LECTURE NOTES 
ON 
BACTERIOLOGY 
For Dental Students 
BY 
HENRY H. JANEWAY, M.D. 
NEW YORK 
PAUL B. HOEBER 
67-69 EAST 59th STREET Copyright, 1915 
By PAUL B. HOEBER LECTURE NOTES ON BACTERIOLOGY 
GENERAL CHARACTERS OF PATHOGENIC ORGANISMS 
Definition of Bacteriology-Bacteriology is that science which 
investigates the morphology and conditions of cultivation, growth 
and develppmepjpand the relation to disease of that class of miero- 
organisms^which are capable of continued propagation within the 
tissues of the body. 
All those microorganisms which are capable of such continued 
propagation within the body are termed pathological microorgan- 
isms, because their propagation within the tissues results in the 
production of diseased conditions which if unrestrained will lead 
to death. 
Different Kinds of Organisms Included in the Study of Bac- 
teriology-Such pathological microorganisms belong for the most 
part to the large group of unicellular microorganisms which are 
called bacteria. But the science of bacteriology also includes the 
study of the other unicellular microorganisms, those, for instance, 
belonging to the pathological yeasts, fungi and protozoa, for a 
wide variety of unicellular microorganisms possess the power of 
living within the tissues of the human body when once introduced 
within them. 
Difficulty of Classification-In fact some difficulty attends the 
attempt to classify these organisms and this difficulty depends 
upon the fact that as the various forms of life are traced back- 
ward from the more complex forms of life to the simplest forms, 
there is a gradual disappearance of the sharp distinctions which 
separate the higher forms of life. 
All higher forms of life may be quite readily classified into 
the plant or the animal kingdoms. With the former we associate 
the absence of the power of movement and therefore the absence 
of muscles, nerves, and circulation, and finally we associate with 
plants the power of the synthesis of complex organic foodstuffs from 
the simple elements. 
4 LECTURE NOTES ON BACTERIOLOGY 
This last criterion serves perhaps the widest use in the sepa- 
ration of plants from animals, but in attempting to apply it as 
a means of classification to the disease-producing organisms so 
many irregularities are met with in respect to this very power 
that the proper classification of these organisms becomes difficult. 
We may, however, place the majority of pathological microorgan- 
isms among the plants. 
Rough Classification of the Plant Kingdom from the Bacteri- 
ological Standpoint-All plant life is divided into two large 
divisions: 
I. Cryptogamia, or plants reproducing themselves without 
flowers or seeds. 
II. Phanerogamia, or plants bearing flowers and seeds. 
Fig/fl.--Streptococcus pyogenes in 
phlegmonous pus. Cover glass prep- 
aration stained with methylene blue. 
Magnified 1000 diam. 
Fig. 2.-Staphylococcus pyogenes 
Cover glass preparation stained with 
Fuchsin. Magnified 1000 diam. 
The Lower Forms to Which the More Strictly Pathogenic 
Organisms Belong-For the purpose of bacteriology, the subdivi- 
sions of the cryptogamia may be limited to: 
I. Schizophyta-Organisms reproducing themselves by fission. 
They are chlorophyll free and reproduce themselves by dividing in 
one, two or three planes of space. Many of them form endospores, 
many of them possess the power of movement by either flagella or 
an undulating membrane. They are divided into the following sub- 
divisions : 
6 LECTURE NOTES ON BACTERIOLOGY 
1. Coccacea?-These are spherical organisms appearing under 
the microscope as minute dots. Very rarely they form endospores. 
According to the number of planes in which they divide they are 
classified as: 
(a) Streptococci-organisms dividing in one plane. They con- 
sequently appear under the microscope as a chain of dots because, 
always dividing in the same plane of space, the individual organ- 
isms in virtue of hanging together for a longer or shorter time 
Fig. 3.-Yellow-green sarcinae from 
the air. Cover glass preparation 
from a -gelatin culture; unstained. 
Magnified 1000 diam. 
Fig. 4.-Anthracis with spores. Cover 
glass preparation from an agar cul- 
ture. Stained with Fuchsin. Mag- 
nified 1000 diam. 
after division must form a chain-like thread. They are non-motile. 
Represented by the streptococcus. (Fig. 1.) 
(b) Micrococci-organisms dividing in two planes of space, and 
consequently they adhere together in the form of squares or irreg- 
ular clusters. They are non-motile and are represented by the 
Staphylococcus pyogenes aureus. (Fig. 2.) 
(c) Sarcinae-organisms dividing in three planes of space. 
They appear under the microscope as cubes or bunches, presenting 
some evidences of solidity. They are non-motile. (Fig. 3.) 
(cZ) Pianococcus-motile organisms dividing in two planes of 
space. Their power of motion is due to the possession of flagella 
or slender whip-like structures, which, by a whirling or vibratory 
or undulatory movement, enable the organisms to move rapidly 
through the fluid in which they are living. 
8 LECTURE NOTES ON BACTERIOLOGY 
(e) Planosarcina-organisms dividing in three planes of space 
and possessing movement. 
2. Bacteriaceas-organisms which are long or short but always 
rod-like or longer in one direction than in the other. They are 
never spiral. They always divide in the transverse diameter. 
They may hang together in chains or appear under the microscope 
as 'single rods. Their ends may be rounded or square. Many of 
Fig. 5.-Anthrax threads. Cover glass 
preparation from the spleen of a 
mouse showing capsule surrounding 
the bacilli. Stained with methylene 
blue and mounted in balsam. Mag- 
nified 1000 diam. 
Fig. 6.-Typhoid bacilli showing the 
flagella. Cover glass preparation 
from an agar culture. Mordant with 
ferri tannas and stained with Fuch- 
sin. Magnified 1000 diam. 
them form endospores and many forms possess the power of 
motion. 
Three subdivisions of the bacteriaceae are recognized: 
(a) Bacterium possessing no flagella; often forming endospores. 
(Figs. 4 and 5.) 
(&) Bacillus possessing peritrichal flagella or flagella around 
the whole organism's sides as well as ends. A few species form 
endospores. (Fig. 6.) 
(c) Pseudomones, possessing only polar or end flagella. 
A few species form endospores. (Fig. 7.) 
3. Spirillacem-organisms longer in one direction than the 
other, but spirally curved or representing a portion or segment 
of a spiral curve. They always divide in the transverse diameter. 
Many possess the power of movement. The majority are rigid 
10 LECTURE NOTES ON BACTERIOLOGY 
organisms and move by means of flagella. The following 'Subdivi- 
sions are recognized: 
(a) Spirosoma-non-motile and rigid organisms. 
Fig. 7.-Pseudomonas bacilli with polar 
flagella. Cover glass preparation 
from hay infusion. Magnified 1000 
diam. 
Fig. 8.-Microspira. Spirillum rubrum 
showing flagella. Cover glass prepa- 
ration from an eight-day gelatin cul- 
ture. Mordant with ferri tannas 
fuchsin and stained with Fuchsin. 
Magnified 1000 diam. 
(6) Microspira-rigid organisms with one or more, rarely two 
or three, polar undulating flagella. (Fig. 8.) 
Fig. 9.-Spirillum. Spirillum serpens 
showing flagella. Cover glass prep- 
aration from stagnant canal water. 
Magnified 1000 diam. 
Fig. 10.-Spirochaste. Spirillum from 
recurrent fever. Cover glass prep- 
aration from human blood. Mag- 
nified 1000 diam. 
12 LECTURE NOTES ON BACTERIOLOGY 
(c) Spirillum-rigid organisms with polar tufts of five to 
twenty flagella curved in the form of a semi-circle or in the form 
of flattened undulating curves. (Fig. 9.) 
(d) Spirochete--flexible organisms possessing no known organs 
of locomotion. They may possess, however, and probably do, a 
marginal undulating membrane. (Fig. 10.) 
The spirochete are classed as plants more as matter of con- 
venience. In their morphology and physiology and relation to 
other forms of life they present characters more allied to those of 
Fig. Ila.-Blastomycosis of the skin. 
Budding organism in tissue. Mag- 
nified 1200 diam. (Ormsby.) 
Fig. 11b.-Blastomycosis of the skin. 
Hanging drop. Magnified 1200 diam. 
From a photograph. (Ormsby.) 
protozoa than to plants, and furnish an illustration of the con- 
fusion of biological characters which is met with as life is traced 
down the scale to these simple unicellular forms. 
To the above classes of bacteria belong the vast majority of 
disease-producing organisms. A few diseases are, however, due to 
other organisms which represent slightly higher stages in plant 
life than bacteria. 
The Higher Forms of Microorganisms-One of these higher 
forms of pathogenic organisms is 
II. Hemiascomycetes, or the yeasts-They are unicellular or- 
ganisms which possess a wall of cellulose showing a double contour 
14 LECTURE NOTES ON BACTERIOLOGY 
under the microscope and multiplying by a process of budding. 
Their protoplasm contains many rather coarse granules. Yeasts, 
therefore, present a greater degree of organization than bacteria, 
and on this account may very properly be said to represent a 
more complex and therefore higher stage of life. 
There are many varieties among which may be mentioned 
1. Saccharomycetes or ordinary yeasts, including, for instance, 
Fig. 12.-Actinomycosis. A cover glass 
preparation from the tumor of the 
jaw of a calf. Magnified 500 diam. 
Fig. 13.--Leptothrix. A cover glass 
preparation made from the mouth 
showing rigid spirilla without fla- 
gella, spirochsete and large slender 
unbranched leptothrix. Stained with 
gentian violet. Magnified 1000 diam. 
such forms as are responsible for the fermentations producing beer 
or the rising of bread. 
2. Blastomycetes, a form of yeast which produces a specific 
disease known as Blastomycosis. When gaining successful entrance 
to the tissues they cause a combination of a productive and exuda- 
tive inflammation, capable of spreading externally and of infecting 
fresh areas, and if unchecked, of causing death. (Figs. 11, a 
and b.) 
A second form of higher pathogenic organism is- 
III. Mycoljacteriacea, including cells which form long or short 
cylindrical filaments often clavate, cuneate or irregular in contour. 
At times they show appearances due to either true or false branch- 
ing. They possess no flagella, no sheath, and form no endospores. 
They divide always in their transverse diameter, but multiply also 
by forming gonidia-like bodies due to a repeated transversal divi- 
16 LECTURE NOTES ON BACTERIOLOGY 
sion in planes close to each other. It is this character which jus- 
tifies the claim that they possess a higher type of organization. 
Two subdivisions may be mentioned: 
1. Actinomycetes. 
2. Mycetoma, or Madura foot. 
Both these parasites are very similar. Actinomycetes are prone 
to infect cattle and next to cattle, human beings are most readily 
infected. The parasite probably originally exists in the straw, 
hay or grass which cattle eat. Infection generally occurs by way 
of the mouth, and the teeth are not infrequent portals of entry. 
Man may become infected from cattle or directly from grass or 
straw. When once actinomyces has successfully invaded the tis- 
sues, it gives rise to a chronic productive and purulent inflamma- 
tion. Nodular swellings form which are prone to break down 
and leave sinuses. When the bones or periosteum become infected 
a variety of pathological processes, including necrosis and rarefying 
ostitis, occur in them. Within the discharge from the broken down 
abscesses are found characteristic yellowish granules, a fraction 
of a m.m. in diameter, which represent minute clusters of the myce- 
lial threads of the actinomyces. 
Examined under the microscope, these clusters appear as radi- 
ating mycelial threads, each thread ending in a little knob-like 
extremity. Between the threads are spherical coccoid bodies. 
Neither the knobs or coccoid bodies are spores, but probably rep- 
resent degenerative changes and contaminations. (Fig. 12.) 
Within infected tissues minute rosettes of radiating mycelial 
threads are found. Each thread is composed of a sheath inclosing 
a granular protoplasm and possesses the thickness of an ordinary 
anthrax bacillus. There are said to be thirteen different varieties 
of actinomycotic fungus. 
Mycetoma is limited to warm climates. It usually gains en- 
trance to the tissues of the body through the foot. The disease 
is characterized by the formation of the same nodular swelling as 
in actinomycosis. These break down and leave suppurating sinuses. 
Within the discharge from the sinuses occur either yellow or black 
granules. 
Probably the form associated with yellow granules is actinomy- 
cosis itself. The black granules examined under tbe microscope 
after crushing them and separating out the fungus with sodium 
18 LECTURE NOTES ON BACTERIOLOGY 
hypochlorite or strong sodium 
hydrate, show a dense network 
of mycelial threads with irreg- 
ular thickenings and many 
branches. 
IV. Chlamydobacteriacece- 
another class of organisms high- 
er than true bacteria, but still 
not presenting the organization 
of true molds, include a number 
of different varieties which it is 
convenient to group together 
under the name of chlamydobac- 
teriaceae, or sheath bacteria. 
Both these and the mycobac- 
teriaceas may be classed as high- 
er bacteria, to all of which the 
name of Trichomycetes may be applied to indicate the common posi- 
tion of these two groups. . The chlamydobacteriaceae all possess a 
surrounding sheath and may be branched or unbranched and 
divide transversely to 
the long axis. They in- 
clude the following sub- 
divisions : 
1. Leptothrix cells, 
which occur as single 
unbranched threads. 
This organism has been 
frequently found in as- 
sociation with inflamma- 
tion in the mouth. It is 
always obtained from 
the surface of the mu- 
cous membrane and 
never causes severe sys- 
temic symptoms, and it 
is even probable that 
when present it is mere- 
ly there as a saprophyte. 
Fig. 14.-Cladothrix. Cover glass 
preparation from a gelatin culture 
of an organism obtained from water. 
Magnified 1000 diam. 
Fig. 15.-Streptothrix enteola. Film prepara- 
tion from peptone-beef-broth culture, fourteen 
days at 37° C. Magnified 1000 diam. (McFar- 
land.) 
20 LECTURE NOTES ON BACTERIOLOGY 
2. Cladothrix. These are cells which are united lengthwise 
and sometimes show a pseudodichtomous branching. The branch- 
ing, in other words, is only apparent and due to a misplacement or 
angulation, caused by the growth of freshly divided threads. In 
alleged infections with cladothrix it is difficult to be sure whether 
one is dealing with true or false branching, and therefore the 
differentiation of this organism from the next, the streptothrix, 
becomes difficult. True cladothrix infections are therefore 
doubtful. (Fig. 14.) 
3. Streptothrix. This organism differs from the last in forming 
Fig. 16.-Crenothrix. Photographed 
in a living condition from a cover 
glass preparation made from City 
water. Magnified 250 diam. 
Fig. 17.-Oidium lactis. From a smear 
preparation on a gelatin plate, 
Magnified 1000 diam. 
threads with true branches in its process of growth and division. 
True infections with organisms presenting all the characters of the 
streptothrix have been described by many observers. They have 
been found in abscesses in various locations and in superficial in- 
fections of mucous membranes, as of the mouth, intestines, and 
within the pulmonary alveoli. (Fig. 15.) 
4. Crenothrix are organisms which occur as unbranched 
threads, but divide in all three directions of space. (Fig. 16.) 
5. Phragmidiothrix. Cells at first united in unbranched 
threads, but dividing in all three directions of space so that they 
appear as a rope of threads. Some of the cells penetrate the 
sheath and thus give rise to branches. 
22 LECTURE NOTES ON BACTERIOLOGY 
Fig. 18.-Oidium albicans. Mycehrl thread with four ripe chlamydospores; and 
conidia in the middle of the picture. (MacNeal.) 
6. Thlothnx. These are simple cells inclosed m delicate 
sheaths. They are non-motile and divide only in one plane of 
space. Their distinguishing feature is the fact that their proto- 
plasm contains sulphur granules. 
V. Thiobacteria, or the Beggia- 
toacere. These are threads which ap- 
pear to possess no sheath and hence 
they form a separate class of the 
higher bacteria. They divide only in 
one plane of space, but they possess 
the power of motion by means of an 
undulating membrane. They contain 
sulphur granules. There are two 
main families and numerous subfam- 
ilies. They are all soil and water or- 
ganisms. 
VI. Hypomycetes. Possessing a 
still higher organization, are the true 
molds or Hypomycetes or Eumycetes. 
A number of these are pathogenic. 
Others are important to the bacteri- 
ologist because they may contaminate 
his cultures. There are two main va- 
rieties : 
1. The Mycomycetes, organisms 
possessing a mycelial network which 
is divided into numerous partitions 
and which reproduces itself asexually 
only. The simplest examples are the 
organisms grouped under the term: 
(a) Oidia, which really bridge the gap between the yeasts and 
the molds. One of these organisms, the Oidium albicans, is path- 
Fig. 19. - Penicillium crusta- 
ceum. Conidiophore with ver- 
ticillate branches, s', s", b, st, 
sterigmata attached chains of 
conidia. Magnified 540 diam. 
(Jordan.) 
24 LECTURE NOTES ON BACTERIOLOGY 
ogemc. It causes an infection or mucous membranes known as 
thrush. It is prone to affect the mucous membranes of poorly 
nourished children. The Oidium albicans appears as budding oval 
cells, but budding cells which under certain conditions grow out in 
the form of mycelial threads and hyphae, from which spores are 
developed. (Fig. 18.) 
(b) Penicillium is an example of a higher form of my corny cetes. 
This organism is grouped in long cells divided by many transverse 
partitions. From the cells be- 
tween the partitions separate 
hyphae are given off and from 
the ends of the latter two or 
three branches called conidio- 
phores^ These latter end in 
two or three branches again, 
which are called sterigmata, 
and on the ends of the latter 
are formed rows of spores. 
The penicillium is a frequent 
contamination of culture me- 
dium. The green mold which 
is frequently met with on 
moist bread is penicillium. 
(c) Aspergillus (Fig. 20) 
is another variety of molds a little more highly organized. It is a 
frequent form of contamination of culture media. It is composed of 
a delicate meshwork of mycelial threads. The threads form cor- 
ridiophores. These develop club-shaped expansions at their ends 
and from the latter radially arranged sterigmata arise, from the 
free ends of which spores are produced. This form may also form 
spores inside the terminal hyphte. Such spore cases are called 
ascospores. When such a spore case develops in the center of a 
mycelial thread, it is called a chlamydospore. The thread on each 
side dies out, setting free the chlamydospore. 
2. Phycomycetes. The second subdivision of the Hypomycetes 
are the Phycomycetes. These possess no partitions, the entire or- 
ganism consisting of a single multinueleated cell. This group mul- 
tiplies by both asexual and sexual methods. A very commonly met 
with example is the Mucor. It appears as a network of mycelial 
Fig. 20.-Aspergillus fumigatus, from the 
lung of a parrot. (Jordan.) 
26 LECTURE NOTES ON BACTERIOLOGY 
sp 
nv 
Sjo 
e- 
1 
6 
c 
TO 
3 
z 
❖ 
Fig. 21.--Mucor mucedo: 1, A sporangium in optical longitudinal section: c, 
columella; m, wall of sporangium; sp, spores; 2, a ruptured sporangium with 
only the columella (c) and a small portion of the wall (m) remaining; 3, two 
smaller sporangia with only a few spores and no columella; 4, germinating spores; 
5, ruptured sporangium of Mucox' mucilaginous with deliquescing wall (m) and 
swollen interstitial substance (s); sp, spores. (Jordan.) 
Fig. 22.-Malassezia furfur. (Besson.) 
28 LECTURE NOTES ON BACTERIOLOGY 
threads. From the mycelial threads hyphae arise and upon the end 
of the hyphae a spherical capsule develops. This is called the spor- 
angium. The sporangium is supported upon the hyphae by the pro- 
jection of the latter into it in the form of a cylindrical rod called the 
columella. Within the sporangium a number of round spore-like 
Fig. 23.-Achorion schbnleinii. Colony developing from a favus scale. End, 
endoconidia on submerged hyphae. (MacNeal.) 
bodies are formed. When the spores ripen, the sporangium bursts 
and sets the spores free. Reproduction in this manner is an asexual 
method, but the mucor may reproduce itself sexually. 
When this occurs, two hyphae or their branches approach each 
other. These approaching branches are termed gametophores. 
When the gametophores come into contact their protoplasm freely 
30 LECTURE NOTES ON BACTERIOLOGY 
mixes. After the mixing process, septa appear, which inclose 
within the united gametophores a central cell called the zygo- 
spore. The zygospore enlarges 
and sends out a branch which 
forms upon its end a non-sexual 
sporangium. 
The three most important 
pathogenic hypomycetes all re- 
semble the penicillium closely. 
They are the 
1. Microsporon furfur, caus- 
ing the skin disease known as 
pityriasis versicolor. (Fig. 22.) 
2. Achorion schbnleinii, 
causing favus. 
3. Trichophyton tonsurans, 
causing ringworm. Scrapings of 
the skin in pityriasis versicolor, 
examination of the crushed yel- 
low cup in favus or scrapings of the skin or examination of diseased 
hairs in ringworm, will show mycelial threads and, mixed with 
them, a large number of spores. In culture media all three organ- 
isms grow mycelia, ascospores and chlamydospores. (Fig. 24.) 
Fig. 24.-Herpes tonsurans. Cover 
glass preparation from an agar plate 
culture. Unstained. Magnified 240 
diam. 
THE MOST IMPORTANT PATHOLOGICAL MICROORGANISMS 
The above outline has roughly indicated the morphology of the 
various forms of pathogenic microorganisms. Of these bacteriology 
is chiefly concerned with the bacteriaceas, as it is to the organisms 
thus classified that by all odds the most important pathogenic 
parasites belong. 
All these organisms consist, as has been explained, of a minute 
mass of protoplasm preserving the shape of the three divisions 
into which bacteria are divided, i. e., the cocci, the bacilli, and the 
spirillge. 
THE STRUCTURE OF BACTERIA 
The Protoplasm of Bacteria-Although performing the great 
physiological functions common to all life, it shows very little dif- 
32 LECTURE NOTES ON BACTERIOLOGY 
ferentiation. No nucleus presenting structural differentiation of 
the cell is present. Either the whole cell must be considered to be 
nuclear material, or the nucleus is diffused throughout the whole 
cell uniformly. 
Granules-Various granules appear within the bodies of certain 
bacteria. Their full significance is not understood. 
Cell membrane-Certainly every bacterium must be consid- 
ered to possess a cell membrane. This membrane may be demon- 
strated in some species by plasmolysis, or in the shadow forms 
resulting from degeneration. In some species, the capsule may 
be stained in a manner to differentiate it from the bacterial 
body. 
Movement and Flagella-Many bacteria possess the power of 
movement. This is accomplished by flagella, the slender whip-like 
structures, previously mentioned. Only a very few cocci possess 
flagella. Flagellated bacteria belong almost exclusively to the 
bacilli and spirilla. As regards the flagella, bacteria are divided 
into the following classes: 
I. Gymnobacteria, possessing no flagella. 
II. Trichobacteria, possessing flagella. 
1. Monotricha, possessing one polar flagellum. 
2. Lophotricha, possessing a tuft of flagella at one pole. 
3. Amphitricha, possessing flagella at both poles. 
4. Peritricha, organisms completely surrounded with flagella. 
Spores-Many bacteria possess the power of transforming them- 
selves into very resistant forms of life. Such bacteria develop 
within their bodies round or oval, highly refractible bodies, which 
are very difficult to stain. These bodies possess the power of per- 
petuating the life of the bacterial cell in a quiescent stage over 
periods during which it is subjected to unfavorable conditions 
for its life. Such conditions, for instance, as are represented by 
unfavorable degrees of temperature, nourishment, or moisture. 
Reproduction-Bacteria multiply by simple fission in their 
transverse diameter. Each daughter organism thus expands to the 
length of the adult forms. 
Karyokinetic changes have never been demonstrated in bacteria. 
Spherical bacteria as a rule lengthen somewhat in one direc- 
tion before they divide, which they may then do in any of the 
three planes of space, according to the variety of coccus concerned. 
34 LECTURE NOTES ON BACTERIOLOGY 
Many bacteria show a tendency to hang together in longer or 
shorter chains after division. 
The life of one generation depends much upon the supply of 
nourishment. When the supply is plentiful it may be as short as 
twenty minutes. At this rate of growth sixteen trillions may be 
produced in one day. 
Many bacteria present characteristic irregularities in their 
shape. Some may be enlarged at one end; others may show a 
beaded appearance. The media and osmotic relations of the 
surrounding fluid in which the bacteria may be growing will 
cause slight variations in size, shape, and contour of bacteria. 
Unfavorable conditions in the media of growth will cause an 
irregular beaded appearance, various distortions of the organisms, 
enlargements at the ends, and poorly staining shadow-like forms. 
All such modifications of the organism are to be regarded as 
evidences of degeneration in it. 
Chemical Composition-Water composes about 80 to 85% of 
the bacterial body. Of the remainder protein matter constitutes 
50 to 80%. 
The fluidity of the media may modify profoundly the quantity 
of water contained within bacteria. 
Among the organic chemical substances are: nucleo protein; 
globulins; fats and waxes, present in large amounts in certain 
bacteria such as the tubercle bacillus; carbohydrates, chiefly in the 
form of cellulose, and entering into the formation of the bacterial 
wall. Glycogen, however, is also contained within bacteria. 
The principal salts in the ash from bacteria are the salts of 
potassium, sodium, calcium and magnesium. 
Nutrition of Bacteria-Bacteria, as a class, do not possess the 
complete synthetic powers of the higher plants, but they differ 
in turn from animals in being able to utilize as food not only 
the complex proteins, carbohydrates, and fats, but also simpler 
substances, such as the amino-acids, urea, ammonia and ketones, 
and even such organic acids as tartaric, citric, acetic, glycerine and 
alcohol. 
There are a few bacteria which can obtain the nitrogen directly 
from the air. These are the nitrifying bacteria which grow in 
symbiosis with leguminous plants. The majority of bacteria obtain 
their nitrogen from diffusible or in other cases from non-diffusible 
36 LECTURE NOTES ON BACTERIOLOGY 
proteins. Many bacteria have become highly specialized in their 
relation to proteins, requiring very special forms of protein. 
There are other bacteria which can get their carbon from the CO2 
of the air. 
Many bacteria display very special relations to oxygen and 
this property is such a prominent character of bacteria that it 
forms the basis of one means of classification. 
Some bacteria will grow with or without the presence of free 
oxygen. These are called facultative bacteria. Other bacteria will 
only grow in the presence of free oxygen. These are called obliga- 
tory aerobic bacteria. 
Still others will not grow unless all free oxygen has been ex- 
cluded from the media in which they exist.' These are termed 
obligatory anaerobic bacteria. By adaptation, a certain amount of 
modification of these relations to oxygen may be brought about. 
The hydrogen required by bacteria is obtained chiefly from 
the water around them, but also from other combustions of hydro- 
gen, which may be assimilated. 
The phosphate is obtained from the phosphates of calcium, 
magnesium, sodium and potassium. 
The chlorides do not seem to be absolutely essential for bac- 
terial growth and yet they are more favorable to growth in culture 
media than the potassium salts. 
The sulphur is derived from the sulphates. For thiobacteria 
sulphur in the form of hydrogen sulphide is necessary. 
The iron is derived from the ferrous salts of the earth. 
CONDITIONS AFFECTING THE GROWTH OF BACTERIA 
1. Food Supply-An abundance of assimilable nourishment 
constitutes an important factor among those influencing the ra- 
pidity of the growth of bacteria, as in the vegetable world in 
general. The food, moreover, must be suited to the bacteria in 
question. Many bacteria can only live on very special forms of 
protein. The gonococcus, for instance, must have the proteins of 
the human blood. Without an admixture of these in the artificial 
culture media, they cannot be cultivated. 
2. The Relation to Other Bacteria-Certain different species 
of bacteria are mutually favorable to the growth of each other. 
38 LECTURE NOTES ON BACTERIOLOGY 
Other species are antagonistic to each other. Bacteria living 
together and exerting mutually a favorable influence upon each 
other, are said to be living in symbiosis with each other. 
3. Temperature-Bacteria display a strong dependence upon 
temperature. The vast majority grow best at 37.5° C., a few will 
thrive at 20° C., and a few others at 70° C. But in general the 
temperature of the body is the optimum temperature for bacterial 
growth. Certain bacteria may grow well in a rather wide range 
of temperature. Most of the vegetative forms will grow fairly well 
at temperatures between 20° and 40° C. 
An unfavorable temperature may alter some of the specific 
properties of bacteria as for instance their virulence or the func- 
tion of pigment formation, or cause the formation of spores. 
An investigation of the temperatures necessary to effect the 
destruction of bacteria is important from the standpoint of sterili- 
zation, either for the purpose of rendering infected articles harm- 
less, or for the purpose of the sterilization of surgical dressings. 
Most of the vegetative forms will be killed by an exposure of 
ten minutes to a temperature between 55° and 60° C. 
The tubercle bacillus, though not forming spores, is very re- 
sistant to heat. An exposure of 10-20 minutes at 70° C. is neces- 
sary for its destruction. For the destruction of spores, exposure 
to temperatures of 121° C. is necessary for at least twenty minutes. 
Such temperatures can only be reached by exposing the organisms 
to steam under pressure. In general the most resistant anthrax 
spores are incapable of resisting boiling water for four minutes 
time. The spores of the red potato bacillus for instance will resist 
100° C. for 5% hours, but will be killed in two minutes by a 
temperature of 127° C. or 20 lbs. pressure. They are killed in- 
stantly at 130° C. or 25 lbs. pressure. 
The following are the temperatures of steam at various 
pressures: 
1 lb. = 102.3°c. 
2 lbs. = 104.2°c. 
3 lbs. = 105.7°c. 
4 lbs. = 107.3°c. 
5 lbs. = 108.8°c. 
6 lbs. = 110.3°e. 
7 lbs. = 111.7°c. 
8 lbs. = 113. °c. 
9 lbs. = 114.3°e. 
10 lbs. = 115.6°e. 
11 lbs. = 116.8°c. 
12 lbs. = 118. °c. 
13 lbs. = 119.l°c. 
14 lbs. = 120.2°c. 
15 lbs. = 121.3°c. 
16 lbs. = 122.4°c. 
17 lbs. = 123.3°c. 
18 lbs. = 124.3°c. 
20 lbs. = 126.2°c. 
22 lbs. = 128.1°c. 
24 lbs. = 129.3°c. 
26 lbs. = 131.5°c. 
28 lbs. = 133.1°c. 
30 lbs. = 134.6°c. 
40 LECTURE NOTES ON BACTERIOLOGY 
The most resistant vegetative organism is the Staphylococcus 
pyogenes aureus. This organism in general is killed by an exposure 
of 1% minutes to boiling water. To insure, therefore, the sterility 
of surgical instruments, they should be 
boiled for at least 2 minutes. Such treat- 
ment may not kill all spores, and for this 
reason and also for the sake of insuring a 
thorough permeation of the steam, surgical 
dressings should be subjected to a tempera- 
ture of 121° C. for 20 to 30 minutes. 
Moist IIeat Versus Dry Heat-These 
facts refer only to moist heat which is far 
more effective against bacteria than dry heat. 
The spores of anthrax while they are 
killed in twelve minutes by moist heat at 
100° C. require for their destruction in dry 
heat a temperature of 140° C. for three 
hours. The explanation probably lies in the fact that the smaller 
the percentage of water in albuminous fluids the higher the tem- 
perature required for their coagulation. Thus ordinary egg albu- 
Fig. 25.-Basketfor hold- 
ing test tubes or Petri's 
dishes. 
Fig. 26.-Sterilizing can for Petri's dishes 
and other small glassware. 
Fig. 27.-Pipette 
sterilizer. 
min is coagulated at 56° C., while anhydrous egg albumin may be 
heated to 170° C. without coagulation. As the percentage of water 
diminishes, higher temperatures are required for coagulation. 
With 25% of water 74-80°c. 
With 18% " " 80-90°c. 
With 6% " " 145°c. 
For another reason, because it is more penetrating, steam is a1 
more effective sterilizing agent than dry heat. Steam is a better 
42 LECTURE NOTES ON BACTERIOLOGY 
conductor of heat than dry air. This has been tested by placing 
self-registering thermometers in varying thicknesses of gauze. The 
layers of gauze were surrounded by hot air at a temperature of 
130°-140° C. for four hours time, and the temperatures registered 
by the thermometers inclosed in similar thicknesses of gauze sur- 
rounded by moist heat at 90°-105.3° C. for three hours time, were 
compared with those placed in the layers of gauze surrounded by 
dry heat. 
Thermometers inclosed in 20 thicknesses of gauze in 
hot air registered 86° In steam, 101° 
Thermometers inclosed in 40 thicknesses of gauze in 
hot air registered 72° " " 101° 
Thermometers inclosed in 100 thicknesses of gauze in 
hot air registered below 70° " " 101.5° 
These facts make clear the necessity of removing all the air 
from such articles as surgical dressings, for instance, during their 
sterilization by steam before the period of sterilization is begun. 
Sterilization by heat is the most useful method of destroying 
bacteria and, as has been explained, sterilization by steam is the 
most effective method of applying heat. It is applicable primarily 
to surgical dressings, the surgeon's instruments and his gloves, 
and to sheets, cotton and linen fabrics. Many delicate fabrics 
may be sterilized without injury by moist heat, if the condensation 
of the water within them is prevented by sterilization under 
pressure. Comparatively little condensation takes place under 
these conditions. 
Woolens undergo a marked shrinkage when sterilized by moist 
heat. They may, however, be sterilized by moist heat if this 
shrinkage is not a matter of great importance. Dry heat may be 
used to sterilize woolens if the temperature is not allowed to rise 
above 140° C. and maintained for three hours. 
Dry heat is also useful. It is used especially in the preparation 
of glassware for bacteriological purposes. It may be used for many 
fabrics which would be injured by steam, but the high tempera- 
tures required will produce some charring and injury of the 
fabrics unless the greatest care is used. The standard temperature 
for sterilization by dry heat is 150° for one hour. Many articles 
cannot be sterilized by heat or any other method without serious 
44 LECTURE NOTES ON BACTERIOLOGY 
injury. Such articles, if badly contaminated in sick rooms, must 
be burned. 
Fractional Sterilization and Pasteurization-Certain albumi- 
nous fluids, such as milk or certain special artificial culture media 
containing the natural proteins of the blood, cannot be sterilized 
by the usual methods of heat sterilization. In order to render such 
fluids sterile, advantage is taken of the fact that all spores will de- 
Fig. 28.-Hot air sterilizer. 
Fig. 29.-Arnold steam sterilizer. 
velop into vegetative stages when surrounded by favorable condi- 
tions of moisture, nutriment and temperature. The fluids are there- 
fore subjected to a temperature of 55° C. for one hour on six succes- 
sive days. On the first day, all vegetative forms are killed and 
only spores remain. The culture media may then be placed in a 
temperature favorable to bacterial growth. This will cause the 
spores to develop into vegetative forms which will be killed by the 
second day's sterilization. The repetition of the process on four 
additional days is for the purpose of insuring the death of the 
spores which may not have transformed themselves into vege- 
tative forms during the first interval of heat application. The 
temperature of 55° C. is adopted because no known proteins of 
46 LECTURE NOTES ON BACTERIOLOGY 
the blood coagulate below this temperature. With certain fluids 
such, for instance, as Loeffler's serum media, higher temperatures 
such as 100° C. may be used with safety and the duration of the 
period of heating may be limited to fifteen minutes and the num- 
ber of sterilizations limited to three. Sterilization at intervals of 
time in this manner is called fractional sterilization, and when it is 
done at low temperatures in a manner to avoid coagulation of 
proteins, it is called pasteurization. 
The exact temperature to be used in sterilization by pasteuriza- 
tion depends in part upon the coagulation point of the proteins in 
the solution to be sterilized, the possibility of producing further 
alterations in the fluid, as, for instance, the destruction of the 
natural enzymes in milk, as well as upon the destruction of bac- 
teria. The subject of pasteurization is of most importance in 
connection with milk. 
Of first consideration is the thermal death point of the various 
pathogenic bacteria which occur in milk. Of these, unquestion- 
ably, the tubercle bacillus is most resistant. It loses its infective 
properties for guinea pigs when heated to 60° C. for twenty min- 
utes. The typhoid bacillus is killed by 60° C. in two minutes. The 
diphtheria bacillus by 55° C. in five minutes, and the same is true 
of cholera. The dysentery bacillus is killed by 60° C. in ten 
minutes. 
A good standard for sterilization, therefore, is 60° C. for twenty 
minutes time. This temperature does not materially alter the chem- 
ical properties of milk. 
Pasteurization, however, as carried out in the home or com- 
mercially is a very inexact process. By the various methods tem- 
peratures varying from 66.5° C. to 85° C. or 150° F. to 185° F. 
are used, and for varying lengths of time. When milk is once 
heated to 158° F. or 70° C. it acquires a cooked flavor. Unques- 
tionably, the clinical results of feeding children on pasteurized 
milk demonstrated that the milk is changed in some manner not 
thoroughly understood. Sill, for instance, states that of 25,000 
infants that came under his charge during five years, those that 
were fed on pasteurized or sterilized milk continuously or for 
part of the time on one and part of the time on the other, no 
less than 97% developed rickets or scurvy or a combination of 
the two. 
48 LECTURE NOTES ON BACTERIOLOGY 
While it is true that the sterilized milk may have been respon- 
sible for the greatest amount of harm among these children, yet 
there can be little doubt that feeding with pasteurized milk con- 
tinuously is also harmful. When, however, the source of the milk 
is a matter of doubt, as in traveling or for short periods in very 
warm weather, it is better to use pasteurized milk. In no case 
is it wise to feed children continuously on pasteurized milk. 
Cold does not destroy bacteria. Most bacteria will withstand 
any degree of cold. Cholera bacilli are viable after one month's 
exposure to -32.5° C., and six or seven days of alternate thawing 
and freezing, a process which is more effective. 
Typhoid fever bacilli have caused infection after having been 
kept in ice for nine months, and diphtheria after having been 
exposed to a temperature of -25°. C. for six months' time. 
Even the temperature of liquid air, -312° F., for seven days, 
has failed to kill the typhoid bacillus or the Bacillus coli communis, 
diphtheria bacillus, the cholera bacillus, or the Staphylococcus 
pyogenes aureus or anthrax. 
Drying-The growth of bacteria is affected by drying. Bac- 
teria, however, display great differences in their ability to with- 
stand the withdrawal of water. Those species forming spores are 
able to withstand drying for long periods, in some instances 
indefinitely. 
On the other hand, such organisms as the gonococcus and 
Pfeiffer's bacillus will succumb in a few hours to drying, and the 
cholera bacillus in from four to twenty-four hours. 
4. Light-Light is a powerful destructive agent to bacteria. 
It affects almost all forms except the thiobacteria. Direct sun- 
light, for instance, will kill the tubercle bacillus in two hours. 
Even anthrax spores will succumb to sunlight in time. 
Diffuse daylight is not so powerful, but typhoid bacillus is 
killed in four hours time by diffuse sunlight: and by direct sun- 
light it is killed in 1^ hours time. In drinking water and sewage 
water the sunlight is efficacious to a depth of 25 or 30 cm. In 
clear water, it is germicidal to a depth of 6 feet. 
The exclusion of oxygen markedly diminishes the power of 
sunlight to kill bacteria. 
The bactericidal power of light aided by the action of fresh 
oxygen may be made use of in disinfecting articles contaminated 
50 LECTURE NOTES ON BACTERIOLOGY 
iii the sick room, which cannot be sterilized by heat. Expensive 
books, hangings, curtains, furniture, mattresses, paintings, etc., 
may be exposed to the open air in sunlight. 
Because of the difficulty, however, in sterilizing such articles, 
they should be removed from the room occupied by an individual 
suffering from an infective disease as soon as the diagnosis is 
made. 
5. Destruction of Bacteria by Chemicals-Of as great impor- 
tance as any method of destroying bacteria is their destruction by 
chemical agents. 
The most important chemical destroyers of bacteria are bi- 
chloride of mercury, potassium permanganate, carbolic acid, lysol, 
creolin, and the cresols; hydrogen peroxide, formaldehyde, sulphur 
dioxide, chlorine, and the hypochlorites and chloride of lime, 
alcohol, iodine, sodium carbonate and soap. 
Bichloride of mercury is the most powerful of all metallic 
antiseptics, yet there are many disadvantages which attend its 
use. 
With organic materials it forms an insoluble coagulum which 
may be an albuminate or sulphide of mercury or both, according 
to the nature of the organic material with which the bichloride of 
mercury is mixed. 
The precipitation of the albuminate of mercury may be pre- 
vented by the addition of five parts of sulphuric, hydrochloric, 
or tartaric acids, or ten parts of sodium chloride for each part of 
sublimate in 1,000; but the formation of the sulphide of mercury 
cannot be prevented if the conditions necessary for its formation 
are present. 
For these reasons solutions of bichloride of mercury are totally 
inappropriate for the disinfection of feces or sputum or the 
majority of organic fluids containing bacteria. Where a minimum 
amount of organic material is present, as, for instance, on con- 
taminated glass or enamel or agate ware, bichloride is an efficient 
antiseptic in strengths of 1 to 500, to 1 to 1,000. 
A solution of 1 to 1,000 is very slow, requiring more than 
ten minutes time to kill the Staphylococcus albus. 
In strengths of 1 to 5,000 a fifteen minute exposure is insuffi- 
cient to kill the Bacillus coli communis and Bacillus pyocyaneus 
and Staphylococcus albus or aureus. 
52 LECTURE NOTES ON BACTERIOLOGY 
A solution of 1 to 500 will kill the staphylococcus in sixty to 
ninety seconds. 
On the hands it is impossible to use bichloride of mercury 
in this strength without creating in a short time a very obstinate 
dermatitis. In fact, in much weaker strengths, 1 to 2,000, few 
hands will escape a disagreeable irritation if not an actual der- 
matitis. Hands injured in this way become more dangerous than 
clean, but unsterilized hands, because their surface, particularly 
about the nails, becomes covered with scales, cracks, and even 
minute granulating areas which form favorite lurking places for 
bacteria. Solutions of bichloride are therefore totally inappro- 
priate for hand disinfection. 
Sublamin-Another form of mercury known as sublamin or 
ethyl enediamine sulphate of mercury, has been recently intro- 
duced to replace the time-honored bichloride of mercury. Sub- 
lamin is free from many of the disadvantages of the bichloride. 
It is relatively non-irritating and forms no precipitate with albu- 
minous or soapy solutions. 
When used in strengths of 3 to 1,000 it destroys bacteria with 
greater rapidity than solutions of bichloride of equal strength. In 
strength of 1 to 1,000 it kills the Staphylococcus aureus in ten 
minutes and the Bacillus pyocyaneus and the Bacillus coli com- 
munis in 1% minutes. 
Carbolic Acid-Next to bichloride of mercury carbolic acid is 
the most used antiseptic. 
As an antiseptic there has been much conflicting opinion regard- 
ing its potency. Certain organisms represented by the typhoid 
bacillus possess a great resistance to carbolic acid. All the common 
pathogenic microorganisms, except the typhoid, such, for instance, 
as the Staphylococcus pyogenes aureus and albus, and the- Bacillus 
pyocyaneus, and Bacillus coli communis are uniformly killed in two 
minutes time by 1 to 20 solution, and in four minutes time by a 
1 to 40 solution. 
Such solutions, however, are far too strong to use on the hands. 
Carbolic acid is a serviceable antiseptic to sterilize fluids con- 
taining organic materials, or to keep albuminous solutions free 
from bacterial growth. For the disinfection of large quantities of 
fluid it is expensive. When used to disinfect typhoid stools the 
special resistance of the typhoid bacillus to it must be remembered. 
54 LECTURE NOTES ON BACTERIOLOGY 
Cresols-Under the term cresols are included mixtures of 
methyl ortho, meta and para carbolic acid. They occur as im- 
purities in crude carbolic acid. Tricresol is a mixture of 40% 
meta, 35% ortho, and 25% para phenol. According to many they 
are more efficient antiseptics than carbolic acid itself. Harrington 
has confirmed this view as regards tricresol. They are all in- 
soluble in water, but may be dissolved by the aid of soap and such 
solutions have double the bactericidal power of carbolic acid, 
against the Bacillus prodigiosus, the Bacillus pyocyaneus, and the 
Staphylococcus pyogenes aureus. 
Creolin-Creolin is a dark brown alkaline liquid containing 
10% of the cresols held in solutions by soap. A 5% solution of 
creolin is an effective antiseptic against cholera, typhoid, and 
anthrax, and the pyogenic cocci. Against these organisms it is 
somewhat more effective than carbolic acid itself. 
Both creolin and cresol make valuable antiseptics with which 
to render typhoid stools non-infectious. 
Lysol is a 50% solution of the cresols held in solution by 
neutral soap. 
A 2% solution is more effective against pyogenic cocci than 
a 3% solution of carbolic acid, though Harrington has not been 
able to confirm this belief. Because of its slippery qualities and 
neutral and bland characters, it makes an excellent surgical anti- 
septic. 
Chlorine-Nascent chlorine is an antiseptic gas when brought 
into sufficiently intimate contact with bacteria. It, moreover, de- 
composes offensive gases, such as ammonia and hydrogen sul- 
phide, resulting from decomposing organic matter. In the pres- 
ence of moisture it unites with the hydrogen of the water vapor, 
setting free nascent oxygen which in itself is capable of oxidiz- 
ing decaying organic matter and to a limited degree of destroy- 
ing bacteria. As a fumigating agent it is too destructive of 
metals and fabrics and far inferior to formaldehyde. As a 
deodorant, however, it is of value. Use has been made of it 
for the sterilization of the hands preparatory to surgical 
operations. 
It may be set free by mixing lime and soda in the hands, with 
water, until the two form a paste. The paste is then spread over 
the hands and arms. The nascent chlorine is set free and is very 
56 LECTURE NOTES ON BACTERIOLOGY 
penetrating. Experiments have demonstrated that this method of 
sterilizing the hands is fairly dependable. 
Chlorinated lime, or bleaching powder, is a combination of 
calcium chloride and calcium hypochlorite. Its exact composition 
is not positively known. It is prepared by passing chlorine over 
dry slaked lime. It is a dry white powder and should possess 
only a faint odor of chlorine. 35% of chlorine should be avail- 
able from it. In solution it is, however, an efficient antiseptic. 
A 0.12% solution will kill cholera and typhoid bacilli in 5 
minutes. 
A 0.10% solution will destroy anthrax bacilli in 1 minute. 
A 0.12% to 0.15% will kill the Staphylococcus p. aureus in 5 
minutes. 
A 0.12% solution will kill the streptococcus in 1 minute. 
A 5.0% solution will kill anthrax spores in 15 minutes. 
A 10% solution will kill anthrax spores in 70 minutes. 
Labarraque's solution is a solution of several compounds of 
chlorine chiefly of NaClO and NaCl. It contains 2.6% of available 
chlorine. 
Hypochlorous acid is another antiseptic preparation contain- 
ing chlorine. It is quite possible that chlorine in solution exerts 
its antiseptic action always as hypochlorous acid. 
It kills anthrax spores in extremely dilute solutions. It 
will kill the staphylococcus growing in broth in strengths of 
1 : 3000. 
The addition of a little weak acid greatly increases its power. 
Thus a saturated solution which contains 5% of calcium hypo- 
chlorite requires 7-10 minutes to kill the anthrax spores, while the 
same solution with the addition of two volumes of a 1.25% solu- 
tion of acetic acid will kill the spores in less than one minute. 
All these preparations of chlorine are cheap and exceedingly 
valuable antiseptics for rendering organic mixtures like decaying 
material or contaminated feces or urine innocuous. 
Oxygen, Ozone and Hydrogen Peroxide-Prolonged aeration is 
rightly to be regarded as a valuable assistant in disinfection. The 
disinfectant action, however, of free oxygen and ozone has been 
questioned. Many have attributed powerful disinfectant action 
to ozone. Ransome and Foulerton have investigated the question 
and conclude that dry ozone has no appreciable action on the 
58 LECTURE NOTES ON BACTERIOLOGY 
vitality of the tubercle, the tetanus, the diphtheria, the anthrax, the 
typhoid bacillus, or the Bacillus coli communis, the Bacillus 
pyocyaneus, the Diplococcus pneumoniae, the Bacillus prodigiosus, 
or the pyogenic coccus. 
Any purifying action which ozone or oxygen may possess is 
due to its oxidizing action upon putrescible matter, an end toward 
which the bacteria themselves are also working. 
It is probable that certain percentages of ozone in solution may 
be antiseptic. By actual experiment, it has been claimed that 2.3 
milligrams of ozone were sufficient to destroy 99% of bacteria 
in 200 c.c. of water from Tejel Lake. With 3 to 4 milligrams Weyl 
obtained complete sterilization of 0.5 liter of water containing 
6000 bacteria to the cubic centimeter. River and sewage water 
are not so strongly affected but when there is only a moderate 
amount of pollution, it is possible that ozone might be a cheap 
and efficient means of purifying drinking water. 
Hydrogen peroxide is a strong oxidizing agent. In the pres- 
ence of organic matter oxygen is set free and attacks and oxidizes 
the organic matter, including the bacteria. 
A 1.0% solution will kill diphtheria and typhoid bacilli in five 
minutes, the streptococcus in ten minutes, and the staphylococcus 
in fifteen to thirty minutes. 
A %% solution will kill diphtheria in fifteen minutes and 
staphylococcus in one hour's time. 
Diluted hydrogen peroxide makes a useful antiseptic to mucous 
membranes. Inside the mouth it is harmless and assists in purify- 
ing this cavity. It must, however, always be remembered that 
in strengths in which it can be used in the mouth considerable 
time is needed to kill bacteria, and this time is too long to make 
it possible to sterilize the mouth by hydrogen peroxide. Its 
action, however, in destroying putrifying organic matter within or 
about the teeth is important in the purification of the oral cavity. 
Sodium carbonate possesses a very definite antiseptic action. 
Moreover, it saponifies grease and dissolves albuminous material. 
It is therefore an important purifying agent. As an antiseptic, 
it is not efficient when the temperature of the solution in which 
it is dissolved is below 132° F. 
When present in 2% solution at 132° F. it kills diphtheria 
bacilli in two minutes and in 5% solution in one minute. The 
60 LECTURE NOTES ON BACTERIOLOGY 
same solution is effective against the staphylococcus at tempera- 
tures of 140° F. At this temperature they are killed in five 
minutes. 
All solutions of sodium carbonate are ineffective against the 
staphylococcus at temperatures below 140° F. 
Typhoid bacilli are killed by a 2% solution in one minute at 
temperatures of 122° F. 
The colon bacilli are killed by a 2% solution at a temperature 
of 122° F. in five minutes and at 95° F. in one hour. 
The addition of sodium carbonate to the water in which surgical 
instruments are boiled greatly increases the efficiency of their 
sterilization both because of its antiseptic power and of its action 
in preventing the rusting of the ' instruments. 
Soap is by no means an unimportant purifying agent from 
the standpoint of antisepsis, as well as because it dissolves grease. 
A 10% solution kills the typhoid bacillus in one minute; a 5.0% 
solution in three to ten minutes, and the colon bacillus in fifteen 
minutes. 
The staphylococcus is unaffected by a 20% solution after one 
hour's exposure. 
Soap, however, greatly enhances the antiseptic power of cer- 
tain other antiseptics. This is particularly true of the carbolic 
acid group. It doubles the antiseptic power of carbolic acid. 
Silver Compounds-Silver nitrate in solution even in such 
dilute solutions as 1 to 2000 is a strong antiseptic. It is also a 
good astringent and hence it makes a valuable agent for use on 
mucous membranes. 
A 0.5% solution of silver nitrate kills the staphylococcus in two 
to five minutes. 
Certain organic compounds of silver, however, are strong anti- 
septics and act as such quite irrespective of the amount of silver 
which they contain. These compounds are of particular value 
upon the mucous membranes. The following are the most im- 
portant, with the indication of the strength of their action: 
Protozol 2-4 % sol. kills the staphylococcus in 3-5 minutes 
Albargin 10-20% sol. kills the staphylococcus in 2-5 minutes 
Ichthargan 1-1 % sol. kills the staphylococcus in 1-4 minutes 
Largin 10-20% sol. kills the staphylococcus in 2-5 minutes 
Argonin 5% sol. kills the staphylococcus in 3-6 minutes 
62 LECTURE NOTES ON BACTERIOLOGY 
A review of all these antiseptics emphasizes the fact that the 
time of exposure of the bacteria to them and the strength of the 
solution are very important factors in their bactericidal power. 
With all of them, these factors approach an amount which unfit 
them in general for use about the body or for surgical work. 
Alcohol-With alcohol the case is somewhat different. The 
staphylococcus will succumb to immersion in 70% alcohol in two 
minutes' time. 70% alcohol is a bland fluid and it is quite within 
the bounds of possibility to immerse the hands or the field of 
operation in 70% alcohol for two minutes. It has been found that 
dilute alcohol is more efficient than strong alcohol. Absolute 
alcohol, for instance, requires one hour to kill the staphylococcus. 
Dilute alcohol, therefore, is a practical surgical antiseptic. 
A mixture of alcohol with bichloride and hydrochloric acid, 
which will kill the staphylococcus in ten seconds, has been devised 
by Harrington. It is a good antiseptic to use in the preparation 
of the skin of the field of operation. Its formula is 
Hydrochloric acid 60 c.c. 
Alcohol 640 c.c. 
Bichloride of mercury 0.8 gram. 
Water 300 c.c. 
Iodine-As a rapid and non-irritating antiseptic alcohol stands 
quite apart. It thus becomes a useful surgical antiseptic and its 
position as such is shared by tincture of iodine, and for the same 
reasons. Iodine is a powerful disinfectant. In solutions of 0.2-1% 
it is superior to solutions of bichloride of mercury in strengths of 
1 to 1000. The latter requiring fifteen minutes to kill the 
staphylococcus while iodine 2 to 1000 kills them in two minutes. 
Iodine does not form insoluble compounds in the tissues and 
in effective strengths it is not irritating. It can be put directly 
into wounds without harm. It is almost always used in the form 
of the tincture so that such solutions furnish a combination of 
alcohol and iodine. Abundant experience has now been collected 
regarding its use in the preparation of the skin for surgical opera- 
tions and this experience is most favorable to its reputation as a 
surgical antiseptic. 
Formaldehyde is a gas and possesses a certain amount of anti- 
64 LECTURE NOTES ON BACTERIOLOGY 
septic power when liberated as such in a confined space. It also 
possesses a strong disinfectant action in solution. 
Commercial formalin is a 40% solution of formaldehyde in 
water. When added to feces and urine to strengths of 1/20 of 
the volume of the fluid to be disinfected, it will be effective in one 
hour's time. In a 5% solution in water formaldehyde requires 
twenty minutes to kill the Staphylo- 
coccus pyogenes aureus. 
2% tablespoonfuls of formaline in a 
quart of water will disinfect sputum in 
two hours' time. Liquid stools will be 
sterilized in the same time by the same 
percentage. 
Formaldehyde finds, however, its 
chief field of usefulness as a gaseous 
antiseptic, though this is perhaps be- 
cause there is no ideal gaseous disinfec- 
tant. It may be generated from methyl 
alcohol or volatilized by boiling its aque- 
ous solutions. Both these methods are 
unsatisfactory. When generated from 
methyl alcohol a large part is converted 
into carbon monoxide and carbon diox- 
ide. 
Attempts to volatilize it from its 
solutions are generally defeated by the 
ease with which it undergoes polymeriza- 
tion. By mixing formalin with calcium 
chloride which forms with the formalde- 
hyde formochlorol, the polymerization of 
formaldehyde may be prevented. Also by rapid volatilization, little 
polymerization takes place. A number of apparatus and methods 
have been devised for generating formaldehyde gas. The most use- 
ful of these is Trillat's Autoclave. 
It consists of a silver lined pot of heavy copper, holding a 
gallon. The cover is secured with turnbuckles. Connected with 
the interior is a pressure gauge and thermometer and an outlet 
nozzle controlled by a valve. The pot is filled to within a quarter 
to three-quarters of its capacity with formalin and calcium 
Fig. 30.--Trillat's autoclave. 
(Harrington.) 
66 LECTURE NOTES ON BACTERIOLOGY 
chloride; 150 grams of calcium chloride are used to the liter of 
formalin. The mixture is known as formochlorol. The mixture 
must be free of methyl alcohol. 
A Swedish lamp, fed with kerosene vapor, is placed under the 
pot and when the gauge shows a pressure of the atmosphere, the 
nozzle of the instrument is turned into the keyhole of the room 
and the valve is gradually opened. For a room 18x15x10 feet, one 
hour's use of this apparatus will furnish a sufficient concentration 
within the room. 
Another method of generating formaldehyde is from pastilles 
Fig. 31.-Breslau regenerator and lamp. (Harrington.) 
of paraform. These may be volatilized over an alcohol lamp as 
in the Shering lamp. Two one-gram pastilles for every thirty- 
five cnbic feet of air space give sufficient concentration of the gas 
to the space to be disinfected. 
Another method is by means of the Breslau apparatus, which 
is so constructed that 3.5 liters of fluid containing formalin diluted 
with four parts of water may be brought to a boil in ten minutes, 
and the whole evaporated in one hour's time. The speed of evap- 
oration and the quantity are controlled by the amount of alcohol 
used in the lamp which is placed below the apparatus. It should 
be one-quarter of the amount of fluid in the boiler. With this 
dilution and speed of evaporation little polymerization of the 
formaldehyde occurs. 
68 LECTURE NOTES ON BACTERIOLOGY 
Still another method of generating formaldehyde consists m 
the mixture of potassium permanganate and formalin in propor- 
tions of 6.5 of the one to one part of the other, in an open deep 
pot within the space to be disinfected. By this method 81% of the 
available formaldehyde gas is liberated from the formalin and 
4/5 of this quantity is set free in five minutes time. 
A similar method consists in quickly mixing paraformaldehyde 
and the metallic peroxide of barium and strongium, which, when 
mixed together, form a powder called autan. When this mixture 
is thrown into water formaldehyde is evolved with such rapidity 
that some experimenters consider 
that the usual precautions of seal- 
ing of the room are unnecessary. 
Huber and Bickel have sug- 
gested the rapid mixture in a large 
tub of two quarts of formalin, four 
pounds of quicklime, and six 
quarts of boiling water for every 
1,000 cubic foot of space to be dis- 
infected. The lime is placed in 
first and then the water poured 
over it and finally the formalin. 
The gas and generous volumes of 
steam are given off with great ra- 
pidity. 
As regards the efficiency of formaldehyde gas, it is generally 
agreed that at best it is only a surface disinfectant. The gas 
cannot, for instance, penetrate efficiently into the interior of a 
closely rolled bundle of clothing or inside of a pillow or mattress. 
It will, however, reach all parts of a loosely rumpled mass of 
cheese cloth and it will sterilize test objects placed inside a laid- 
open pillow or mattress. It will sterilize the surface of walls, floors 
and ceilings, but it can not render innocuous dust and dirt which 
has collected in cracks. Moisture within the air of the space to 
be disinfected is quite necessary to the successful action of formal- 
dehyde. A relative humidity of 65% is essential, and yet exposed 
objects must not be wet, as a covering of moisture will prevent 
the penetration of the gas. The temperature of the room to be 
disinfected must be about 69° F. Dogs and cats left accidentally 
Fig. 32.-Vertical section of Breslau 
regenerator. (Harrington.) 
70 LECTURE NOTES ON BACTERIOLOGY 
in rooms which have been disinfected have been killed. All 
flies, roaches and bedbugs are also killed. Formaldehyde is said, 
however, to be less effective against mosquitoes than sulphur 
dioxide. 
In a series of experiments in air-tight glass cabinets, smears of 
anthrax spores of staphylococci and diphtheria bacilli were ren- 
dered sterile after two and a half hours' exposure to an atmos- 
phere containing the full yield of formaldehyde from 110 c.c. of 
formalin for every 1,000 cubic feet of space. 
Sulphur Dioxide-Only one other gaseous disinfectant has been 
used to any extent. It is sulphur dioxide and is generated by 
burning flowers of sulphur in an open pot, or from candles pre- 
pared for the purpose. If the powder is used, 1-6 lbs. are burned 
for every 1,000 cubic feet. The combustion of this quantity is 
never complete. To avoid the burning of sulphur liquefied sulphur 
dioxide may be used. Candles are sold which may simply be 
lighted and burned in requisite number according to the size of 
the space to be disinfected. 
In the absence of moisture, the bactericidal action of sulphur 
is practically nil, and notwithstanding some favorable reports, the 
general consensus of opinion is that sulphur dioxide is a very 
superficial antiseptic of far less potency and penetrating power 
than formaldehyde. It is, however, a very efficient agent against 
mosquitoes. In the presence of moisture it is to some extent oxi- 
dized to sulphuric acid, and tarnishes metals, corrodes fabrics, 
reduces organic matter and destroys colors. It leaves a very 
persistent odor. Its use as a gaseous disinfectant in view of the 
greater advantages and efficiency of formaldehyde is only to be 
condemned. 
THE INFLUENCE OF BACTERIA UPON THE MEDIA IN WHICH THEY GROW 
1. Fermentation-The fermentation of carbohydrates is car- 
ried on chiefly by yeasts, but many bacteria possess the power of 
fermenting carbohydrates. This is accomplished by the produc- 
tion of the appropriate ferment by these different bacteria, each 
bacterium producing its own ferment, either amylase or cellulase, 
gelase, invertase, maltase, lactase, as the case may be. 
72 LECTURE NOTES ON BACTERIOLOGY 
Another form of fermentation is the production of lactic acid. 
This may be produced as an almost pure product from both 
disaccharides as lactose, or from monosaccharides as dextrose, by 
certain lactic acid ferments produced by bacteria. 
Oxidizing Ferments-These are termed oxidases. The produc- 
tion of acetic acid from cider or wine consists in the oxidation of 
the weak alcohol contained in the cider or wine by bacteria pos- 
sessing the specific power of producing oxidases. 
Alcoholic Fermentation-This change is accomplished by zymase 
and the production of zymase is a specific property of many yeasts 
and bacteria. Either the mono- or disaccharides may be trans- 
formed into ethyl alcohol. 
Alcohol and Carbon Dioxide-The many varieties and the speci- 
ficity of the strains of yeasts and bacteria are illustrated by the 
care which is necessary in order to produce the various wines 
and liquors, the flavors of which are largely dependent on the by- 
products of specific ferments. 
2. Protolysis-All the changes produced by proteolytic en- 
zymes are produced by bacteria and by various methods a variety of 
proteolytic enzymes may be extracted. All the usual amino-acids, 
leucin, tyrosin and the fatty acids and mercaptans, indol and 
skatol, carbon dioxide, hydrogen sulphide, ammonia and free hydro- 
gen are produced by these ferments. Moreover, some of the prod- 
ucts formed by the action of certain bacteria on proteins are exces- 
sively poisonous. 
Such substances are called ptomaines. These are not toxins, 
soluble products formed within the bacterial body and representing 
a product of the life of the bacterial cell, but a product formed 
from proteins external to the bacterial cell by the action of the 
bacteria upon proteins. Examples of these ptomaines are putrescin, 
cadaverin, kreotoxin, tyrotoxin (from cheese), silotoxin (from 
vegetables). 
3. Lytic Substances for Animal Excreta-An organism pro- 
ducing such a change specifically in urea is the Micrococcus ureae, 
which splits urea (NH2)2CO-|-2H2O=2NH34-CO2-|-H2O. Such a 
reaction as the transformation of nitrogen of the organic proteins 
into ammonia is necessary for all life on the earth. It is a step in 
the chain of processes: preparing nitrogen for assimilation by 
plants which assimilation in turn is essential to prepare forms of 
74 LECTURE NOTES ON BACTERIOLOGY 
nitrogen which are capable of assimilation by man. Few plants, 
however, can assimilate nitrogen in the form of ammonia. 
By a special variety of bacteria the nitromonas ammonia may 
be oxidized into ammonium nitrite, and the nitrites in turn may 
be transformed by nitromonas into nitrates. 
These bacteria are termed nitrifying bacteria, and are to be 
looked upon as forming a most important relation to the con- 
tinuation of life on the earth. 
Still other bacteria, the Bacillus radicicola, in symbiosis with 
the roots of leguminous plants, are capable of enriching the soil's 
content of nitrogen. These bacteria accomplish the reduction of 
nitrates to nitrites and the latter to ammonia, and finally the 
liberation of free nitrogen from the ammonia. 
A different variety of bacteria is responsible for each individual 
change in this series of reactions. Although these bacteria have 
been termed the wasters of nitrogen, and are working toward an 
end which is opposed to that end toward which those which trans- 
form nitrogen into a form available for plant life are working, yet 
they fulfill a useful purpose. They form links of the chain by 
which decaying organic matter is completely disposed of. 
Bacteria also exist which possess the power of splitting fat 
into its chemical components, glycerine and fatty acid. This power 
is due to the elaboration of a lipase; a simple illustration of this 
power is the change by which butter becomes rancid. 
The Production of Lab Ferments-A Lab ferment is a ferment 
which will coagulate certain proteins. The precipitation of casein 
in milk is an illustration of the effect of a Lab ferment. Certain 
varieties of bacteria produce it. 
Special Activities of Bacteria-The liberation of light energy 
by bacteria-Certain bacteria possess the power of producing light. 
This light is not a true phosphorescence as it exists apart from 
previous illumination and develops as well in cultures left in the 
dark. Much of the so-called phosphorescence at sea is due to these 
bacteria, though a large share is also produced by coelenterates 
and other invertebrates. 
The Formation of Pigment by Bacteria-Many bacteria form 
pigment. In only a few species is this pigment contained within 
the bacterial cell. It is usually external to the cell within the 
surrounding media. 
76 LECTURE NOTES ON BACTERIOLOGY 
In the thiobacteria, the bacteria which form sulphur granules, 
the yellow granules are found within the cells and the formation 
of these granules appears related to the nutrition of the bacteria. 
Pigments of all colors are formed. The Bacillus prodigiosus, for 
instance, stains the surrounding media a brilliant red. The 
Bacillus pyocyaneus stains it a green color. 
The pigment formation is dependent closely upon cultural con- 
ditions of temperature, quality and reaction of the media and the 
supply of oxygen. It may be considered to be an index to the 
biological activities in general of the pigment-forming bacteria. 
METHODS OF STUDY OF BACTERIA 
Different Methods of Identification of Bacteria-In order to 
study bacteria and to become familiar with their vital activities 
and the consequences of these activities upon their environment, 
it is necessary to separate them from other bacteria, so that each 
variety may be studied by itself. 
The different varieties of bacteria are so numerous and the 
morphological differences between them so few that the identifica- 
tion of different varieties on morphological criteria alone is of 
very restricted value. "With few exceptions, different varieties 
and species of bacteria can only be distinguished by studying not 
only their- 
1. Different morphology, but also their 
2. Different staining reactions, 
3. The different manner in which they grow upon various 
artificial culture media, 
4. Their various infective characters when inoculated into 
animals, or when accidentally infecting human beings, 
5. Their reactions toward specific substances which are pro- 
duced in the living animal as a consequence of infection by 
them. 
In order to study any one of these five classes of characters 
of any one variety of bacteria, it is necessary to separate such 
bacteria from all other varieties of bacteria with which they may 
happen to be mixed. 
Methods of Isolating Bacteria-This may be accomplished with 
78 LECTURE NOTES ON BACTERIOLOGY 
certain kinds of pathogenic bacteria, by animal inoculation, and 
with others by special culture media. 
If an animal is inoculated with a variety of pathogenic bacteria 
to which it is susceptible, it is seldom, even if other bacteria are 
present in the mixture, that the special bacteria in question will 
not go on to produce its specific infection. At the death of the 
animal, if it succumbs to the infection, the specific bacteria may be 
recovered unmixed with other bacteria from the animal's blood or 
tissues. They may then be inoculated into appropriate culture 
media and grown in pure culture. 
In the case of other bacteria, a separation of a suspected 
variety from other varieties with which it may be mixed, 
can be accomplished by inoculating culture media containing 
melted gelatin or agar. After inoculation, the bacteria may be 
evenly distributed through the media by twirling around the 
tube containing the inoculated media while still warm and 
melted. The culture media may then be poured out upon 
shallow glass dishes, upon the surface of which it quickly 
hardens. 
Each bacterium within the tube, and therefore a representative 
of all within the original mixture, is separated from its fellows 
and held imprisoned by the solid media. 
Each bacterium immediately starts to divide and produce a 
small colony so that soon the whole surface of the agar or gelatin 
becomes covered with minute colonies which are visible to the 
naked eye. Inasmuch as each bacterium gives rise to a colony 
which is characteristic of itself, the different number of bacteria 
present may be picked out by the differences between the various 
colonies. 
With the aid of the low powers of the microscope, the differ- 
ences between the colonies become easily recognized and by means 
of a platinum wire one end of which can be dipped into the colonies, 
some of the bacteria can be removed and planted into a fresh tube 
of media. Inasmuch as the transplant consists only of bacteria 
from one colony it will give rise to a pure culture of the bacteria 
which gave rise to the colony from which the transplant was 
taken. 
In the same manner, bacteria may be taken from other col- 
onies, and in this manner pure cultures obtained from all the 
80 LECTURE NOTES ON BACTERIOLOGY 
different colonies, and thus the different kinds of bacteria in the 
original mixture separated. 
The Identification of Bacteria Separated Upon Culture Media 
-Having in this manner isolated in pure culture a bacterial 
strain, the bacteriologist is in a position to attempt to identify 
the strain. 
Morphology-He should begin first by studying its morphology. 
Is the particular organism motile or non-motile? Is it a coccus, 
bacillus, or spirillum? A minute speck of the culture may be 
placed in a drop of normal saline fluid on a glass slide and covered 
with a cover slip, and examined under the microscope. 
Motility-This procedure will determine whether the organism 
is motile or non-motile. 
Staining-The shape of the organism is better studied after 
staining. A minute speck of the culture is mixed upon a glass 
slide with plain water and allowed to dry. Next, the glass slide 
is quickly passed three times through the flame of a Bunsen burner 
in order to fix the bacteria firmly to the slide, and finally a drop of 
5% aqueous mixture of a saturated alcoholic solution of either 
fuchsin, gentian violet, or methylene blue is placed over the spot 
on the slide containing the bacteria. Fuchsin will dissolve in 
water to the extent of 1.5%, in alcohol (96%) to 3%; Gentian 
violet in water to 1.5%, in alcohol (96%) to 1.5%; Methylene 
blue in water to 6.7%, in alcohol (96%) to 7%. 
The stain is left on the bacteria from one-half to one and a 
half minutes, washed off with water, and the slide blotted dry and 
the stained area on the slide mounted in Canada balsam. 
When examined under the microscope, the individual bacteria, 
aside from exceptional instances, will appear stained in a manner 
which far more clearly brings out their shape and contour than 
when examined in a fresh state. 
All the various morphological characters must be noted, but 
such a method of staining will not bring out flagella or capsules, 
nor if the bacteria contain spores will these be stained. 
Spores may reveal their presence by showing within the organ- 
ism as clear unstained spherical bodies, but to stain flagella and 
spores special staining processes are necessary. 
Staining Flagella-A number of methods are used to stain 
flagella. The cover slips must be scrupulously cleansed. By 
82 LECTURE NOTES ON BACTERIOLOGY 
Loffler's method, the following mordant, freshly filtered, is poured 
on the fixed bacteria: 
20% aqueous solution of tannic acid 10 parts 
Ferrous sulphate, saturated solution 5 parts 
Saturated alcoholic solution of fuchsin 1 part 
The solution is heated, but not boiled, upon the slide for one-half 
to one minute. The slide is then washed in water and the bacteria 
stained again with 5% aniline water fuchsin, or aniline water 
gentian violet, made slightly alkaline by addition of 1/10% sodium 
hydrate. The stain should be 
freshly filtered directly upon 
the cover glass and warmed gen- 
tly for one to two minutes. It 
may then be washed in water, 
dried and mounted in balsam. 
The aniline oil water is made 
by shaking up one part of ani- 
line oil with nine parts of dis- 
tilled water. After shaking, 
1/10 part of volume of a satu- 
rated alcoholic solution of fuch- 
sin or gentian violet is added 
and the mixture is filtered. {See 
Fig. 6.) 
Staining Capsules-T his 
method is a very satisfactory 
method for staining capsules. 
A smear containing the organism is made by spreading on a 
cover slip a drop of beef blood serum. 
The preparation is dried and fixed and stained for a few sec- 
onds with 5% mixture of saturated alcoholic fuchsin or gentian 
violet in water. The preparation is flooded with the dye and 
steamed over the flame for a few seconds. 
The dye is now washed off in a 20% aqueous solution of copper 
sulphate solution. The cover slip is then blotted without washing, 
and mounted in balsam. 
Staining Spores-Spores may be stained satisfactorily by Ab- 
Fig. 33.-Anthrax bacilli. Cover glass 
preparation from the spleen of a 
mouse showing the capsules. Mag- 
nified 1000 diam. 
84 LECTURE NOTES ON BACTERIOLOGY 
bot's method. The bacteria and spores are smeared on the cover 
slip or slide and fixed with heat. 
1. They are then covered with Loffler's alkaline methylene blue, 
a stain containing 
30 c.c. of saturated alcoholic solution of methylene blue to 
100 c.c. of a 1 : 10,000 solution of potassium hydrate in water 
The stain, on the slide is brought to a boil and kept hot, but not 
boiled again for one minute. 
2. It is now rinsed off in 
water. 
3. And covered with a mix- 
ture of 80% alcohol, 98 c.c., and 
nitric acid 2 c.c., until all the 
blue color has disappeared. 
4. Again rinsed in water. 
5. Now dipped in saturated 
alcoholic solution of eosin 10 
c.c., water 90 c.c. 
6. Rinsed in water, blotted 
and mounted. By this method 
the spores are blue and the bac- 
terial bodies are pink. 
Differential Stains-T w o 
staining methods are of special 
use in differentiating bacteria. 
These are: 
I. Moller's method, which gives a very good counterstain: 
1. Smear is made and fixed. 
2. Wash in chloroform for two minutes. 
3. Rinse in water. 
4. Cover with 5% chromic acid one-half to two minutes. 
5. Wash in water and invert cover slip in carbol-fuchsin in 
a small dish and heat gently with a flame until it steams, and 
continue the steaming for three to five minutes. 
6. Remove most of the color with 0.5% aqueous solution of 
sulphuric acid for five to ten seconds. 
7. Wash in water. 
Fig. 34.-Bacillus subtilis, showing the 
spores. Cover glass preparation 
from an agar culture. The spores are 
stained with Fuchsin, the bacilli with 
methylene-blue. Magnified 1000 
diam. 
86 LECTURE NOTES ON BACTERIOLOGY 
8. Counterstain with aqueous methylene blue for one-half to 
one minute. 
II. Gram's Stain and Stains for Acid-fast Bacteria-All bac- 
teria may be classified according to their reaction toward Gram's 
stain into two classes: those which are Gram positive and Gram 
negative, and one of the first stains to apply in dealing with any 
unknown bacterium is Gram's stain. 
The staining is performed as follows: 
1. Smears are fixed on cover slip or slides by heat. 
2. They are covered with fresh aniline water-gentian-violet 
made as follows: 
5 c.c. of aniline oil shaken with 125 e.e. of water and filtered through moist 
filter paper. 
100 e.c. of this solution and 
12 e.c. of saturated alcoholic solution of gentian violet. 
The stain acts best when twenty-four hours old, but may be used 
at once. It lasts for three to five days if kept corked. This is 
allowed to remain on the smear for five minutes. 
3. Pour off the stain and cover with Gram's iodine for two 
to three minutes. Gram's iodine is: 
Iodine 1 gram 
Potassium iodide 2 grams 
Distilled water 300 e.c. 
4. Rinse in 97% alcohol until the stain is washed out of the 
smear. This takes thirty seconds to two minutes. 
5. Wash in water. 
6. Counterstain with Bismarck brown or in aqueous fuchsin 
or in weak carbol-fuchsin aqueous solution of Bismarck brown by 
heat, allowing to cool, filtering and diluting with nine parts of 
water. 
When any bacterium exposed to this staining method retains 
the gentian-violet stain, it is said to be Gram positive. 
The ordinary pathogenic organisms may be divided into the 
following two classes: 
88 LECTURE NOTES ON BACTERIOLOGY 
(Gentian-violet stain retained) 
Micrococcus pyogenes aureus 
Micrococcus pyogenes albus 
Streptococcus pyogenes 
Micrococcus tetragenus 
Pneumococcus 
Bacillus subtilis 
Bacillus anthracis 
Bacillus diphtherias 
Bacillus tetanus 
Bacillus tuberculosis 
Bacillus aerogenes capsulatus 
Bacillus botulinus 
Gram Positive 
(Stained with counterstain) 
Meningococcus 
Gonococcus 
Micrococcus catarrhalis 
Bacillus coli 
Bacillus typhosus 
Bacillus fecasis alkaligenes 
Bacillus enteritidis 
Bacillus proteus 
Bacillus mallei 
Bacillus pyocyaneus 
Bacillus influenzae 
Bacillus mucosus capsulatus 
Bacillus pestis 
Bacillus maligni cedematis 
Spirillum choleras 
Bacillus Koch Weeks 
Bacillus Morax Axenfeld 
Gram Negative 
Special stains for acid-fast bacilli-A few bacilli are known 
as acid-fast because when once successfully stained they yield 
up their stain to acid-decolorizing fluids very slowly. The prin- 
cipal member of this group is the Bacillus tuberculosis-other 
members are the bacillus of leprosy and the smegma bacillus, al- 
though the latter yield their stain more readily to alcohol. A 
staining method, therefore, embodying a decolorization with both 
acid and alcohol, is diagnostic of the tubercle bacillus, as acid- 
fast bacilli are encountered so rarely that where one variety is 
suspected, others are usually not present. 
The following is the staining method: 
1. The smears are made and fixed by heat. 
2. Stained in hot carbol-fuchsin up to the steaming point and 
continuing the steaming for the five minutes. 
3. Rinse in water. 
4. Decolorize with either 
5% aqueous nitric acid, 
5% aqueous sulphuric acid, 
1% hydrochloric acid, 
for two minutes or until washing with water ceases to bring away 
the pink color. 
5. Wash with 90% alcohol until no more color can be re- 
90 LECTURE NOTES ON BACTERIOLOGY 
moved. Where the presence of lepra or smegma bacilli are sus- 
pected, the washing with alcohol must be very carefully done, and 
where these organisms are searched for, only a second or two of 
washing with alcohol must be permitted. 
6. Wash in water and counterstain in aqueous methylene blue 
for one-half to one minute. 
7. Wash in water, blot, dry and mount. The acid-fast bacilli 
will be red and other bacteria blue. 
Stains for Spirochceta Pallida-For staining the Spirochaeta 
pallida an intense nuclear stain is necessary. None is better than 
any of the blood stains applied for a prolonged period. The Nocht, 
Ramanowsky or Jenner or Hasting or Wright method is very ex- 
cellent, particularly if the fluid is made a little alkaline. Of 
special value is the Grenisa method. 
The commercial Grenisa stain consists of 
Azur II eosin 3 grains 
Azur II 8 grams 
dried over sulphuric acid powdered and dissolved in glycerine and 
methyl alcohol. The smears are fixed in methyl alcohol and allowed 
to dry. 
Ten drops of the staining fluid are added to 10 c.c. of dis- 
tilled water made slightly alkaline with one to two drops of a 
1% solution of potassium carbonate. This mixture is allowed to 
cover the smear for ten to fifteen minutes for ordinary bacteria or 
protozoa, but for three to four hours for Spirochaeta pallida or for 
Negri bodies. 
Staining Bacteria in Sections-In order to stain bacteria in 
tissues, the tissue should be preferably fixed in formalin and 
embedded in paraffin. 
1. The sections then should be stained with Lbffler's alkaline 
methylene blue for 5 to 15 minutes, and the excess of color re- 
moved by 
2. 1 to 1000 acetic acid for about 10 seconds. 
3. They may then be dehydrated by short emersion in absolute 
alcohol and 
4. Cleared in xylol 
5. And mounted in balsam. 
92 LECTURE NOTES ON BACTERIOLOGY 
For staining bacteria by Gram's method in tissues: 
1. The aniline-water-gentian-violet is applied for five minutes. 
2. Excess poured off and replaced with Gram's iodine. 
3. Decolorized with 85% alcohol. 
4. Counterstained with weak aqueous methylene blue. 
5. Blotted and dehydrated in xylol and mounted. 
The Relative Value of Staining and a Study of the Morphol- 
ogy of Bacteria in the Identification of Bacteria-As has been 
emphasized, staining alone and a study of morphology rarely 
gives sufficient information for this differentiation of the dif- 
ferent varieties of bacteria. In many instances given the source 
of the bacteria and the clinical features of the lesion from which 
the bacteria have been obtained, it does give sufficient informa- 
tion. For, however, more accurate identification and in the ma- 
jority of instances for identification alone, the cultural characters 
of bacteria must be known. These comprise the different peculiari- 
ties of the growth of bacteria on the various artificial culture 
media. 
The Composition of Artificial Culture Media-The most im- 
portant of these are: 
1. Meat extract broth made by dissolving 5 grams of Liebig 
meat extract and 10 grams of Witte's peptone and 5 grams of 
salt in 1000 c.c. of water and brought to a reaction of +1% acid- 
ity (+1% acidity means that 1% of a 1/10 normal sodium 
hydrate solution is required to neutralize 10 c.c.). The mixture 
is then cleared by boiling with a solution white of egg in 60 c.c. of 
water, filtered and run into test tubes and sterilized. 
2. Meat infusion broth made in exactly the same manner except 
that 500 grams of lean meat are extracted in the refrigerator for 
24 hours with 1000 c.c. of distilled water. The infusion is then 
strained and used as the basis of the media instead of the Liebig's 
extract. This makes a more widely useful culture media. 
Sugar Broth-In the ordinary beef extract or infusion broth 
some sugar is present and it is desirable for the study of some 
varieties of bacteria to have a broth containing only known sugars. 
The sugar may be removed from broth by growing the colon 
bacillus in it within the incubator for eighteen hours. The number 
of colon bacilli which should be added will be correct if 10 c.c. 
of a twenty-four hour fresh culture of Bacillus coli communis is 
94 LECTURE NOTES ON BACTERIOLOGY 
added to 1000 of uncleared broth. After fermentation has been 
complete, the Bacillus coli communis may be killed by sterilizing 
the broth, which may then be filtered. 
To separate portions of this sugar-free broth, there may then 
be added only one of the sugars, dextrose, lactose or saccharose. 
In this manner a pure sugar broth is made and a means ob- 
tained for testing the power of various bacteria to ferment the 
different sugars. 
Glycerine broth is made by adding 6% of c.p. glycerine. 
Calcium carbonate broth is made by 1% of calcium carbonate 
and 1% of glucose. 
Dunham's peptone solution is merely a solution of 10 grams of 
peptone and 5 grams of sodium chloride in 1000 c.c. of water. 
Nitrate Solution-A solution of- 
10 grams of peptone 
0.2 grams of potassium nitrate in 
1000 c.c. of water 
Uschinsky Protein Free Medium-A solution of- 
3.4 grams of asparagin 
10 grams ammonium lactate 
5. grams sodium chloride 
0.2 grams magnesium sulphate 
1.0 grams potassium phosphate 
Solid Media Capable of Inoculation in a Liquid State-Many 
bacteria will not grow at all in these last mixtures. It is most 
essential for the separation of bacteria to possess some media which 
can be inoculated in a liquid state and kept at either room tem- 
perature or, better, at incubator temperature in a solid state. 
The addition of gelatin or agar-agar to meat extract or in- 
fusion broth in proportions of 120 to 130 grams of the finest 
French sheet gelatin or 15 grams of agar-agar will produce such 
a solidifying medium. 
In preparation of the gelatin care must be used to avoid heating 
too long when sterilizing as it easily loses its solidifying power 
if so heated. Gelatin is easily melted by immersing the test tubes 
containing it in hot water, and will remain solid at the room 
96 LECTURE NOTES ON BACTERIOLOGY 
temperature but not in the incubator or above 2°-25°. Agar-agar, 
on the other hand, must be heated in the sterilizer and cooled 
rather accurately to 42°C. for inoculation. Below this temperature 
it quickly becomes solid. After solidifying it may be placed in 
the incubator without danger of melting. 
Litmus Media-In order to test the effect of bacteria upon the 
reaction of the media in their immediate neighborhood, enough of 
a pure 5% litmus solution may be added to develop a purple color 
when cool to agar-agar, to which 2% of lactose has been added. 
'Welch's Modification of Guernieri's Medium-A media con- 
taining 5 grams of agar, 80 grams of gelatin, 5 grams of sodium 
chloride and 10 grams of peptone to 1000 of water, is very useful 
for storing bacteria and for stab cultures and for the pneumococcus. 
Dorsett Egg Medium-Useful for the cultivation of the tubercle 
bacilli. It consists of eggs broken and shaken up under sterile 
conditions in a sterile flask, diluted with 25 c.c. of sterile water 
for every four eggs. The eggs are then strained and run into 
tubes, sterilized on a slant at 13°C. for four to five hours for 
two days, at 76°C. on third day, and on the fourth day at 100°C. 
for fifteen minutes. 
Potato Media-Merely halved cylindrical cores of potatoes neu- 
tralized by washing in running water over night, or better, by 
immersing in 1% sodium carbonate for a half hour. The half 
cores are put into test tubes and sterilized by the fractional 
method. 
Milk Media-Fresh milk heated in Arnold's sterilizer for fif- 
teen minutes and set in ice chest twelve hours to allow cream to 
rise. The cream is then separated and the milk tubed either with 
or without the addition of litmus, and sterilized. 
Serum Media-Blood is collected at the slaughter house, al- 
lowed to coagulate; set in the ice chest for twenty-four to thirty- 
six hours to permit separation of serum, which is then pipetted 
off. 
Then, to three parts of clear serum there is added one part of 
a 1% glucose beef infusion or real infusion bouillon. The media 
is then tubed and coagulated and sterilized in the inspissator at 
70°-75°C. for two hours on six successive days. After preliminary 
coagulation it may be sterilized in Arnold's sterilizer at 100°C. for 
two days. 
98 LECTURE NOTES ON BACTERIOLOGY 
Serum Water Media-For the detection of fermentation clear 
beef serum is diluted with two to three times its bulk of distilled 
water sterilized in the Arnold's sterilizer for fifteen minutes at 
100° and colored with litmus 1% of a 5% aqueous solution. To 
various fractions the desired sugar is added (when indulin is 
added it must be previously dissolved and sterilized in autoclave, 
fifteen minutes at fifteen pounds, to destroy its spores). The water 
serum may then be tubed and sterilized at 100° by fractional 
method. 
Special culture media-A number of special media have been 
devised for the isolation of the typhoid bacilli. Among these may 
be mentioned Hiss' floating media: 
Agar 15 grams 
Gelatin 15 grams 
Liebig's meat extract 5 grams 
Sodium chloride 5 grams 
Dextrose 10 grams 
Distilled water 1000 c.c. 
The typhoid bacillus grows out in characteristic long filaments, 
while the colon calories remain compact. 
Piorkowski Prine Gelatin-Normal urine of sp.gr. of about 1020 
is collected for several days, allowed to become alkaline and mixed 
with 5% pepton and 3.3 of gelatin. The mixture is heated, filtered, 
and tubed and sterilized by fractional methods. The typhoid bacilli 
grow out in long filaments while the colon bacilli form compact 
colonies. 
Conradi Drigolski Media-Three pounds of meat are infused 
in two liters of water for twelve hours, strained and boiled. 20 
grams of pepton, 20 grams of nutrose, 10 grams of sodium chloride, 
all boiled and filtered, and 69 grams of agar added. To this 
mixture separately prepared solutions of litmus and crystal violet 
are added. On this media typhoid colonies are small, blue and 
transparent; colon colonies are large, red and opaque. 
Bile Media: 
Ox bile 900 c.c. 
Glycerine 100 c.c. 
Pepton 20 grams 
100 LECTURE NOTES ON BACTERIOLOGY 
Jackson Lactose Bile Media: 
Sterilized undiluted ox bile or 11% solution of dry, fresh ox bile.. . . 100 c.c. 
Pepton 1 gram 
Lactose 1 gram 
It is filled into fermentation tubes holding 40 c.c. and sterilized 
by the fractional method. It is useful for isolating typhoid and 
colon bacilli in water and for enriching other media for the pur- 
pose of isolating these organisms from feces. 
102 THE CULTURAL CHARACTERS OF SOME OF THE MORE 
IMPORTANT PATHOGENIC BACTERIA, WHICH 
ARE OF SURGICAL INTEREST 
A description of the cultural characters of a few of the more 
important bacteria, those chiefly of surgical interest, will serve 
to illustrate the uses of culture media in bacteriology. 
The Staphylococcus-No organisms are of greater surgical in- 
terest than the pyogenic cocci. These are more especially the 
Staphylococcus pyogenes aureus and the Streptococcus pyogenes. 
The Staphylococcus is a spherical organism having an average 
diameter of 0.8 micra., but varying between 0.4 and 1.2 micra. 
They appear singly or in pairs or more frequently grouped in 
grape-like clusters (see Fig. 2). They stain readily with all the 
usual basic aqueous anilin dyes and are Gram positive. 
1. They grow equally well on meat extract or infusion broth 
upon which 
(a) They produce uniform clouding. 
(b) Form a surface pedicle and sediment. 
(c) Impart an acrid odor. 
2. Optimum temperature of growth is 30°C. 
3. They are facultative anaerobic and hence grow well in stab 
culture. 
4. A slight alkaline reaction is most favorable to their growth. 
5. On gelatin they grow readily. 
(a) In 36-48 hours pinhead, shining, gray raised colonies 
which later become yellow. 
(b) The gelatin becomes liquefied by the ferment gelatinage 
produced by the staphylococcus. 
6. On agar the growth is much like that in gelatin except for 
the liquefaction. 
7. In milk they produce coagulation and lactic and butyric 
acid. 
8. On potato they grow well, forming a dry looking and yellow 
growth. 
104 LECTURE NOTES ON BACTERIOLOGY 
9. On coagulated animal serum, the growth is abundant and 
eventually liquefaction takes place. 
10. The nitrates in nitrate solutions are reduced to nitrites. 
11. In Dunham's broth indol is formed. 
12. In sugar media acid fermentation without the production 
of gas takes place. 
13. In litmus solutions the color is lost as a result of re- 
duction. 
The Streptococcus Pyogenes-The streptococcus is responsible 
for the more diffused, rapidly spreading forms of suppurating in- 
flammation in contrast to the tendency toward localized abscess 
formation of the staphylococcus. 
The streptococci are also spherical organisms measuring .5 to 1 
micra. in diameter. They show a strong tendency to occur in 
chains. They easily take the usual basic anilin dyes (see Fig. 1) 
and are Gram positive. Their cultural characteristics present cer- 
tain contrasts to those of the staphylococcus. 
1. They grow better on meat or veal infusion broth than on 
extract broth; diffuse clouding is rare. They manifest a strong 
tendency to sink and lactic acid rapidly forms. 
2. Delicate strains may even require the addition of some 
animal albumin like blood serum or pleuritic fluid. 
3. A moderate alkalinity is favorable to the growth. 
4. The optimum temperature is 37° C. 
5. They grow better in free access of oxygen, but complete 
exclusion of oxygen will not prevent growth. 
6. In milk streptococcus grows readily and forms acid and a 
coagulum. 
7. Gelatin shows small, grayish, delicately opalescent, raised 
round and finely granular colonies, with slightly corrugated edges. 
In stab cultures the growth takes place slowly as a very thin white 
line. The gelatin is not liquefied. 
8. On agar, the cultures have much the same characters. 
9. On broth, gelatin, or agar or in broth which has been en- 
riched with animal serum, the growth is far more luxuriant, and 
very good on Loffler's serum. 
10. No growth takes place in potato. 
11. On media containing red cells some discoloration of the 
media takes place from hemolysis. 
106 LECTURE NOTES ON BACTERIOLOGY 
12. In the indulin serum media of Hiss streptococci do not 
produce acid or coagulation. 
Many varieties of streptococci occur. The Streptococcus 
brevis frequently found in the mouth occurs in short chains and 
is probably largely, if not entirely, saprophytic. It is more apt to 
cause diffuse clouding in broth and to stain somewhat irregularly 
by Gram's method. 
The Pneumococcus-The pneumococcus is capable of causing 
a variety of inflammations in the body, some of which are accom- 
panied by severe and acute symptoms attended by general bac- 
teraemia as any general bacterial infection. It is an organism fre- 
quently found in the mouth. Just as streptococcus is more 
difficult to cultivate artificially than the staphylococcus, so the 
pneumococcus is more difficult to cultivate than the streptococcus. 
Morphologically, the pneumococcus is a lancet-shaped coccus oc- 
curring in pairs and surrounded by a wide capsule. 
It may occur rarely singly or even in short chains. It takes 
the usual basic anilin stains. It is Gram positive. 
1. On meat extract broth it does not grow. 
2. A sparse growth may be obtained on veal broth. 
(a) A slight alkalinity is favorable to its growth. 
(b) It slightly clouds the media. 
(c) The cloudiness disappears as organism sinks to 
bottom. 
3. No growth as a rule takes place on gelatin or in stab 
cultures. 
4. On agar plates the colonies resemble the streptococcus. 
5. The addition of animal fluids greatly increases the amount 
of growth. 
6. The growth in milk is rapid; profuse results in the produc- 
tion of acid and coagulation. 
7. In potato a very thin, moist, hardly visible growth ap- 
pears. 
8. On Lbffler's coagulated blood serum, the growth is moist 
and discreet and in colonies. After some days the colonies tend 
to dry out and disappear and in this particular differs in rather 
a characteristic way from streptococci, which remain for a longer 
time and are also more opaquely white. 
9. Upon media made of whole rabbits' blood and agar, 
bottom. 
108 LECTURE NOTES ON BACTERIOLOGY 
the pneumococcus grows with great luxuriance, after 4-5 days 
forming thick black surface colonies. They produce no hemo- 
lysis on media containing blood and thus differ from strepto- 
cocci. 
The Gonococcus-The gonococcus is also a diplococcus but the 
two organisms forming one pair are flattened upon the surfaces 
facing each other. This gives them a coffee bean appearance 
which is quite characteristic. Their size is 1.6 micra. x 0.8 micra. 
(Fig. 31). It is not difficult to find them in the pus of fresh cases 
of gonorrhea, in which they are readily stained and are found 
both extracellularly and crowded within the cycloplasm of the 
leucocytes. They differ from the 
previous cocci studied in being 
Gram negative. In chronic gon- 
orrheal urethritis, vaginitis, or 
conjunctivitis it is often very 
difficult to find them and they 
may be confused with other 
Gram negative cocci, also found 
on these membranes. 
The gonococcus is even more 
difficult to cultivate than the 
pneumococcus. They may be 
grown upon a media consisting 
of two or three parts of meat in- 
fusion agar mixed with one part 
of uncoagulated human ascitic 
fluid, hydrocele fluid, or blood serum. The agar must be cooled to 
45 before the serum is added. The medium is still further improved 
by adding 6% of glycerin or 1% glucose. Whole rabbits' serum 
may be used in place of human serum. Plate streak cultures may 
also be made by streaking a drop of blood drawn from the finger 
over the surface of the glycerin or glucose infusion agar. The most 
favorable reaction is neutrality or slight acidity. 
It grows sparsely under anaerobic conditions. The optimum 
temperature for the growth is 37.5° C. and no growth takes place 
below 30° C. or above 38.5°. The colonies appear as very delicate 
gray opalescent spots at the end of twenty-four hours. There is 
no tendency toward confluence. Their margins are slightly un- 
Fig. 35.-Gonococcus. Cover glass 
preparation stained with methylene 
blue. Magnified 1000 diam. 
110 LECTURE NOTES ON BACTERIOLOGY 
dulated. Their consistency is sticky. There is no clouding of 
fluid media. 
The Bacillus Tetani-This is a slender bacillus 2 to 5 micra. 
in length by 0.3 to 0.8 in breadth. The vegetative forms of some 
young cultures have slight mobility by means of numerous peri- 
trichal flagella. It readily develops spores which appear at one end 
of the bacillus and give it a drum stick appearance from the 
enlargement of the body of the bacillus around the spore. The 
bacillus stains easily with anilin dyes and is Gram positive. 
The bacillus only grows in 
the absence of oxygen, with ha- 
bituation to gradually increas- 
ing quantities of oxygen. Such 
habituated organisms, however, 
suffer a diminution of their tox- 
in forming power and their 
pathogenicity. -Under anaerobic 
conditions the Bacillus tetani: 
1. Grows well in meat infu- 
sion broth. 
2. It clouds the broth quick- 
ly. 
3. It grows well on meat in- 
fusion gelatin at 20°-22° C. 
4. It fluidifies the gelatin 
slowly. 
5. On agar it grows well at 33.5° C. The colonies show a com- 
pact center surrounded by a loose network of wavy, radiating 
filaments. 
6. It grows well on milk and does not coagulate it. 
7. It forms a very delicate growth on potato. 
The most favorable temperature for growth is 37.5° C. The 
most favorable reaction is slight alkalinity or neutrality. 
The addition to all media of %% of glucose, maltose or sodium 
formate greatly favors growth. In carbohydrate media carbondi- 
oxide gas is formed. It is mixed with H2S, which gives the cul- 
tures a characteristic unpleasant odor. Live steam will kill the 
spores of tetanus in five minutes time and 5% carbolic in fifteen 
hours. They will remain virulent for many years. 
Fig. 36.-Tetanus bacilli, with spores. 
Cover glass preparation from agar 
culture stained with Fuchsin. Mag- 
nified 1000 diam. 
112 LECTURE NOTES ON BACTERIOLOGY 
Tetanus is the cause of the disease called lockjaw. In infected 
human beings, mules and horses it is very fatal; but infection 
by no means always follows the introduction of the bacilli. This 
is borne out by the comparatively few cases of tetanus occurring 
and the very wide distribution of the organisms in the soil. 
The Bacillus of Anthrax-This is a large bacillus 4 to 6 micra. 
long and .5 to .6 micra. broad. It is usually seen simply and 
though the daughter bacilli adhere together to a certain degree, 
they never form long chains. Their ends are rounded. Gunther 
describes it as a non-motile bacillus. In artificial media it forms 
spores which are oval and broader than the bacillus so that the 
body of the bacillus is slightly smaller at the site of the spore. The 
spores are usually near but never as in tetanus at the end of the 
bacillus. It stains readily with the anilin dyes, but is Gram neg- 
ative. (See Figs. 4 and 5.) 
1. The bacillus is strictly aerobic. 
2. It grows well on all ordinary laboratory media. 
3. The addition of glucose, glycerin or neutrose favors growth. 
4. In all media it forms gas. 
5. It grows equally well in slightly acid or alkaline media. 
6. On gelatin plates the cultures are round or oval with com- 
pact centers and fine radiating filaments around the periphery. 
7. It liquefies the gelatin. 
8. On agar the colonies are circular with a granular periphery 
composed of a tangle of threads. 
9. In agar and gelatin stabs the growth appears in a short 
time as a diffuse cloud. Gas forms near the bottom. 
Anthrax is pathogenic to cattle, sheep and goats and very fatal 
to infected guinea pigs. Horses are not very susceptible. Dogs, 
cats, rabbits and birds are immune. Man is relatively immune, 
though many cases of death have been recorded in men who have 
been infected. 
Diphtheria-The diphtheria bacilli possess rather striking 
morphological characters. They are slender, straight or slightly 
curved, varying in length from 1.2 to 6.4 micra. and in breadth 
from .3 to 1.1 micra. They are rarely of uniform thickness but 
show club shaped thickenings at one or both ends. In consequence 
of thickening at one end they are often wedge shaped. 
The bacillus stains with the aqueous anilin dyes. It stains in 
114 LECTURE NOTES ON BACTERIOLOGY 
rather a characteristic manner with Loffler's methylene blue. 
Stained with this stain for 5 to 10 minutes, the bacilli often appear 
beaded, due to transverse unstained portions. Deeply staining oval 
bodies may appear at the ends. These are the so-called polar or 
Babes-Ernst bodies. 
Diphtheria bacilli are Gram positive. "When stained directly 
from the throat the bacilli often lie in clusters of four or five 
roughly parallel to each other or tending to converge by their small 
ends. The diphtheria bacillus is non-motile, possesses no flagella 
and does not form spores. It 
shows decided preference for 
oxygen but may grow in the ab- 
sence of oxygen if suitable car- 
bohydrates are present. 
1. The diphtheria bacillus 
grows readily in all the richer 
laboratory media. 
2. It grows on meat extract 
broth but better on uniform 
broth. 
3. The most favorable reac- 
tion is slight alkalinity about 
.5% in terms of 1/10 normal 
OH. 
4. Animal proteins greatly 
favor its growth. 
5. Lbffler beef blood media is more favorable to its growth. 
Its colonies appear as minute gray, glistening spots in twelve to 
twenty-four hours. 
6. In meat infusion broth diphtheria grows rapidly, forming 
a pedicle on the surface. The broth remains clear. 
7. On gelatin the growth is slow because of the low temperature 
at which this media is kept. 
8. On agar diphtheria grows well but far less so than on beef 
serum. The agar colonies are quite characteristic. They are 
round, or oval, with a dark, heaped up center surrounded with 
a loose, coarsely granular disk. The edges are particularly irregu- 
lar, almost as if torn. 
9. In milk it grows very readily but does not coagulate milk, 
Fig. 37.-Diphtheria bacilli. Cover 
glass preparation from an agar cul- 
ture stained with Fuchsin. Mag- 
nified 1000 diam. 
116 LECTURE NOTES ON BACTERIOLOGY 
10. On potato it grows very poorly. 
11. It forms no indol in pepton solutions. 
Diphtheria causes a superficial fibro purulent inflammation of, 
most frequently, the throat, nose and larynx. It is often fatal. 
Man and the horse are very susceptible. Rats and mice are re- 
sistant. Dogs, cats, fowls, rabbits and guinea pigs are sus- 
ceptible. 
The Bacillus Mallei-The Bacillus mallei is the cause of 
glanders. This is a disease characterized by catarrhal ulcerative 
inflammation of the mu- 
cous membrane of the res- 
piratory tract. It is char- 
acterized by a formation 
of multiple abscesses and 
adenitis. Horses are most 
susceptible to it. It is 
very fatal. Man may be 
infected from horses and 
rarely recovers. The ba- 
cilli may be obtained 
from the enlarged glands 
in pure culture. 
Asses, colts, guinea 
pigs, and more rarely 
dogs, are susceptible. 
Cattle, hogs, rats and 
birds are immune. 
The glanders bacillus 
or Bacillus mallei is a small bacillus, 3-4 micra. long and 0.5 to 7.5 
micra. in breadth. 
There are characteristic variations in the size of the bacilli. 
It stains readily with the usual anilin dyes, but shows char- 
acteristic irregularity in staining qualities, deeply staining por- 
tions alternating with unstained portions. It is negative to Gram's 
stain. It has no flagella and forms no spores. 
1. It grows well on all meat infusion media. 
2. It is fairly independent to the reaction of the media. Glyc- 
erin and glucose favor its growth. 
3. It is aerobic. 
Fig. 38.-Bacillus mallei. Pure culture from 
glucose-agar. Carbol-fuchsin. Magnified 
1200 diam. (Jordan.) 
118 LECTURE NOTES ON BACTERIOLOGY 
4. The most favorable temperature is 37.5° and it fails to grow 
at temperatures below 22° C. 
5. In broth there is at first a diffuse clouding and later a 
heavy shiny tough sediment and a similar scum. The broth 
darkens. 
6. On agar yellowish-white colonies appear in 23 hours at 
37.5° C. They possess a round even border and are finely granu- 
lar. The older the culture, the more yellow the color which is 
developed. 
7. On gelatin, colonies are greyish-white, never very abundant 
and no liquefaction occurs. 
8. In milk, acidification and 
coagulation slowly take place. 
9. On potatoes, if not too 
acid, the growth is abundant in 
48 hours, and the surface is cov- 
ered with a yellowish transpar- 
ent slimy layer, which gradually 
darkens to a deep reddish- 
brown. 
The Bacillus of Tuberculo- 
sis-The tubercular bacillus is 
the cause of a rarely acute, more 
often sub-acute or chronic in- 
flammation which may attack 
any tissue in the body. The ten- 
dency to chronicity depends 
upon the resistance of the body 
to the growth of this organism 
within it and results in a tendency toward localization of the in- 
flammatory process excited and only a very gradual extension or 
reappearance of the process in other parts of the body. 
Tuberculosis is the most frequent disease affecting human 
beings. It stands second to pneumonia only as a cause of death. 
Naegeli in 1900 showed that it is very rare before one year of 
age. It is rare between one and five years, but very fatal at this 
time of life. 
From the first to the fourteenth year tubercular lesions are 
present in one-third of the bodies subjected to autopsies; from 
Fig. 39.-A tuberculous tubercle con- 
taining giant cells from the section of 
the lymph gland. The tuberculi are 
stained with Fuchsin and other 
bacilli with methylene blue. Mag- 
nified 500 diam. 
120 LECTURE NOTES ON BACTERIOLOGY 
the fourteenth to the eighteenth year, one-half showed tubercular 
lesions; from the eighteenth to the thirtieth, almost all cases showed 
some tuberculous infection. 75% of these were healed, and 25% 
active. Moreover, two-fifths of all deaths occurring at this time 
were due to tuberculosis. After the age of thirty-five active 
lesions decreased in number and healed lesions increased. 
The tubercle bacillus is a slender rod 2 to 4 micra. long and 
0.2 to 0.5 micra. broad. The ends of the rods are rounded; the 
rods may be straight or slightly curved. Frequently they will 
appear beaded or irregularly stained. 
It is claimed that the bacilli are surrounded by a membrane 
which gives a cellulose reaction and contains waxy substances and 
is very resistant to stains and to drying. Bronchial forms have 
been demonstrated by some observers and, therefore, the tubercle 
bacillus approaches the higher orders of bacteria. 
Tubercle bacilli may be isolated from the sputum only with 
considerable difficulty. The sputum should be washed free from 
other organisms in eight to ten changes of sterile water, and the 
material selected from the center of caseous masses for cultiva- 
tion. It is best obtained from the center of caseous lymph nodes. 
On blood serum at 37.5° C. the colonies become visible usually 
at the end of eight to fourteen days. They appear then as small, 
dry, grayish-white scaly spots with corrugated surfaces. After 
three to four weeks' cultivation these join, covering the media 
with a dry whitish wrinkled membrane. 
Coagulated dog-serum is one of the most favorable media. 
Slants of agar, containing % c.c. of whole rabbits' blood, are very 
useful. 
Tuberculosis grows well on glycerin agar at 37.5° C. Colonies 
becoming visible at 10 days to two weeks as the same dry whitish 
corrugated membranes. 
Glycerin veal infusion broth is an excellent medium. Bacilli 
will spread out as a thin, opaque, floating membrane which thickens 
and becomes wrinkled. The cultures emit an aromatic odor. 
Tubercle bacilli will also grow on glycerin potato. 
The Two Pathogenic Varieties of Tubercle Bacilli-From the 
standpoint of infection in man, two varieties of the tubercle bacilli 
are of special importance. One of these is the bovine type and 
the other is known as the human type. Repeated attempts have 
122 LECTURE NOTES ON BACTERIOLOGY 
been made to infect cattle with the human type but without suc- 
cess. On the other hand, the bovine type has been demonstrated 
to be the cause of many infections in the human being. The 
majority of these infections are, however, in individuals below 
sixteen years of age. Below five years the danger is greatest. 
Morphologically the bovine bacilli are shorter than the human 
type and grow less luxuriantly upon artificial media. 
An important cultural difference 
is the fact that the bovine type when 
grown upon slightly acid glycerin 
bouillon reduces the acidity until the 
reaction reaches neutrality or even a 
slight alkalinity, whereas the hu- 
man type scarcely alters the acidity 
of the culture media. Guinea pigs 
and rabbits die much more rapidly, 
two to five weeks when inoculated 
with the bovine type than when in- 
oculated with the human type, which 
usually causes a disease lasting for 
six months. 
The recognition, however, of the 
two types and the fact that children 
can become infected with bovine tu- 
berculosis is of great practical im- 
portance as it makes it mandatory 
to exclude the use of tuberculous 
cows for the supply of milk. 
The great frequency of tubercu- 
losis and the recognition at the same 
time that this curse could be abso- 
lutely eliminated if proper care was 
exercised in the nursing of the tuberculous sick, makes the exercise 
of such care of supreme importance to the human race. Every 
tuberculous animal should be sought out and cremated; all indi- 
viduals dying of tuberculosis should be disposed of in the same way. 
Tuberculous sick who will not or cannot properly care for 
themselves should be sequestrated in properly arranged colonies 
set apart for this disease, where all contaminated excretions, par- 
Fig. 40.-Film preparation from a 
genital syphilitic papule; in the 
center are two specimens of 
Spirochaeta pallida, the other 
three are specimens of Spiro- 
chaeta refringens. (MacNeal.) 
124 LECTURE NOTES ON BACTERIOLOGY 
ticularly the sputum in pulmonary cases, can be properly steri- 
lized or burned. 
Though such a regime against the disease seems extravagant, 
yet if successfully carried out, it would stamp out in a compara- 
tively short time a disease which disables a third of the human race 
and kills more individuals, with the exception of pneumonia, than 
any other disease, and before the present amount of care was ex- 
ercised, which medical agitation has now stimulated, tuberculosis 
stood first in the death rate. 
Fig. 41.-Spirochaeta pallida stained by Levaditi method. The section shows an 
infarcted lymph vessel at the junction of two branches. The lumen is filled 
with leukocytes. The spirochetes follow the lymph vessel for the most part, 
but are also penetrating into the surrounding tissue. (MacNeal.) 
Syphilis-General Description-In 1905, the Spirochaete pal- 
lida, otherwise called Treponema pallidum, was discovered and 
demonstrated to be the cause of syphilis. 
This is an extremely delicate organism measuring from 4 to 
10 micra. in length and varying up to 0.5 micra. in thickness. Its 
126 LECTURE NOTES ON BACTERIOLOGY 
body is spirally twisted like a corkscrew, with rather sharply bent 
spirals varying in number from 2 to 12. The ends of the organism 
taper delicately. There is a delicate flagella at each end and on 
each margin a delicate undulating membrane. The organism is 
very motile, rotating in its movements on its long axis and occa- 
sionally bending its whole body It moves by gliding movements 
backward and forward. It may be seen readily with the use 
of the dark field illuminating condenser. 
The Spirochsete pallida stains with great difficulty with any 
of the ordinary anilin dyes. 
Special nuclear stains, as the Giemsa stain, must be used and 
the organism stained for a very long time. 
The organism will not grow upon artificial culture media. 
Pathogenic Features-No more important subject comes up 
for discussion in hygiene than the prophylaxis of syphilis. No 
disease is accompanied with more disastrous consequences nor so 
surely terminates life by a variety of chronic productive inflam- 
mations. Any organ in the body may be attacked, including not 
infrequently the heart, the central nervous system and the blood 
vessels. Its prophylaxis, however, must be based entirely upon 
moral issues, but, in the interest of the hygiene of the community 
in general, the dangers of sexual transgressions should be made the 
subject of a broad educational campaign, beginning with the chil- 
dren in school. 
The Factors Favoring the Entrance of Bacteria into the Tis- 
sues of the Body-The possibility of bacteria to live and multiply 
depends almost solely upon their being surrounded with a sufficient 
food supply and moisture and a suitable temperature. These con- 
ditions are so universally present that bacteria may be said almost 
to be ubiquitous; certainly they are in all parts of the earth 
usually inhabited by man and animals. 
Man himself may be said to constitute a living culture tube 
for bacteria. Few portions of his economy, which come into con- 
tact with the things of the outside world, are free from bacteria, 
many of which are pathogenic. The more intimate the contact of 
a portion of the body with the outside world, the greater the num- 
ber and variety of bacteria found in such a portion. 
SUSCEPTIBILITY AND RESISTANCE TO DISEASE 
128 LECTURE NOTES ON BACTERIOLOGY 
On the Part of the Skin-The skin always contains bacteria 
within its crevices and within the openings of the pores. The 
Staphylococcus epidermis albus is a constant inhabitant of the 
skin. It is not infrequently the cause of stitch abscess in surgical 
work. 
On the Part of the Nails-No portion of the surface of the body 
comes into more intimate contact with the external world than the 
finger nails. Moreover, the cleft between the nail and the end 
of the finger favors the accumulation of many varieties of bacteria. 
These clefts are all the more apt to lodge dangerous bacteria be- 
cause the hands, particularly the dentist's and surgeon's hands, are 
so prone to handle regions already infected with dangerous bac- 
teria. 
Bacteria in the Ear-Special varieties of bacteria inhabit cer- 
tain locations. In the wax of the ear there is normally found a 
bacterium named Micrococcus cereus flavus. 
Bacteria in the Stomach-In the stomach, under normal con- 
ditions, few bacteria are to be found. The acidity of the stomach 
contents kills many of the bacteria passing through it. It forms a 
very efficient filter to the intestinal tract. Within the intestines 
the Bacillus lactus erythrogenes and the Bacillus coli communis are 
present in such overwhelming proportions that they practically 
represent the sole species inhabiting the intestines. 
Bacteria of the Intestines-There are also found in the intes- 
tines the Streptococcus coli gracilis and the Bacillus enteritiditis 
sporogenes. The mucus of the large intestines is inimical to most 
bacteria. Moreover, the exhaustion of nourishment and collection 
of bacterial products also results in the death of many of the 
bacteria within the intestines, so that although 38 to 46% of the 
total nitrogen of the feces is in that form of bacteria, the majority 
of them are dead. 
Bacteria of the Mouth-The conditions within the mouth, how- 
ever, are very different. Standing as it does next to the outside 
world and, with the nose, constituting the only gateway through 
which all that enters the body, including both food and air, must 
pass, it receives a great variety of bacteria. These multiply within 
the mouth in proportion to the presence of resting places within 
the mouth in which they may lodge and find food. Any disease 
within the mouth will furnish retention places for food or the 
130 LECTURE NOTES ON BACTERIOLOGY 
oral discharges, and thus become so many incubated culture tubes 
within the mouth. Any irregularity in the shape of cavities in 
the teeth or concretions forming around the base of the teeth will 
also form still more dangerous resting places for bacteria. 
From even relatively clean mouths there may be isolated some 
100 species of bacteria, many of which are well known pathogenic 
bacteria. There are constantly to be found within the mouth the 
Leptothrix innominata 
Bacillus buccalis maximus 
Leptothrix buccalis maxima 
Idiococcus vaginatus 
Spirillum sputigenum 
Spirochmte dentium. 
The microorganisms associated especially with dental caries 
have been divided by Goodby into 
A. Those which produce acid, which include: 
1. Streptococcus brevis 
2. Bacillus necrodentalis 
3. Sarcina alba 
4. Sarcina lutea 
5. Sarcina aurantia 
6. Staphylococcus pyogenes aureus 
7. Staphylococcus pyogenes salvarius 
B. Those which liquefy gelatin and produce pigment 
1. Bacillus mesentericus rubus 
2. Bacillus mesentericus vulgatus 
3. Bacillus mesentericus fuscus 
4. Bacillus fuscus 
5. Bacillus gingivalis pyogenes 
6. Bacillus liquefacium motilis. 
Of these organisms the Streptococcus brevis and the Bacillus 
necrodentalis are said to be particularly associated with dental 
caries. 
Recently there has been described as constantly present in the 
suppurating margins of the gum of patients with Pyorrhea alveo- 
laris an ameba which has been termed Endamoeba buccalis (Fig. 42). 
While its exact relationship to the etiology of pyorrhea is a matter 
of doubt, it is certain that the killing of this ameba by doses of 
132 LECTURE NOTES ON BACTERIOLOGY 
Fig. 42.-Endamoeba buccalis. 
Fig. 43.-Diagonal section of decayed dentine. Infection with micrococci, bacilli 
and screw forms. (Miller.) LECTURE NOTES ON BACTERIOLOGY 
Fig. 44.-Longitudinal section of decayed dentine, showing infection with micro- 
cocci, bacilli and threads (Mixed infection). (Miller.) 
Fig. 45.-Longitudinal ^section of decayed dentine, showing infection with 
micrococci. (Miller.) Fig. 46.-Longitudinal section of dentine artificially decayed. Infection with 
bacilli. (Miller.) 
Fig. 47.-Decayed dentine, showing that laterally the boundary between the 
infected and non-infected parts may be very regular. (Miller.) LECTURE NOTES ON BACTERIOLOGY 
emetin % grain, hypodermically for three days, is of material assist- 
ance in the cure of pyorrhea. 
FACTORS CONTROLLING THE DECAY OF THE TEETH 
Although many attempts have been made to isolate and identify 
the bacteria which are responsible for tooth decay and although 
Fig. 48.-Portion of a longitudinal ground section through the crown of a molar, 
showing two cavities of decay, with the transparent zones at c. (Miller.) 
lists of such specific organisms represented by the one given above 
from Goodby have been prepared, yet up to the present time no 
certainty exists that tooth decay is always associated with any one 
of even a few specific bacteria. 
As Miller has long ago pointed out the essential factor in tooth 
decay is a solution of the inorganic salts composing the teeth and 
140 LECTURE NOTES ON BACTERIOLOGY 
the entrance through the defects thus created of bacteria between 
the fibers of the teeth. The primary defects are due to the forma- 
tion of acids from particularly the carbohydrate constituents of 
the food. The process is continued by bacteria gaining entrance 
to these defects and to the action of these acid producers is added 
the peptonizing action of other bacteria upon the albuminous con- 
stituents of the canaliculi of the teeth, the intercellular substance 
separating the enamel prisms and the dentine tubules and Sharp- 
ley's fibres. 
Preparations of teeth which are undergoing decay show lines 
of bacteria invading the intercellular intervals between the pris- 
matic fibres of the teeth, the canaliculi of the eementum and the 
dentinal tubules of the dentine. 
The process of decay may then be caused by any bacterium 
capable of producing acid fermentation or peptonization of the 
albuminous intercellular material of the teeth. In how far these 
processes are limited to specific, bacteria has not yet been settled but 
the whole process must be regarded rather as saprophytic than as 
infectious and occurs in dead as well as live teeth. Quite different 
from the process of decay are the various infections occurring in 
the mouth. These may be due to any one of the various pathogenic 
bacteria capable of giving rise to a local or general infection of the 
tissues. 
These facts have been developed more clearly by Miller and his 
deductions from them have been expressed so clearly that there is 
little excuse for not repeating them in his own words. 
PREDISPOSING CAUSES OF DENTAL CARIES 
Iii contradistinction to the exciting causes of caries, we charac- 
terize as predisposing such conditions of the individual or of the 
teeth which divest them of their normal power of resisting excit- 
ing causes, or by which they offer to the exciting causes especial 
points of attack. 
Predisposing conditions are only found in the teeth themselves, 
in their development, position, etc., while, on the other hand, all 
external agencies are to be considered as exciting causes. It is 
therefore not logical to regard gout, e.g., as a predisposing cause, 
because it is accompanied by an acid reaction of the saliva. The 
142 LECTURE NOTES ON BACTERIOLOGY 
action of an acid on the teeth will always be the same, whether it 
is secreted by the mucus or salivary glands, or formed in the mouth 
by fermentation, or introduced from without; it is invariably an 
exciting, not a predisposing cause. 
1. The structure of the teeth plays the most important part as 
a predisposing cause of dental caries. Poorly developed, soft, por- 
ous teeth, with many large interglobular spaces, are highly predis- 
posed to caries. As a lump of table-salt dissolves more rapidly in 
water, on account of its porosity, than an equally large piece of 
rock-salt, porous dentine is more rapidly decalcified than well-devel- 
oped, firm dentine, because the acid may the more readily penetrate 
the tissue, and because less acid is required to decalcify a porous 
than a hard tooth. It may easily be proved by experiment that 
poorly developed dentine is much more rapidly attacked by acids 
than sound dentine. Not only does the decalcification, but also the 
destruction of the cartilage, advance more rapidly in the former 
case, because the microorganisms, in many cases at least, enter the 
interglobular spaces and more readily pervade and destroy the 
entire softened tissue. 
2. As a second predisposing factor I designate abnormally deep 
fissures or blind holes (foramina caeca) in molars and superior lat- 
eral incisors, especially in cases where the enamel also is poorly 
developed. By their continual retention of food-particles, such 
points directly induce caries, and offer but little resistance to it in 
consequence of the absence of an intact protecting cover of enamel. 
3. In the third place, fissures or cracks in the enamel are re- 
garded as a predisposing cause. I have not, however, been able to 
convince myself that decay frequently starts from these enamel- 
cracks, often found in senile teeth. They are usually too narrow 
to permit the entrance of food-particles, and consequently do not 
serve as points of retention. 
4. In the fourth place, teeth are predisposed to decay by a 
crowded, irregular position. An instructive example is furnished 
in cases where the first bicuspid stands inside of the arch, so as to 
form a triangle with the second bicuspid and the canine; or the 
second bicuspid forms a triangle with the first bicuspid and first 
molar. It is impossible to keep the space between these three teeth 
clean, and fermentation and acid formation continually occur there 
and attack the teeth. Not only in such cases, but wherever a 
144 LECTURE NOTES ON BACTERIOLOGY 
crowded position of the teeth favors the retention of food-partieles 
or renders their removal difficult, a predisposition to caries prevails. 
The form of a tooth is not without influence; teeth with convex 
approximal surfaces touching each other at one point only are, 
caeteris paribus, less subject to caries than teeth with flat or slightly 
concave surfaces, because the latter cannot be kept so clean, either 
spontaneously (by the tongue, etc.) or with the brush. 
5. A recession or loosening of the gums from the neck of the 
tooth not only lays bare the dentine, but also permits the entrance 
of food-particles between the necks of the teeth or into the pockets 
formed by the loosening of the gums, by which means a further 
predisposing cause for caries is furnished. 
6. Many consider pregnancy as a predisposing cause. It is not 
to be denied that during pregnancy women are particularly subject 
to caries. The reason for this is, however, probably to be sought in 
the fact that the patients generally neglect the care of the mouth 
during that time, and that the buccal secretions assume an acid 
reaction; these are both exciting causes of decay. Pregnancy itself 
can only be regarded as a predisposing cause in so far as it effects 
a loosening of the gums or a change in the structure of the teeth 
by a withdrawal of the lime-salts to build up the foetal skeleton. 
Whether such an extraction takes place has not been definitely 
ascertained. 
7. Many believe that a predisposition to caries may be in- 
herited. It cannot be denied that badly developed, irregular teeth 
may be and are inherited, and in so far inheritance may be con- 
sidered as a predisposing cause of caries. 
8. Wedl, Tomes, and others mention as predisposing causes 
various general diseases, as rheumatism, gout, diabetes, gastro- 
enteritis, dyspepsia, cancer of the stomach, scrofula, rachitis, and 
tuberculosis. These diseases may indeed furnish the exciting juices, 
but how they can predispose the teeth themselves to caries is not 
readily apparent, unless they render them more easy of access to 
the exciting causes by concomitant gum-diseases as described un- 
der 5. 
I doubt whether climatic or geological conditions have much 
to do with the origin of caries. Esquimaux, Lapps, Icelanders, 
Arabs in Nubia, Patagonians, etc., have the best teeth in spite of 
unfavorable climatic conditions. 
146 LECTURE NOTES ON BACTERIOLOGY 
INFLUENCE OF CIVILIZATION ON DECAY 
That decay of the teeth is not a disease peculiar to civilization is 
proved by the manifold observations which have been made on the 
skulls of ancient and modern uncivilized races in Europe and 
America. A visit to any anatomical or anthropological museum 
and an examination of a large number of race-skulls will con- 
vince every one of the correctness of this assertion. Such exami- 
nations have been made by Broca, M'agitot, Mummery, Barrett, 
myself, and many others, and invariably led to the same conclusion, 
that decay has occurred in all races, civilized as well as uncivilized, 
and at all times. 
Races subsisting solely on meat (Greenlanders, etc.) come near 
forming an exception to the rule, yet they appear to be not abso- 
lutely exempt from decay. 
That the frequency of decay is greater in civilized races than 
among savages has also been established by numerous observations. 
Mummery's interesting communications are especially worthy of 
mention. He found decay among the old Britons of the dolicho- 
cephalous type in 2.94 per cent., among the brachycephalous 
Britons in 21.87 per cent., among the Romano-Britons in 28.67 per 
cent., and among the ancient Egyptians in 41.66 per cent. 
There is no doubt that a deterioration of the teeth accom- 
panies the progress of civilization. The reasons for this are many. 
The mode of life of most uncivilized races not only conditions a 
sound, well-developed body, but the osseous system, of course in- 
cluding the teeth, shows the same vigorous development, and above 
all a compact structure. An individual whose youth is spent in 
the open air, unrestricted in his bodily freedom, is likely to possess 
a body better developed in all its parts than one who has been 
brought up in a modern school-room. 
The quality of the food also exerts an influence on the teeth 
not to be underrated. They form no exception to the rule that 
an unused member will be less perfectly developed than one con- 
stantly used. 
The pressure brought to bear upon the teeth by mastication 
causes a more lively circulation in the periosteum in the pulp, 
thereby inducing an increased deposit of lime-salts or a more com- 
plete calcification. Practical experience also teaches that chil- 
148 LECTURE NOTES ON BACTERIOLOGY 
dren brought up on soft foods (broths, paps, etc.) generally have 
bad teeth. If a race of human beings or of animals were to make 
no use of their teeth for several generations, we should expect to 
find a gradual deterioration of the dental structure. It is, to say 
the least, highly probable that the soft quality of many of our 
foods, as compared to those of uncivilized races, conditions a soft, 
porous dental substance, as well as an imperfect development of 
the jaw-bone, and a concomitant crowded position of the teeth. 
Then again, the chemical composition of the food is of great 
influence upon the origin and extension of caries. 'Whoever grants 
the truth of our proposition,-no caries without acid,-and recog- 
nizes the fermentative processes in the mouth as the chief source 
of the decalcifying acids, and has verified our table, will hardly 
deny the correctness of this statement. 
A person living on such foods only as undergo no acid fer- 
mentation in the mouth (meat, raw vegetables, roots, etc.) will, I 
am convinced, be but comparatively little afflicted with caries. If 
this supposition were well founded, a comparison of the frequency 
of caries among races subsisting on meat alone with that of races 
who consume vegetable or mixed foods would yield higher figures 
for the latter. Caries should also then be more frequent in phy- 
tophagous animals than in carnivora. 
That such investigations are connected with enormous difficul- 
ties is apparent. It is extremely difficult or altogether impossible 
to eliminate other simultaneously present, especially predisposing 
causes. In the second place, the statements of certain authors con- 
cerning the food of savage tribes do not always agree, and further- 
more suitable material for these examinations is extremely scarce 
in most anatomical collections. 
Very interesting and valuable figures have been gathered by 
Mummery, which are meant to establish the relation of caries to 
the healthy or unhealthy manner of life of a given race. 
These figures, with a few changes which concern the nourish- 
ment of the races specified, and with the addition of those which 
I have deduced from various anatomical and anthropological col- 
lections, are presented in the following table: 
150 LECTURE NOTES ON BACTERIOLOGY 
Ancient Races. 
No. of 
Skulls. 
Caries. 
Percentage 
of 
Caries. 
Food. 
Ancient Britons 
68 
2 
2.94 
Meat (beef, wild boar). 
(dolichocephalous.) 
Ancient Britons 
32 
7 
21.87 " 
(brachycephalous.) 
Ancient Britons 
59 
24 
40.68 
(exploration of Greenwell.) 
Mixed food (meat, fish, 
Ancient Britons 
44 
9 
20.45 
- 
oats, wheat, beans, roots, 
(mixed.) 
etc.). 
Romano-Britons 
143 
41 
28.67 
Anglo-Saxons 
76 
12 
15.78 
Ancient Egyptians... 
36 
15 
41.66 
Modern Races. 
No. of 
Skulls. 
Caries. 
Percentage 
of 
Caries. 
Food. 
Esquimaux 
81 
2 
2.46 
Meat and fish. 
North Americans 
63 
2 
3.17 
Meat and fish, probably not 
(coasters.) 
North Americans 
22 
2 
9.09 
quite exclusively. 
Mostly meat, some vege- 
(interiors.) 
South Americans 
26 
7 
27.00 
tables. 
Principally meat. 
Feejee Islanders 
38 
2 
5.26 
Human flesh and mixed 
Polynesians 
79 
8 
10.12 
food. 
Mixed food. 
Sandwich Islanders.. 
21 
3 
14.28 
" cc 
New Zealanders 
66 
2 
3.30 
Human flesh, pork, fish, 
Australians 
132 
27 
20.45 
roots. 
Mixed food. 
Tasmanians 
33 
9 
27.27 
cc cc 
Chinese 
50 
21 
40.20 
" " mostly vege- 
East Indians (north) 
152 
9 
5.92 
table. 
Mixed food. 
East Indians (south) 
71 
10 
14.84 
cc cc 
Africans (east) 
32 
8 
25.00 
cc cc 
Kaffirs 
49 
7 
14.28 
cc cc 
Africans (west) 
236 
66 
27.96 
cc cc 
Lapps 
22 
1(?) 
4.54(?) 
Meat or fish, milk, cheese. 
The Gauchos, a cattle-breeding tribe, inhabiting the pampas of 
La Plata, and subsisting on meat, are said to be free from caries, 
while a related tribe in Chili, that subsists on bread, beans, meat, 
etc., showed 19.3 per cent, of caries. Again, those Gauchos who 
152 LECTURE NOTES ON BACTERIOLOGY 
live in cities, and who eat mixed food and much sugar, also suffer 
much from decay of the teeth. 
A hasty examination of several skulls led Black to the supposi- 
tion that those races which consume much sour fruit are less af- 
flicted with caries than those living on meat and grain. But when 
we remember how the teeth are destroyed by a grape cure, we 
can only regard the result of Black's investigation as accidental, 
particularly as it was but a "hasty" one. 
In one point, however, all examinations coincide. All authors 
call attention to the fact that the Esquimaux, certain meat-eating 
tribes of North American Indians, Icelanders, and, as far as I have 
observed, Lapps also, are almost entirely exempt from caries. 
The immunity of these races cannot, it seems to me, be ex- 
plained by the favorable hygienic mode of life, climatic influences, 
etc., alone. They often suffer from famines; various diseases are 
frequent, especially among the Lapps, and the latter, as well as 
the Esquimaux, are becoming extinct. That the number of inhab- 
itants of Iceland has remained stationary for the last few cen- 
turies is said to be due to "volcanic eruptions, frequent epidemics, 
unhealthy mode of life, famines, etc." 
I think, therefore, every one will agree with me that the con- 
ditions prevailing in the countries named are not to be regarded 
as conducive to a perfect development of the human body. 
Those factors which contribute to restrict the occurrence of 
decay of the teeth, are, in my opinion, (1) a mode of life favorable 
to the development of the whole body, (2) the use of food which 
is sufficiently hard to afford the teeth the exercise necessary for 
their vigorous development, (3) the use of food which does not 
undergo an acid fermentation in the mouth. 
PROPHYLAXIS OF DENTAL DECAY 
To every one at all acquainted with the nature of that condi- 
tion of the teeth denominated as decay, caries, etc., and with the 
causes by which it is produced, it must be apparent that there 
are four ways by which we may counteract or limit the ravages 
of this disease. We may endeavor (1) by hygienic measures to 
secure the best possible development of the teeth; (2) by repeated, 
thorough, systematic cleansing of the oral cavity and the teeth, to 
154 LECTURE NOTES ON BACTERIOLOGY 
so far reduce the amount of fermentable substances as to materially 
diminish the production of acid, as well as to rob the bacteria of 
the organic matter necessary to their rapid development; (3) by 
prohibiting or limiting the consumption of such foods or luxuries 
which readily undergo acid fermentation to remove the chief source 
of the ferment-products injurious to the teeth; (4) by the proper 
and intelligent use of antiseptics to destroy the bacteria, or at 
least to limit their number and activity. 
That a great influence is exerted upon the process of fermenta- 
tion in the human mouth by the mechanical cleansing mentioned 
under 2 may be easily proved by the following experiment: Take 
10.0 c.c. saliva from the mouth in the morning before cleansing 
it, add 0.5 gr. starch, and place the mixture in the incubator. Then 
cleanse the mouth and teeth most thoroughly with the brush, tooth- 
pick, floss silk, etc., after which take 10.0 c.c. again (easily obtained 
by chewing a quill toothpick or in the manner described), add 0.5 
gr. starch as before and place also in the incubator. The first mix- 
ture not only shows signs of fermentation sooner than the second, 
but also forms much more acid in a given time. That different 
kinds of foods and luxuries play vastly different roles in the fer- 
mentations of the human mouth must be apparent to every one 
from the table given. 
The substances which give rise to fermentation in the mouth 
accompanied by the development of acid, belong almost without 
exception to the group of the carbohydrates. The general opinion 
that putrefying meat gives rise to products which attack the teeth 
is, I repeat it, entirely unfounded and erroneous. The products 
of a putrefying mixture of saliva and meat (whether cooked or 
raw) are always alkaline, and when meat has remained for some 
time between the teeth it may even act as a preventive to decay 
in so far as it tends to neutralize the acids produced by the fer- 
mentations of carbohydrates. The latter, however, are, as a rule, 
unless the albuminous substances preponderate to a great degree, 
more than sufficient to satisfy the basic products of the albuminous 
fermentation (putrefaction), so that in case of mixed diet the 
reaction will still be acid, not so strongly, however, as in purely 
amylaceous diet. 
Most authors give sugar the chief place among those foods 
which exert an injurious action upon the teeth-again a conception 
156 LECTURE NOTES ON BACTERIOLOGY 
which is not quite right. It is true that the constant breathing in 
of sugar-dust exerts a very destructive effect upon the front teeth 
in particular, known as sugar decay'(Zuckercaries). In general, 
however, the chief role in the production of decay is performed 
by bread, potatoes, etc., not only because they produce more acid, 
but because they, on account of their insolubility, may remain for q 
long time sticking to or between the teeth, whereas the readily 
soluble sugar is soon diluted or carried away. In my opinion 
sugar can equal bread in its destructive action upon the teeth 
only when it is consumed as an ingredient of sticky, insoluble sub- 
stances. 
Naturally, we cannot think of making the attempt to banish 
the carbohydrates from the list of foods and luxuries of civilized 
races; but we may accomplish a great deal for the teeth if we 
prevent the constant and unnecessary consumption of sweets, etc., 
indulged in by many young and not a few adult persons. 
USE OF ANTISEPTICS WITHIN THE MOUTH 
Not all antiseptics are of real value within the mouth. Many 
experiments have been made with a view to finding a satisfactory 
one, but little advance has been made over the conclusions origi- 
nally drawn by Miller. Of all the antiseptics he places the greatest 
reliance on bichloride of mercury and salicylic acid. 
He concludes: "From the above experiments only few of these 
substances are serviceable for the purpose of cleansing the mouth. 
The bichloride of mercury is the most active, not only because it 
has the highest antiseptic power, but because its action continues 
for a longer time. Even after the solution has been removed from 
the mouth, traces remaining retain their antiseptic action, even 
when they have been diluted from one to two hundred times by 
the fluids of the mouth. None of the other materials possess this 
property in so high a degree. Furthermore, sublimate appears 
to penetrate particles of food, deposits, etc., more rapidly than the 
other materials given. I have satisfied myself by many experiments 
that it is possible, after a complete mechanical cleansing of the 
mouth, to obtain by means of sublimate (1-2500) an almost per- 
fect sterilization of the mouth. Unfortunately, the application of 
158 LECTURE NOTES ON BACTERIOLOGY 
the bichloride of mercury is limited, on account of its very poison- 
ous properties. 
"As for salicylic acid, many are of the opinion that it attacks 
the teeth (decalcifies them), and that it consequently should never 
be used in the mouth. On the other hand, others deny this action. 
I myself have seen it used for years without any evil conse- 
quences, and do not fear to use it now and then in the strength of 
1-200 or 1-300. 
"In all diseases of the human mouth in which antiseptics are 
indicated, particularly in acute infectious diseases, salicylic acid 
may be used for a short time without any danger to the teeth. 
For continual use perhaps the milder, though somewhat weaker 
benzoic acid in the concentration of 1-200 is preferable, unless 
it should turn out that this also may have an injurious effect upon 
the teeth. 
"A solution of bichloride of mercury which I advocate is the 
following: 
Acid, thymic 0.25 
Acid, benzoic 3.00 
Hydrarg. bichloride 0.80 
Tine, eucalypt 15.00 
Alcohol absolut 100.00 
01. gaultheria gtt. XXV. 
"One naturally hesitates to prescribe a mouth-wash which con- 
tains bichloride of mercury, but I think a more thorough considera- 
tion of the question will show that it is not so reprehensible an 
act as may at first appear. 
"The strength in which the bichloride is used in the mouth is 
about 1 /2000. Let us suppose that the patient swallows of the 
solution two grams daily (as a matter of fact, one need not swallow 
any at all) ; it would require one hundred days to have swallowed 
0.1 gram of the salt, which is the maximum dose for one day. In 
this matter, however, reasoning is of little value; nowhere is the 
saying in medicine, experience is of greater value than reasoning, 
truer than in questions dealing with the psychological action of the 
salts of mercury. I, myself, have made extensive use of the above 
formula without a trace of any physiological or toxicological action, 
160 LECTURE NOTES ON BACTERIOLOGY 
and if a sufficient number of members of the profession would 
make a trial of this solution upon themselves, and report the results, 
a great deal would be done toward solving the question of the 
advisability of recommending the wash in practice. 
' ' The taste of the bichloride is exceedingly disagreeable, even in 
dilute solutions; it may to a certain extent be disguised by the use 
of rose-water in place of aqua destillata as a solvent, as suggested 
by Allan. 
"Unfortunately, our pharmacopoeia is not yet so rich that the 
physician or dentist can restrict himself to the use of good-tasting 
medicaments. 
'41 have been informed by some who have used the bichloride as 
a mouth-wash, that whereas it has most excellent effect in all sup- 
purative diseases of the gums, it discolors the teeth. This of 
course would be a serious disadvantage if it should prove to be 
true. All the discoloration I have ever observed could be readily 
removed with brush and powder. 
' ' On the whole, however, the fear of a possible toxic effect from 
the bichloride, which in consideration of the fact that certain 
individuals are extremely sensitive to the action of this drug, we 
shall scarcely be able to escape from, will probably prevent its ever 
being extensively introduced except for occasional use in acute in- 
fectious and putrid conditions of the mouth. I personally have 
never prescribed it for prolonged use, except for a few friends 
who were in a position to control its action. From such the only 
complaint I have heard has been of its bad taste." 
Hygienic Care of the Mouth - The hygienic care of the mouth 
bears, therefore, a very direct relation to the health of the indi- 
vidual, and may all be summed up in the words, "Cleanliness of 
the oral cavity." As the first step toward this the digestion 
and any general causes of ill health should be corrected. Such 
a step goes far to keeping the surface of the tongue and the 
inside of the mouth clean. The second step includes the preven- 
tion of the formation of any cavities or irregularities around or in 
the teeth, cavities in which food remnants or bacteria may be re- 
tained. The teeth must be carefully cleaned, all cavities within 
them carefully filled and polished off even with the surface of the 
teeth, all deposits around the base of the teeth removed. Very little 
can be accomplished by the use of antiseptics within the mouth. 
162 LECTURE NOTES ON BACTERIOLOGY 
While they may impart a clean odor to the breath and leave a clean 
taste within the patient's mouth, the length of time required by 
the usual antiseptic in strengths appropriate to be used within 
the mouth in order to destroy pathogenic bacteria is far too long 
to make it possible to use them within the mouth for the purpose 
of rendering this cavity aseptic. 
We must regard the mouth, therefore, as abounding, even under 
the best of conditions, with bacteria, but that these rarely cause 
harm, if they are, so to speak, kept on the move through the ali- 
mentary tract by the failure to permit of the formation of reten- 
tion places in the mouth. Much the same obtains of the rest of the 
body. 
PROPHYLAXIS AGAINST BACTERIA IN THE BODY IN GENERAL 
Its surface is covered with bacteria, but if these are not per- 
mitted to grow in retention places, little harm can come from 
them. 
Infection-"When, however, bacteria are allowed to propagate 
to any degree in retention places on the surface of the body, 
whether this be on the external surface of the body or mucous sur- 
face in connection with the alimentary or respiratory tracts, the 
products formed by the bacteria may be responsible for the en- 
trance of the bacteria into the tissues of the body, through wounds 
made by contaminated instruments. When introduced by either of 
these methods within the tissues of the body the bacteria find the 
most favorable conditions of temperature and food supply. In 
fact, all the conditions from these standpoints are necessary for 
their unlimited propagation. The successful introduction of bac- 
teria into the tissues of the body is called infection, and were it 
not that the tissues of the body are capable of a reaction which not 
infrequently is successful in ultimately killing and eliminating the 
bacteria from its tissues, there would never be such a phenomenon 
as recovery from an infectious disease. 
Factors Controlling Recovery-The ease with which recovery 
takes place from the effects of the invasion of the tissues of the 
body by bacteria, depends upon two factors: the invasive power of 
the bacteria on the one hand, and the promptness with which the 
body is able to rally its defensive forces against the bacteria, on 
the other hand. 
164 LECTURE NOTES ON BACTERIOLOGY 
Invasive Power, Virulency and Fastness-By the invasive 
power of a strain of bacteria is meant its power of progressively in- 
vading the tissues of the body after it has once effected its entrance 
into them. It depends in part upon the number of bacteria intro- 
duced and in part upon the virulency of the particular strain of 
bacteria in question. 
Virulence is the disease-producing power of bacteria. Or the 
capacity of a strain of bacteria to grow or multiply within the 
tissues of the body independently of, or unrestrained by, the forces 
of resistance to them on the part of the tissues of the body. 
Different strains of the same variety of bacteria, as for in- 
stance different strains of the streptococcus, may possess widely dif- 
fering degrees of virulence. A high degree of virulence on the part 
of a strain of bacteria may depend in part upon a highly developed 
power of progressive growth within the body or the development 
within the bacteria in question of a high power of resistance 
against the opposing forces of the body. Such qualities are termed 
fastness. 
Immunity-The factors limiting the growth of pathogenic bac- 
teria within the tissues for which the body itself is responsible, rep- 
resent a reaction on the part of the tissues themselves against the 
bacteria, and depend, with the possible exception of phagocytosis, 
upon the development of substances within the blood and lymph 
which are destructive to the life and growth of the bacteria or their 
products. 
The presence within the body of these forces resisting infection 
constitutes the state of immunity. Immunity, in other words, is 
the power of the body to resist a progressive growth of bacteria 
within its tissues. The mechanism by which this resistance is ac- 
complished is complicated and affords a problem of unusual interest 
for study. 
Toxemia-Toxins and Other Harmful Products Produced by 
Bacteria-Aside from the presence within the blood of specific sub- 
stances resisting infection by bacteria, many secondary factors 
enter into the problem. These concern the particular manner in 
which the bacteria in question may produce their harmful effect 
upon the body. 
Some bacteria growing chiefly upon the surface of the body, as 
diphtheria upon the surface of a mucous membrane, or at least in 
166 LECTURE NOTES ON BACTERIOLOGY 
a limited area at the site of their entrance into the tissues, as 
tetanus, will cause all the symptoms of their specific disease in 
virtue of the absorption of soluble poisons from this site. 
Such bacteria possess in other words the power of producing 
soluble poisons. These soluble poisons are called extracellular 
toxins, and the condition is called Toxemia. These soluble and dif- 
fusible toxins are only one of a variety of substances found in any 
culture media containing the bacteria producing them. There may 
be, in addition, lysins, acids, enzymes, pigments, etc., and other 
toxins in addition to those responsible for a specific train of symp- 
toms which are regularly associated with the disease caused by the 
bacteria in question. 
Sapremia-Sometimes bacteria have no power actually to gain 
a foothold within the tissues, but may live entirely upon dead ma- 
terial still attached to the body or retained within a natural cavity 
of the body. The absorptions of the products formed by the bac- 
teria living under these conditions can cause a train of symptoms, 
the condition resulting being termed sapremia. 
Septicemia-When the bacteria not only enter the tissues but 
quickly invade the circulation so that they become uniformly dis- 
tributed in the blood throughout the body, the condition is known 
as bacteremia or septicemia. 
Pyemia-When again the bacteria enter the circulation, but 
show a disposition to become especially entrenched at multiple 
sites in the tissues of the body due to the sweeping away of masses 
of bacteria and their lodgment at multiple locations, the condition 
is known as pyemia. 
Other bacteria produce their toxic symptoms only after the 
solution of their bodies has taken place. Substances contained 
within their bodies which are not, in other words, diffusible, are 
responsible for the poisoning which constitutes the disease produced 
by such bacteria. Such substances are termed endotoxins. 
Secondary Factors Modifying the Severity of Infection on the 
Part of the Body-Among the secondary factors on the part of the 
body which serve to modify infection apart from the presence of 
specific immune substances, are: 
1. The avenue of infection. 
This must be appropriate for the entrance of the bacteria. 
168 LECTURE NOTES ON BACTERIOLOGY 
The gonococcus, for instance, cannot enter through the unbroken 
skin, respiratory or alimentary tracts. 
2. Any condition lowering the general physiological activity, as 
(a) fatigue, 
(&) exposure to cold, 
(c) improper diet, illustrated by the mouth infections 
common in scorbutus. Carnivorous animals are not 
liable to many diseases affecting herbivorous ani- 
mals, 
(d) intoxication of all kinds, 
(e) traumatism, seriously mutilating the tissues, 
(f) certain morbid conditions illustrated by diabetes or 
arterio-sclerosis, 
(g) An associated infection with other bacteria. The 
metabolic products of some bacteria serve to in- 
tensify the powers of growth of others. 
Immunity-Immunity proper means only a specific resistance 
to disease. It may be 
I. Natural-And there are numerous facts which demonstrate 
the presence of a natural resistance to disease. There is, for 
instance, a marked difference between individuals in this power. 
There is a still more marked difference in this particular between 
races, so that we may speak of 
II. Racial immunity-The difference displayed toward the ex- 
anthemata between the aborigines of the South Sea Islands and 
the European races is a familiar example. 
III. Species immunity-Animals differ greatly among them- 
selves and from human beings in their ability to resist certain 
diseases. 
A spontaneous infection of an animal with typhoid, cholera 
or meningococcus is unknown. Gonorrhea and syphilis are 
produced only with the greatest difficulty in animals. Lep- 
rosy and influenza have never been produced experimentally in 
animals. 
Acquired Immunity-Differing from all these forms of immun- 
ity is an immunity which is acquired by the individual or animal. 
Such a state is illustrated by the difference presented in the suscep- 
tibility of the individual to the disease before and after recovery 
from it. For instance, few unprotected individuals will escape 
170 LECTURE NOTES ON BACTERIOLOGY 
smallpox or measles, if intimately exposed to them. On the other 
hand, an individual who has once had these diseases presents a 
striking resistance to them after recovery under similar condi- 
tions of exposure. 
Active Immunity-The state of immunity which is thus in- 
duced by an animal contracting and recovering from a disease 
toward which it subsequently becomes immune, is known as active 
immunity because it depends upon some activity on the part of 
the tissues of the body, on the production by the body cells, in 
other words, of substances which are responsible for this immune 
state. 
Passive Immunity-In contrast to active immunity is the state 
of immunity toward a disease which may be conferred instantly 
upon an animal by the transfer to it of certain immune sub- 
stances from the blood of another animal which is immune. This 
form of immunity is called passive immunity. 
Antitoxin Immunity-It is exemplified best by antitoxin im- 
munity to diphtheria and tetanus and offers the most clean-cut 
and simplest example of immunity reactions. If horses are given 
diphtheria and have recovered, it is found that their blood serum 
will both protect other animals or human beings against infection 
by the diphtheria bacillus and induce a rapid state of recovery in 
an animal or human being already suffering from diphtheria. It 
is found, moreover, that the production of whatever the sub- 
stances are which are produced in the horse, and which confer 
protection on another animal, they are not dependent directly 
upon the diphtheria bacillus. They may be stimulated to pro- 
duction within the horse by the injection of appropriate quan- 
tities of a broth culture of the diphtheria bacillus in which all 
the bacilli have been killed. In these sterilized broth cultures is 
the diphtheria toxin which results in the production of the protec- 
tive substances. 
The protective substances produced by the injection of the 
toxins into the animal, which becomes immune, are called the 
antitoxins, and it is the antitoxin, produced in the immune animal 
in excess of the quantity needed to protect the animal from any 
ill effects of a second injection of the same amount of toxin that 
is responsible for the development of a high state of immunity 
so that, after the production in this manner of antitoxin in an 
172 LECTURE NOTES ON BACTERIOLOGY 
animal, an injection of the same or larger amount of toxin no 
longer will produce any symptoms whatever. If the injection of 
toxin is repeated at intervals in increasing doses, the animal 
develops so much antitoxin that it may receive many times the fatal 
dose of toxin without any symptoms resulting. In the same man- 
ner, the animal is no longer capable of becoming infected with the 
diphtheria bacillus. 
More than this, it is found that if a fatal dose of toxin is 
mixed with an appropriate quantity of antitoxin in a test tube, 
and subsequently the mixture injected into a susceptible animal, 
no symptoms will result. 
Character of the Toxin-Antitoxin Reaction-It appears then 
that the substance termed antitoxin developed in the blood of an 
animal by the injection of a specific toxin, possesses the power of 
rendering the toxin inert when the two are mixed together. 
The fact that certain mixtures of toxin and antitoxin may 
again become toxic after the application of correct degrees of 
heat, demonstrates that the antitoxin does not destroy the toxin. 
The action is rather one of chemical union by which the anti- 
toxin is united to the toxin and when the toxin is so bound it is 
incapable of exerting any toxic action. 
The reality of the combination of toxin and antitoxin is made 
more evident by the fact that if the toxin and antitoxin are 
standardized, the two may be titrated against each other in a man- 
ner demonstrating that definite quantities of toxin require definite 
quantities of antitoxin for their exact neutralization. 
Standardization of Antitoxin-In order to standardize the anti- 
toxin and toxin, a unit is necessary. Inasmuch as the antitoxin 
may be dried and kept in a stable condition while the toxin is un- 
stable, the unit of both toxin and antitoxin should be expressed 
in terms of antitoxin. The antitoxin unit is that quantity which 
is sufficient to protect a guinea pig weighing 350 grams from 100 
fatal doses of toxin. 
Toxoids and the Combining Groups of the Toxin-While the 
quantitive relations between toxin and antitoxin are well pre- 
served, the toxin solution with age may undergo a marked diminu- 
tion in their toxic power, though the combining power for anti- 
toxin is not altered. 
The altered toxins are called toxoids and their formation from 
174 LECTURE NOTES ON BACTERIOLOGY 
toxins with unchanged affinity for the antitoxin indicates that 
the toxin must possess two combining groups: one, the so-called 
haptophorous group which unites with the antitoxin, and under- 
goes no change in the transformation of toxin into toxoids, and 
a second combining group, the toxophorous group, which becomes 
modified in the transformation of toxin into toxoids and which 
represents the atom group of the toxin molecule which is respon- 
sible for the toxic action of the toxin. 
Other Combining Affinities for the Antitoxin in Toxin Solutions 
or Toxins-In completely neutralized mixtures of toxin and anti- 
toxin, there exist combining affinities which will unite with fresh 
toxin if this should be added. In fact, in a completely neutralized 
mixture of 100 units of toxin and 1 unit of antitoxin there must 
be added 100 fresh toxin units or an amount represented by the 
quantity of antitoxin present in the mixture, before one addi- 
tional toxin unit will be set free to cause the death of an injected 
guinea pig. 
In old or deteriorated toxin and antitoxin mixtures, the toxin 
having deteriorated, there will be in the completely neutralized 
mixture fewer fatal doses of toxin, but as measured by the anti- 
toxin unit the binding quantity after neutralization is complete 
before toxin is free to cause symptoms, in unchanged. In other 
words, the same binding substances must be present in the fresh 
toxin-containing bouillon. They must be different, therefore, from 
the toxoid and possess a weaker affinity for the antitoxin than 
either the toxin or toxoid possesses. They have been called toxons 
or epitoxoids because of their weaker affinity for the antitoxin. 
The toxoids themselves are to be classed as different bodies from 
the toxons or epitoxoids, and according to Whetter they possess a 
stronger or equal affinity for the antitoxin than the toxin, and 
hence are called protoxoids or syntoxoids. 
The fact, however, that in the fresh toxin-containing broth there 
are unsatisfied affinities capable of union with antitoxin may be 
more simply explained, not by assuming that they are different 
bodies from the toxin itself in the broth, but that the combining 
power of the toxin and antitoxin for each other varies, as in any 
mixture of acid and alkali, proportionately to the amount of ionic 
disassociation of the toxin and antitoxin. 
In attempting to neutralize a weak alkali with an acid the 
176 LECTURE NOTES ON BACTERIOLOGY 
amount of acid capable of combining with the alkali would depend 
upon the amount of disassociation in this acid and this would 
decrease with the diminution in the strength of the acid. As, 
however, the disassociated acid unites with the alkali, more dis- 
association would take place in the undisassociated portion and 
this added disassociation would then act as the toxons. 
Summary of the Mechanism of Antitoxin Immunity-The es- 
sential facts, however, concern only the phenomenon that when 
a diffusible toxin such as is almost solely responsible for the acute 
illnesses of diphtheria and tetanus, is injected into an animal in 
increasing doses, beginning, of course, with sublethal doses, there 
is produced in excess in the injected animal by the cells, naturally 
by those attacked by the toxin, a substance capable of neutraliz- 
ing, according to exact chemical relations, the toxin. And in 
virtue of the presence of this substance or antitoxin, the serum 
of such an animal is capable of rendering inert the toxin in an 
individual suffering from diphtheria or tetanus, provided the anti- 
toxin is injected early enough in the disease. It is natural to assume 
that the antitoxin is simply a portion of the body cell which is 
thrown off into the circulation, and that portion of the cell which 
has a chemical affinity for the toxin. In other words, when the 
cell is deprived of the function of this atom group or receptor, as 
it is called, because of its union with the toxin the cell so attacked 
makes good the loss and produces another receptor; but not simply 
one, but more than one, so that an excess of these receptors are 
thrown off into the circulation and exist there free, capable of 
uniting in the future with toxin. The body remains, therefore, 
protected for a long period. 
Duration of the Period of Protection-In diphtheria the time 
limit during which the use of antitoxin is successful is long, so 
that the treatment of diphtheria by antitoxin is of the greatest 
service in this disease. In tetanus, on the other hand, the value 
of its antitoxin is chiefly limited to the incubation period of the 
disease. It is chiefly of use, therefore, in the prophylactic treat- 
ment of tetanus. 
Chemical Characters of Antitoxin-The antitoxin is a more 
stable body than the toxin. It is more thermostabile, the toxin being 
comparatively thermolabile. It probably has a larger molecule, as 
from mixtures of the toxin and antitoxin subjected immediately 
178 LECTURE NOTES ON BACTERIOLOGY 
after mixing to filtration through dense filters, the pores of which 
are filled with gelatin, less and less toxin will come through the 
filter, as it is bound to the antitoxin the molecule of which is too 
large to come through the filter. 
The Preparation of Antitoxin-In the preparation of anti- 
toxin, the horse is made use of. The first dose of antitoxin made 
is weakened either by heating the toxin, mixing it with antitoxin 
or lysol solution, or by simply using small quantities. 
The intervals between the injections are five days to a week. 
At these intervals, stronger and stronger doses of toxin are given 
until in two to three months the horse's blood serum contains 
to each c.c. 250-800 or 1,000 antitoxin units. 
It is a decided advantage to have a concentrated antitoxin as 
under these conditions there is less danger of various toxic symp- 
toms developing which are dependent upon other substances in 
the serum. Less than 200 units to one c.c. are not allowed. 
There are special ways of concentrating and purifying anti- 
toxin solutions dependent upon their precipitation with ammonium 
sulphate. 
Immunity by Bacteriolysis-The production of antitoxin is 
protective only because it disposes of the poisonous products of 
bacteria, thus reducing the bacteria to harmless parasites and 
maintaining a state of health notwithstanding the presence within 
the body of dangerous pathogenic bacteria, until the body can 
dispose of the bacteria themselves. 
Bacteriolysis-If cholera bacilli are injected into the peritoneal 
cavity of an immune guinea pig or mixed with the peritoneal fluid 
of an immune guinea pig in a test tube outside the body, the 
cholera bacilli will be destroyed and their bodies can be seen under 
the microscope to undergo dissolution. The guinea pig may be 
made immune by either infecting it with cholera or injecting it 
with the dead cholera bacilli. Evidently, as in the formation 
of antitoxin, the presence within the guinea pig of the cholera 
bacilli, the bodies of which contain substances toxic to the guinea 
pig, has resulted in the production of a substance in the serum 
capable of destroying the proteins of the cholera bacilli. 
Mechanism, of Bacteriolysis-This destroying or lytic substance 
is extremely thermolabile as it is destroyed by an exposure of 
thirty minutes to a temperature of 56° C. Such inactivated serum 
180 LECTURE NOTES ON BACTERIOLOGY 
may again recover its lytic power by aciding to it a little fresh 
blood serum taken from any animal whatever. Obviously then, 
two substances must be concerned in the process of bacteriolysis; 
one substance present in the blood of all animals and a second 
substance present only in the blood of the immune animal. 
Much light has been thrown upon the nature of the process 
involved by the similar reaction of hemolysis. 
Hemolysis--It has been discovered that an animal may be 
made immune to the red blood cells of another species by the 
injection of these red cells of the other species, in the sense that 
the animal made so immune develops in its blood a substance 
which is lytic or destructive to these red blood cells. 
If, for instance, an appropriate amount (5 or 10 or 15 c.c.) of 
a 5% suspension of the red blood cells of one animal are injected 
intravenously into another animal and the injection repeated three 
or four times at intervals of every five days, the blood serum of the 
injected animal acquires the power of destroying, in vibrio or vivo, 
the red cells of the species of animal from which the injected red 
blood cells were obtained. 
Method of Testing the Reaction-The reaction may be tested in 
a test tube by mixing together a little serum of the immune animal 
and 1 c.c. of a 5% suspension of washed red blood cells of the 
species against the red blood cells of which the animal furnishing 
the immune serum is immune. This mixture is set aside for a half- 
hour in the incubator and then placed in the cold until the red 
blood cells in the mixture have settled. 
At the end of this time, if the serum has been taken from an 
animal immune to the red blood cells used for the test, the red 
blood cells will have been destroyed and their stroma will have 
settled to the bottom of the test tube. 
The supernascent fluid will be red and transparent due to the 
solution of the liberated hemoglobin. If the animal furnishing 
the serum for this test had not been made immune, the red blood 
cells still retaining their color will be found as a red sediment 
in the bottom of the test tube, while the supernascent fluid will 
be clear and colorless. 
Mechanism of the Reaction-This reaction is really a lytic reac- 
tion and corresponds in every way to the reaction of bacteriolysis. 
In the blood of the immune animal in the one case a new substance 
182 LECTURE NOTES ON BACTERIOLOGY 
or immune body has been developed which is capable in conjunc- 
tion with the thermolabile substance present in the serum of all 
animals, of destroying the bacteria to which the animal has been 
made specifically immune. The immune body in this instance is 
termed a bacteriolysin. 
In the blood of the immune animal in the second case a new 
substance has been developed which, it is found also, must act in 
cooperation with the same thermolabile substance present in the 
blood of all animals and with such cooperation is capable of 
destroying the red blood cells to which the animal is specifically 
immune. In this instance the new substance is termed a hemolysin. 
The two reactions resemble each other in all particulars and 
facts explanatory of one may be applied to the other. It is found, 
for instance, that heating the serum containing specific hemolysins 
for half an hour at 56° C. will destroy the hemolytic power of 
the serum, but that the serum may be reactivated by adding a 
little fresh serum of another animal. 
At least two substances, therefore, are concerned in the reac- 
tion of hemolysis: a thermola- 
bile substance destroyed at 56° 
C., normally present in all 
bloods, and a more thermosta- 
bile substance which is only 
present in the immune animal. 
The thermolabile substance 
has been termed the comple- 
ment or alexin and the ther- 
mostabile substance is termed 
the immune body or ambocep- 
tor, or by the older French au- 
thors, the substance sensibiles- 
satrice. 
Method of Separating Am- 
boceptor and Complement- 
These two substances may be 
separated from each other by mixing the red blood cells used for 
the production of immunization with an inactivated serum contain- 
ing the immune body or specific hemolysin to these red cells and 
allowing the red cells and inactivated serum to remain in contact 
Fig. 49.-Enclosed electric centrifuge. 
184 LECTURE NOTES ON BACTERIOLOGY 
with each other for an appropriate length of time. After the lapse 
of such a period, the red cells may be separated by centrifugaliza- 
tion and the supernascent serum poured oft. 
If the supernascent serum is now tested for the presence of the 
hemolysin by mixing it with other red blood cells from the same 
species, the cells of which were used to produce immunity, and 
with a little fresh serum from another animal, in order to supply 
the complement, it will be found that it contains no immune body 
or hemolysin. If, however, the centrifugalized red cells are tested 
in the same manner by the addition of a little fresh complement, 
hemolysis will result, showing that the immune body has attached 
itself to the red cells during the time in which they remained in 
contact before centrifugalization. 
The immune body may in the same way become bound to the 
corpuscles in the presence of the complement, if the three com- 
ponents are mixed at 0° C. and kept for' a length of time at this 
temperature. If, after such a period, the red cells are separated 
by centrifugalization, it will be found that the immune body has 
become attached to them. It has been concluded from these facts 
that the destroying body in a lytic reaction is the complement but 
that the complement cannot unite directly with the bacteria or red 
cells, but only through the intermediation of the immune body. 
The immune body has been held to possess therefore a cytophile 
haptophore atom group, by which it attaches itself to the red cell 
or bacterium, and another, the complementophil haptophore atom 
group, which receives the complement. In other words, the com- 
plement is united to the red cell and bacterium by means of the 
immune body and the whole purpose served by the immune body 
is to unite the complement to the red cell or bacterium and thus 
make possible the action of the complement on the red cell or 
bacterium. Whether or not this is the correct explanation, it is 
certain that the reaction cannot occur without the participation 
of both complement and immune body, and that the immune body 
possesses the power of uniting with the red cell or bacterium under 
conditions impossible for the complement. 
Specific Agglutination-Before entering into any further dis- 
cussion of the relation of these lytic reactions to immunity, two 
more specific reactions will be mentioned. 
It is found that if bacteria are injected into an animal that the 
186 LECTURE NOTES ON BACTERIOLOGY 
injected animal's serum acquires the power of clumping or ag- 
glutinating this variety of bacteria when mixed with them in ap- 
propriate proportions. The clumping or agglutinating power of 
the serum may be tested under the microscope or in an open 
test tube. 
Technic of Agglutination-In the microscopical test various 
dilutions of the serum up to 1 : 1000 are made, and the more 
specific the greater may be the dilution; these are mixed with 
bacteria on a microscopical slide, preferably kept warm. In a 
short time, the bacteria will come together in little clumps and, 
if they have been freely swimming about, they will cease their 
movement. 
Microscopically similar dilutions are made in test tubes by 
mixing proper quantities of the serum with suspensions of fresh 
cultures in normal saline fluid. The tubes may be set aside in 
the incubator and, if agglutination takes place, the clumping of 
the bacteria will cause them to be precipitated to the bottom of the 
tube and the previously uniform cloudy suspension will become 
clear and transparent. This re- 
action of agglutination is due to 
the development in the blood of 
the injected animal of specific 
agglutinins. 
Mechanism, of Agglutination 
-Agglutinins, as is the case 
with bacteriolysins and haemoly- 
sins, do not require the presence 
of complement, as they are ac- 
tive after exposure to 55° C. 
and even to 65° C. or 70° C. 
After destruction by higher de- 
grees of temperature their solu- 
tions cannot be reactivated by 
the addition of fresh comple- 
ment. Specific agglutinins have nothing to do with bacterioly- 
sins; in fact, the mere agglutination of bacteria by them does not 
result in the death of bacteria and a serum may be strongly aggluti- 
nable but feebly lytic and vice versa. Moreover, they will aggluti- 
nate dead as well as living bacteria. This fact makes it possible to 
Fig. 50.-Agglutination of the typhoid 
bacillus by a specific serum. Jen- 
ner's stain. (Besson.) 
188 LECTURE NOTES ON BACTERIOLOGY 
prepare permanent emulsions of bacteria for use in testing serum 
for the presence of agglutinins and this greatly facilitates the 
use of such tests for diagnostic purposes. 
Group Agglutination-The use of agglutinating tests for diag- 
nostic purposes is somewhat interfered with by the fact that an 
agglutinin will produce some agglutinating action on allied va- 
rieties of bacteria but never in such dilutions as upon the specific 
bacteria which has stimulated its production in the animal body. 
Thus the serum of a typhoid immune animal will agglutinate the 
typhoid bacillus in dilutions of 1 :1000 and the colon bacillus in 
dilutions of 1:200, whereas the agglutinating power of normal 
serum for the colon bacillus is never over 1:20. Such a reaction 
is termed "group agglutination." For accurate diagnostic work 
by the agglutination test the specific quality of the reaction de- 
pends solely upon the degree of dilution with which it is possible 
to secure the reaction. It must also be remembered that some- 
times an agglutinin will act in a dilute solution when it will not 
do so in a strong solution. This phenomenon has been explained 
by the assumption that proagglutinoids are present in sufficient 
amounts in the concentrated solution to interfere with the reaction. 
Permanency of Agglutinins-The life of agglutinins is not 
permanent in the immunized animal. After inoculation, they ap- 
pear from the third to the sixth day and increase from seven to 
thirteen days, and then fall off to a variable lower level, represent- 
ing amounts which are present for long periods without change. 
Agglutinins may be absorbed and thus removed from the serum 
containing them by the species of bacteria giving rise to them. It 
is concluded, therefore, that they unite with the bacteria when 
producing agglutination and hence that the reaction of agglutina- 
tion is chemical. 
Cytolysins-Not only do organized cells such as bacteria and 
red blood cells, but any cell, as the cells of the various organs 
of the body injected into the circulation, possess the power of 
creating auto-destructive substances. In the case of cells, the lytic 
substances are called cytolysins. 
Precipitins-But more than this, even protein injected into 
the circulation of an animal will stimulate the formation of a 
body which, in extreme dilutions, will precipitate this protein 
from its solutions. The animal may thus be said to be immune to 
190 LECTURE NOTES ON BACTERIOLOGY 
the protein producing the precipitating substance, and the new 
substance responsible for the precipitation is called a precipitin. 
Precipitin reactions are most specific and specific precipitins 
may be produced by the widest varieties of proteins. Thus far 
these reactions have an almost unlimited range of application. By 
means of them the identity of almost any protein may be estab- 
lished. While group precipitin reactions certainly exist, the pre- 
cipitin, caused by the injection of the filtrate of a typhoid bacillus 
causing a precipitate with filtrates of a colon bacillus, yet like 
agglutination tests, these reactions never occur in such high dilu- 
tions as when the specific precipitin is tested against its own anti- 
gen-protein. When using proper dilutions human blood serum may 
be identified in such dilutions as 1:50,000, the only known possible 
source of error being the blood serum of such a closely related 
species as the anthropoid ape. 
Precipitins have never been found in normal blood serum 
though naturally-occurring agglutinins have been found. 
Mechanism of the Precipitin Reaction-Like agglutinins, how- 
ever, the precipitins act apparently without the participation in 
the reaction of a complement, at least of a complement which is 
thermolabile in temperatures below 60° or 70° C. A precipitin 
containing sera is inactivated by a temperature of 60°-70° C., but 
if a precipitin heated to this temperature has been mixed with a 
protein giving rise to it, a union probably takes place between the 
protein and heated precipitin because the subsequent addition of 
fresh precipitin will not precipitate the protein. 
This fact indicates that precipitins are composed in all proba- 
bility of two atom groups: a labile haptophore group which unites 
the precipitin to the mother protein, and a stable precipitophore 
or zymophore group. 
Antigens, Antibodies and Cell Receptors-The above descrip- 
tion of antitoxins, lysins, agglutinins, and precipitins demonstrates 
that the animal body possesses the power of producing protective 
bodies against a wide variety of substances which may enter the 
circulation. In fact, it is almost permissible to enunciate as a 
general law that any foreign substance or body, theoretically 
harmful to the body, stimulates the body to produce protecting anti- 
bodies within itself so that when this body is again met with in 
the circulation, it is altered and thus presumably rendered inert. 
192 LECTURE NOTES ON BACTERIOLOGY 
prepare permanent emulsions of bacteria for use in testing serum 
for the presence of agglutinins and this greatly facilitates the 
use of such tests for diagnostic purposes. 
Group Agglutination-The use of agglutinating tests for diag- 
nostic purposes is somewhat interfered with by the fact that an 
agglutinin will produce some agglutinating action on allied va- 
rieties of bacteria but never in such dilutions as upon the specific 
bacteria which has stimulated its production in the animal body. 
Thus the serum of a typhoid immune animal will agglutinate the 
typhoid bacillus in dilutions of 1:1000 and the colon bacillus in 
dilutions of 1:200, whereas the agglutinating power of normal 
serum for the colon bacillus is never over 1:20. Such a reaction 
is termed "group agglutination." For accurate diagnostic work 
by the agglutination test the specific quality of the reaction de- 
pends solely upon the degree of dilution with which it is possible 
to secure the reaction. It must also be remembered that some- 
times an agglutinin will act in a dilute solution when it will not 
do so in a strong solution. This phenomenon has been explained 
by the assumption that proagglutinoids are present in sufficient 
amounts in the concentrated solution to interfere with the reaction. 
Permanency of Agglutinins-The life of agglutinins is not 
permanent in the immunized animal. After inoculation, they ap- 
pear from the third to the sixth day and increase from seven to 
thirteen days, and then fall off to a variable lower level, represent- 
ing amounts which are present for long periods without change. 
Agglutinins may be absorbed and thus removed from the serum 
containing them by the species of bacteria giving rise to them. It 
is concluded, therefore, that they unite with the bacteria when 
producing agglutination and hence that the reaction of agglutina- 
tion is chemical. 
Cytolysins-Not only do organized cells such as bacteria and 
red blood cells, but any cell, as the cells of the various organs 
of the body injected into the circulation, possess the power of 
creating auto-destructive substances. In the case of cells, the lytic 
substances are called cytolysins. 
Precipitins-But more than this, even protein injected into 
the circulation of an animal will stimulate the formation of a 
body which, in extreme dilutions, will precipitate this protein 
from its solutions. The animal may thus be said to be immune to 
190 LECTURE NOTES ON BACTERIOLOGY 
the protein producing the precipitating substance, and the new 
substance responsible for the precipitation is called a precipitin. 
Precipitin reactions are most specific and specific precipitins 
may be produced by the widest varieties of proteins. Thus far 
these reactions have an almost unlimited range of application. By 
means of them the identity of almost any protein may be estab- 
lished. While group precipitin reactions certainly exist, the pre- 
cipitin, caused by the injection of the filtrate of a typhoid bacillus 
causing a precipitate with filtrates of a colon bacillus, yet like 
agglutination tests, these reactions never occur in such high dilu- 
tions as when the specific precipitin is tested against its own anti- 
gen-protein. When using proper dilutions human blood serum may 
be identified in such dilutions as 1:50,000, the only known possible 
source of error being the blood serum of such a closely related 
species as the anthropoid ape. 
Precipitins have never been found in normal blood serum 
though naturally-occurring agglutinins have been found. 
Mechanism of the Precipitin Reaction-Like agglutinins, how- 
ever, the precipitins act apparently without the participation in 
the reaction of a complement, at least of a complement which is 
thermolabile in temperatures below 60° or 70° C. A precipitin 
containing sera is inactivated by a temperature of 60°-70° C., but 
if a precipitin heated to this temperature has been mixed with a 
protein giving rise to it, a union probably takes place between the 
protein and heated precipitin because the subsequent addition of 
fresh precipitin will not precipitate the protein. 
This fact indicates that precipitins are composed in all proba- 
bility of two atom groups: a labile haptophore group which unites 
the precipitin to the mother protein, and a stable precipitophore 
or zymophore group. 
Antigens, Antibodies and Cell Receptors-The above descrip- 
tion of antitoxins, lysins, agglutinins, and precipitins demonstrates 
that the animal body possesses the power of producing protective 
bodies against a wide variety of substances which may enter the 
circulation. In fact, it is almost permissible to enunciate as a 
general law that any foreign substance or body, theoretically 
harmful to the body, stimulates the body to produce protecting anti- 
bodies within itself so that when this body is again met with in 
the circulation, it is altered and thus presumably rendered inert. 
192 LECTURE NOTES ON BACTERIOLOGY 
The body giving rise to an antibody is termed an antigen; and an 
antigen may be defined as any substance which when injected into 
the circulation of an animal is capable of stimulating, in virtue 
of a physiological chemical reaction, the production of a self- 
neutralizing, self-lytic, self-agglutinating, self-precipitating or 
otherwise self-destroying substance. 
The antibodies, immune bodies or amboceptors are always 
specific and thus multiple. The complement, however, is always 
the same body. There is but a single complement. The production 
of antibodies by the injection of antigens is a phenomenon of such 
generality that it is even possible to prepare antiamboceptors 
and anticomplements. 
An antibody is produced in excess and acts by forming a 
chemical combination with the antigen. It may be regarded as a 
thrown off atom group from the body cells attacked by the antigen 
and presumably those atom groups in the cell which are attacked 
by the antigen. Such thrown off atom groups are termed cell 
receptors. 
Varieties of Receptors-So far as the present knowledge goes, 
it has been convenient to divide receptors into three orders: 
1. Receptors of the first order are represented by antitoxins 
in which only one free combining group exists, that which anchors 
the toxin. 
2. Receptors of the second order represented by agglutinins 
and precipitins which possess free combining atom groups, one 
of which, like the haptophore of the toxin, unites with the antigen; 
while the other produces the specific change on the antigen which 
results in precipitation or agglutination. 
3. Receptors of the third order, in which three free combining 
atom groups are concerned in the reaction: The haptophore and 
complementophore groups of the amboceptor and the lytic atom 
group of the complement. 
Distinction between Facts and Theory in the Explanation of 
the Mechanism of Immunity Reactions-It must never be for- 
gotten, however, that the above explanation of the mechanism 
by which antibodies are produced and by which they act on antigens 
are only theories serving as conveniences in dealing with the facts. 
Though these theories explain the facts they may not be the whole 
194 LECTURE NOTES ON BACTERIOLOGY 
explanation, as the reactions may depend in large part on un- 
known facts of physical chemistry. 
It has never been positively shown that the antibodies floating 
free in the blood are identical with the atom group of the cells 
which are attacked by the antigens. 
One illustration from the field of agglutination will suffice to 
demonstrate the relation of physical chemistry to some phase re- 
actions. While the reaction of agglutination depends certainly 
upon the union of the agglutinin with its antigen, yet no agglutina- 
tion will take place in the absence of certain salts in solution. 
In fact bacteria which have absorbed other substances entirely 
different from agglutinins, such as uranium compounds, or col- 
loidal silicic acid, can be agglutinated by the subsequent addition 
of these salts to the solution containing them. We do not know 
how much the physical conditions alone have to do with these 
reactions. 
Practical Use Made of Immunity Reactions in Vitro-These 
various immunity reactions are not only of importance in ex- 
plaining the protective mechanism of the body against infection 
by bacteria and the manner in which recovery from disease is 
possible, but much practical use is made of them for the identifica- 
tion of strains of bacteria. 
The diagnosis of the various proteins by the precipitin test 
and the utilization of the agglutinating reaction and the lytic 
reaction for the identification of bacteria have already been re- 
ferred to. The agglutinating reaction is a very valuable one for 
the separation of various strains of bacteria. 
Complement Fixation Test-Of equal importance, perhaps 
of greater importance for diagnostic purposes, is that reaction 
which is known as the complement fixation test. 
This reaction depends 
1. Upon the fact that there is one complement for all im- 
munity reactions which require a complement for their completion. 
2. Upon the fact that it is possible to determine by titration 
exactly how much complement is totally consumed in the reaction, 
though this quantity bears within limits an inverse ratio to the 
amount of amboceptor used in the reaction. In other words, an 
antigen may be mixed with just enough amboceptor and comple- 
ment to bind all the complement. If then to this mixture there 
196 LECTURE NOTES ON BACTERIOLOGY 
is added another amboceptor and antigen, the second antigen will 
not be attacked by its amboceptor because there is no unbound or 
available complement. If, however, there had been either no 
antigen or no immune body in the first mixture, the second antigen 
would have been attacked because its amboceptor being present, 
the complement in the first mixture would be free to attack it. 
Complement Fixation for the Identification of an Antigen- 
Such a reaction may be used to demonstrate the presence or absence 
of an unknown antigen or immune body, and therefore the presence 
in the body of bacteria acting as such an antigen or stimulating 
the production of the immune body. 
The Wassermann Reaction-The most extensive use of this 
reaction is made in the diagnosis of syphilis. For the demonstra- 
tion of the presence or absence of this disease it is not an in- 
fallible guide but nevertheless a fairly dependable one. 
The plan of the reaction is as follows: In one test tube there 
is placed an appropriate quantity (0.2 c.c.) of the inactivated 
serum of the individual whose blood it is desired to test for the 
presence or absence of the immune body which exists in the blood 
of patients harboring the Spirochsete pallida. 
To this is added 0.1 c.c. of a guinea pig's serum and the re- 
quired amount of a substance acting as syphilitic antigen, dis- 
solved in 1 c.c. of normal saline fluid. 
In the early days of the application of this test, alcoholic ex- 
tracts of chopped up syphilitic livers were used for antigens. These 
livers were most frequently obtained from still-born syphilitic 
fetuses which were rich in the Spirochsete pallida. Subsequently, 
it has been found that the liquids, and hence the extracts of 
normal livers or of the guinea pig's heart or commercial lecithin 
itself, will fulfill the function of antigen in this reaction. As such 
antigens are easier to procure, they are now used by preference, 
but their use transforms the reaction from the sphere of immunity 
reactions possessing a logical basis throughout to the field of em- 
pirical though still specific reactions. 
Technic of the Wassermann Test. Quantity-The correct 
amount of antigen to use in this reaction must be determined by 
titration, for instead of using an experimentally adjusted quan- 
tity of complement, a constant quantity of complement is used 
and sufficient antigen to bind completely this amount of comple- 
198 LECTURE NOTES ON BACTERIOLOGY 
ment. The constant quantity of complement selected is that con- 
tained in 0.1 c.c. of guinea pig serum. To this amount in two series 
of separate tubes is added graded quantities of the antigen. To 
the mixture in one series of tubes is added 0.2 of a c.c. of inac- 
tivated blood serum from a patient having syphilis and in the 
other series the 0.2 of a c.c. of the inactivated serum from a normal 
individual. These mixtures are allowed to remain in contact for 
one hour and then each is tested for unbound complement by mix- 
ing with red blood cells and an inactivated serum, which is 
hemolytic to these red blood cells and therefore one which con- 
tains amboceptor. The amount of antigen which is correct to 
use in the Wassermann reaction is that amount giving hemolysis 
with the normal serum but just failing to give hemolysis with the 
syphilitic serum. With this amount of antigen when .2 c.c. of sus- 
pected serum replaces the known syphilitic serum, as in the titra- 
tion tests when we add the red blood cells and their hemolytic 
amboceptor, we will fail to get hemolysis if the suspected serum 
contains syphilitic amboceptor or, in other words, comes from an 
individual having syphilis. Whereas if the suspected serum is 
normal, no hemolysis will be obtained. 
The Amboceptor-In this reaction the only other ingredient 
which it is necessary accurately to measure is the hemolytic am- 
boceptor, for as has been mentioned, in any immune reaction an 
excess of amboceptor may inhibit the reaction and the optimum 
quantity of amboceptor varies with the quantity of complement 
used. The hemolytic amboceptor is obtained by injecting into a 
rabbit the washed red blood cells of a sheep. 5-15 c.c. of a 5% 
suspension of the washed red blood cells of a sheep are injected 
at five or six day intervals into a rabbit for three or four times. 
The serum of this rabbit is markedly hemolytic to the red blood 
cells of the sheep. In other words, to obtain the correct quan- 
tity of the rabbit's serum to use in the test, graded quantities are 
mixed with 0.1 c.c. of complement and 1 c.c. of a 5% suspension of 
red blood cells and the quantity chosen as the unit which is just 
strong enough to give hemolysis. For the test at least double this 
quantity is used. 
The Controls-In order to exclude error in conducting a Was- 
sermann test, it is necessary to arrange a number of controls; 
one with a serum known to be syphilitic in order that we may be 
200 LECTURE NOTES ON BACTERIOLOGY 
sure that our conditions will work when syphilitic immune bodies 
are actually present in the tested serum; another with normal 
serum, in order that we may be sure that the test will be negative 
with a normal serum. A control in which the hemolytic system 
alone is used with the antigen in order that we may be sure that 
it is working as would be expected. 
And finally, all four tubes duplicated without antigen, in order 
that we may be sure that there is nothing introduced by the anti- 
gen which would lead to error. 
Method of Setting up the Reagents for the Reaction-In per- 
forming the test therefore, the following tubes with the contents 
which are indicated, should be prepared: 
1 
3 
5 
7 
2 c.c. of suspected ser- 
um 
1 c.c. of complement 
Required amt. of an- 
tigen in 1 c.c. of salt 
sol. 
Enough salt sol. to 
make 3 c.c. 
2 c.c. of known syphi- 
litic serum 
1 c.c. of complement 
Required amt. of an- 
tigen in 1 c.c. of salt 
sol. 
Enough salt sol. to 
make 3 c.c. 
2 c.c. of normal serum 
1 c.c. of complement 
Required amt. of an- 
tigen in 1 c.c. of salt 
sol. 
Enough salt sol. to 
make 3 c.c. 
1 c.c. of complement 
Required amt. of an- 
tigen in 1 c.c. of salt 
sol. 
Duplicate of above row, but without the antigen. (See page 204.) 
All these tubes are placed in the incubator for one hour and 
then to them are added 2 units of rabbit's amboceptor for the 
sheep's corpuscles, 1 c.c. of a 5% suspension of the sheep's cor- 
puscles, and everything set aside on ice until the next morning. 
If syphilis is present, and the controls have excluded error, 
tubes 1 and 3 will show no hemolysis; all the others should show 
hemolysis. If the test is negative only tube 3 should show no 
hemolysis. 
202 LECTURE NOTES ON BACTERIOLOGY 
2 
4 
6 
8 
2 c.c. of suspected ser- 
um 
1 c.c. of complement 
Enough salt sol. to 
make 3 c.c. 
2 c.c. of known syphi- 
litic serum 
1 c.c. of complement 
Enough salt sol. to 
make 3 c.c. 
2 c.c. of normal serum 
1 c.c. of complement 
Enough salt sol. to 
made 3 c.c. 
1 c.c. of complement 
Enough salt sol. to 
made 3 c.c. 
Forensic Identification of Proteins by the Fixation of Com- 
plement-Just as the Wassermann reaction identifies an unknown 
immune body, as has been mentioned, the same principle may be 
made use of to identify an unknown antigen with a known im- 
mune body. The test is sufficiently delicate to detect .0001 of a c.c. 
of animal proteins. The technic of the test is practically the 
same as the technic of the Wassermann reaction except that instead 
of titrating the antigen .0001 of a c.c. of the suspected serum is 
used. The immune body containing serum (a rabbit serum is 
usually made use of) must be titrated in diminishing quantities 
with antigen and complement and then tested against the hemolytic 
system. The smallest quantity successfully inhibiting hemolysis is 
the unit and one and one-half to two times this quantity may be 
used for the test. The correct quantity of hemolytic amboceptor 
must also be ascertained as in the Wassermann reaction by titration. 
Anaphylaxis-The consequences resulting from the various im- 
munity reactions are in general beneficial to the body and yet the 
early attempts to artificially induce immunity by the injection of 
serum containing immune bodies, even in such a disease as diph- 
theria in which the immune serum contains a body capable of so 
directly neutralizing the toxin, were soon discovered to give rise 
occasionally to very serious symptoms. 
204 LECTURE NOTES ON BACTERIOLOGY 
In fact, a number of deaths attributable to the use of diph- 
theria antitoxin occurred. 
It was noted that even when animals also were highly immune 
they sometimes showed an increasing susceptibility to toxins. 
Further experimentation developed the fact that certain ani- 
mals would become very susceptible to repeated injections of any 
foreign protein. For instance, if a guinea pig is given 1/10000 
of a gram of egg or .001 c.c. of horse serum and 6-8 days after- 
wards an injection of 1/100 of these quantities, it will die almost 
immediately with explosive symptoms, among which general con- 
vulsions and dyspnea are the most prominent. This phenomenon 
was termed anaphylaxis or allergy (altered state) and was found 
to have a rather general application although the symptoms and 
severity of the condition varied greatly in different animals. 
Provocation of Anaphylaxis-It was found that the state was 
always induced by at least a relatively small but definite quantity 
of protein. The guinea pig, which is the most susceptible animal 
to anaphylaxis, requires only one initial dose. Rabbits and dogs 
which do not respond so easily, require several preliminary injec- 
tions. There then follows an incubation period of 6-8 days when 
the animal enters the allergic state, which is most highly developed 
during the next 10-21 days. During this period the typical symp- 
toms can be provoked by the injection of a single dose of 1/100 
of the quantity originally necessary to sensitize the animal. 
Symptoms in Different Animals-In the guinea pig these symp- 
toms come on with great rapidity after intravenous injection and 
consist of great dyspnea and convulsions and death within a 
minute or so. If the injection has been made intraperitoneally, 
the symptoms come on more slowly. The animal scratches its 
nose and body, becomes restless, and chokes. Convulsive move- 
ments occur about the mouth and throat. The animal retches; 
there is a discharge of feces and urine; finally there are general 
convulsions, depression of temperature of 7-13° C., respiration 
and death. On autopsy, the lungs fail to contract but remain dis- 
tended, forming a perfect cast of the interior of the thoracic 
cavity. Their failure to contract is due to an extreme spasm of 
the small bronchi. 
If the animal survives there is a close relation between the 
extent of the fall of the temperature and the severity of the 
206 LECTURE NOTES ON BACTERIOLOGY 
symptoms, and the length of time which it takes for the tem- 
perature again to reach normal. There is also a leucopenia and 
a temporary loss of complement in the blood. 
In dogs, the symptoms are somewhat different. They be- 
come very restless, whine or bark, and finally become very weak 
and fall over; discharge their feces and urine and suffer a marked 
fall in blood pressure. In dogs the dyspnea is not a marked 
feature. 
In rabbits there is a striking edema and even necrosis of various 
areas in the body, particularly of the skin surface. The respiratory 
disturbance, the marked prostration and fall in blood pressure and 
increased peristalsis are all marked features. 
In man there are very few cases characterized with the alarm- 
ing symptoms of collapse and death, but the immediate symptoms 
are not unlike those of the guinea pig. It is possible for symptoms 
to develop after the first injection and in such instances one must 
assume that antibodies responsible for the reaction are already pres- 
ent in the blood. Reinjection of these individuals may mean 
death. When milder symptoms occur, urticarial and erythematous 
eruptions, local and general edema, pains in the joints, headache, 
weakness, fever, leucopenia, and sometimes bloody diarrhoea are 
commonly met with. 
Group Reactions and Degree of Specificity of the Reaction- 
In the allergic state, an animal may respond to some degree to the 
reinjection with other animals' sera but it always responds with 
smaller doses to the serum used for sensitization. For instance, a 
guinea pig sensitized to sheep serum responded to the following 
quantities of other sera: 
Sheep sera 004 c.c. 
Goat sera 004 c.c. 
Beef serum 01 c.c. 
Swine serum 6 c.c. 
Human serum 1.0 c.c. 
Horse serum 2.0 c.c. 
Chicken serum No reaction 
The species specificity is well marked in the case of bacterial 
proteins. 
The specificness of the various fish proteins is not well marked. 
208 LECTURE NOTES ON BACTERIOLOGY 
The serum of man and the ape, or the horse and the ass, or 
the sheep and the goat, or the rat and the mouse are indistinguish- 
able by anaphylactic shock, though the serum of the rabbit and 
hare are. 
Passive Anaphylaxis--Anaphylaxis may be induced passively 
by the injection of a second animal not necessarily of the same 
species with the serum of a sensitized animal. If optimal quan- 
tities of serum are transferred and proper provocative doses given, 
the passive anaphylactic reaction may be excited immediately. 
If an animal recovers from the anaphylactic state it becomes 
refractory to a second injection of the serum toward which it was 
previously sensitive. This refractory state may also be induced 
by very large doses of the antigen, or serum causing the ana- 
phylaxis, administered during the later stages of the incubation 
period, i. e., just before the onset of the sensitive period. 
Sometimes it is possible to induce the refractory state pas- 
sively, i.e., by the injection of the serum of a refractory animal, 
but usually the injection of the serum of a refractory animal 
induces sensitiveness instead of the refractory state. 
Explanation of the Phenomenon of Anaphylaxis-The explana- 
tion of anaphylaxis can only be satisfactorily made upon the gen- 
eral principles of immunity, and many of the facts regarding the 
conditions of anaphylaxis support such a view. Due to the injection 
of the foreign protein, an immune body to this protein becomes 
produced, which, through the action of the complement in the blood, 
results in the proteolysis of these proteins when next injected. 
Such a hypothesis would explain the temporary loss of complement 
in the reacting animal. It would also explain the possibility of 
inducing passive anaphylaxis. Moreover, it has been discovered 
that there is a specific element in the allergic state which is ther- 
mostabile, while the active element can be inactivated by heat and 
such serum reactivated by fresh serum. Furthermore, there is a 
close analogy between symptoms induced by the injection of the 
albuminoses and peptones, which represent the proteolytic products 
of animal proteins and the symptoms of anaphylaxis. 
The discovery of the facts relative to anaphylaxis has cast quite 
a new light upon the cause of many of the symptoms of certain 
diseases. The striking skin eruptions in the exanthemata, for in- 
stance, are of such a character both as to their time of onset in 
210 LECTURE NOTES ON BACTERIOLOGY 
relation to the incubation period, and their general character, as 
to suggest that their cause may be due to proteolytic products. 
The same may even be said of such a disease as typhoid fever 
and many other diseases as regards their rather uniform incuba- 
tion period, the period of illness, and the eruptions accompanying 
them, etc. 
PHAGOCYTOSIS 
The power of the body to produce within itself specific immune 
bodies capable of making possible reactions which inhibit the 
growth and development of bacteria in the body or render their 
products innocuous, or actually destroy them and their proteins, 
is unquestionably the strongest means of defense which the body 
possesses against disease-causing organisms. Nevertheless, it is not 
the only means of defense. 
Certain cells of the body have retained during the process of 
development of the animal body the power of intracellular inges- 
tion and digestion of foreign bodies and especially of bacteria-a 
power possessed by so many unicellular protozoa and the cells 
lining the body cavity of some of the higher multicellular animals, 
as the coelenterates. 
The cells in the animal body possessing this power are some of 
the white blood corpuscles, especially the polynuclear leucocytes, 
and to a lesser degree, the large mononuclear leucocytes and 
eosinophiles, but probably not the lymphocytes. And in addition 
to these free cells certain fixed cells in the body, those in particular 
lining the serous cavities and blood and lymph spaces. The smaller 
leucocytes are called microphages, the larger mononuclear cells and 
the fixed cells are termed the macrophages; all these cells are col- 
lectively termed, when spoken of in connection with this digestive 
function, as phagocytes, and the processes by which harmful or- 
ganized solid material is removed from the circulation or tissue 
spaces by them, is termed phagocytosis. 
The process of phagocytosis may be studied by injecting cul- 
tures of bacteria into the peritoneal cavity of an animal such as a 
guinea pig, and removing the peritoneal fluid at intervals after the 
injection and examining the same under the microscope. At first 
there is a diminution of the white cells in the fluid. Later there 
212 LECTURE NOTES ON BACTERIOLOGY 
is a marked increase, i. e., in case the conditions are favorable to 
phagocytosis. 
The cells of the exudate, when examined under the microscope, 
are seen to contain the bodies of bacteria within them. By ap- 
propriate stain it can be demonstrated that these bacteria are in 
most cases undergoing degeneration and subsequently dissolution 
in consequence of intracellular digestion. In other cases, in which 
the bacteria have been engulfed but yet have resisted digestion, the 
staining reactions of the bacteria may remain little changed, but 
the body of the leucocyte shows disintegration. Moreover, certain 
bacteria may resist digestion but not ingestion, and various bacteria 
differ in the type of phagocytic cell which they attract. 
Chemiotaxis-The power of bacteria to repel the phagocytes, 
illustrated by the temporary preliminary period of diminished 
cellular exudation, is called negative chemiotaxis. In the case of 
some particularly virulent organisms it is the sole influence pro- 
duced upon the phagocytes. 
The power of bacteria to attract the phagocytes, the more usual 
reaction against the introduction of bacteria into the body, is called 
positive chemiotaxis. 
Relation of Phagocytosis to the Recovery from Disease-It can 
readily be appreciated that this property of the free and fixed 
cells of the body to respond to the presence of bacteria by ingest- 
ing and digesting them, thus removing and killing them, must 
bear a very direct relation to the power of the body to recover 
from infection. 
Metschnikoff long ago showed that there was a direct parallelism 
between the degree of phagocytosis excited and the power of rab- 
bits and guinea pigs to recover from infections by anthrax. In in- 
fections of human beings the degree of leucocytosis is a rather 
accurate guide to the resistance against infection. 
The power of the phagocytes to dispose of harmful bacteria 
by a process of ingestion and digestion of their own, independent 
of other factors, may be by no means the whole explanation of 
phagocytosis. 
Opsonins-It has been demonstrated that substances are pres- 
ent in the sera of animals in which phagocytosis is actively progress- 
ing which act upon the bacteria and render them a more easy 
prey for the phagocytes. These substances are termed opsonins. 
214 LECTURE NOTES ON BACTERIOLOGY 
While phagocytes can proceed without the assistance of the 
opsonins inasmuch as washed leucocytes are capable of phagocytic 
activity, yet these same washed leucocytes are phagocytically far 
more active if to the mixture of bacteria with them there is added 
also serum containing opsonins. 
Specificity of Opsonins-The opsonins moreover are specific; 
that is, each variety of bacteria upon ingestion will cause its own 
specific opsonin, rendering it a more easy prey to the phagocytes 
and having no particular action upon other bacteria or at least 
a very much diminished action on other bacteria. 
In a serum containing a number of opsonins which may have 
been excited by the injections into an animal of several varieties 
of bacteria, each opsonin may be separately absorbed and removed 
by the bacteria for which it is specific. 
Opsonins excited in the serum of an animal by the injection 
of bacteria, specific opsonins, in other words, are called immune 
opsonins. Opsonins, however, may be found in normal serum 
quite apart from immunization. These are called natural opsonins. 
The Relation of the Humeral Explanation of Immunity to 
Phagocytosis-Attempts have been made to identify opsonins with 
both complement and amboceptors. Certain it is that some op- 
sonins, both natural and immune or excited opsonins, are thermo- 
stabile, while others are thermolabile. Moreover, the failure of the 
thermolabile opsonins to correspond with true immune bodies has 
been confirmed by a number of observers. Still other observers, 
particularly those who attempt to explain all immunity as a 
process of phagocytosis, claim that the immune body and comple- 
ment are really derivatives of the leucocytes. 
They have shown, for instance, that the immune body is par- 
ticularly plentiful in such organs as the spleen, lymph nodes and 
bone marrow. 
Levaditi claims to have demonstrated the absence of comple- 
ment in blood plasma in which no destruction of leucocytes has 
taken place. 
The general evidence, however, supports the view that in the 
fight against infection phagocytosis constitutes a force which is 
independent of that power of the body which produces the various 
immune substances which are found in the serum. IIow far inde- 
pendent these two activities of the body are, cannot positively be 
stated. , . 
216