.35 
23 
8.67 
6 
12.47 
15 
10.14 
24 
8.51 
7 
12.16 
16 
9.94 
25 
8.35 
8 
11.86 
17 
9.75 
CIRCUMFERENCES AND AREAS OF CIRCLES 
Formula for Area 
A = ^ = .785398 D2 
4 
Formula for Diameter 
D = 2 a/- = 1.128379 VX 
* 7T 
Diameter. 
Circumference. 
Area. 
Diameter. 
Circumference. 
Area. 
1 
8 
.39270 
.012 
7 
21.991 
38.485 
1 
4 
.78540 
.049 
' 8 
25.133 
50.265 
3 
8 
1.1781 
.110 
9 
28.274 
63.617 
1 
2 
1.5708 
.196 
10 
31.416 
78.54 
5 
8 
1.9635 
.307 
20 
62.832 
314.16 
i 
2.3562 
.442 
30 
94.248 
706.86 
i 
2.7489 
.601 
40 
125.664 
1256.6 
1 
3.1416 
.785 
50 
157.080 
1963.5 
2 
6.2832 
3.142 
60 
188.496 
2827.4 
3 
9.4248 
7.069 
70 
219.911 
3848.5 
4 
12.566 
12.566 
80 
251.328 
5026.5 
5 
15.708 
19.635 
90 
282.744 
6361.7 
6 
18.805 
28.274 
100 
314.160 
7854.0 APPENDIX 
565 
TABLE SHOWING EQUIVALENTS OF PRESSURE AND HEAD OF WATER 
Head in Feet and Equivalent Pressrue in Pounds 
5 to 60 Feet. 
70 to 180 Feet. 
200 to 1000 Feet. 
Feet Head. 
Pounds Press. 
Feet Head. 
Pounds Press. 
Feet Head. 
Pounds Press. 
5 
2.17 
70 
30.3 
200 
86.6 
10 
4.33 
80 
34.6 
250 
108.2 
15 
6.50 
90 
39.0 
300 
129.9 
20 
8.66 
100 
43.3 
350 
151.5 
25 
10.83 
110 
47.6 
400 
173.2 
30 
12.99 
120 
52.0 
500 
216.5 
35 
15.16 
130 
56.3 
600 
259.8 
40 
17.32 
140 
60.6 
700 
303.1 
45 
19.49 
150 
65.0 
700 
346.4 
50 
21.65 
160 
69.2 
900 
389.7 
60 
26.09 
180 
78.0 
1000 
433.0 
5 to 60 Pounds. 
70 to 170 Pounds. 
180 to 500 Pounds. 
Pounds Press. 
Feet Head. 
| Pounds Press. 
Feet Head. 
Pounds Press. 
Feet Head. 
5 
11.5 
70 
161.6 
180 
415.6 
10 
23.0 
80 
184.7 
190 
438.9 
15 
34.6 
90 
207.8 
200 
461.7 
20 
46.2 
100 
230.9 
225 
519.5 
25 
57.7 
110 
253.9 
250 
577.2 
30 
69.3 
120 
277.0 
275 
643.0 
35 
80.8 
130 
300.1 
300 
692.7 
40 
92.3 
140 
323.2 
325 
750.4 
45 
103.9 
150 
346.3 
350 
808.1 
50 
115.4 
160 
369.4 
400 
922.6 
60 
138.5 
170 
392.5 
500 
1154.5 
Pressure in Pounds and Equivalent Head in Feet 566 
APPENDIX 
U. S. GALLONS TO LITERS 
(Metric) 
CUBIC FEET TO LITERS 
(Metric) 
Gallons. 
Liters. 
1 
Gallons. 
Liters. 
1 
3.78544 
51 
193.05744 
2 
7.57088 
52 
196.84288 
3 
11.35632 
53 
200.62832 
4 
15.14176 
54 
204.41376 
5 
18.92720 
55 
208.19920 
6 
22.71264 
56 
211 98464 
7 
26.49808 
57 
215.77008 
8 
30.28352 
58 
219.55552 
9 
34.06896 
59 
223.34096 
10 
37.85440 
60 
227.12G40 
11 
41.63984 
61 
230.91184 
12 
45.42528 
62 
234.69728 
13 
49.21072 
63 
238.48272 
14 
52.99616 
64 
242.26816 
15 
56.78160 
65 
246.05360 
16 
60.56704 
66 
249.83904 
17 
64.35248 
67 
253.62448 
18 
68.13792 
68 
257.40992 
19 
71.92336 
69 
261.19536 
20 
75.70880 
70 
264.98080 
21 
79.49424 
71 
268.76624 
22 
83.27968 
72 
272.55168 
23 
87.06512 
73 
276.33712 
24 
90.85056 
74 
280.12256 
25 
94.63600 
75 
283.90800 
26 
98.42144 
76 
286.69344 
27 
102.20688 
77 
291.47888 
28 
105.99232 
78 
295.26432 
29 
109.77776 
79 
299 04976 
30 
113.56320 
80 
302.83520 
31 
117.34864 
81 
306.62064 
32 
121.13408 
82 
310.40608 
33 
124.91952 
83 
314.19152 
34 
128.70496 
84 
317.97696 
35 
132.49040 
85 
321.76240 
36 
136.27584 
86 
325.54784 
37 
140.06128 
87 
329.33328 
38 
143.84672 
88 
333.11872 
39 
147.63216 
89 
336.90416 
40 
151.41760 
90 
340.68960 
41 
155.20304 
91 
344.47504 
42 
158.98848 
92 
348.26048 
43 
162.77392 
93 
352.04592 
44 
166.55936 
94 
355.83136 
45 
170.34480 
95 
359.61680 
46 
174.13024 
| 96 
363.40224 
47 
177.91568 
97 
367.18768 
48 
181.70112 
98 
370.97312 
49 
185.48656 
99 
374.75856 
50 
189.27200 
| 100 
378.54400 
Cu.ft. 
Liters. 
Cu. ft. 
Liters. 
1 
28.3166 
51 
1444.1466 
2 
56.6332 
52 
1472.4632 
3 
84.9498 
53 
1500.7798 
4 
113.2664 
54 
1529.0964 
5 
141.5830 
55 
1557.4130 
6 
169.8996 
56 
1585.7296 
7 
198.2162 
57 
1614.0462 
8 
226.5328 
58 
1642.3628 
9 
254.8494 
59 
1670.6794 
10 
283.1660 
60 
1698.9960 
11 
311.4826 
61 
1727.3126 
12 
339.7992 
62 
1755.6292 
13 
368.1158 
63 
1783.9458 
14 
396.4324 
64 
1812.2624 
15 
424.7490 
65 
1840.5790 
16 
453.0656 
66 
1868.8956 
17 
481.3822 
67 
1897.2122 
18 
509.6988 
68 
1925.5288 
19 
538.0154 
69 
1953.8454 
20 
566.3320 
70 
1982.1620 
21 
594.6486 
71 
2010.4786 
22 
622.9652 
72 
2038.7952 
23 
651.2818 
73 
2067.1118 
24 
679.5984 
74 
2095.4284 
25 
707.9150 
75 
2123.7450 
26 
736.2316 
76 
2152.0616 
27 
764.5482 
77 
2180.3782 
28 
792.8648 
78 
2208.C348 
29 
821.1814 
79 
2237.0114 
30 
849.4980 
80 
2265.3280 
31 
877.8146 
81 
2293.6446 
32 
906.1312 
82 
2321.9612 
33 
934.4478 
83 
2350.2778 
34 
962.7644 
84 
2378.5944 
35 
991.0810 
85 
2406.9110 
36 
1019.3976 
86 
2435.2276 
37 
1047.7142 
87 
2463.5442 
38 
1076.0308 
88 
2491.8608 
39 
1104.3474 
89 
2520.1774 
40 
1132.6640 
90 
2548.4940 
41 
1160.9806 
91 
2576.8106 
42 
1189.2972 
92 
2605.1272 
43 
1217.6138 
93 
2633.4438 
44 
1245.9304 
94 
2661.7604 
45 
1274.2470 
95 
2690.0770 
46 
1302.5636 
96 
2718.3936 
47 
1330.8802 
97 
2746.7102 
48 
1359.1968 
98 
2775.0268 
49 
1387.5134 
99 
2803.3434 
50 
1415.8300 
100 
2831.6600 APPENDIX 
567 
USEFUL FACTORS FOR WATER 
Based on Weights at 62° F. (Standard Temperature) 
U. S. gallonsX 8.3356 = pounds 
" " X .13368 = cubic feet 
" " X231. = cubic inches 
" " X .83356 = English gallons 
" " X 3.78544 = Liters 
" " X . 020985 = Kokus (Japan) 
" " X .30815 = Bedeps (Russia) 
English gallons X 10. = pounds 
" " X . 160372 = cubic feet 
" " X277.12 = cubic inches 
" " X 1.1997 = U. S. gallons 
" " X 4.5413 = Liters 
" " X .025175 = Kokus (Japan) 
" " X .36969 =Bedeps (Russia) 
LitersX 2.202 = pounds 
" X • 035302 = cubic feet 
" X61.023 = cubic inches 
" X .26417 = U. S. gallons 
" X .2202 = English gallons 
" X .0055435 = Kokus (Japan) 
" X .081405 =Bedeps (Russia) 
KokusX 397.22028 =pounds 
" X 6.3703 = cubic feet 
" X11008.00 = cubic inches 
" X 47.6535 = U. S. gallons 
" X 39.722028 = English gallons 
" X 180.39 = Liters 
" X 14.6847 = Bedeps (Russia) 
Bedeps X 27.05 = pounds 
" X .43381 = cubic feet 
" X749.618 = cubic inches 
" X 3.24512 =U. S. gallons 
" X 2.705 = English gallons 
" X 12.2843 = Liters 
" X . 0680983 = Kokus (Japan) 568 
APPENDIX 
Cubic feet of waterX 62.355 = pounds 
" " XI728.00 = cubic inches 
" " X 7.4805 = U. S. gallons 
" " X 6.2355 = English gallons 
" " X 28.317 = Liters 
" " X . 15698 = Kokus (Japan) 
" " X 2.3052 = Bedeps (Russia) 
Pounds of water X .016037 = cubic feet 
" " X27.712 = cubic inches 
" " X .11997 = U. S. gallons 
" " X . 1 = English gallons 
" " X .45413 = Liters 
" " X . 0025175 = Kokus (Japan) 
" " X .036969 = Bedeps (Russia) 
SURVEYOR'S MEASURE 
7.92 inches = 1 link 
25 links = 1 rod 
4 rods = 1 chain 
10 square chains or 160 square rods = 1 acre 
640 acres = 1 square mile 
36 square miles (6 miles sq.) = 1 township 
CUBIC MEASURE 
1728 cubic inches = 1 cubic foot 
128 cubic feet = 1 cord (wood) 
27 cubic feet = 1 cubic yard 
40 cubic feet = 1 ton (shipping) 
2150.42 cubic inches = 1 standard bushel 
268.8 cubic inches = 1 standard gallon 
1 cubic foot = about four-fifths of a bushel APPENDIX 
569 
METRIC EQUIVALENTS 
Linear Measure 
1 centimeter = 0.3937 inch 
1 decimeter =3.937 inches =0.328 feet 
1 meter =39.37 inches = 1.0936 yards 
1 dekameter = 1.9884 rods 
1 kilometer =0.62137 mile 
1 inch = 2.54 centimeters 
1 foot =3.048 decimeters 
1 yard = 0.9144 meter. 
1 rod =0.5029 dekameter 
1 mile =1.6093 kilometers 
Square Measure 
1 square centimeter = 0.1550 square inch 
1 square decimeter =0.1076 square foot 
1 square meter =1.96 square yards 
1 are =3.954 square rods 
1 hektar =2.47 acres 
1 square kilometer =0.386 square mile 
1 square inch =6.452 sq. centimeters 
1 square foot =9.2903 sq. decimeters 
1 square yard = 0.8361 sq. meter 
1 square rod =0.2529 are 
1 acre =0.4047 hektar 
1 square mile =2.59 square kilometers 
1 cubic centimeter = 0.061 cubic inch 
1 cubic decimeter =0.0353 cubic feet 
Measure of Volume 
1 cubic inch = 16.39 cubic centimeters 
1 cubic foot =28.317 cubic decimeters 
1 cubic yard =0.7646 cubic meter 
1 cord =3.624 steres 
1 quart dry =1.101 liters 
1 quart liquid = 0.9463 liter 
1 gallon =0.3785 dekaliter 
1 peck =0.881 dekaliter 
1 bushel =0.3524 hektoliter 
1 cubic meter 
1 stere 
1.308 cubic yards 
0.2759 cord 
1 liter = 
0.908 quart dry 
1.0567 quart liquid 
1 dekaliter = 
f 2.6417 gallons 
I .135 pecks 
1 hektoliter =2.8375 bushels 
Weights 
1 gram = 0.0527 ounce 
1 kilogram = 2.2046 lbs. 
1 metric ton = 1.1023 English ton 
1 ounce = 28.85 grams 
1 lb. = 0.4536 kilogram 
1 English ton = 0.9072 metric ton. 
Approximate Metric Equivalents 
1 decimeter =4 inches 
1 meter =1.1 yards 
1 kilometer = f of mile 
1 hektar = 2| acres 
1 stere, or cubic meter = f of a cord 
1.06 quart liquid 
0.9 quart dry 
1 liter = 
1 hektoliter = 2f bushels 
1 kilogram =21 pounds 
1 metric ton = 2200 pounds 570 
APPENDIX 
Equivalents of grams or c.c. in apothecary's weight. (From Merck's 
Materia Medica): The following are approximate equivalents. 
0.001 gm. or c.c. =^& grn. or min. 
0.003 " " =A " " 
0.004 " " =A " " 
0.008 " " =1 " " 
0.01 " " = i " " 
0.015 " " = i " " 
0.03 " " = 1 " " 
0.05 " " = t " 
0.6 " " =1 " " 
0.1 " " =1$ " " 
0.2 " " =3 " " 
0.25 " " =4 " " 
0.3 gm. of c.c. = 5 grn. or min. 
0.5 " " =8 " " 
0.6 " " =10 " " 
0.8 " " =12 " " 
1 " " =15 " " 
4 " " = 1 dr. or fl-dr. 
15 " " =4 " " 
30 " " = 1 oz. or fl-oz. 
120 " " =4 " " 
237 " " =8 " " 
475 " " = 1 lb. or pint 
950 " " =2 " 
COMPARISON OF THERMOMETER SCALES 
Centi- 
grade. 
Reaumur. 
Fahren- 
heit. 
Centi- 
grade. 
Reaumur. 
Fahren- 
heit. 
Centi- 
grade. 
Reaumur. 
Fahren- 
heit. 
-30 
-24.0 
-22.0 
14 
11.2 
57.2 
58 
46.4 
136.4 
-28 
-22.4 
-18.4 
16 
12.8 
60.8 
60 
48.0 
140.0 
-26 
-20.8 
-14.8 
18 
14.4 
64.4 
62 
49.6 
143.6 
-24 
-19.2 
-11.2 
20 
16.0 
68.0 
64 
51.2 
147.2 
-22 
-17.6 
- 7.6 
22 
17.6 
71.6 
66 
52.8 
150.8 
-20 
- 16.0 
- 4.0 
24 
19.2 
75.2 
68 
54.4 
154.4 
-18 
-14.4 
- 0.4 
26 
20.8 
78.8 
70 
56.0 
158.0 
-16 
-12.8 
3.2 
28 
22.4 
82.4 
72 
57.6 
161.6 
-14 
-11.2 
6.8 
30 
24.0 
86.0 
74 
59.2 
165.2 
-12 
- 9.6 
10.4 
32 
25.6 
89.6 
76 
60.8 
168.8 
-10 
- 8.0 
14.0 
34 
27.2 
93.2 
78 
62.4 
172.4 
- 8 
- 6.4 
17.6 
36 
28.8 
96.8 
80 
64.0 
176.0 
- 6 
- 4.8 
21.2 
38 
30.4 
100.4 
82 
65.6 
179.6 
- 4 
- 3.2 
24.8 
40 
32.0 
104.0 
84 
67.2 
183.2 
- 2 
- 1.6 
28.4 
42 
33.6 
107.6 
86 
68.8 
186.8 
0 
0.0 
32.0 
44 
35.2 
111.2 
88 
70.4 
190.4 
2 
1.6 
35.6 
46 
36.8 
114.8 
90 
72.0 
194.0 
4 
3.2 
39.2 
48 
38.4 
118.4 
92 
73.6 
197.6 
6 
4.8 
42.8 
50 
40.0 
122.0 
94 
75.2 
201.2 
8 
6.4 
46.4 
52 
41.6 
125.6 
96 
76.8 
204.8 
10 
8.0 
50.0 
54 
43.2 
129.2 
98 
78.4 
208.4 
12 
9.6 
53.6 
44.8 
132.8 
100 
80.0 
212.0 APPENDIX 
571 
CONVERSION OF PARTS PER MILLION TO GRAINS PER UNITED STATES 
AND IMPERIAL GALLONS 
Parts per 
Million. 
Grains per 
Imperial 
Gallon. 
Grains per 
United States 
Gallon. 
Parts per 
Million. 
Grains per 
Imperial 
Gallon. 
Grains per 
United States 
Gallon. 
1 
.0700 
.0583 
40 
2.8000 
2.3327 
2 
.1400 
.1166 
50 
3.5000 
2.9129 
3 
.2100 
.1749 
60 
4.2000* 
3.4990 
4 
.2800 
.2332 
70 
4.9000 
4.0822 
5 
.3500 
.2915 
80 
5.6000 
4.6654 
6 
.4200 
.3499 
90 
6.3000 
5.2486 
7 
.4900 
.4082 
100 
7.0000 
5.8318 
8 
.5600 
.4665 
200 
14.0000 
11.6630 
9 
.6300 
.5248 
300 
21.0000 
17.4950 
10 
.7000 
.5831 
400 
28.0000 
23.3270 
20 
1.4000 
1.1663 
500 
35.0000 
29.1290 
30 
2.1000 
1.7495 
* Other values may be computed from the above table. 
140 
Sp. Gr. = o for Baume lighter than water. 
n ~|" 130 
140 
B° = ~~ for Baume lighter than water. 
Sp. Gr. 
145 
Sp. Gr. = for Baume heavier than water. 
Io4 - /5 
145 
B°= 145~-for Baume heavier than water. 
Sp. Gr. 572 
APPENDIX 
SPECIFIC GRAVITIES IN DEGREES BAUME 
Liquids Lighter than Water 
Degrees 
Baum6. 
Specific 
Gravity. 
Degrees 
Baum6. 
Specific 
Gravity. 
Degrees 
Baume. 
Specific 
Gravity. 
10 
1 .000 
15 
0.966 
20 
0.933 
11 
0.993 
16 
0.959 
21 
0.927 
12 
0.986 
17 
0.952 
22 
0.921 
13 
0.979 
18 
0.946 
23 
0.915 
14 
0.972 
19 
0.940 
24 
0.909 
25 
0.903 
30 
0.875 
35 
0.849 
26 
0.897 
31 
0.870 
36 
0.843 
27 
0.892 
32 
0.864 
37 
0.838 
28 
0.886 
33 
0.859 
38 
0.833 
29 
0.881 
34 
0.854 
39 
0.828 
40 
0.824 
48 
0.787 
58 
0.745 
41 
0.819 
50 
0.778 
60 
0.737 
42 
0.814 
52 
0.769 
65 
0.718 
43 
0.805 
54 
0.761 
70 
0.700 
46 
0.796 
56 
0.753 
75 
0.683 APPENDIX 
573 
SPECIFIC GRAVITIES IN DEGREES BAUME AND TWADDLE 
Liquids Heavier than Water 
Hydrometer 
Reading Degrees. 
Specific 
Gravity. 
Hydrometer 
Reading Degrees 
Specific 
Gravity. 
Twaddle. 
Baume. 
Twaddle. 
Baum6. 
0 
.0 
1.000 
43 
25.5 
1.215 
1 
.7 
1.005 
44 
26.0 
1.220 
2 
1.4 
1.010 
45 
26.4 
1.225 
3 
2.1 
1.015 
46 
26.9 
1.230 
4 
2.7 
1.020 
47 
27.4 
1.235 
5 
3.4 
1.025 
48 
27.9 
1.240 
6 
4.1 
1.030 
49 
28.4 
1.245 
7 
4.7 
1.035 
50 
28.8 
1.250 
8 
5.4 
1.040 
51 
29.3 
1.255 
9 
6.0 
1.045 
52 
29.7 
1.260 
10 
6.7 
1.050 
53 
30.2 
1.265 
11 
7.4 
1.055 
54 
30.6 
1.270 
12 
8.0 
1.060 
55 
31.1 
1.275 
13 
8.7 
1.065 
56 
31.5 
1.280 
14 
9.4 
1.070 
57 
32.0 
1.285 
15 
10.0 
1.075 
58 
32.4 
1.290 
16 
10.6 
1.080 
59 
32.8 
1.295 
17 
11.2 
1.085 
60 
33.3 
1.300 
18 
11 9 
1.090 
61 
33.7 
1.305 
19 
12.4 
1.095 
62 
34.2 
1.310 
20 
13.0 
1.100 
63 
34.6 
1.315 
21 
13.6 
1.105 
64 
35.0 
1 320 
22 
14.2 
1.110 
65 
35.4 
1.325 
23 
14.9 
1.115 
66 
35.8 
1.330 
24 
15.4 
1.120 
67 
36.2 
1.335 
25 
16.0 
1.125 
68 
36.6 
1.340 
26 
16.5 
1.130 
69 
37.0 
1.345 
27 
17.1 
1.135 
70 
37.4 
1.350 
28 
17.7 
1.140 
71 
37.8 
1.355 
29 
18.3 
1.145 
72 
38.2 
1.360 
30 
18.8 
1.150 
73 
38.6 
1 365 
31 
19.3 
1.155 
74 
39.0 
1.370 
32 
19.8 
1.160 
75 
39.5 
1.375 
33 
20.3 
1.165 
76 
39.8 
1.380 
34 
20.9 
1.170 
77 
40.1 
1.385 
35 
21.4 
1.175 
78 
40.5 
1.390 
36 
22.0 
1.180 
79 
40.8 
1.395 
37 
22.5 
1.185 
• 80 
41.2 
1.400 
38 
23.0 
1.190 
81 
41.6 
1.405 
39 
23.5 
1.195 
82 
42.0 
1.410 
40 
24.0 
1.200 
83 
42.3 
1.415 
41 
24.5 
1.205 
84 
42.7 
1.420 
42 
25.0 
1.210 
85 
43.1 
1.425 574 
APPENDIX 
SPECIFIC GRAVITIES IN DEGREES BAUME AND TWADDLE. 
(Continued) 
Liquids Heavier than Water 
Hydrometer 
Reading Degrees. 
Specific 
Gravity. 
Hydrometer 
Reading Degrees. 
Specific 
Gravity. 
Twaddle. 
Baumd. 
Twaddle. 
Baumi. 
86 
43.4 
1 .430 
129 
56.6 
1.645 
87 
43.8 
1 .435 
130 
56.9 
1.650 
88 
44.1 
1.440 
131 
57.1 
1.655 
89 
44.4 
1.445 
132 
57.4 
1.660 
90 
44.8 
1.450 
133 
57.7 
1.665 
91 
45.1 
1.455 
134 
57.9 
1.670 
92 
45.4 
1.460 
135 
58.2 
1.675 
93 
45.8 
1.465 
136 
58.4 
1.680 
94 
46 1 
1.470 
137 
58.7 
1.685 
95 
46.4 
1.475 
138 
58.9 
1.690 
96 
46.7 
1.480 
139 
59.2 
1.695 
97 
47.1 
1.485 
140 
59.5 
1.700 
98 
47.4 
1.490 
141 
59.7 
1.705 
99 
47.8 
1.495 
142 
60.0 
1.710 
100 
48.1 
1.500 
143 
60.2 
1.715 
101 
48.4 
1.505 
144 
60.4 
1.720 
102 
48.7 
1.510 
145 
60.6 
1.725 
103 
49.0 
1.515 
146 
60.9 
1.730 
104 
49.4 
1.520 
147 
61.1 
1.735 
105 
49.7 
1.525 
148 
61.4 
1.740 
106 
50.0 
1.530 
149 
61.6 
1.745 
107 
50.3 
1.535 
150 
61.8 
1.750 
108 
50.6 
1.540 
151 
62.1 
1.755 
109 
50.9 
1.545 
152 
62.3 
1.760 
110 
51.2 
1.550 
153 
62.5 
1.765 
111 
51.5 
• 1.555 
154 
62.8 
1.770 
112 
51.8 
1.560 
155 
63.0 
1.775 
113 
52.1 
1.565 
156 
63.2 
1.780 
114 
52.4 
1.570 
157 
63.5 
1.785 
115 
52.7 
1.575 
158 
63.7 
1.790 
116 
53.0 
1.580 
159 
64.0 
1 795 
117 
53.3 
1.585 
160 
64.2 
1.800 
118 
53.6 
1.590 
161 
64.4 
1.805 
119 
53.9 
1.595 
162 
64.6 
1.810 
120 
54.1 
1.600 
163 
64.8 
1.815 
121 
54.4 
1.605 
164 
65.0 
1 820 
122 
54.7 
1.610 
165 
65.2 
1.825 
123 
55.0 
1.615 
166 
65.5 
1 830 
124 
55.2 
1.620 
167 
65.7 
1.835 
125 
55.5 
1.625 
168 
65.9 
1.840 
126 
55.8 
1.630 
169 
66.1 
1.845 
127 
56.0 
1.635 
170 
66.3 
1.850 
128 
56.3 
1.640 
171 
66.5 
1.855 APPENDIX 
575 
Chlorine Data 
Chlorine [yAwpos = green] a greenish yellow gas, easily compressed 
to a liquid, was discovered in 1774 by Scheele, a Swedish Chemist. 
Atomic weight 35.45, molecular weight 70.9, vapor density 35.8. 
Liquefies at-33.60 C. (-28.5° F.), solidifies at -102° C. (-151.36° F.). 
Under pressure of 6 atmospheres liquefies at 0° C. (32° F.). 
Temperature in Degrees Fahrenheit 
Io ® o I © 
Temperature In Degrees Centigrad s 
CURVE SHOWING PRESSURE 
OF 
CHLORINE GAS AT VARIOUS TEMPERATURES 
Pre isure in Pounds [er Square jhch 
WEIGHTS OF CHLORINE 
Datum. 
Gaseous Chlorine. 
Liquid Chlorine. 
Specific gravity 
2.49 (air = l) 
1.44 (water = 1) 
Weight of 1 liter 
3.167 gms. 
1440 gms. 
Weight of 1 cu. ft. 
0.198 lb. 
89.752 lbs. 
Weight of 1 gal. 
0.026 lb. 
11.999 lbs. 
One volume of liquid chlorine is equivalent to 444.4 volumes of chlorine gas. 576 
APPENDIX 
Temperature. 
Solubility Ratio by Volume. 
Pounds of Chlorine Soluble 
in 1,000,000 Gals, of Water. 
. C. ° 
F. 0 
0 
32 
1.5 
20,000 
10 
50 
3.0 
40,000 
30 
88 
1.8 
24,000 
SOLUBILITY OF CHLORINE 
One part per million =8.34 lbs. per million gallons of water =0.058 grain per gallon. One 
grain per gallon =17.12 parts per million =142.86 lbs. per million gallons of water. 
Chlorine -Pounds Per 24 Hours. 
Seconds Per Pulsation 
Chlorine -Pounds Per 24 Hours'. 
Chlorine -Pounds Per 34 Hours. 
Chlorine - Parts Per Million. 
Water - Million Gallons Per 24 Hours. 
Fig 86.-Chlorine Data for the Treatment of Water Supplies. (Wallace-Tiernan 
Co.) See also data on another page. APPENDIX 
577 
Transportation of Bacteriological Specimens. The preparation and 
shipment of specimens for bacteriological examinationis a very impor- 
tant procedure. Unfortunately the bacteriologist and 'chemist may not 
have much to do with this step in the examination of any substance; 
usually, he is expected to handle the specimen after it has been delivered 
to his laboratory. At times, ho may be quite justified in refusing to 
handle certain specimens which are sent because it would be dangerous 
to himself or to those with whom he comes in contact. Since much of 
the material for bacteriological examination is sent through the United 
States mails it is important that he have a knowledge of the postal 
rules and regulations which concern these shipments. Hasseltine 
(1918)* has given a good resume of the subject. The following quota- 
tion is cited by this author from the Postal Regulations issued by the 
Post Office Department. Sections 472 and 473 read as follows: 
Sec. 472. All kinds of poison, and all articles and compositions 
containing poison, and all poisonous animals, insects and reptiles, and 
explosives of all kinds, and inflammable materials and infernal machines, 
and mechanical, chemical, or other devices or compositions, which may 
ignite or explode, and all disease germs or scabs, and all other natural or 
artificial articles, compositions or materials of whatsoever kind which 
may kill, or in any wise hurt, harm or injure another, or damage, deface, 
or otherwise injure the mails or other property, whether sealed as first- 
class matter or not, are hereby declared to be non-mailable matter, and 
shall not be conveyed in the mails or delivered from any post office or 
station thereof, nor by any letter carrier; but the Postmaster General 
may permit the transmission in the mails, under such rules and regula- 
tions as he shall prescribe as to preparation and packing, of any articles 
hereinbefore described which are not outwardly or of their own force 
dangerous or injurious to life, health or property. (The rest of this 
section is not concerned with shipment of bacteriological materials.) 
Sec. 473. 1. Specimens of diseased tissues may be admitted to the 
mail for transmission to United States, State, municipal, or other labora- 
tories in possession of permits referred to in paragraph 3 of this section 
only when inclosed in mailing cases constructed in accordance with this 
regulation: Provided, That bacteriologic or pathologic specimens of 
plague and cholera shall under no circumstances be admitted to the 
mails. 
2. Liquid cultures, or cultures of microorganisms in media that'are 
* Hasseltine, H. E. 1918. Public Laboratory Specimens, Their Preparation 
and Shipment. Public Health Reports 32, 2016-2032. Reprint from the same No. 
438-1918. 578 
APPENDIX 
fluid at the ordinary temperature (below 45° C. or 113° F.), are unmail- 
able. Such specimens may be sent in media that remain solid at ordi- 
nary temperature. 
3. No package containing diseased tissue shall be delivered to any 
representative of any of said laboratories until a permit shall have first 
been issued by the Postmaster General, certifying that said institution 
has been found to be entitled, in accordance with the requirements of 
this regulation, to receive such specimens. 
4 (a) Specimens of tubercular sputum (whether disinfected with 
carbolic acid or not disinfected) shall be transmitted in a solid glass vial 
with a mouth not less than 1 in. in diameter and capacity of not more 
than 2 oz., closed by a cork stopper or by metallic screw top protected 
by a rubber or felt washer. Specimens of diphtheria, typhoid, or other 
infectious or communicable diseases or diseased tissue shall be placed in 
a test tube made of tough glass, not over | in. in diameter and 
not over 7| ins. in length, closed with a stopper of rubber or cotton and 
sealed with paraffine or covered with a tightly fitting rubber cap. 
(6) The glass vial or test tube shall then be placed in a cylindrical 
tin box, with soldered joints, closed by a metal screw cover with a rubber 
or felt washer. The vial or test tube in this tin box shall be completely 
and evenly surrounded by absorbent cotton, closely packed. 
(c) The tin box, with its contents, must then be inclosed in a closely 
fitting metal, wooden, of papier-mache block or tube, at least in. 
thick in its thinnest part, of sufficient strength to resist rough handling 
and support the weight of the mails piled in bags. This last tube shall 
be tightly closed with a screw-top cover with sufficient screw threads to 
require at least one and one-half full turns before it will come off, and 
fitted with a felt or rubber washer. (See Fig. 8.) 
5. Specimens of blood dried on glass microscopic slides for the diag- 
nosis of malaria or typhoid fever by the Widal test may be sent in any 
strong mailing case which is not liable to breakage or loss of the specimen 
in transit. 
6. Upon the outside of every package of diseased tissues admitted 
to the mails shall be written or printed the words " Specimen for bac- 
teriological examination. This package to be pouched with letter 
mail." APPENDIX 
579 
Frost's Plate Counter. 
Each of the larger squares represents an area of 1 sq. cm. The figures 60 and 40 indicate the 
number of square centimeters in the circles bounded by the respective lines. Each sector is 
one-tenth the area of its circle. 
Department of Bacteriology, University of Illinois. 580 
APPENDIX 
Frost's Plate Counter. 
Each of the larger squares represents an area of 1 sq. cm. The figures GO and 40 indicate the 
number of square centimeters in the circles bounded by the respective lines. Each sector is one- 
tenth the area of its circle. 
Department of Bacteriology, University of Illinois. INDEX 
A 
Acetaldehyde reaction for ethyl alcohol, 
194 
Acid fast staining, theory of, 87 
, Ziehl-Niehlsen method, 86 
Acidity, degrees recognized in milk, 114 
- degrees recognized in clear media, 115 
Aciduric bacteria, 236 
Action of bacteria on carbohydrates, 
193 
glucosamine, 191 
polypeptides, 190 
proteins, 189 
bile on bacteria, 234 
certain bacteria on proteins, 367 
Adjusted milks, defined, 374 
Adulteration of compressed yeast with 
brewer's yeast, 220 
Advantages of "bleach" in water treat- 
ment, 325 
pasteurization of milk, 416 
Aerobic respiration, 241 
Aeroscope, 264 
Agar, 43 
- colonies, study of, 110 
- for Ps. radicicola, 50 
- gelatin medium (North), 51 
- medium, plain, 48 
- stroke, Study of, 110 
Age of milk, 377 
Agglutination of bacteria, 320 
Alanin, 184 
Albuminoid ammonia, determination in 
water, 282 
Alcohol as a disinfectant, 140 
- table, 561 
Alcoholic fermentation, 194 
Alkaline blood agar, 70 
- hemoglobin agar, 70 
- methylene blue, 90 
Alkalinity, determination in water, 285 
Allen's microscopic method of milk 
analysis, 404 
Alwood fermentation valve, 220, 221 
Analysis of mud deposits, 352 
• sewage sludge, 352 
Anderson's method for tubercle bacilli 
in milk, 413 
Amino acids in gelatin, 41 
proteins, 184 
Ammonia production by pure cultures, 
116 
Ammonium chloride in bread, 218 
Anaerobic methods, 17 
- respiration, 242 
Analysis of Liebig's meat extract, 41 
Aniline gentian violet, 90 
Anti-protein sera, 491 ' 
Antiseptic values, 143 
Albargin, disinfectant, 135 
Aperture, numerical, 28 
Aqueous alcoholic stains, 81 
- stains, preparation, 81 
Argentamin, disinfectant, 135 
Argentose, 135 
Arginine, bacterial decomposition, 244 
-, chemical structure, 244 
Arnold steam sterilizer, 126 
Arnold's test for heated milk, 372 
Ascomycetes, 202 
Asepsis in food preservation, 495 
Ash in milk, 368 
Aspartic acid, 186 
Atomic weights, 563 
Autoclave, 125 
Available chlorine, 136 
- oxygen in water, 344 
Ayer's milk tube method, 408 
B 
Basidiomycetes, 202 
Babcock test for fat in milk, 369 
581 582 
INDEX 
Bacillus anthracis, isolation from water, 
322 
- colon index, 314 
test in water analysis, 299 
- diphtheria) in milk, 414 
- dysenteri®, isolation from water, 320 
- enteritidis in water, 297 
- tuberculosis in feces, 258 
milk, 412 
water, 323 
- typhosus in butter, 426 
cream, 437 
feces, 254 
isolation from water, 317 
, Carnot and Halle's method, 256 
, Holt-Harris-Teague method, 
255 
, Kendall and Day's method, 255 
, Lumsden and Stimson's meth- 
od, 254 
, Morishama and Teague's 
method, 257 
, Teague and Clurman's method, 
257 
- -•, longevity in water, 343 
Bacteria and life, 233 
-, Eberle-Klein method, 248 
- in baked bread, 224 
cream and cream ripening, 423 
egg w hite, 459 
yolk; 459 
feces, 238 
fresh eggs, 460 
self-rising bread, 223 
the stomach, 230 
-, MacNeal's procedure for Winterberg 
method, 247 
-, Matill and Hawk's procedure, 252 
-, Steel's modification of the Strass- 
burger method, 250 
Bacterial count in milk, significance, 385 
water analysis, 290 
- examination of butter, 423 
eggs, 463 
ice, 337 
milk, 375 
- groups in ice cream, 435 
raw and pasteurized milk, 417 
- vs. chemical examination of water, 271 ' 
Bacteriological specimens, transporta- 
tion, 577 
Bacteriology of butter, 423 
cheddar cheese, 429 
ice, 336 
Balaz's test for boiled milk, 373 
Barn conditions in milk production, 377 
Barsiekow's medium, 59 
Baskets, various types of, 7, 8 
Bean agar, 50 
Beattie's method for anaerobic bacteria, 
22 
Beer wort, 57 
Beijerinck's medium for nitrate reduc- 
tion, 67 
Benedict's method for estimation of 
dextrose, 197 
reducing sugars, 196 
Benzoic acid, detection in milk, 371 
Bergey's method for typhoid bacilli in 
butter, 426 
Besson's method for tubercle bacilli in 
milk, 413 
Bile, germicidal action, 234 
- salt broth, 46 
Biochemical oxygen demand ol sewage, 
350 
Bismuth compounds in disinfection, 135 
Blanching, 507 
Blood agar, 69 
, alkaline, 70 
for streptococci, 69 
- films, staining, 87 
- serum, 69 
Boiling in sterilization, 126 
Boric acid in disinfection, 139 
milk, determination, 371 
Bottcher's counting chamber, 10 
- moist chamber, 76 
Botulism, 533 
Bread making, 222 
•- medium, 59 
Breed's capillary pipettes, 397 
Breed smears in milk analysis, 397 
Bromine as a disinfectant, 134 . 
- reaction for tryptophane, 192 
- water test for skatol, 247 
Brown's fermentation tube, 1 
Buchner's anaerobic methods, 17 INDEX 
583 
Bushnell and Mauer's method for egg 
analysis, 465 
Butter flavor, 423 
c 
Cadaverine, 529 
Calcium compounds in disinfection, 135 
- oxide, 138 
Calibration of micrometer oculars, 36 
Campbell's method for tubercle bacilli 
in milk, 413 
Candling eggs, 461, 463 
Canned meats, 481 
Canning, 506 
Cannon's hydrolized casein medium, 63 
Capaldi's egg medium, 58 
Capsules, determination on bacteria, 109 
Capsule stain, Huntoon's method, 84 
-, Muir's method, 83 
, Welch's method, 84 
Carbohydrate agar, 48 
- broths, 44 
Carbohydrates, action of bacteria on, 193 
-, classification, 193 
Carbol fuchsin, 90 
Carbonated broth, 47 
Carbon in disinfection, 140 
Carnot and Halle's method for typhoid 
bacilli in feces, 256 
Carriers and food infection, 530 
Carrot infusion medium, 60 
Case history of botulism, 535 
Casein agar after Ayers, 70 
- medium, 63 
Causes of food poisoning, 533 
Cellulose agar, 61 
Certificates for chemical examination of 
water, 276, 277 
Characteristics of a good indicator of 
pollution, 294 
. anaerobic water bacteria, 298 
fermentation, 240 
milk-borne epidemics, 541 
water-borne epidemics, 541 
Cheddar cheese, bacteriology, 429 
Cheese bacteriology, 428 
- poisoning, 431 
-- ripening, 428 
Chemical analyses of ice, 337 
- and bacterial examination of water, 
271 
- changes in milk from souring, 366 
- constituents of milk, 363 
- examination of milk, 367 
- standards for milk, 373 
Chicago drainage canal and Illinois 
River, 341 
Chicken broth medium, 46 
Chinese ink preparations, 78 
Cholera red reaction for M. choleras in 
water, 322 
Chlorine data, 575 
Chloramine, 139 
- in water treatment, 326 
Chloride of lime in disinfection, 135 
Chlorine as a disinfectant, 132 
-, determination in water, 280 
Cholera, M. microspira in feces, 257 
Chopped meats, 480 
Chromatic aberration, 29 
Classification of water bacteria, 193, 286 
proteins, 182 
yeasts, 213 
Cleaning apparatus, 6 
- hemocytometers, 16 
- solution, 6 
Coagulation coefficient of disinfectants, 
171 
- of milk by rennin, 365 
Coarse adjustment on microscope, 25 
Cohn's classification of bacteria, 94 
- solution, preparation, 63 
, characteristics of bacteria in, 112 
Cold storage, 505 
Cole's method for microscopic agglutina- 
tion, 321 
Collection of milk samples, 393 
water samples, 273 
Colloidal silver in disinfection, 135 
Color, determination in water, 278 
- reactions of proteins, 191 
Coloring matter in milk, 371 
Combined acidity of the stomach, 233 
Comparison of thermometer scales, 570 
Composition of hens' eggs, 453 
milk serum, 365 
• tomato pulp, 514 584 
INDEX 
Completed test for 7?. coli in water, 302 
Compounds in goats' and human milk, 
364 
Compressed yeast, 216 
, microscopic examination, 220 
, outline of manufacture, 217 
Concave slides, types, 77 
Concentrated solutions in food prepara- 
tion, 498 
Condensed milk, 433 
Conjugated proteins, 183 
Conn's method for bacteria in butter, 428 
Constituents of media, 39 
Coplin's jars for staining, 80 
Copper salts in disinfection, 139 
Corn agar medium, 50 
Cover glasses, sizes and thicknesses, 5 
Cresol as a disinfectant, 141 
Cystine, decomposition, 245 
-, structure, 185 
Czapek's solution, 66 
D 
Dairy score card, 380 
Dakin-Carrel solution in disinfection, 137 
Dark ground illumination, 78 
Deaminization, 243 
De Bary's classification of bacteria, 95 
Decarboxylation, 243 
Decoction of dried fruits, 59 
Delepine's method for tubercle bacilli 
in milk, 412 
Derived proteins, 183 
Descriptive chart, 100 
Description of microorganisms, 101 
Detection of foreign proteins, 490 
Determination of the origin of epidemics, 
544 
Development of the Descriptive Chart, 
1(M) 
Dextrose potassium phosphate broth, 47 
Diagnosis of food poisoning, 535 
Dialysis sterilization, 128 
Dialyzed milk, 72 
Diet and the intestinal bacterial flora, 235 
Differential staining of blood films, 87 
Dilution bottles, 2 
- pipettes for blood counts, 15 
Dilutions, 10 
Direct vs. plate count on milk, 396 
Dirt in milk, 384 
Disadvantages of "bleach" in water 
treatment, 325 
Disinfectants, standardization, 144 
Disinfection, 131 
- vs. periodic system, 133 
Dissolved oxygen in water, 344 
Doane-Buckley method for leucocytes 
in milk, 410 
Dolt's agar medium, 50 
- medium, 65 
Dorset's egg medium, 58 
Dough raising power of bread yeasts, 220 
Dox's solution for fungi, 65 
Draw tube on microscope, 27 
Dried eggs, 46 
Drigalski and Conradi's medium, 62 
Dry heat sterilization, 121 
Drying in food preservation, 504 
Dunham's solution, 47 
Durham's fermentation tube, 1 
E 
Effect of baking on microorganisms in 
bread, 224 
low temperatures on bacteria, 504 
pasteurization on bacteria in milk, 
417 
pressure in food preservation, 506 
storage on ice cream bacteria, 436 
yeast on flavor of bread, 222 
Efficiencies of peptone, 40 
Efficiency of bread yeasts, 220 
Egg-meat mixture for putrefaction, 58 
Ehrenberg's work on classification of 
bacteria, 94 
Ehrlich's reaction for indol, 115 
Elements in inorganic compounds, 187 
- of "quality" in milk, 381 
Elsner's potato medium, 60 
Endo's agar medium, Kendall's method, 
52 
, Hygienic Laboratory method, 53 
, Levine's method, 54 
- medium for isolating &. typhosus from 
water, 318 INDEX 
585 
Endospore, observation of, 109 
Enzymes, 118 
- in eggs, 460 
Eosin-brilliant green agar medium, 54 
methylene blue agar medium, 54 
Epidemics of paratyphoid fever, 532 
Errors in staining flagella, 85 
Erythrocytes, enumeration, 13 
Esten and Mason's method for bacteria 
in ice cream, 437 
Estimation of bacteria in tomato prod- 
ucts, 517 
dextrose (Allihn's method), 196 
molds in tomato products, 515 
Ethyl acetate reaction for alcohol, 195 
- alcohol, aldehyde reaction, 194 
, iodoform reaction, 194 
Evaluation of bleaching powder, 328 
Examination of bread, 224 
canned foods, 511 
clams, 489 
Hamburger steak, 483 
meat, 482 
molds, 212 
oysters, 485 
sausage, 483 
shell fish, 483 
tin cans for leaks, 513 
water for available chlorine, 329 
F 
Factors influencing bacteria in milk, 375 
• eggs, 458 
disinfection, 133 
Fasting and intestinal bacteria, 236 
Fats, 197 
- in milk, 368 
Fecal and non-fecal B. coli, 308 
Fehling's solution, preparation, 195 
- test for reducing sugars, 195 
Fermentation in food preservation, 497 
the intestinal tract, 240 
- of sugars by streptococci, 295 
- reactions of pure cultures, 117 
- tubes, types, 1 
Fermented milks, 433 
Fermenting powers of yeasts, determina- 
tion of, 220 
Fermi's culture fluid, 68 
Fernbach's antitoxin flask, 5 
Ferric chloride test for phenol, 247 
Ferrous sulphate in disinfection, 139 
Filtration through liquids, 128 
- sterilization, 127 
Fine adjustment of the microscope, 26 
Flagella staining, 86 
Flaming sterilization, 122 
Flasks, types of, 5 
Flat sours in canning, 511 
Flavor of butter, 423 
Flours used in bread making, 222 
Fluorescein for tracing water pollution, 
323 
Food-borne epidemics of disease, 542 
Food poisoning, 528 
- preservation, 494 
Formaldehyde in disinfection, 142 
Forms of growth, 102 
Frankel and Voges' solution, 68 
Frankel's solution, 64 
Free ammonia in water, determination, 
282 
Freudenreich's flask, 5 
Frost and Ravenel's test for heated 
milk, 372 
Frost's microscopic method for milk 
analysis, 401 
-- plate counter, 579, 580 
Frozen eggs, 463 
Fructification in molds, 203 
Fuller and Johnson's classification of 
water bacteria, 289 
Fungi, 202 
- imperfecti, 202 
G 
Gases in canned foods, 510 
Gastric analysis, 233 
-- juice, germicidal action, 229 
Gedding's method for M. cholerce in 
feces, 257 
Gelatin, preparation and composition, 
42, 43 
- agar, 56 
-, colonies, study of, 111 
- stab, study of, 111 586 
INDEX 
Gerber's test for milk fat, 369 
Germicidal action of saliva, 228 
gastric juice, 229 
Giblet broth, 46 
Giltay and Aberson's solution, 67 
Glossary of terms, 105 
Glucosamines, bacterial action on, 191 
Glucose formate broth, 47 
agar, 49 
Glutamic acid, 186 
Glycerol in disinfection, 142 
- potato broth, 60 
Glycocoll, 184 
Grades on milk, 391 
- milk by bacterial count, 387 
Grading eggs, 455 
Gram positive and negative bacteria, 83 
- stain, Nicoll's modification, 83 
, theory of, 81 
Graves' modification of the Durham 
fermentation tube, 2 
Griess method for nitrites, 116 
Groups, bacteriological, 99 
- of bacteria in intestines, 236 
H 
Halogens as disinfectants, 134 
Ham souring, 499 
Hanging blocks, 77 
- drops, 77, 78 
Harding and Prucha's method for bac- 
teria in cheese, 431 
Haricot decoction, 58 
Harrison's method for bacteria in cheese, 
432 
Hay infusion, 57 
Hazen theorem, 269 
Heated milk, determination of, 371 
Hemocytometer rulings, 14 
- chamber, 12 
Heese agar medium, 51 
- and Niedner's agar, 50 
High-pressure steam sterilization, 122 
Hiss agar-gelatin medium, 51 
- medium, 56 
Histidine, 187 
Holt-Harris-Teague method for B. typho- 
sus in feces, 255 
Hot air oven sterilization, 122 
Houston's method for isloating strepto- 
cocci from feces, 296 
Huntoon's method for staining capsules, 
84 
Hydrogen sulphid formation, 116 
Hydrolysis of carbohydrates during 
sterilization, 44 
Hyphae, structure of, 212 
I 
Ice and typhoid fever, 339 
- cream, 434 
plant score card, 439 
Identification of bacteria by agglutina- 
tion, 321 
Illuminating power of the microscope, 29 
Illumination of the microscope, 31 
Incidence of tubercle bacilli in market 
butter, 427 
milk, 414 
Incineration in sterilization, 122 
Inclination joint on the microscope, 25 
Indol from tryptophane, 244 
-, test for, 115 
Indicator solutions, 553 
Indicators of pollution, 294 
Infection of food by carriers, 530 
Influence of salt on bacteria, 499 
Initial contamination of milk, 375 
Inorganic disinfectants, 133 
- elements in organic compounds, 187 
Inspissator, 127 
Instructions for collecting water samples, 
274 
Intermittent sterilization, 127 
Interpretation of bacterial tests on milk, 
387 
results in water analysis, 315 
Intervals before analysis of water 
samples, 273 
Intestinal bacteria, effect of protein 
diet, 236 
water drinking, 237 
- flora, classification, 238 
Invigoration of cultures, 108 
Iodine absorption number of fats, 198 
■- in disinfection, 134 INDEX 
587 
lodoformogen, 134 
Iodoform reaction for acetone, 195 
ethyl alcohol, 194 
lodo-hemol, 134 
Iodol, 134 
lodomuth, 134 
Isolation of B. enterilidis sporogenes from 
water, 299 
B. typhosus from water, 317 
pure cultures, 10 
Isoleucine, 185 
J 
Jeffer's counting plate, 9 
Jensen's system of classification of bac- 
teria, 97 
Jones' anaerobic method, 23 
Jordan's classification of water bacteria, 
290 
- non-protein medium, 66 
K 
Kendall and Day's procedure for B. 
typhosus in feces, 255 
Ketchup, 514 
Key to Aspergillus molds, 210 
genera of budding fungi, 215 
true yeasts, 214 
penicillia, 207 
Koch's concave slide, 76 
- culture flask, 5 
Koettstorfer number of fats, 199 
Kolle culture flask, 5 
Kusserow's method for determining 
fermenting power of yeasts, 221 
Kuster's anaerobic method, 18 
L 
Labarraque's solution, 137 
Lactic acid fermentation, 193 
in the stomach, 233 
Lactose bile in water analysis, 311 
Lafar's counting plate, 9 
Leavening agent in self-rising bread, 223 
Leeuwenhoeck's work on bacteria, 94 
Leffman-Beam test for milk fat, 369 
Lehmann and Neumann's work on 
classification, 97 
Lellendahl's staining dish, 80 
Lentz's anaerobic apparatus, 18 
Leucine, structure, 185 
-, enumeration, 15 
Leucocytes in milk, 408 
Levine's eosin-methylene blue agar, 54 
Liebermann's reaction, 192 
Liebig's meat extract, 41 
Life cycles of bacteria, 99 
Light, sterilization, 128 
Lime in disinfection, 136 
Lipman-Brown medium, 66 
Liquid chlorine, addition to water, 331 
Lister culture flask, 5 
Lister's work on milk, 95 
Litmus gelatin, 56 
- lactose agar, 48 
- milk, preparation, 71 
, cultural characteristics in, 115 
- whey, 71 
Lohnis' method for bacteria in butter, 
426 
cheese, 432 
Loss of virulence on culture media, 118 
Low temperature, effect on bacteria, 504 
Lugol's iodine solution, 90 
Lumsden and Stimson's method for 
B. typhosus in feces, 254 
Lymphocytes, staining of, 89 
Lysine, decomposition, 245 
-, structure, 186 
Lysol in disinfection, 141 
M 
MacKenzie's culture flask, 68 
McLeod's anaerobic method, 18 
Magnifying power of microscope, 29 
Magnification tables, microscopes, 33, 34 
Malachite green medium, 52 
Malt extract medium, 58 
Mayer's culture fluid, 62 
Meat extract, composition of, 40, 41 
, partition of nitrogen in, 42 
- infusion, preparation, 40 
- juice (infusion), 41 
Medium for fungi, 65 588 
INDEX 
Meissl's method for determining fer- 
menting power of fungi, 220 
Metals, action of on bacteria, 133 
Methods of adding "bleach" to water, 
327 
water treatment, 324 
- for study of yeasts, 218 
Methyl red test in water bacteriology, 
307 
Micro-analysis of tomato products, 515 
Micrometer, ocular, 436 
Micrometry, 34 
Microscope, 23 
Microscopic agglutination, 321 
- examination of gastric contents, 233 
- method for milk analysis, 395 
Microspira cholera, in feces, 257 
milk, 413 
water, 322 
Migula's classification of bacteria, 95 
Milk agar, preparation of, 72 
- borne epidemics, 541 
- medium, characters of bacteria in, 112 
, preparation of, 71 
- of lime, 138 
- proteins, 365 
- samples, 396 
- standards, 392 
Millon's reaction, 191 
Mills-Reincke phenomenon, 268 
Modified aeroscope, 265 
Mohler's method for bacteria in cheese, 
432 
Moist chamber preparations of yeasts, 
219 
-■ heat sterilization, 122 
Molds in butter, 425 
-, structure, 203 
Moore's staining dish, 80 
Miquel culture flask, 5 
Mordants, 81 
Morishaina and Teague's method for 
B. typhosus in feces, 257 
Motility, 109 
Mucor, 205 
- mucedo, 204 
Muir's method for staining capsules, 83 
Muller's classification of bacteria, 94 
My coderma in bread yeast, 221 
N 
Naegeli's medium, 63 
Naples jar for staining, 80 
Neisser's method for spore staining, 85 
Nesslerization, 281 
Neutral red agar, 50 
bile salt agar, 49 
broth, 46 
Nicoll's modification of Gram's stain, 83 
Nitrate broth, 45 
Nitrates, determination in water, 283 
Nitrite formation, 116 
-, test for, 116 
Nitrites, determination in water, 283 
Nitrogen-free medium for bacteria, 65 
-, significance in water analysis, 280 
Nitroprusside reaction for acetone, 195 
Nitrosoindol test, 115 
North's agar-gelatin medium, 51 
Novy's anaerobic method, 23 
Number of bacteria in feces, 238 
Numerical aperture, 28 
Nutrient broth, preparation, 44 
, characteristics of bacteria in, 110 
- gelatin, 55 
Nylander's test for reducing sugars, 196 
O 
Objections to pasteurization, 416 
Objectives on microscope, 27 
Oculars on microscope, 31 
Odor, determination of, in water, 279 
Oidium lactis, 205 
Omelianski and Winogradski's medium, 
67 
Opinions on bacterial count in milk, 389 
Organic acids in the stomach, 233 
Organic halogens, test for, 189 
- nitrogen, test for, 187 
- sulphur, test for, 188 
Origin of bacteria in meat, 479 
- epidemics, 544 
Ox-bile medium, 59 
Oxygen consumed, determination of, in 
water, 284 
- relations, study of, 110 
- solution in water, 345 INDEX 
589 
P 
Paratyphoid fever, 531 
Parietti's solution, 62 
Partially confirmed test for B. coli in 
water, 301 
Pasteur's medium, 66 
Pasteurization of milk, 414 
Pathogenic bacteria in river water, 343 
7 and pasteurization of milk, 419 
- streptococci in milk, 422 
Pathogenicity of pure cultures, to ani- 
mals, 117 
, to plants, 118 
Pea-flour extract, 57 
Penicillium, 207 
Peptone, 40 
- sucrose solution (Buchanan), 57 
Permeability of egg shells, 459 
Petri dishes, types of, 2, 3 
Pettenkofer's experiment on cholera, 23Q 
Phenol in disinfection, 141 
- from tyrosine, 243 
Phenylamin, 185 
Phenyl-hydrazine reaction, 196 
Phycomycetes, 202 
Physiological method for dilutions, 210 
Pickling in food preservation, 503 
Piorowski's culture flask, 5 
Plain milk medium, 71 
Plan of modern corn packing plant, 508 
Plate vs. microscopic count on milk, 396 
Platinum wires, 7 
Polynuclear neutrophilic leucocytes, 89 
Polypeptids, bacterial action on, 190 
Potato agar, 61 
- gelatin, 60 
- infusion, 60 
- mash, 60 
- slant, study of, 111 
- starch jelly, 111 
Pouring plates, 9 
Prazmowski's culture flask, 63 
Precipitin test for proteins, 491 
Preparation of common stains, 90 
standard reagents, 554 
Preservation of food by asepsis, 495 
by concentrated solutions, 498 
Preservatives in milk, 371 
Pressure and head of water, 565 
Presumptive test for B. coli, 301 
Procedure for B. coli identification at 
Cincinnati, 313 
studying water bacteria, 287 
Process of canning, 507 
Processing canned foods, 509 
Proline, 187 
Properties of ice, 336 
Proskauer and Beck's culture medium, 
68 
Protargol in disinfection, 135 
Proteins, bacterial action on, 189 
-, color reactions of, 191 
-, in milk, 371 
-, precipitation reactions, 192 
-, structure of, 184 
Prune decoction, 57 
Pseudo yeasts, 213 
Ptomaines, 529 
Pure cultures, invigoration of, 108 
-, isolation of, 10 
, methods of study, 108 
, transferring, 10 
- water, 271 
Putrefaction in the intestines, 243 
Putrescine, 529 
Q 
Qualitative tests for elements in pro- 
teins, 189 
- estimation of fecal indol, 246 
"Quality" in milk, 379 
Quinone in disinfection, 143 
R 
Rack for fermentation tubes, 1 
Raisin gelatin, 56 
Rating of oysters, 487 
Raulin's solution for molds, 66 
Reaction in milk cultures, 114 
Reagents, preparation of, 554 
Red corpuscles, 13 
Reddening of cod, 503 
Redfield's medium, 59 
Reed's methods for leucocyte in milk, 409 
Reichert-Meissl number of fats, 199 590 
INDEX 
Relation between bacteria and dirt in 
milk, 384 
- of microscopic to plate count, 407 
pressure to temperature, 122 
the 37° to the 20° C. count, 291 
water filtration to typhoid fever, 
270 
Relative stability of streams, 347 
Reliability of bacteria tests on milk, 386 
Reporting milk analysis, 395 
Required oxygen in water, 345 
Residue, determination of, in water, 279 
Rettger's aeroscope, 264 
- method for egg analysis, 467 
Revised method for tomato products, 
520 
Rhizopus, 205 
Rice milk medium, 72 
Ropy bread, 224 
Roszahey's counting flask, 9 
Routine milk analysis, 393 
- steps in bacterial water analysis, 305 
Roux culture flask, 5 
Russell's medium, preparation of, 52 
, use in water analysis, 318 
S 
Sabourand's agar for yeasts, 51 
Salicylic acid in milk, detection, 371 
Saliva, germicidal action of, 228 
Salt in food preservation, 498 
Sample bottles, 5 
Sampling of milk, 367 
Sanitary inspection in water hygiene, 269 
Saponification number of fats, 199 
Sausage bacteriology, 481 
Savage's method for bacteria in ice 
cream, 437 
leucocytes in milk, 409 
tubercle bacilli in butter, 426 
Scheme for identifying yeasts, 219 
Schneider's method for bacteria in 
butter, 428 
Score card, 380 
Sediment in milk, 384 
Self-improvement of streams, 340 
Self-rising bread, 223 
Seliwanoff's reaction for ketoses, 195 
Separation of B. coli and streptococci in 
water, 297 
Serine, 185 
Serum agar, 70 
Serum bouillon, 47 
Sewage streptococci, 296 
Shell membrane of eggs, 453 
Significance of bacterial count in milk, 
385 
Silicate jelly, 117 
Silver citrate, 135 • 
-, colloidal in disinfection, 135 
- salts as disinfectants, 135 
Sizes and capacities of tin cans, 510 
Slaked lime, 138 
Slides for mounting bacteria, 5 
Slimy bread, 224 
Smear, preparation, 80 
Smillie's method for anaerobic bacteria, 
20 
Smith's fermentation tube, 1 
Smoking in food preservation, 497 
Soil extract agar, medium, 49 
gelatin medium, 55 
Solubility of oxygen in water, 564 
Sources of water supply, 269 
Souring of milk, 365 
Specific gravities of common acids, 559 
- gravity of milk, 368 
Spherical aberration, 29 
Spore staining, 85 
Springers in canning, 511 
Spronck's peptone yeast extract, 57 
Stability in streams, 347 
Stabilized gentain violet, 91 
Staining, methods, 81 
-, theory, 79 
Standard aeroscope, 264 
- loops, 7 
Standardization of the microscope, 398 
Starch agar, 60, 62 
- jelly, 61 
Steam sterilization, theory of, 124 
Steel's method" for fecal bacteria, 252 
Sterilization methods, 121 
Stewart's counting apparatus, 11 
Stiles' method for egg examination, 463 
Stokes' method for leucocytes in milk, 
409 INDEX 
591 
Storage of food in tin .cans, 513 
Storch's method for heated milk, 371 
Strassburger method for fecal bacteria, 
250 
Streaming steam sterilization, 126 
Strength of yeasts, 220 
Streptococci in water, 297 
Structure of eggs, 453 
hyphae, 212 
Structure of molds, 202 
Substage on the microscope, 27 
Sugar in food preservation, 503 
Sullivan's culture fluid, 64 
Swells in canning, 510 
Synthetic agar, 49 
T 
Teague and Clurman's eosin-brilliant 
green agar, 54 
method for B. typhosus in feces, 
257 
Travis' method for M. choleras in 
feces, 258 
Temperature of milk, 378 
- relations of bacteria, 112 
Theory of pasteurization, 415 
staining, 79 
Thermal death point, 118 
Thymol, disinfectant, 141 
Tin cans, 509 
Tobacco smoke, effect on mouth bac- 
teria, 228 
Toleration of bacteria to acids and 
alkalis, 118 
Torrey's anaerobic method, 19 
Total acidity of the stomach, 233 
Toxicity coefficient of disinfectants, 175 
Toxin of Bacillus botulinus, 533 
Tracing pollution in water, 323 
Transporting bacteriological specimens 
in the U. S. mail, 577 
Treasury Department Standard for 
water examination, 311 
True yeasts, 213 
Trypsin broth, 45 
Tryptagar, 49 
Tryptophane, bromine reaction, 192 
- broth, 47 
Tryptophane, decomposition of, 244 
-, structure, 186 
Tubercle bacilli in butter, 425 
Turbidity, determination in water, 278 
Typhoid bacilli in ice, 338 
- death rate in American cities, 271 
- fever case record, 548 
-' - from oysters, 487 
in Pittsburgh, 268 
Tyrosine, Decomposition, 243 
-, structure, 185 
U 
Ultra-violet light, action on microor- 
ganisms, 130 
Urine medium, 58 
Uschinsky's medium, characteristics of 
bacteria in, 112 
Use of the microscope, 24 
Utensils influencing milk production, 377 
V 
Valine, 185 
Van Del den's solution, 64 
Van Ermengen's procedure for staining 
flagella, 86 
Vegetable bouillon, 46 
Vegetative cells, 109 
Ventilation tube, 220 
Vincent's method for tomato products, 
528 
Voges-Proskauer reaction in water anal- 
ysis, 307 
W 
Ward's classification of water bacteria, 
288 
Water bacteria, classification, 286 
Water-borne epidemics of disease, 541 
- drinking and bacteria in intestines, 
237 
Welch's method for staining capsules, 84 
Whey agar, 71 
- gelatin, 55 
White corpuscles, enumeration, 15 
Wiley's method for streptococci in ice 
cream, 437 592 
INDEX 
Wine medium, 57 
Winogradski's medium for nitrite for- 
mation, 67 
Winterberg method for fecal bacteria, 247 
Working distance of the microscope, 30 
Wolffhugel's counting apparatus, 9 
Wording reports on water analysis, 317 
Wort gelatin, 56 
Wright's stain, preparation, 88 
- anaerobic method, 18 
- procedure for staining blood films, 87 
X 
Xantho-proteic reaction, 191 
X-ray fluorescence, bactericidal prop- 
erties, 129 
Y 
Yeast foods, 216 
-, genera of true, 214 
- in bread making, 222 
- in feces, 258 
Yeasts, classification of, 213 
Z 
Ziehl-Niehlsen method of acid fast 
staining, 87 
Zinc chloride in disinfection, 139 
Zinsser's anaerobic method, 19