Laboratory 
Technician’s Manual 
Medical Department 
Enlisted Technicians School 
Brooke General Hospital 
Ft. Sam Houston, Texas 
Third Edition, 31 May 1944. 
1290—SAaSFD—^S-5-44—250 LABORATORY BOOK laboratory teg hnici ans manual 
THIRD EDITION 
TABLE OF CONTENTS 
Chapter 
PART I 
Page 
The Microscope (Illustration Courtesy Spencer Lens Co,), ... . 1 
Anatomy and Physiology 2-15 
Hematology. , . . . * 16-34- 
Blood Typing 35-4-0 
Cerebrospinal Fluid 41-4-5 
Urine. 46-58 
Sputum . 59-61 
Gastric analysis . ....... 62-64 
Antigens, antibodies & Immunity ) , 65-90 
Serological Tests for Syphilis ) 
Notes 91-96 
PaET II 
Bacteriology ....... 98-198 
FART III 
General Chemistry 1-43 
Blood Chemistry 44.-52 
PaRT IV 
Parasitology . * Sec, I & II 
Parasitological Methods Sec, III 
Examination of Feces ) 
Some Arthropod Vectors of Disease, Sec, IV 
COMPILED BY LABORATORY SECTION 
FRUITED AND BOUND BY PUBLICATIONS SECTION 
MEDICAL DEPARTMENT ENLISTED TECHNICIANS SCHOOL 
BROOKE GENERAL HOSPITAL, FORT SAM HOUSTON, TEXAS 
31 May 1944 MICROSCOPE 
The microscope is an instrument which magnifies minute 
objects for visual inspection, and is to be considered an 
instrument of precision. 
Binocular microscope is a microscope with two eye pieces 
permitting the use of both eyes. 
A compound microscope is one that contains two or more 
lenses. 
Simple microscope is one that consists of a single lense 
or of several lenses that act as one. 
The constituent parts of a laboratory G.I. compound micro~ 
scope are; 
1. piece; may be cf any magnification, the usual ones 
are; 
a. 6X. 
b. 10X. 
2. Body tube; the body of the microscope. 
3. Draw tube; an extending tube at the upper end of the 
body tube. 
4. Pinion Heads. 
a. Coarse adjustment. 
b. Fine adjustment is made by the micrompter head, 
5. The Rack; is a type of gear attached to the tube 
and is the means by which the pinion can make the adjustments. 
6. The Revolving Nose Piece; is at the lower end of the 
body tube and holds the various objectives. 
7. The objective is a group cf lenses contained in the 
small piece at the lower end of the body tube, 
a. Lower power, 
b. High power (dry). 
c. Oil immersion. 
8. The stage is that part of the microscope on which is 
placed the object that is to be viewed. 
9. Mechanical stage is the mechanism attached to the stage 
which enambles by the use of pinions the movement of the object 
which is being viewed with accuracy and ease. 
10. The condenser is the mechanism under the stage, the 
lenses of which focus the light on the object on the stage. Also 
contains an iris mechanism by which the quantity of light can 
be accurately controlled. 
11. The mirror is below the stage at some distance and 
contains both a concave and a flat surface, 
12. The entire mechanism is held together by the handle, 
pillar and base of the microscope. Mechanical Features of the Microscope 
First, place the micrometer disc in the proper position in the eyepiece. The engraved scale should be downward or in the 
plane of the diaphragm. Place a stage micrometer (Cat. No. 400) beneath the objective. Count the number of divisions of the 
image of the stage micrometer that lie between either adjacent lines of the eyepiece micrometer or a known number of gradua- 
tions of the eyepiece micrometer. It is preferable to use as many graduations of the eyepiece micrometer as is convenient. 
DIVIDE THE LENGTH USED OF THE STAGE MICROMETER BY THE LENGTH USED OF THE EYE- 
PIECE MICROMETER. 
It is important to recognize that the graduations of the stage micrometer usually are hundredths of a millimeter and the 
graduations of the eyepiece micrometer usually are tenths of a millimeter or twentieths of a millimeter. As an example, 
if an eyepiece micrometer that is graduated in tenths of a millimeter is used, and five divisions of the eyepiece micrometer 
are found to take in two and one-half divisions of the stage micrometer, then what is usually called the reduction factor of 
.025 
this eyepiece micrometer is determined by the fraction ——- which is .05. 
This reduction factor must be determined for each combination of objective and eyepiece that is used. Once determined 
it can be used continually with this combination, with the same length of draw tube. 
In using this factor, the number of graduations of the eyepiece micrometer, that measure the length of an unknown 
object on the stage, is determined. This number of graduations is multiplied by the reduction factor, giving the actual length 
in millimeters of the unknown object. 
METHOD OF CALIBRATING MICROMETER DISCS FOR EYEPIECES Path of light rays through the Microscope 
M-81-IV 343 
PRINTED IN U. S. A. ANATOMY AND PHYSIOLOGY 
I. Definitions 
1. Cell - a cell is the simplest unit from which all living things 
are built. Each cell has an outer membrane, cytoplasm and 
a nucleus. 
2. Tissue - a tissue is a group of cells, similar in origin, 
structure and function, together with the substance between 
the cells, 
3. Organ - an organ is a group of tissues which are united together 
in one unit for the performance of a special function or 
work. 
4-. A system - a system is a group of organs associated together to 
perform a special function (work). 
5. Anatomy - anatomy is the study of the structure of the body and 
the relation of the different tissues and organs of the 
body to one another. 
6. Physiology - physiology is the study of the workings of the vari- 
ous organs and systems of the body, both independently 
and together in life. 
II. The Systems. 
1. The Skeletal System is composed of bones and joints. The skeleton 
is the bony framework of the body, gives it stability and form, 
and protects the organs, while the joints permit of motion. Bone 
is composed of about one third animal matter, mostly gelatin, and 
two-thirds mineral matter, chiefly lime salts. The animal 
matter gives bone its toughness and elasticity, while the mineral 
matter gives it its hardness. Bones may be classified as long. 
short, flat and irregular. ' 
The long bones, of which the thigh bone, and shin bone are 
examples, form a system of levers which support the weight of the 
body and provide means of moving about. The short bones, as those 
of the wrist and ankle, are found where strength and limited 
motion are needed. The flat bones, as those of the skull, serve 
chiefly for protection. The irregular bones are illustrated by 
those of the pelvis. 
2. The Muscular System is composed of muscles. The muscles provide 
the power to move the bones and thus make it possible for us to 
move about, pick up food, chew it and swallow it and so forth. 
Muscles have the power of contracting only, as a rubber band. 
They cannot push or shove, only pull. What then stretches them? 
For each muscle that pulls one way, there is another muscle which 
pulls in the opposite direction by system of levers and pulleys. 
For example, take the flexors and extensors of the forearm. The 
flexor muscles flex or bend the forearm at its joint, the elbow, 
while the extensors straighten or extend the forearm. There are, 
of course, other types of muscles which can only be mentioned and 
not explained, because of the shortness of the time. They are 
abductors, adductors and rotators. The aforementioned muscles all come under the general heading of voluntary muscles or those moved at 
will. The involuntary muscles are those like the heart and muscles of 
the stomach and intestine, which work without our thinking about them, 
3.The Nervous System is composed of the brain, spinal cord, nerves and 
ganglia. This is a very complicated system (far beyond the scope of this 
course) which enables man to think and to make the complicated machine, 
which our body is, work together as a coordinated whole. The brain, 
situated within the cranium, is the ’’seat" of all intellect and will, and 
the central station from which all the orders for motion are sent out and 
to which all the reports called sensations are forwarded. The spinal 
cord extends downward from the brain through the spinal canal and is 
largely a bundle of nerves or wires like a big cable. The nerves branch 
off from the spinal cord to all parts of the body. The ganglia are 
small masses of nervous tissue arranged in pairs along the spinal 
column and in groups about the heart and great viscera; they have to do 
with the involuntary musculature of the heart, lungs, blood vessels, 
gastro-intestinal tract, and the great viscera. 
Wien a person wants to walk, for example, messages are sent from the 
brain down the spinal cord, out the nerves to the muscles of the legs and 
feet; also by different nerves to the muscles of the arms, so that they 
will swing with the legs; and by yet other nerves to the muscles of the 
back for balance. 
The special senses are special modifications of the nervous system. 
They are - touch, located generally in the skin, but more especially in the 
finger tips; taste, smell, hearing and sight, 
4-.The Circulatory System is made up of the heart, blood vessels, lymph 
vessels, the blood and the lymph. This system brings food and water to 
the various organs, tissues and cells, as well as carries off their waste 
products. The driving power, of course, for all of this is the heart, 
the great pump. The human heart, as well as the heart of the higher 
animals, is a four-chambered muscular organ, which weighs about 3/4- of a 
pound. It may be divided into a right and a left side, each with two 
chambers. The right side receives the impure blood returning from all 
parts of the body, and forces it through the pulmonary circulation in the 
lungs, where the blood is purified. The left side of the heart receives 
the pure blood from the lungs and forces it cut the great artery called 
the aorta, which breaks up into numerous smaller arteries, taking the blood 
to all parts of the body. The right auricle or atrium has a thin muscular 
wall and acts chiefly as a reservoir to receive the impure blood returning 
through the great veins. Tfith the contraction of the auricle, the valve 
leading into the right ventricle is opened and the blood flows into the 
right ventricle. Very shortly afterwards the thicker walled right 
ventricle contracts. This closes the valve between the ventricle and the auricle, at the same time opening the valve in the 
pulmonary artery, so that the blood is forced out the 
pulmonary artery to the lungs. The left auricle or atrium 
then receives the blood from the lungs through the pulmonary 
veins and when it contracts the blood passes through a valve 
into the left ventricle. The left ventricle, since it has 
to force the blood to all parts of the body, has a thicker 
muscular wall than the right ventricle. When the left ven- 
tricle contracts it closes the valve to the left auricle 
and opens the valve into the aorta and forces the blood out. 
Although the right and left' sides of the heart have been 
discussed separately, it must be remembered that both auri- 
cles contract together and both ventricles about 0.16 of a 
second later. 
The blood vessels may be classified as arteries, ca- 
pillaries and veins. The arteries carry blood away from 
the heart| they have muscular and elastic walls. The ca- 
pillaries are fine caliber, thin walled vessels which form 
a network for the exchange of food, vrter and gases, with 
the cells of the tissues and organs of the body. The veins 
carry the blood back to the heart. The veins differ from 
the arteries in that they have valves in their walls to 
prevent the back flow of blood and also have thinner muscu- 
lar walls with less elastic tissue. The capillaries form 
a bridge between the small branches of the arteries and the 
smaller veins. Later, when you prick fingers for blood, 
it is from this capillary network that the blood will come. 
The lymph vessels are entirely separate from the blood 
vessels and carry a thin white milky fluid called lymph. 
We cannot take time to discuss this here. The composition 
of the blood will be taken up in another lecture. 
5. The Respiratory System is composed of the air passages, the 
nose, mouth, pharynx, larynx (voice box), trachea (wind 
pipe) and the lungs. The respiratory system is very closely 
associated with the muscular and circulatory systems. It 
is the muscular system, chiefly the diaphragm, which causes 
the air to be drawn into the lungs and it is the circulatory 
system which effects the exchange of carbon dioxide gas 
and water vapor for the oxygen in the fresh air. 
The diaphragm is a dome-* shaped muscle (top of the 
dome up) which lies between the thoracic cage (in*which the 
lungs and heart lie) and the abdomen. The diaphragm sucks 
air into the lungs very much the same as the plunger in a 
pump sucks air into a pump. Since it is dome-shaped, when 
the diaphragm contracts, it flattens out and pulis down like 
the plunger. Since the lungs are very elastic they expand 
as the air rushes in. At the same time the lungs expand 
laterally as the inside of the thoracic cage is made larger 
by the expansion of the chest. The lungs are two in number, 
a right and a left. The right lung is divided into three 
lobes: upper, middle and lower lobes, while the left lung 
is composed of two lobes only: the left upper and left lower 
lobes. The lungs may be compared to a fine sponge. In the 
thin walls separating the numerous air spaces, the pulmonary capillaries run so that there is only a very thin wall 
between the blood and the air. Since there is relatively 
much oxygen and very little carbon dioxide and moisture in 
the fresh air in the lungs, while there is the reverse, 
that is, more carbon dioxide and moisture and less oxygen 
in the impure blood, there is an equalization which takes 
place through the thin membrane separating the blood and 
air. The oxygen passes into the', blood while carbtn dioxide 
and moisture pass out into the air in the lung spaces. In 
this manner, the blood is purified. 
6. The Digestive System is made up of the alimentary canal 
(30 feet), the mouth, pharynx, esophagus or gullet, stomach, 
small intestine and large intestine; and accessory organs 
which produce digestive juices as the liver, pancreas and 
salivary glands. Since the nutritive constituents of the 
blood are constantly being used up in the repair of tissue 
and the production of energy, either as heat or work, it 
is necessary that there should be a constant supply of new 
material. This is done by the digestive system which takes 
food and breaks it down into simpler products for absorption 
into the blood. Foods may be classified into five headings 
according to the alimentary principles which they contain: 
a. Proteins or nitrogenous substances. 
b. Fats. 
c. Carbohydrates or starches and sugars. 
d. Minearals including water and salts, 
e. Accessory food substances or vitamins. 
All of these alimentary principles are necessary for 
Hi u, 
In the mouth, provision is mc.de for the mastication 
or chewing of the food and its admixture, with saliva; be- 
yond this is the apparatus for swallowing, the pharynx and 
esophagus, which convey the food to the stomach, where a 
partial reduction and solution of it takes place. In the 
small intestine the digestion and solution are completed 
with the aid of bile salts from the liver and pancreatic 
juice from the pancreas. The nutritive principles, composing 
the chyme (digested food) ere taken up by the blood capil- 
laries which lie in the intestinal membranes. The unab- 
sorbable or undigested material together with a large amount 
of water, pass on into the large intestine where water is 
absorbed into the blood, thus leaving a more dry waste or 
formed feces which is expelled through the anus. The spleen 
has no direct part in digestion, but it does serve indirectly 
by acting as a reservoir for the storage, in the intervals 
of digestion, of the additional amount of blood needed du- 
ring digestion. Other important functions of the spleen 
are the production of leucocytes, or white blood cells, and 
the destruction of erythrocytes or rod blood cells. 
7. The Excretory System is composed of the kidneys, ureters, 
bladder, urethra, and to a less extent, skin, lungs and 
intestine. In all life processes waste products and poi- 
sons are produced, which, if rot eliminated, are fina.T’v fatal even to the life which produced them. The yeast 
fungus growing in a sugar solution produces a poison, al- 
cohol, which when it reaches a certain concentration, des- 
troys the life of the yeast; so with the human, it produces 
poisons which must be thrown off if the body would live, and 
the apparatus by which these poisons are eliminated is known 
as the excretory system. 
The kidneys, one on each side, are situated in the 
loins, at the back of the abdomen, on either side of the 
spinal column, and just below the last rib. They are about 
four inches long, and weigh about five ounces each. They 
consist of two portions, a cortex and a medullary portion. 
The cortex or outer portion consists of mime rous groups of 
capillaries called glomeruli where water and salts are filter- 
ed from the blood into the tubules. Farther down these tubu- 
les, the secreting epithelium, with which the tubules are lined, 
takes from the blood urea and other waste products to complete 
the urine. The medulla or inner portion of the kidney consists 
largely of a number of urinary tubules which collect the urine 
from the various units of the cortex and empty the urine into 
a funnel-shaped sac, situated in a depression on the inner 
side of the kidney, called the pelvis. The pelvis has a con- 
stricted neck which is the starting point of the ureter. The 
ureters are two muscilo-membranous tubes about the size of a 
goose quill, which extend from the pelvis of the kidney to the 
urinary bladder. 
The urinary bladder is a muscular bag situated behind 
the pubis and directly in front of the rectum. When moderately 
full it holds one pint and under certain conditions may be 
extended to hold a quart or more. The urethra is eight or 
nine inches long in the male and extends from the neck of the 
bladder to the meatus which is the external opening; surround- 
ing the urethra at the neck of the bladder is a pear-shaped 
gland called the prostate. This can be felt through the rec- 
tum which is directly behind it. In gonorrhea or "clap" it 
is this gland, the prostate, which retains the gonoccocci or 
the causative organisms. 
The excretions of the skin will be taken up later. The 
excretion of the lungs, moisture and carbon dioxide has already 
been mentioned. The excretion of the intestine, namely, feces, 
is only mentioned in passing. 
The average man passes about fifteen hundred cubic cen- 
timeters or three pints of urine a day. The composition of 
urine will be taken up later. 
8, The Reproductive System is included here only for completeness. 
In the male it consists of paired organs; the testes, tho 
epididymis, the vas deferens, the seminal vesicles, the ejacu- 
latory ducts; unpaired parts are the prostate, urethra and penis. 
In the female they consist of the paired organs, the 
ovaries and oviducts; and the unpaired, uterus, or womb, the 
cervix and vagina. 
9. The Endocrine System or the glands of intevnal secretion are 
the islet cells of the pancreas, thyroid gland, parathyroid 
glands, suprarenal or adrenal glands, the hypophysis cerebri 
or pituitary, and certain tissues of the gonads or testes and ovaries. This system also is included only for complet- 
ness and need not concern us here. These glands secrete 
certain chemical substances, called hormones, directly in- 
to the blood which have to do with the growth and develop- 
ment of the body and its control in relation to its environ- 
ment. 
10. The Integument is the skin or covering of the body with its 
glands, sebaceous and sweat glands, and its derivatives, 
hair, nails and teeth. The skin is a tough, elastic mem- 
brane which covers the entire body and is continuous at the 
various orifices, as the nose, mouth and anus, with the 
mucous membrane. Anatomically it consists of two layers, 
the epidermis or cuticle, .and the derma or true skin. The 
cuticle is, that part which is raised when a blister occurs 
and which pulls off after Scarlet Fever. It serves as the 
protection for the true skin. 
The derma constitutes the greater part of the thick- 
ness of the skin, and contains the blood vessels, nerves, 
sebaceous (oil) and sweat glands. 
The sebaceous glands secrete an oily substance which 
gives to the skin its softness and pliability; the orifices 
of the ducts of the sebaceous glands are particularly large 
about the face and nose, and when plugged with dirt from 
the familiar "black-heads”. 
The sweat glands are in vast numbers all over the 
body and their orifices constitute what are known as the 
pores. They secrete a variable amount of water, averaging 
about two pints a day, and the water contains organic mat- 
ter and salts, and constitutes the perspiration or sweat. ANATOMY AND PHYSIOLOGY OF THE URINARY SYSTEM 
Now that we are about to delve into the field of urine analysis, 
it is fitting that we should first learn a little about the anatomy 
and physiology of the urinary system. 
To sum it up briefly, the urinary system consists of the kidneys, 
the ureters, the bladder and the urethra whose function it is to eliminate 
from the body certain nitrogenous wastes of the body diluted in water 
and to help regulate the acid base balance, of the blood. 
The kidneys, two in number, are located at the back of the abdomen, 
one on either side of the spinal column just in front of the short or 
floating ribs. The kidneys are bean-shaped and together weigh 300 grams 
or about 2/3 of a pound. Each kidney is covered by a thin connective 
tissue capsule and is surrounded by fat whose connective tissue holds the 
kidneys in place. The side of the. kidney, directed toward the spinal 
column, is indented and is called the hilum. It is through the hilum that 
the artery and vein of the kidney enter. They are also called the renal 
artery and vein since ’•renal” refers to the kidney. Each kidney consists 
of a cortex and a medulla. The cortex is the outer portion in which is 
located the smaller blood vessels, the glomeruli and the small tubules. 
The medulla is the central portion in which the pyramids with their 
collecting tubules, and the larger blood vessels lie. A cross section 
of a fresh kidney appears light reddish brown. In the outer band, which 
is the cortex, numerous small red pin points are seen. These are the 
glomeruli. The pyramids of the medulla, so called because they are shaped 
like an inverted pyramid with their bases toward the cortex and their 
apices toward the hilum, are a little darker brown and are striated, the 
striations being the collecting tubules which converge toward the apex of 
the pyramid. Of course, there is connective tissue which holds the blood 
vessels, glomeruli, small and large tubules together, (Gee Figure I.) 
Surrounding the apex of each pyramid is a cup-shaped structure made 
of connective tissue lined with epithelium, called a minor calyx, which 
receives the urine from the collecting tubules of the pyramids. Several of 
these minor calyces are received into a similar but larger structure called 
a major calyx. There are three of these major calyces which are received 
into a.funnel-shaped structure called the renal pelvis, ’which lies partly 
within the hilum of the kidney. The renal pelvis consists of three layers, 
an inner epithelial layer, a middle muscular layer and an outer connective 
tissue layer. 
The small end of the "funnel” of the renal pelvis is continuous with 
the ureter, which is a small structure about the size of a goose quill which 
extends down along the side of the spinal column at the back of the abdomen 
to the brim of the pelvic cavity where it passes along the lateral wall of 
the pelvis, then dips downward, then toward the mid line and finally up- 
ward to enter the bladder on its under surface. The two ureters enter the 
bladder about 1-1/2 inches apart. The ureter also consists of three layers; 
an inner epithelial layer, a middle muscular layer and an outer connective 
tissue layer. The muscular layer is composed of an inner longitudinal and 
an outer circular layer of muscle. The urinary bladder is a muscular bag lined with epithelium which is 
situated behind the pubis and in front of the rectum. In the female the 
uterus is between the two. The muscular coat of the urinary bladder is 
composed of an inner longitudinal, a middle circular and an outer longi- 
tudinal layer of involuntary muscle. The ureters pass through the muscu- 
lar wall of the bladder in a diagonal course, so that when the bladder con- 
tracts the ureters are shut off to prevent the back flow of urine to the 
kidneys. 
The opening of the urethra at the front of the bladder with the two 
openings of the ureters form a triangle called the trigone of the bladder. 
The urethra, which conveys the urine to the exterior, consists of a 
connective tissue tube lined with epithelium. In the female the urethra 
is slightly over one inch long, while in the male it varies up to 10 or 
more inches. The male urethra is subdivided into three parts; the prostatic 
urethra, which is completely surrounded hy the prostate gland; the mem- 
branous portion, about one centimeter long between the prostate and the 
voluntary muscles in the floor of the pelvis, which form the external- 
sphincter; and the longest part, the cavernous or uenile urethra situated 
in the penis. 
Now to go back again, we shall discuss in more detail the structure 
of the kidney so as to get an idea of the physiology of urine secretion, 
can best be understood by a careful study of Figure XI. 
The renal artery and vein enter the hilum (indented portion of the 
kidney) just above the renal pelvis, where they soon break up into 
numerous branches* Now we shall follow the course of the blood. 
The renal artery gives off numerous branches called interlobar ar- 
teries, which pass up between the pyramids. At the top of the pyramid 
the interlobar artery gives off a branch which curves off like an arch 
and is therefore called the arcuate artery. The arcuate artery gives off 
two types of branches; one, the interlobular artery, which is involved in 
the secretion of urine only; and, the interstitial artery which nourishes 
the tissues of the kidney. 
The interlobular arteries pass up in the cortex giving off branches 
called the afferent arteries which break up into capillaries in the glo- 
merulus. Unlike arteries elsewhere, the glomerular capillaries are re- 
ceived into arteries called efferent arteries. Each efferent artery again 
breaks up into capillaries about the proximal and distal convoluted tubules. 
These capillaries are finally received into the stellate veins. The stel- 
late veins empty into the interlobar veins and so on back into the renal 
vein. 
Bowihan’s capsule is really the beginning of the tubular system and is 
really the blind end of a tube which has been invaginated by the 
glomerular capillaries. A study of Fig. II will reveal clearly the tubular 
system. 
RENAL FUNCTION 
In the past 20 or 30 years there has been a great deal of work done 
on renal function. The complete story of the way in which urine is pro- 
duced is still unknown. So far, we know that water, salts, uric acid, 
urea, creatinine, chloride and glucose are filtered out through the glo- 
merular capillaries into Soman’s capsule. In the tubules there is a selective reabsorption back into the blood of glucose, chloride and water 
called threshold substances, by the cells lining the tubules, while non- 
threshold substances, such as uric acid, urea and creatinine are not re- 
absorbed and are passed on out in the urine. At the same time, the cells 
of the tubules form Ammonia from urea which, as the basic radical (NH,) re- 
places the fixed bases, sodium and potassium, in the salts, so that the 
sodium and potassium is saved for the blood. This enables the kidney to 
eliminate acid radicals, partly neutralized by the radical, and 
thus helps to maintain the acid-base balance of the blood. 
Normally the capillary walls are not permeable to tho large pro- 
tein or albumin molecules, but in disease when the supply of oxygen is low, 
the permeability of the capillary walls is altered, so that albumin passes 
through and we have albuminuria or albumin in the urine. 
In diabetes mellitus, when sugar is not burned properly in the blood, 
due to the lack of insulin, the amount of sugar in the blood becomes very 
high. This sugar concentration exceeds the renal threshold so that sugar 
is excreted in the urine. Fig .1 — Sche™ at cc JlAyam 0/ a /ong «rtal median 
Of <x k »d*%ej u reiar. P I JH ScK e** »C JuAq o-f grti 
of a ANATOMY AIM'D PHYSIOLOGY OF THE DIGESTIVE SYSTEM 
Now that we are about to take up the study of stool examinations, 
it is fitting that we take up in more detail the digestive system. By 
this time you should know that the digestive system consists of the 
alimentary canal and such accessory organs as those which produce diges- 
tive enzymes, or ferments, and assist in the handling of the food. The 
accessory organs include the teeth, tongue, salivary, gastric, and intes- 
tinal glands, liver and pancreas* 
Alimentary Canal - This is a long tube, some 30 or more feet in 
length, which extends from the mouth to the anus. It is made up of the 
mouth, pharynx, esophagus, stomach, small and large intestine. 
The general structural plan of the canal is: (1) a lining of mucous 
membrane, in which there are numerous glands; (2) a submucous layer of 
loose connective tissue, into which the glands may penetrate, and in which 
the blood vessels are located; (3) layers of involuntary muscular tissue 
( an inner circular, an outer longitudinal, and, in the stomach alone, 
an innermost oblique layer); and (A) and outer layer which is fibrous in 
nature above the diaphragm and serous below the diaphragm* 
The Peritoneum - This may be defined as the serous membrane wnich 
linos the abdominal cavity and is invaginatod (pushed in) by, or reflec- 
ted over, the viscera. The different viscera in the abdomen lie within 
the peritoneum in the same manner as your fist'would be within a partially 
inflated rubber balloon, if you were to push your fist into it. Layers 
of peritoneum which extend from the body wall to a viscus and suspend it, 
form the mesentery. The great omentum which coversthe viscera like an 
apron is a long double fold of the peritoneum. Unlike the peritoneum 
elsewhere, however, the omentum contains a variable amount of fat tissue 
and lymphatics. The omentum affords protection for the viscera by its 
bulk, acting as a cushion, and through its power to move about it can help 
wall off infection such as from a ruptured appendix. For this reason the 
omentum is often called the “policeman” of the abdomon* See Fig, II and III, 
The Mouth - The mouth is the beginning of the alimentary canal, it 
is composed of the vestibule and the oral cavity. The vestibule is that 
part between the lips and cheeks without, and the teeth within. The two 
communicate just behind the last molar tooth. The oral cavity is that 
part in front of the pharynx, and between the teeth, in which the tongue 
lies. 
The tooth are composed of an outor hard substance called enamel 
which covers the exposed part, and a loss hard substance called comontum, 
covering the root of the tooth, and the ground substance called dentine. 
In addition there is a central canal which contains the pulp tissue and 
the nerve. The full adult sot of teeth is 32 in number or 28 without 
the wisdom teeth. 
* Tongue - The tongue is a mass of muscle covered with a rough 
pappilated mucous membrane. The tongue aids in chewing and swallowing 
the food and is important in talking. The sense organs of taste aro 
located in the tongue. Salivary Glands - These consist of three pairs of glands, namely: 
the parotid, the submaxiliar7 and sublingual. The parotids are located, 
one in front of the lobe of each ear. It is inflammation of the parotid 
gland which we call mumps. The submaxillary glands are located in front 
of and below the angle of the jaw while the sublingual glands are under 
the tongue. The salivary glands secrete saliva which contains an enzyme, 
amylase. Amylase aids in the digestion of starches. 
Pharynx - The pharynx is a muscular tube lined with raucous membrane 
which extends from the back of the nasal cavity to just below the thyroid 
cartilage where the esophagus begins. It communicates with the nose above; 
with the mouth in the middle, and with the larynx below. The pharynx is 
what is commonly called the throat. 
Esophagus - The esophagus is a muscular tube lined with mucous mem- 
brane which extends from just below the thyroid cartilage to the upper 
end of the stomach. It passes through the lower part of the neck where 
it is between the trachea in front and the muscles overlying the verte- 
bra behind. It passes through the thoracic cavity right in front of the 
vertebral column. In the lower part of the thoracic cavity it turns to 
the left slightly and passing in front of the aorta, it passes through 
a hole in the diaphragm, just to the left of the midline, to join the 
stomach on the abdominal side. 
The upper 1/3 of the, esophagus has a little voluntary muscle which 
is continuous with that of the pharynx. The musculature of the lower 2/3 
consists entirely of involuntary muscle - an inner circular and an outer 
longitudenal layer. The mucous membrane of the esophagus contains mucus 
glands only, which lubricate the tube but have no direct connection with 
digestion. 
The Act of Swallowing - In swallowing the bolus of food is flipped 
into the pharynx by the tongue, the nasopharynx being closed off by the 
soft palate, the muscles of the pharynx contract to pass the food on in- 
to the esophagus, where its involuntary muscles pass it on to the stomach. 
At the opening of the larynx there is a structure called the epiglottis, 
which in the act of swallowing, projects out over the opening of the 
larynx to prevent food from going down the trachea or wind pipe. 
The Stomach - The stomach is a large muscular bag lined with 
mucous membrane, which is interposed between the esophagus and the small 
intestine. The upper end of the stomach, called the fundus, lies against 
the concavity of the diaphragm. See Figure I. Most of the stomach lies 
in the upper left quadrant of the abdomen. Its lesser curvature is in 
close relation to the liver. The lower end or phylorus is about in the 
midline and it joins the small intestine just to the right of the midline 
at the pyloric valve. 
The mucous membrane or mucosa of the stomach is thrown up into 
numerous folds called rugae, which give more surface. The gastric glands 
lie in the mucosa; they are composed of two main types of cells: The 
parietal and chief cells. The parietal cells produce hydrochloric acid 
while the chief cells produce protease (pepsin). The muscular coat of 
the stomach consists of three layers: an inner oblique layer; a middle 
circular layer and an outer longitudinal layer. The middle circular lay- 
er becomes thicker at the pylorus to form the pyloric sphincter. Small Intestine - The small intestine has the greatest length of the 
entire alimentary canal and is approximately 23 feet long. It has numerous 
coils. The small intestine is subdivided into three parts: tho duodenum, 
the jejunum, and the ileum, from tho top down. The small intestine also 
has a mucous membrane and a muscular coat composed of an inner circular 
and outer longitudinal layer of involuntary muscle. In tho mucosa, chiefly 
of tho duodenum, arc tho glands which produce the digestive enzymes or fer- 
ments, which complete tho process of digestion. The mucosa is thrown up 
into numerous folds called plicae and those arc further divided into projec- 
tions called villi, all of which tond to increase the digestive and absorp- 
tive surface. 
The lower end of the small intestine or the ileum, enters the large 
intestine in the right lower quadrant of the abdomen. This is called the 
ileo-caocal .junction. 
The Lgrfie Intestine - Starts in the right lower quadrant of the ab- 
domen extends upwards along the right side of the abdomen to the liver, 
where it turns to the left, goes across to the left side, passing just be- 
low the stomach to the spleen where it turns downwards, passing along the 
left side of the abdomen to the pelvis where it takes an tf3” curve toward 
the midlinej then passes downward to tho anus. 
The large intestine or Colon is subdivided into several parts. The 
first part is a blind pouch, about 3 or U inches long, called the caecum to 
which the appendix is attached. The ileum enters the caecum at its upper 
end at the ileo-caocal valve. That part from the iloo-caecal valve to the 
liver is called the ascending colon. The bond at the liver is called the 
hepatic flexure, That part which extends across the abdomen to the spleen 
is called the transverse colon. The bend at trie spleen is called the sploe- 
nic flexure. That part from the spleen down to the pelvis is called the 
descending colon. The ,?S’’ shaped part in the pelvis is the sigmoid colon. 
The straight portion extending from the sigmoid to the anus is the rectum. 
The anus is the external opening only. 
The mucosa of the colon appears smooth to tho naked eye instead of 
being rough like tho small intestine. It has, however, microscopically 
numerous muccous glands. Tho muscular coat has the usual inner circular 
and outer longitudinal muscular layers except that tho longitudcnal layer 
is concentrated into three narrow bands, visible to the naked eye, which 
are called tenia cell. At the lower end of the rectum tho circular layers 
become thickened to form the internal (involuntary) sphincter* At the anus 
there is a sphincter of voluntary muscle. 
Accessory Organs - The salivary glands have already been mentioned. 
The accessory organs situated in the abdomen are the liver and pancreas. 
Liver - The liver is the largest gland in the body* It weighs nor- 
mally to 1500 grams or about 6,5 lbs. The liver is closely applied to 
the under surface of the diaphragm. It consists of two main lobes, a right 
and a left lobe which are demarcated on the surface by the falciform liga- 
ment of the liver. The right lobe is the largest. The left lobe extends 
a short distance to the left of the midline. Biliary System - The biliary system consists of the bile ducts 
both inside the liver and outside of it, and the gall bladder. The right 
and left hepatic ducts. one from each lobe of the liver join to form the 
common hepatic duct. The gall bladder, you might say, is a large out- 
pouching of the common hepatic duct. The gall bladder is connected to 
the common hepatic duct by a narrow duct called the cystic duct. From 
the junction of the cystic duct and the common hepatic duct down, it is 
known as the common bile duct. The common bile duct and the pancreatic 
duct join to enter the duodenum together about 4- inches from the pyloric 
sphincter of the stomach. The gall bladder is a thin muscular bag lined 
with mucous membrane, where the bile, which is formed in the liver, is 
concentrated and stored until needed for digestion. In addition a thin 
mucus is added to the bile by the mucous glands of the gall bladder. 
See Fig. I. 
Pancreas - The pancreas is a long narrow organ which normally weighs 
90-150 grams or about one quarter of a pound. The pancreas lies at the back 
of the abdomen against the vertebral column in the upper part of the abdo- 
men behind, and parallel with, the transverse colon. The head of the pan- 
creas lies in the first bend of the duodenum and the tail lies against the 
spleen. The pancreas has two separate types of glandular tissue. One 
type secretes digestive juices into the pancreatic duct and the other type 
the islets of Langerhans. secrete insulin directly into the blood stream. 
Insulin is the substance which has to do with carbohydrate metabolism and 
it is the lack of it that causes Diabetes meHitus. Thus, the pancreas 
is a gland of both internal and external secretion. 
PHYSIOLOGY OF DIGESTION 
Digestion is the process of getting foods into soluble form, so 
as to fit them for absorption into the blood, so that they may be carried 
to all tissues. This alteration is brought about by enzymes. 
An enzyme is an organic catalyst, produced by a living organism. 
An enzyme increases the speed of a reaction without adding in any way to 
the energy changes involved in the reaction or taking part in the forma- 
tion of the end products. 
Enzyme action is specific. That is, a certain enzyme will act on 
one type of food stuff alone and no other. Enzymes also have an optimum 
temperature and optimum reaction. The optimum temperature of course is 
body temperature. The optimum reaction is acid for the gastric enzymes 
and alkaline for the intestinal and pancreatic enzymes. 
The types of food may be classed as inorganic and organic.- The 
inorganic foods are water and salts, which need no digestion. The organic 
foods are: carbohydrates (sugars and starches), fats, and proteins. In 
addition there are accessory foods (also organic) which are known as 
vitamins. 
Digestion begins in the mouth by the process of chewing the food 
and the salivary enzyme amylase which begins the digestion of starch. After 
the voluntary act of swallowing, which has already been discussed, the bolus 
of food is passed down the esophagus by the involuntary muscles to the 
stomach by peristaltic waves. In the stomach gastric enzymes, protease 
and lipase, with the aid of hydrochloric acid, begin the breakdown of 
proteins and fats respectively. This is done as the food is churned about 
and mixed with the gastric juice. - 14. - The presence of food in the stomach sets up reflexes which cause 
bile to be released from the gall bladder and pancreatic juice from the 
pancreas into the duodenum. When the food in the stomach is well mixed 
with gastric juice the pyloric valve opens and the food passes into the 
duodenum. 
In the duodenum and small intestine the food is mixed with the bile, 
intestinal and pancreatic juice and digestion is completed. The bile 
emulsifies the fats so that they can be broken down into fatty acids and 
glycerol by pancreatic lipase. The proteins are further broken down into 
amino acids by pancreatic and intestinal proteases. The digestion of 
starches and complex sugars into glucose is completed by various enzjnnes 
from both the pancreas and small intestine. 
Now the fatty acids, amino acids and glucose are ready for absorp- 
tion. This takes place through the small capillaries which lie in the 
villi of the small intestine. The majority of the fatty acids are taken 
up by small lymphatics, called lacteals, also situated in the villi. 
The digested, as well as the undigested food and water, is passed 
along the small intestine by peristaltic waves. By the time the contents 
of the small intestine reach the ileo-caecal valve, digestion and absorp- 
tion of the food products is complete. 
In the large intestine the undigested and unabsorbable food is also 
passed along by peristalsis, but at a much slower rate; taking from 2U to 
hours to pass through. In the large intestine the excess water is absor- 
bed and more or less dry formed stools are produced. Anything which in- 
creases the rate of passage through the colon will give a watery stool or 
diarrhea, 
There are numerous bacteria which normally live in the intestinal 
tract chiefly in the colon. These are chiefly the Bacillus Coli group and 
a few gas forming bacilli. As long as these bacteria remain within the 
intestinal tract, they do no harm, but, once free in the peritoneal cavity, 
as from a ruptured appendix or gun shot wound of the abdomen, they set up 
a very serious inflammation called peritonitis, which causes death more 
often than not. 
Defecation. The dried feces, having accumulated in the rectum, 
sets up a defecation reflex. The internal sphincter is automatically 
released. At the convenience of the individual, the external, voluntary 
sphincter is released, the internal abdominal pressure is increased by 
tightening the voluntary abdominal muscles and the contraction of the 
diaphragm. At the same time, the levator ani, a voluntary muscle, which 
is attached to the anal or voluntary sphincter, is contracted, thus lift- 
ing the lower end of the rectum up over the formed stool so that it is 
expelled. ixgure I. A diagram of the digestive system, showing the 
alimentary canal and accessory organs. The 
liver and gall bladder have been turned up, 
toward the head and to the right. Figure II, Reflections of the peritoneum as seen in 
transverse section at two different levels. 
Figure III. Reflections of the peritoneum as seen 
in sagital section. BLOOD 
The functions of the blood and blood-vascular system are to 
receive from the lungs and alimentary tract and to carry to all 
parts of the body the materials necessary for its nutrition and 
proper temperature and moisture, and to carry away to the excretory 
organs the waste matters which, if retained, would prove 
poisonous. It has also important functions in the protection of 
the body from the invasion of the bacteria of disease. 
The total quantity of blood is usually estimated at one- 
twelfth of the weight of the body, or an average of about a gallon 
and a half or six quarts. 
The blood is red in color; bright red in the arteries; dark 
red in the veins. It is composed of cells or corpuscles floating 
in a liquid called the plasma. 
The cells are of three sorts, the red cells or erythrocytes, 
the white cells or leukocytes, and the blood-plates or platelets. 
The red cells are much the more numerous, there being about five 
hundred times as many as there are white. The red cells are very 
small, about one three-thousandth (1/3000) of an inch in diameter, 
and, though red in mass, the individual cells are seen under the 
microscope to be light yellow in color. They are round,flattened 
discs, like a copper cent, except that they are concave on each side. 
They are composed largely of hemoglobin, a substance containing 
iron which has a great oxygen-carrying power. 
Soon you will learn to count the red cells or erythrocytes. 
There are so many in the human body that it is beyond our compre- 
hension; therefore, we count only a very small fraction of them 
and multiply by a factor which gives us an index of the number of 
red cells. The average number for women is four and a half million 
(4.500.000) per cubic millimeter and for men it is five million 
(5,000,000) per cubic millimeter. In order to impress upon you the 
total number of red cells in the body, assuming six quarts or 
approximately six liters for the total volume of blood, there are 
about thirty trillion (30,000,000,000,000) red cells in the body. If 
a person were to count one red cell per second, ten hours a day 
every day, it would take two million seven hundred and forty thousand 
(2.740.000) years to do it. 
To get back to business, the leukocytes or white cells are not 
flat like a cent, but spherical like a ball, a little larger than 
red cells, composed of protoplasm, and capable of changing their 
own form and of making their way through the unbroken walls of the 
blood vessels. The leukocytes may be divided into two main groups; 
these are the non-granular leukocytes, or agranulocytes. which have 
no specific granules in their cytoplasm, and the granular leukocytes 
or granulocytes, which contain specific granules in their cytoplasm. 
Each group will be considered. 
The non-granular leukocytes are divided into lymphocytes and 
monocytes. The lymphocytes are a little larger than the red cells; 
'the distinguishing feature is a large round nucleus surrounded by 
a thin layer of blue cytoplasm in the stained smear. These consti- 
tute twenty to fifty percent of the total number of leukocytes. • V 
The typical monocytes are a little larger than the lymphocytes• 
The nucleus is relatively large and usually indented; the cytoplasm 
is far more abundant than it is in the lymphocytes, has a pale blue, 
ground glass appearance in the stained smear and may have a few 
small red granules. These constitute- three to eight per cent of 
the leukocytes. 
The granular leukocytes always contain granules in their cyto- 
plasm, The granules differ in various classes of granulocytes. The 
nucleus of the granulocytes is polymorphus (having many shapes) and 
is subdivided into lobes. The classes of granulocytes are: (1) 
acidophil or eosinophil; (2) basophil and (3) neutrophil leukocytes. 
The eosinophils are slightly larger than lymphocytes. The 
nucleus usually has two oval lobes. The cytoplasm contains coarse, 
spherical granules which stain red. These cells constitute 1 to U% 
of the leukocytes, 
The basophils are the same size as the eosinophils, have a faint 
shadowy nucleus and are characterized by large, round, dark blue 
granules in the cytoplasm. These constitute 0,25$ to 1% of the 
leukoc3rbe count, 
The neutrophilic leukocytes are about the same size as the 
eosinophils. The nucleus consists of two to five lobes in the adult 
cell and may be half moon band in the young cells. The cytoplasm 
contains fine granules which stain with neutral dyes and have a pale 
lavender color in stained smears. The neutrophils form fifty to 
seventy-five per cent of the leukocyte count. 
The blood-plates or platelots are small, much smaller than a red 
cell, non-rr-ioleatecf, circular discs. They tend to adhere to one 
another and to all surfaces with which they come in contact except the 
undamaged walls of blood vessels. The blood platelets play an im- 
portant part in the coagulation of blood. They release a substance, 
which, together with tissue juice initiates the process. In stained 
smears the platelets appear as pale yellow or pink discs with fine 
blue granules. The average number is 800,000 per cu, mm, with a 
ran?e of 100,000 to 1.. 500,.000 per cu. mm. 
Origin - in the embryo, the erythrocytes are formed in the blood 
islands in the liver and in. the bone marrow. In the adult they are 
formed only in the red bone marrow;. The granular leukocytes and 
platelets also originate from the' red bone narrow, while the lympho- 
cytes -and monocytes originate from the lymphoid tissue. 
Yhe plasma is the fluid ..port ion of the blood. It contains mono 
than one lunarod constituents. The most abundant constituent is 
water. Other constituents are proteins, carbohydrates, fats, inor- 
ganic-salts, gases, waste products, enzymes, immune bodies, hormones, 
etc, -The proteins are fibrinogen, prothrombin, serum albumin and serum 
globulin. The carbohydrate is glucose,. The fats are neutral fats, 
cholesterol esters, and phospholipids. The inorganic constituents 
include the chlorides, carbonates, phosphates and sulfates of sodium, 
potassium, calcium and magnesium. The gases present in plasma are 
oxygen, carbon dioxide and nitrogen. The waste products include 
urea, uric acid and creatinine, 
Seram is the clear straw colored fluid which results when blood 
clots in a flask or test tube. It is plasma less fibrin. The coagulation of blood is necessary to the preservation of life. 
The doty which consists mainly of fibrin, closes the openings of the in* 
jured vessels and prevents long continued bleeding. 
THEORY OF COAGULATION 
It is thought that when blood is shed, large numbers of blood 
platelets disintigrate and release a substance called thromboplastin 
(also obtained from tissue juice). Thromboplastin combines with calcium 
and prothrombin to form thrombin. Thrombin, which is believed to be an 
enzyme, causes a precipitation of the fibrinogen as fibrin. The following 
schema illustrates the events which occur in coagulation. 
Calciumprothrombin -bthromboplastin->thrombin 
Thrombin t-fibrinogen->fibrin 
Fibrin, of course, forms the clot. 
The time required for coagulation or clotting varies from two to 
six minutes in normal individuals. Oxylates and citrates prevent the coa- 
gulation of blood by uniting with the calcium so that the chain of events in 
coagulation is interrupted. 
To summarize, in a general way, the bleed current may be likened to 
a river and the cells to boats floating upon it: the red cells are the 
freight boats loaded with oxygen which they receive in the lungs and carry 
to all parts of the body; the white cells are the war ships, always on 
the alert for an attack by disease germs; when such an attack occurs the 
leucocytes hurry to the invaded point and a battle ensues in which there 
are killed and wounded on both sides; the blood platelets act as a group 
of skilled workmen to stop leaks in the river banks; the dead white cells, 
when in large number, constitute what is known as pus or matter. The blood 
serum itself not only carries nourishment to all parts of the body, but, 
coming back, acts as a sewer, bringing away the waste products, both liquid 
and gaseous. LABORATORY SECTION 
CELLS IN THE NORMAL DIFFERENTIAL FILM 
We have already gone over a little about the anatomy and phy- 
siology of the blood and have been introduced to the various types 
of cells. Now we shall take up the various types of cells a little 
more in‘detail with particular reference to their appearance in the 
film stained with Wright’s stain. 
It has been said with much truth that an intelligent study of the 
stained film, together with an estimation of hemoglobin, will yield 90 
per cent of all the diagnostic information obtainable from a blood ex- 
amination, The stained films furnish the best means of studying the 
morphology of the blood and blood parasites, and, to the experienced 
they give a fair idea of the amount of hemoglobin and the number of 
red and white cells. An oil immersion objective is required. 
Erythrocytes. - Normally, the red corpuscles are acidophilic. This 
means that they take the acid stain, which, in Wright’s stain is eosin, 
a red dye. However, in well stained smears tjie red cells are a pale 
yellow or pink. When not crowded together, they appear as circular, 
homogeneous disks, of nearly uniform size, lb any normal blood, how- 
ever, there may be a slight individual variation. The center of each 
cell is somewhat paler than the periphery (outside). Red cells are apt 
to be crenated, that is, wrinkled from loss of water, when the film has 
dried too slowly. 
The depth of staining furnishes a rough guide to the amount of he- 
moglobin in the corpuscles. When hemoglobin is diminished, the central 
pale area becomes larger and paler. This condition is known as hypochro- 
mia. In pernicious anemia, on the other hand, as a result of the increa- 
sed hemoglobin content, many ofthe red cells may stain deeply and lack 
the pale center entirely. 
Lymphocytes are small, mononuclear ( i.e,, one nucleus) cells, 
without specific granules in the cytoplasm. Some of the larger lympho- 
cytes, however, may have a few, usually 5 to 10, rounded, discrete, red- 
dish-purplo granules in the cytoplasm. The lymphocytes are about the sizi 
of a red cell or slightly larger, although their diameter is influenced 
to a great degree by the thickness of the film, being greatest in very 
thin films where the cells are flattened out. The typical lymphocyte 
is a cell with a single, sharply defined nucleus containing heavy blocks 
of chromatin, staining blue with Wright’s stain, while the parachromatin 
stains pink, and the cytoplasm a robin's egg blue. The characteristic 
feature of the lymphocyte nucleus is that there is a gradual transition 
between the chromatin and the parachromatin, so that it is practically 
impossible to tell where one stops and the other begins. The nucleus 
is generally round, but is sometimes indented at one side. Larger lym- 
phocytes, nearly twice the size of a red cell, with paler nuclei and 
mpre abundant cytoplasm, are frequently found, especially in the blood 
of children, and are difficult to distinguish from monocytes. It is be- 
lieved that the larger forms are young lymphocytes which become smaller 
as they grow older. Lymphocytes form 20 to 50 per cent of the normal 
differential count. 
Monocytes - Under this heading we include the two typos which have 
long been known as large mononuclear and transitional leukocytes. They 
are merely different forms or ages of the same cell. 
19 The monocyte is the largest cell of normal blood, being generally 
two to three times the diameter of a red cell, although smaller indivi- 
duals are sometimes encountered. It contains a single nucleus, which 
is lobulated, deeply indented, or hersehoeshaped, or less often, rounded 
or oval, and which is commonly located away from the center. 
The zone of the cytoplasm surrounding the nucleus is relatively wide. 
With bright1s Stain the characteristic feature of the nucleus is for the 
chromatin to be in strands. There is also a relatively sharp distinc- 
tion between the chromatin and the parachromatin, which results in a less 
densely stained nucleus than that seen in the lymphocyte, while the cyto- 
plasm is slate colored or has a ground glass appearance. The cytoplasm 
sometimes appears dusted, uniformly or in patches, with fine reddish granu- 
les which are much less distinct than the granules of neutrophiles, and 
smaller than those occasionally seen in lymphocytes. The size of the 
cell, the width of the zone of cytoplasm, and the depth of color (greater 
in lymphocytes) of the mucleus, are points to be considered in distinguish- 
ing between those monocytes which have a round nucleus and lymphocytes, 
but it must be remembered that the thickness of the film has a marked in- 
fluence upon the apparent size of all leukocytes. They appear larger and 
paler when flattened out in very thin films. Also, another differential 
point is that the chromatin of the lymphocyte nucleus appears to be in 
larger blocks while that of the moncyte nucleus tends to be in strands. 
The monocytes constitute, normally, from three to eight per cent of the 
differential count. 
Neutrophils - There is usually no difficulty in recognizing these, 
cells. Their average diameter is slightly larger than that of a lymphocyte. 
The nucleus stains rather deeply, and is very irregular, often assuming 
shapes comparable to letters of the alphabet, E,Z,S, and so forth. Fre- 
quently there appears to be several nuclei, hence the widely used name, 
"polynuclear leukocyte". Upon careful inspection, however, delicate nu- 
clear bands connecting the parts can usually be seen. The cytoplasm is 
relatively abundant and contains great numbers of fine, neutrophilic gran- 
ules. With Wright1s Stain the chromatin of the nucleus is purple and the 
cytoplasmic granules are lilac, while in the well-stained preparation the 
cytoplasm itself is light pink or acidophilic. 
In infections and inflammatory conditions, notably in pneumonia and 
appendicitis, a comparison of the percentage of neutrophils with the total 
leukocyte count yields more information than a consideration of either alone. 
In a general way, as was pointed out by Sondern, the percentage represents 
the severity of the Infection or, more correctly, the degree of toxic ab- 
sorption; while the total count indicates the patient's power of resistance. 
With moderate infection and good resisting powers the leukocyte count and 
the percentage of neutrophiles are increased proportionately. When the 
neutrophilic percentage is increased to a notably greater extent than is 
the total number of leukocytes, no matter how low the count, either very 
poor resistance or a very severe infection may be inferred. The neutro- 
phils constitute 50-75 per cent of the differential count. 
Eosinophils - The structure of these cells is similar to that of a 
neutrophils, with the striking difference that, instead of fine neutrophi- 
lic granules, their cytoplasm contains coarse, round or oval granules hav- 
ing a strong affinity for acid stains. They are easily recognised by the 
size and color of the granules which stain bright red. Their cytoplasm 
has generally a faint, sky-blue tinge, and the nucleus stains somewhat 
less deeply than that of the neutrophil. The eosinophils constitute 1-A 
per'cent of x.he differential count. Basophilfe - In general, these resemble neutrophils, except that the 
nucleus is less irregular (usually merely indented or slightly lobulated) 
and the granules are larger and have a strong affinity for basic stains. 
They are easily recognized by the large, round or oval, dark blue or deep 
purple granules. The nucleus is much paler and often nearly or quite hidden 
by the granules. These constitute 0.25 - 1 per cent of the differential 
count. 
Platelets - With Wright's stain they appear as spheric or ovoid, red- 
dish to violet, granular bodies, about half the size of a red cell. The 
granules in the platelets usually are dark blue or purple. In ordinary 
blood smears they are usually clumped in masses. A single platelet lying 
on a red cell may be easily mistaken for a malarial parasite. 
Platelets are not counted in the differential count, but it is a 
good thing to notice them and become familiar with the normal relative number 
in the blood film, so that marked decreases will be readily noticed. The 
normal platelet count is 500,000-1,500,000 per cu mm., with an average 
normal of 800,000 per cu. mm. 
MOTS? This figure varies somewhat, depending on the method #f counting 
used. The figures given here are those for the method given #n 
page 30, COLLECTION OF BLOOD COUNTS 
Read the slip carefully - avoid collecting counts not ordered; 
as for example, no red count is wanted when a WBC is ordered. 
Blood counts are ordered as follows: 
CBC means a complete blood count; red, white, hemoglobin and two 
blood smears; 
RBC & Hg ~ a red count and hemoglobin; 
WBC - a white count and two blood slides; 
Leukocyte and differential - the same as a WBC. 
COLLECTION OF THE COUNT 
(1) Always be certain you are sticking the right patient. 
(2) Get apparatus ready before sticking patient. 
(3) Sterilize the needle before sticking patient. 
(4) Wash patient’s finger with alcohol. 
(5) Make a quick but deep stick into the finger. 
(6) Wipe away the first drop of blood because it is mixed with 
alcohol and makes the count inaccurate. 
(7) Moderate pressure on the finger should bring enough blood to 
collect the various counts. Do not press too hard as extreme pressure 
causes tissue juice to become mixed with blood. If necessary stick the 
patient again. 
(8) It is best to make the blood smear first because some tissue 
juice tends to come with the first few drops of blood and make the red 
and white counts inaccurate. 
(9) The red, white and hemoglobin may be collected in any order. 
There is no special reason for collecting a white before a red or vice 
versa. 
(10) For the white count - draw .the blood to the ,5 mark on a white 
pipette and dilute to the 11 mark with 3% Acetic acid. 
(11) For the red count - to the ,5 mark on a red pipette and dilute 
to the 101 mark with Hayems fluid. 
(12a) Talquist. 
(12b) For hemoglobin - draw the blood to the 1.0 mark on a hemoglobin 
pipette and dilute to the mark with Sahli. If the patient's blood is very 
pale, draw it to the 20 mark. 
(13) Always write the patients name and date on tha blood smears 
before you leave the bedside of the patient. (14.) Wrap the slip around the pipettes and blood smears and fasten 
with rubber band. This is especially important, if you are collecting 
several counts so as to avoid the error of getting counts mixed. 
(15) If you have collected a count containing hemoglobin, always 
write the time of collection on the slip when you come into the laboratory, 
as hemoglobin estimation must stand 30 minutes before they can be read in 
the colorimeter.* 
SOLUTIONS IN HnLOTQLQGY 
1. Hayems - for RBC 
5 grms of Mercury Bichloride 
10 grms of Sodium Chloride 
50 grms of Sodium Sulfate 
2 liters of distilled water 
2, acetic i-icid Jfo for RBC 
30 cc. Glacial Acetic acid 
970 cc. distilled H20 
3. Sahli - for hemoglobin 
10 cc. hcl to 990 cc. 
distilled rUO 
THa ERYTHROCYTE COUNT - R.3.C. 
The Hayems solution preserves all the cells, so that both red and white 
cells are counted but as there is only about one white cell to 1000 red cells 
this does not make the count so inaccurate. The total number of red cells 
counted is multiplied by a factor to make up for the dilution and for the 
fraction counted. 
1, Shake the pipette vigorously for one minute to insure an even dis- 
tribution of cells. This is especially important in red cell counts. 
2. Blow out the contents of the stem of the pipette and fill the 
counting chamber by holding the pipette at the margin of the cover slip 
and letting a drop run under. Avoid letting the fluid run into the grooves 
of the chamber. 
3. Count red cells under high dry lens. 
4. Count 5 of the squares, one at each corner and the one in the 
center. There should not be difference of more than 10 in any of the 
squares counted. Calculate the red cells by adding 4 ciphers (0000) to the 
total counted. 
Note: Corpuscles which touch the lower and right sides should be counted as 
if within the squares, those touching the upper and left sides, as outside. TECHNIQUE OF BLOOD COUNTING - W.B.C. 
The leukocyte count - The acetic acid used in diluting the blood 
for the leukocyte count dissolves the red cells and leaves the white 
cells to be counted. This solution is placed in a hemocytometer, or 
counting chamber and counted under the microscope. The total number is 
multiplied by a factor to make up for the dilution and for the fraction 
counted in the chamber. 
1, Shake the white pipette vigorously for about one minute to in- 
sure thorough distribution of the cells. 
2, Fill the counting chamber by holding the pipette against the 
edge of the coverslip and letting enough fluid run under to just cover 
the rulings on the chamber. Avoid letting the fluid run into the grooves 
of the chamber. Always expel a few drops of the fluid before filling the 
chamber because the solution in the stem of the pipette does not have the 
accurate dilutions as that in the bulb. 
3, Count the white cells under low power lens. 
X, Count the cells in the A outer corners of the chamber and multiply 
the total number by 50. There should not be a difference of more than 10 
in any of the A squares counted. If this happens refill the counting 
chamber and make another tabulation. 
SOURCES OF ERROR 
The most common sources of error in making a blood count are; 
(a) Inaccurate dilution, usually from faulty technic, occasionally 
from inaccurately graduated pipets. Only an instrument of standard make 
can be relied upon, and it is best to purchase one which has been tested 
by the United States Bureau of Standards. 
(b) To slow manipulation, allowing a little of the blood to coagulate 
and remain in the capillary portion of the pipet. 
(c) Inaccuracy in depth of counting chamber usually due to imperfect 
application of the cover glass, but sometimes to faulty manufacture or to 
softening of the cement by alcohol or heat. A cemented slide should not be 
cleaned with alcohol or left to lie in the warm sunshine. 
(d) Uneven distribution of the corpuscles. This results when the 
blood has partially coagulated, hen it is not thoroughly mixed with the 
diluting fluid, 
(e) The presence of yeasts, which may be mistaken for corpuscles, 
in the diluting fluid. 
color; index 
This means the amount of hemoglobin in the average red blood cell of the 
patient compared with the normal amount. p , . , Hemoglobin per cent 
number of reel blood cells per cent 
Considering 5,000,000 per cu. mm.of red blood cells as 100$, the per- 
centage of normal may, be obtained by multiplying the first two figures 
of the red count by two. 
Example 
1. Red blood cells 
2. Hemoglobin 
Color index 50x2 
5,000,000 
100$ 
A normal color index ranges from 0.85 to 1.15. 
MAKING AND STAINING BLOOD FILMS OR SMEARS 
SPREADING THE FILM - Properly spread films are essential to 
accurate work. They more than compensate for the time spent in learning 
to make them. There are certain requisites for success with any method. 
(a) The slides and covers must be perfectly clean; new slides should be 
soaked in a 10 per cent solution of acetic acid, and then rinsed in clean 
distilled water, and dried; old slides are cleaned by thoroughly washing 
with soap and water, rubbinb with alcohol and drying on a clean towel; (b) 
the drop of blood must not be too largo; (c) the work must be done quickly, 
before coagulation begins. 
The blood is obtained from the finger tip, as for a blood count; 
only a very small drop is required, usually about twice the size of a pin- 
head. The size of the drop largely determines the thickness of the film. 
The proper thickness will depend upon the purpose for which the film is 
imade. For the structure of blood cells and the malarial parasite it should 
be so thin that throughout the greater part of the film, the red corpuscles 
lie in a single layer, close together but not overlapping. For routine 
differential counting of leukocytes a film in which the red cells are piled 
up somewhat is best because the leukocytes are more evenly distributed, and 
because the number of leukocytes in a given area is greatly increased and 
the tedium of counting is correspondingly lessened. The film must not, upon 
the other hand, bo so thick that identification of the various leukocytes 
becomes difficult. In some cases of severe anemia it is very difficult to 
make good films owing tp the large proportion of plasma, which leads to 
slow drying, with consequent distprtion of the red cells and the appearance 
of artifacts. To overcome this the films should be made very thin and dried 
quickly over a low flame. 
WRIGHT'S STAIN 
1, 0.1 grm Wright’s stain. - Eosin-red-acid dye 
Methylene Blue - Basic dye 
2, „60 cc. Methyl Alcohol, THE DIFFERENTIAL COUNT - STAINING OF 
THE, BLOOD SMEAR 
1. Cover the slide with Wright’s stain and let stand for 3 or 
U minutes (depending on the ago of the stain, the older the stain, the 
less time required.) 
2. Add about half the amount of buffer solution as you did the 
stain. Leave buffer on for the1,same length of time as the stain. 
3, Wash the slide with distilled H2Q, remove excess stain from 
the back of the slide and let dry. 
In case of over-staining, pour a few drops of methyl alcohol 
on the slide and wash with distilled water. 
EXAMINATION ‘OF THE STAINED SMEAE 
1. Place a drop of cedar oil on the slide and examine with the 
oil immersion lens. 
2. Pick out an area with good distribution and staining of the 
cells. 
. 3. Using the mechanical stage work from the top of the slide to 
the bottom in a straight line then move the slide to one side with the othei 
adjustment the width of one field and then work up to the top thus: 
until 100 white cells have been counted and classified. 
A. In order to make it easier to count exactly 100 w.b.c, the 
following scheme should be used. Make a rough chart with $ horizontal 
columns and 10 vertical columns. The tally of each kind of w.b.c, seen is 
kept in the appropriate horizontal column. If you are careful not to put 
more than 10 tallies in any one vertical column, by the time you have 10 
tallies in the 10th vertical column, you will have counted 100 cells. See 
example below; THE SCHILLING COUNT 
The Schilling count is a special kind of a differential leuko- 
cyte count where, in addition to classifying the five types of leuko- 
cytes, the immature cells of the myeloblastic series are also recorded. 
In cases with leukocytosis the Schilling count tells the doctor the re- 
lative maturity or immaturity of the leukocytes. See the following chart. 
Nucleoli 
blue. 
No granules 
Nucleoli gone 
Few granules 
in cytoplasm 
Nucleus still round 
Many granules 
in cytoplasm 
Slightly indented 
Nucleus 
Deep Indentation 
Two lobed Nucleus 
* 
Thpee lobed 
Four lobed 
Five or more lobes 
Myeloblast 
Premyelocyte - 
Myelocyte - - 
Juvenile - - 
Band - - 
Segmenter - - 
Segmenter - - 
Segmenter - - 
Segmenter - - EXAMINATION OF RED CELLS ON THE BLOOD SMEAR 
This slide is stained the same as for the differential count. 
Red cells are examined for; 
1. Hypochromasia-paleness in the center of the cells. 
2. Poikilocytosis-irregularity in shape. 
3. Anisocytosis-irregularity In size, 
2U Basophilic stippling-small bluish-black dots on the red colls, 
5- Poly-ehroraatophilia-slate grey color of the cells. 
6. Appearance of immature red cells-erythroblast, pronormoblast 
and normoblasts. 
Erythroblast - stem cell of red series; large cell with a 
blue-grey mottled nucleus and deep blue cytoplasm. 
Pronormoblast - cell size a little smaller, nucleus more 
compact, cytoplasm relatively larger and brownish-blue. 
Normoblast - cell size, same as mature erythrocyte; nucleus 
small, dark and compact, cytoplasm pink, same as mature 
erythrocyte 
Nucleated red cells are premature cells thrown into the 
blood stream to replace used up erythrocytes. They are most abundant 
in myelogenous leukemia and severe secondary anemia. 
ERYTHROPOISSIS 
Erythropoiesis or red cell production takes place in the bone 
marrow. At birth all the bone marrow of the body is active. As the 
person grows older, there is a change to inactive fatty bone marrow. 
The active bone marrow of the adult is limited to the vertebra, bones 
of the skull, ribs, sternum and innominate or hip bones. Blood 
vessels in the bone marrow run longitudinally until they split into 
branches and run vertically. These branches are called blood sinuses. 
Bed cell formation takes place within the inter-sinusoidal capillaries 
or sinuses. Conditions favorable to red cell formation are low oxygen 
supply and reduced circulation. Endothelial cells line the sinuses. 
These cells swell and divide. If the mitotic spindle is in one 
direction, the cells form two more endothelial cells; if in the other 
direction it forms the next stage, the megaloblast - young 
erythroblast - normoblast reticulocyte - normal red cell. 
CLOTTING TIME 
1. Cleanse patient’s finger with alcohol and stick. 
2. Discard the first drop of blood. 
3. Hold a small capillary pipette against the finger so that a 
drop of blood flows into the capillary pipette. 4. Note the time the sample of blood was taken. 
5. Break.off small bits of the pipette at intervals of 30 
seconds until a thread of fibrin can be seen. Note the time this 
Occurs, The clotting time is figured by the length of time from 
the moment the pipette is filled to the time the thread forms. 
FACTORS IN CLOTTING TIME 
(Bancroft & Quick) 
Prothrombin, platelets (which release thrombokinase) calcium 
and'fibrinogen are the blood clotting factors in normal blood. 
Prothrombin plus calcium plus thrombokinase equals thrombin. 
Fibrinogen plus thrombin equals fibrin which is the substance which 
produces the clot. 
BLEEDING TIME 
1, Cleanse the lobe of the patient's ear with alcohol and 
puncture so that blood will flow without pressure. 
2. Start time when blood begins to appear; 30 seconds from 
that time, blot up all the blood with filter paper and repeat every 
30 seconds, until bleeding stops. 
3. Figure the time by counting the blots on the paper and 
divide by two, which gives the time in minutes. 
MALARIAL SMEARS 
Collection of Smear - "Thick Drop" 
1. Place several large drops of blood on a slide. 
2. With the corner of another slide, spread the blood in a 
circular motion, apply rather firm pressure in spreading the blood so 
as to bring the parasites out. 
3. Let stain for 30 minutes. 
Examination of Stained Smear 
1. Examine under oil immersion lens. 
2. Parasites appear as red dots with blue rings. The red cells 
have been dissolved leaving the parasites on the slides. 
3. Never report a malarial smear as "positive" unless you nan 
find at least 3 parasites on the thick drop. Platelet Count: 
THE BRILLIANT CRESYL BLUE MOIST PREPARATION METHOD, A saturated 
solution of brilliant cresyl blue in 95$ alcohol is smeared on a clean 
slide and allowed to dry. The dried stain on slides keeps well and a 
number of slide preparations can be made at the same time and stored un- 
til needed. Before use the film of dye is gently polished with lens 
paper. A small drop of blood from a skin puncture wound is collected en 
a clean coverslip. The coverslip is immediately dropped onto the stain 
and the drop allowed to spread without pressure except for the weight of 
the coverslip. If pressure is required to make the blood spread the pre- 
paration is not suitable for platelet counts. The drop of blood should be 
small so that the margins extending outward do not touch more than two of 
the coverslip edges. The edges of the coverslip are rimmed with vaseline. 
After waiting at least 10 minutes for the cells and platelets to become 
stained, 1,000 red blood cells are counted and the platelets in the same 
fields noted. The count is made under oil immersion, using a bright light, 
A tally counter and a disc in the eyepiece to reduce the size of the field 
are of aid in counting. The platelets appear as spherical or Vesicular 
btdies, blue with darker bluish granules, l/4 to l/2 the size of red blaod 
cells and sometimes larger. The number of platelets is estimated from the 
red cell count taken at the same time the moist preparation is made and is 
computed according to the following equation. 
Number #f platelets: 1,000 r,b,c, x: Number of r.b.c, per cu, mm. 
Examplei 
Red cells counted 1,000 
Number of platelets counted 72 
Red cell count 4-,300,000 
4x122x222 Y 72 a no, platelets per cu, mm, 
1 1,000 
The normal number of platelets with the brilliant cresyl blue moist 
preparation varies from 500,000 to 1,5000,000 per cu. mm., with the average 
around 800,000, The higher count obtained with this method than with other 
commonly used methods is due to the fact that there is a minimum of manipu- 
lation, the platelets are not destroyed in such large numbers and the plate- 
let fragments remain visible. Since the platelets are themselves fragments 
any method which preserves the fragments best is a superior method. The 
main sources of error are the unevenness of distribution, the small number 
of red blood cells counted and failure to make a satisfactory preparation. 
The most common errors in technic are the use of glassware that is not scru- 
pulously clean, the use of too large a dr«p of blood, insufficient stain 
and improper light. 
Reticulocyte Count; 
Slides prepared with brilliant cresyl blue as for the platelet count 
above, may be used. The same procedure is followed in making the prepara- 
tion, One thousand red cells are counted and the number of reticulocytes 
noted. The normal number is 0,1 to 1.0 per cent or 1 to 10 per thousand. Results are reported in per cent. 
SEDIMENTATION RATE 
There are so many methods now in use for performing this test that 
results have not been comparable. Only one method will be given here with 
the normals for this method only. 
This test is useful in determing the severity of the following dis- 
eases: Tuberculosis, Rheumatic Fever and Salpingitis. 
Materials 
1, Veinpuncture equipment, 
2, Cutler tube, this must be purchased from a supply house, it is 
a tube graduated at 1 cc, capacity and marked into fifty 1 mm, 
divisions with 0 at the top, 
3, 2 cc. sterile syringe,- * * 
4-, 3% Sodium Citrate solution (sterile) 
Procedure 
1. Draw 0,1 cc. of 3% Sodium Citrate into the 2 cc, syringe, 
2. Draw into the same syringe 0.9 cc. of blood from the vein, 
3. Mix and pour into the upright tube. 
4-. Read the height of the blood cell column every 5 minutes, 
for one hour, plot results as a graph. 
The normal for men is under 8 mm. and for women under 10 mm. in one hour, 
with a horizontal line. 
1, Diagonal line with a fall greater than normal indicates a mild 
condition, 
2, Diagonal curve with the fall continued in the last half hour 
indicates an active condition, 
3, Vertical curve with the entire fall in. the first half-hour in- 
dicates a more severe condition. 
In an effort to use materials normally on hand in the laboratory 
the modified Cutler method Select test tubes from 75 x 10 
mm, stack of such caliber that 4- cc, gives a column 50 mm, high, etteh the 
tube at this point. Put exactly 0,4. cc. of 3% Sodium Citrate in the tube. 
Fill to the 50 ram, mark with blood. Mix by inverting, avoid air bubbles. 
Set the tube in the vertical position. Measure with a millimeter ruler 
and record at 5,10,15,30 minutes and 1 hour. Chart on a graph. 
Normal for men 2-8 mm, in 1 hour; for women 2-10 mm,- VEIKPUNCTURE 
For Blood Culture 
Materials 
1. Luer syringe, 10 cc., sterile 
2. Needle, 20 gauge, sterile 
3. Flask of appropriate culture medium 
4. Tincture of iodine 
5 , Alcohol, 70$ 
6. Tourniquet 
7. Cotton or gauze pledgets, sterile 
8. Alcohol lamp if no gas burner is available 
Procedure 
1. Thoroughly cleanse skin over the vein and surrounding area of the 
arm for about three inches, with alcohol. 
2. Paint over the vein with iodine and leave on for 2 or 3 minutes. 
3. Light alcohol lamp or burner. 
4. Unwrap syringe and insert plunger into barrel. Do not touch inside 
of barrel or shaft of plunger. 
5. Remove plug from needle tube and flame mouth of tube. 
6. Insert neck of syringe into mouth of tube and tilt tube so that 
needle will slide down over the neck. 
7. Remove syringe and needle and set the needle firmly on the neck, being 
careful to touch only the hub of the needle. 
B. Flame both the needle point and the mouth of the tube that contained 
the needle. Cover the needle YJith the flamed tube and set aside 
while completing the preparation of the arm, 
9. Apply tourniquet above the elbow, not too tightly. If the vein does 
not distend well, have patient clench fist. 
10. Sponge off the iodine with alcohol, 
11. Puncture the skin with needle a little to one side of the vein and 
parallel to it; then enter the vein from that side about half an 
inch above the skin puncture. 
12. After securing the desired amount of blood, loosen the tourniquet and 
have patient open fist. 
13. Press an alcohol-soaked pledget firmly over the puncture and withdraw 
needle quickly. Have patient flex elbow tightly to hold pledget 
in place. 
14. Open flask and flame mouth thoroughly, holding the syringe near but 
not in the flame at the same time, 
15. Insert needle into flask and force blood directly into culture medium 
without touching sides of flask with either needle or blood. 
Flame neck of flask again, replug and incubate. For Other Purposes 
In taking the large number of routine blood samples required for 
other purposes which do not demand sterile blood, it is simpler to use 
only the sterile needle, as so many syringes are seldom available. The 
method is simple and takes little time, provided directions are followed 
and a little patience is exercised. 
Procedure 
1. Swab the site with iodine, followed by alcohol or acetone. Acetone 
alone may be used. 
2. Tighten tourniquet about the upper arm enough to dilate the vein firmly. 
3. Remove the needle from its tube and take the stylet out, being careful 
to touch only the hub. 
4.. Hold the needle tightly between the thumb and index finger at the hub. 
Insert the tube for the blood specimen below the needle, grasping it 
with the third and fourth fingers, so that the hub of the needle is 
just within the mouth of the tube. This is much easier to accomplish 
if the patient's arm is allowed to hang straight down, 
5. Puncture the skin a little to one side of the vein and parallel to it. 
If the needle is sharp, this can be done with one quick motion and is 
not at all painful. 
6. Turn the needle point slightly toward the vein and enter with a quick, 
short stab. If you turn the point too squarely toward the vein you 
risk puncturing both walls. 
7. bhen you have collected 10.to 15 cc, of blood, loosen the tourniquet. 
Bl Press a pledget of cotton soaked in alcohol or acetone over the 
puncture and withdraw the needle quickly, maintaining the pressure 
until the bleeding, if any, has stopped. 
Care of Keedles and Syringes 
1. as soon as the blood sample is taken, shake as much of the blood 
from the needle as possible and drop it into a beaker of water to lake 
the blood. On return to the laboratory, clean thoroughly with cold water 
and dry by forcing alcohol followed by ether through the bore, Never 
put a wet needle away, as rust is dangerous. 
2, Replace the stylet, leaving the loop of wire outside the point 
for the protection of the latter. Slide the needle, point down, into a 
b'atterraan tube, plug tube with cotton and sterilize by dry heat or in the 
autoclave. 
3. Sharpening the needles is best cione on the finest grade of emery 
cloth stretched on a flat surface. Finish on a fine blue water stone. 
Even the finest grade of emery or carborundum, if used alone will leave 
a slight raw edge that may cause too much pain. 4. Syringes must never be left with the plunger in the barrel after 
use, no matter what has been in the syringe. Always wash out the syringe 
with water immediately after use and leave the plunger out until both it 
and the barrel have been carefully dried. Once a plunger has been ’’frozen” 
in the barrel, it may not be possible to remove it. Often the best way to 
free it is to force cold water through the neck of the barrel against the 
head of the plunger, using another syringe with a needle small enough to 
insert into the neck. Warming the barrel in hot water may loosen it. 
Soaking in cold water for several days may be necessary. Never use force. 
5. To sterilize, wrap the plunger and barrel separately in gauze, 
with an outer wrapping of heavy paper. Secure the wrapping with a turn or 
two of ordinary twine, tied in a slip knot to expedite unwrapping. 
Cautions 
1. Certain dangers and discomforts to the patient must be avoided. 
These are (a) Infection, (b) Injury to the vein wall, (c) Hematoma, (d) 
Needless pain, 
2. Infection is due to carelessness. Be certain your needles and 
syringes are sterile. Never touch the shaft of the needle to anything that 
is not sterile before taking the blood. 
3. Injury to the vein wall may cause a clot to form on the wall. 
This may break free in the blood stream and death may result. Causes: Dull 
or rusty needles, too much movement of the needle point while in the vein, 
passing of the stylet through the needle while still in the vein in the 
attempt to free the needle from clots. Never pass the stylet through the 
needle'before withdrawal. If clots plug the needle, withdraw it and try 
the other arm. 
4. Hematoma (blood tumor) is often very paiaful and may become 
infected. Causes: Too large a needle in a delicate vein, withdrawal of 
the needle before tourniquet is loosened, making insufficient pressure over 
the puncture after withdrawal. 
5. Needless pain is often due to excess of care and slowness in 
making the puncture. Dull needles most often cause it. A sharp 17 gauge 
causes less pain than a dull 20 gauge. Remember that those ill enough to 
be in a hospital may be greatly set back by even a slight painful shock, 
especially if many blood tests have to be taken. It is always better to 
make patients lie do?;n or sit in comfort while blood is bei»g taken. BLOOD TYPING 
The typing and cross-matching of human blood for transfusions 
is the most exacting procedure which you as laboratory technicians 
will be called upon to do. A mistake, or perhaps just a little 
carelessness, in carrying out this procedure, can very easily cause 
the death of a patient. As the procedure of blood typing and cross- 
matching, and the theory behind it, becomes clear to you, you can 
readily see the truth of this statement. 
Before plunging into the procedure itself, there are a few 
fundamental principles which it is necessary to know. First, what is 
agglutination? Agglutination means sticking together. It is the 
process of being clumped together. In the logging business up north 
logs are cut and thrown into rivers and are floated downstream to saw 
mills. Now as long as these logs remain separate, they float merrily 
down the stream, but, once they become jammed or agglutinated, the 
logs can no longer get through the narrow places and all are held up. 
So, in the human blood vessels, if the red cells become agglutinated, 
the cells become jammed in the narrow vessels, and the unfortunate 
person dies. Placing the wrong type of blood in a patient's veins 
by transfusion may cause such a tragedy. Another factor to be con- 
sidered in the transfusing of blood is hemolysis. Hemolysis is the 
process by which the red blood cells are broken up or dissolved so 
that they give up their hemoglobin to the plasma. Hemolyzed blood 
is bright cherry red and does not have adequate oxygen carrying power. 
Hemolysis of blood in the body also will cause death and again 
transfusion with the wrong type of blood will cause hemolysis. How- 
ever, since hemolysis does not occur without agglutination, it is 
sufficient to test for agglutination only. 
There are two factors which are necessary to cause agglutination. 
A factor called an agglutinin contained in the serum and a factor 
called agglutinogen, contained within the rod cells. Whenever the two 
like factors come together, agglutination takes place. For example, 
a blood having agglutinin (a) in the serum will cause agglutination 
when it comes in contact with red cells having agglutinogen (a). For 
convenience the agglutinogens are designated by a capital letter and 
the agglutinin with a small letter. 
There are four main types or classes of human blood which we 
shall classify according to the method of Landsteiner in which the 
blood type is designated by the agglutinogen contained in its red 
cells, namely, AB, A, 3, and 0. The last type is called 0 since 
there are no agglutinogens in its cells. See figure below: 
I2E® 
Agglutinogen 
Aeelutinin 
o ) 
Moss 
Jansky 
Incidence 
4B ) 
I 
IV 
3% 
A ) Rbc 
b )serura 
" II 
II 
B ) 
a 
III 
III 
10% 
0 ) 
ab 
IV 
I 
V>% 
From the above figure, it can be seen that each type has an 
opposite kind of agglutinin in the serum, so that, the bleed does not 
become agglutinated within itself. If you look at the figure you can see that type AB cannot be 
given to another type except its own because in the serum of the other 
three types, there is a corresponding agglutinin designated by a small 
letter which will cause agglutination of the .blood. Likewise, type A 
may not be given to B or 0, because of the corresponding agglutinins in 
the serum and type B may not be given to A or 0. 
Again looking at the figure you can see that if this is so then 
type AB may take blood from any of the other three types because there 
"hre no agglutinins in the serum. That is true and for this reason type 
AB is called the Universal Recipient, but, the Serum from anyone of the 
other three bloods contains agglutinins which will agglutinate the cells 
of the'recipient (or patientjTHowever, in an emergency, if type AB donor 
cannot be found and if the blood is given very slowly, any of the three 
other types of blood may be given to a type AB recipient since the volume 
of serum of the blood of the donor is so* small as compared to that of the 
recipient, that the foreign agglutinins are rapidly diluted beyond doing 
any harm. . , 
Likewise, a study of the figure will reveal that type 0 blood 
could be given to any of the other three types since it contains no agglu- 
tinogens in the cells. That is true and for this reason type 0 blood is 
called the Universal Donor. Again the same reasoning holds true. Type 
0 blood may be given to or used as a donor for any of the other three types 
in an emergency, if given slowly and with care. Again the donors serum 
contains agglutinins which will agglutinate the cells of all other types 
but the donors serum is considered to be so diluted by the greater volume 
of serum of the recipient as to make transfusion possible, if done slowly 
and carefully. 
Before I go further, it may be well to explain that blood trans- 
fusion is the process of introducing blood from a donor into a vein of a 
recipient. 
To avoid any unwanted reactions, which might prove disasterous, 
transfusions are always given with blood of the same type when possible. 
Although there are only four main types of human blood, there are 
certain subtypes which often give bad reactions, For this reason, it is 
necessary to cross-match the blood of the recipient with that of the donor 
in addition to having the same type of blood. There are two kinds of 
cross-matching - a major and a minor. The major cross-match is a com- 
parison of the donor1s cells with the serum of the recipient while the 
minor cross-match is a comparison of the recipient’s cells with the serum 
of the donor. Each time a transfusion is given a major and a minar cross 
match is done just before the operation regardless of whether the donor 
has given the recipient blood before or not. This eliminates any possi- 
bility of a mistake. 
Method 
A. Materials needed 
1. Wassermann tubes - 1 for each. 
2. Vein puncture needle - sterile, 1 for each, 
3. Small pipetes or medicine droppers, 
4. Solution of 0.85% saline or 2% sodium citrate in 
s aline. 
5i Khbwh type A ahd type B Serum, B, Procedure. 
1« First make a list of the donors giving each a number, 
then mrnber the tubes accordingly. Then place 1 cc, 
of saline in each of the numbered tubes. 
• > 2. Puncture the finger of the first man on the list and 
place one or two drops of blood in test .tube number one; 
then one or two. drop's of blood from second man in tube 
number two, etcr'This gives cell suspensions in numbered 
• tubes corresponding to the numbers of the men on the list. 
3. On a clean glass slide make two large rings with a china 
marking pencil, Mark the first ring A and the second 
ring B, Number the slide to correspond to the number of 
the donor. Then place one drop each of Type A and B serum 
on their respective rings. Then place one drop of the cell 
suspension on each drop of serum and tilt the slide in a 
rotary motion to mix the cells and serum. Then cover with 
a petri dish ( to avoid evaporation) and allow to stand 
for 20 minutes. Then read with the low power objective 
of the microscope. Agglutination in A only means type B. 
Agglutination in B only means type A. Agglutination in A 
and B means type AB. Agglutination in neither A or B means 
type 0, 
• 4., Procedure for cross-matching, - blood from a vein is need- 
ed from both the donor and the recipient. Draw about B cc. 
of blood from a vein of the donor and from the recipient. 
Place each in a dry tube. Then by dipping the butt end of 
the needle into a tube containing 1 cc of saline, a cell 
suspension can be made from each, 
5. Allow the specimens of whole blood to stand for 10-15 
*■ minutes until a clot is formed. Then carefully slip a 
* wooden applicator stick between the side of the tube and 
; -the clot to Separate it« Then centrifuge to separate the 
clear serum. The serum can then be used for both serolo- 
;gical tests and cross-matching. 
6, Cross-matching - again make two rings on a slide. Mark 
the first R and the second D and number the slide to cor- 
respond with the number of the donor. In R place one drop 
of the recipient’s serum and in D place one drop of the 
donfr’s serum. Then to R add one drop of the donor’s cell 
suspension end to D add one drop of the recipient’s cells. 
Then cover and read in 20 minutes as before. Agglutina- 
tion in either R or D or both, means the donor is not com- 
patible and cannot be used. Of course, if there is no 
agglutination in either R or D the donor is compatible 
and may be used. 
NOTE'S "R” is the major cross-match 
”D” is the minor. The Hevj method of Blood Typing. Using The 
International (Landsteiner) Classifi- 
cation and The flew Pondered Rabbit 
anti derum 
1, As it has been found to be impractical to supply human sera for 
performing these tests, especially prepared dried rabbit sera will be used. 
The two reagents required consist of mixtures of sucrose and dried sera from 
rabbits which have been immunized with human erythrocytes of groups A and 
B respectively. Such sera are standardized and tested by the manufacturer, 
and when used as recommended will cause prompt macroscopic agglutination 
(usually within 1/2 minute) of the respective types of human erythrocytes. 
Medical Department purchase specifications require of the manufacturer that 
the potency of serum be such that when mixed according to directions issued 
by the manufacturer specific macroscopic agglutination A, B, and aB human 
red blood cells respectively will occur in a period not to exceed one minute, 
(To avoid occasional errors they should be rechecked after 10 min.) 
2. It should be borne in mind that when such sera from rabbits immu- 
nized with human red blood cells are used for the determination of blood 
groups the agglutination reaction is direct and is not reversed as when 
human typing sera are used. For yjhen using the rabbit sera, red 
blood cells which agglutinate in Anti A serum belong to group A. and red 
blood cells which agglutinate in the Anti B serum belong to group B. 
3. Technique of blood typing: 
a, Materials: 
Anti A, powdered serum, rabbit (colored with methylene blue) 
Anti B, powdered serum, rabbit (colored with eosin) 
Physiologic salt solution 
Small test tubes 
Microscopic slides 
Wooden toothpicks or applicators 
Wax pencil 
b. The Test 
(l) Carefully cleanse with alcohol and puncture the finger of the 
individual whose blood is to be typed and collect one or two drops of blood 
in a small test tube containing 1,0 cc. of physiologic salt solution. This 
makes an erythrocyte suspension of about ten percent. (2) with a wax pencil draw a line across the middle of a clean 
glass slide and label one end uAn and the other "3". Using a clean medi- 
cine dropper (washed with three changes of saline between each test) place 
in the center of each of the two ends of the slide, "A” and nBu, a large 
drop of the ten percent blood suspension to be tested. Then, with the small 
end of the blue-tipped toothpick, dip up a small mound (about 2mm, long) of 
group A powdered serum (colored with methylene blue) and add this to the 
drop of blood suspension on the "AfI end of the slide. Likewise, using the 
red-tipped toothpick, add a similar amount of group B powdered serum (co- 
lored with eosin) to the "B" drop of blood, Lix the preparations thoroughly 
by stirring each with the unused end of a clean new wooden toothpick. Allow 
the preparations to stand one minute and read the results. 
A. Interpretation of results: 
a. If no agglutination occurs either in serum A or B, the blood 
being tested belongs to group 0 (universal donor). 
b. If agglutination occurs in the group a serum only, the blood 
belongs to group A. 
c. If agglutination occurs in the group B serum only, the blood 
belongs to group B. 
d. If agglutination occurs in both the A and B area, the blood 
belongs to group AB. 
Note: It will be noticed that the Anti A serum imparts to the 
saline suspension of blood cells a bluish green color, while the Anti B 
serum gives an eosin red color. This should prevent confusion in reading 
the results of the test, as the colors remain constant, and thus errors in 
the labeling of slides should be eliminated. 
5• A Suggested Procedure for Determining and Recording the Blood 
Types of Military Personnel. 
The test to be used in determining the blood types of military 
personnel is so simple that elaborate laboratory facilities will not be re- 
quired. In small posts or isolated detachments the tests can be done by 
the surgeon with a few enlisted assistants. In large organizations it is 
suggested that they may be performed expeditiously by using one or more 
"blood-typing teams" composed of medical Department personnel as outlined 
below. The individuals to be typed should be required to report for the 
examination at some convenient central place and to bring with them their 
identification tags, lach "blood-typing team" should be supplied with a 
Graphotype machine for recording the results of the tests on the tags. It 
is estimated that a single team should be able to determine the blood types 
and record the findings for 4,00 individuals in an eight-hour day. ORGANIZATION OF A BLOOD-TYPING TEAM 
The individuals whose blood is to be typed should report by organiza- 
tions to a place designated by the officer in charge of the blood-typing 
team, after which they will pass through the following "stations” in 
sequence* * 
STATION I At this station an enlisted man seated at a table, adding 
to small glass vials 1.0 cc. amounts of physiological salt 
solution by means of a calibrated medicine dropper, will give 
one tube of salt solution to each individual to be tested. 
STATION 2 At this station two (2) enlisted men, hking alternate indivi- 
duals, will collect from the finger of each individual four 
drops of blood, adding this directly to the vial of salt 
solution. The vial will bo returned to the individual who will 
carry it to station 3. 
STATION 3 At this station two (2) enlisted men, taking alternate indivi- 
duals, will prepare the tests by placing with a clean* medicine 
dropper a drop of the blood cell suspension on each of the two 
labeled ends of a glass slide, and then adding the dried sera 
A and B respectively as outlined in paragraph 5 c above. The 
slide containing the individual's blood grouping test and his 
vial will be returned to him, and he will then carry them to 
Station A where the results will be read and recorded. 
* Note; A single medicine dropper may be used continuously 
provided it is rinsed three times with clean physiological saline 
between tests. This can be done by using a beaker or glass 
filled with clean physiological saline and a small container in 
which to discard the washings. (Care should be exercised to 
prevent contamination of the clean saline with the blood washings.) 
STATION A At this station one (1) enlisted man will read the result of 
the test to the Graph©typo operator who will record the result 
on the individual's identification tag* The individual will 
then carry his vial of blood cells, his blood grouping test slide, 
and his identification tag to the next station. 
STATION 5 Here the medical officer will verify the result of the test and 
check this against the record made on the identification tag 
in order to prevent errors in recording. 
STATION 6 At this station the individual who has been examined will discard 
his glass vial and test slide in suitable containers provided 
for that purpose. Two enlisted men will wash and dry the used 
glassware and prepare them to be used again* Cerebrospinal Fluid 
Now we shall take up the cerebrospinal fluid with particular 
reference to the routine tests which you may be called upon to run . 
on the fluid. 
Before going into'the various procedures, it might be well to 
get a little background. The brain is a mass of specialized nerve 
tissue, composed of white and gray matter and a special type of 
connective tissue called glial tissue, which is contained within the 
bony cranium or brain case. The chief parts of the brain are the 
cerebrum, the mid-brain, the cerebellum, and the brain stem, or 
medulla. The cerebrum is divided into two cerebral hemispheres and 
is the seat of intelligence, that is, the center of thought, memory 
and association, etc. The mid-brain is the seat of pain, touch, 
water balance and temperature control. The cerebellum is the seat 
of balance and the coordination of muscles. The medulla is where 
the vital centers of respiration and circulation are located. The 
spinal cord is a continuation of the medulla. The brain and spinal 
cord are covered by three continuous membranes, called the meninges; 
namely, the Dura Mater, the Arachnoid and the Pia Mater, going from 
the outside in. The Pin Mater is quite thin and is very closely 
applied to the surface of the brain and spinal cord and dips into all 
of the convolutions or wrinkles of the brain. It is between the 
Arachnoid and the Pia Mater that the cerebrospinal fluid lies. 
The brain has four ventricles or spaces within its substances. 
The first two are called the lateral ventricles and are situated one 
in each cerebral hemisphere. The third ventricle is situated in the 
mid-brain and the fourth ventricle is situated in the medulla. All 
of the four ventricles are connected and the fourth ventricle is 
continuous with the small central canal of the spinal cord. 
The cerebrospinal fluid forms a "shock absorber" for the brain 
and spinal cord, since they are both completely surrounded by the 
fluid or you might say, the brain and spinal cord float in the cere- 
brospinal fluid. In addition, this fluid forms a sewer into which 
the waste products of the brain and spinal cord are discharged. 
The cerebrospinal fluid is secreted by the choroid' plexuses, 
which are situated in the ventricles of the brain, chiefly the lateral 
ventricles. The fluid is more than just a filtrate of the blood since 
it contains normally about half the concentration of the blood sugar; 
traces of protein and chlorides, etc. 
The cerebrospinal fluid passes out of the ventricles through 
small holes in the roof of the fourth ventricle to enter the sub- 
arachnoid space where it circulates slowly about the brain and spinal 
cord. The volume of spinal ;fluid varies between 100 and 150 cc. 
normally and has a specific gravity of from 1.003 to 1,008. The 
cerebrospinal fluid is absorbed into the blood stream by structures 
called Arachnoid Villi or Pacchionian Bodies which are situated in 
the Dura at the top of the brain in the mid-line above the groove 
between the cerebral hemispheres. The cerebrospinal fluid for diagnostic and therapeutic purposes 
is withdrawn from the sub-arachnoid space in the spinal canal by- 
means of inserting a long hollow needle, usually under local 
anesthesia, between the spinous processes of the third and fourth 
lumbar vertebra. I wish to emphasize that the needle is not intro- 
duced into the spinal cord. As a matter of fact the spinal cord 
ends at the first lumbar vertebra. The meninges extend down farther 
than the cord and form a sac which contains spinal fluid and the 
numerous nerves which form the cauda equina or horses tail. 
Spinal fluid is normally clear like water and has a specific 
gravity, as was already mentioned, of 1.003 to 1.008, or slightly 
heavier than water. Usually not more than 10 or 15 cc. are with- 
drawn at one time. The specimen submitted to the technician is 
usually submitted in three test tubes, numbered from 1 to 3 in the 
order in which they are withdrawn. The first specimen may be 
slightly pink from contamination by blood picked up by the needle on 
the way in; the 'second specimen and surely the third should be 
clear, if the cerebrospinal fluid is normal. In cerebral hemorrhage 
or stroke, or cases of skull fracture with lacerations (tears) of 
the brain, all three specimens may be bloody. In cases of brain 
tumor with slow chronic bleeding in which the blood has become de- 
pigmented, the fluid may be straw-colored or xanthochromic. In 
cases of meningitis, the fluid may be cloudy. 
Routine Tests 
The routine tests on cerebrospinal fluid are total and 
differential cell counts, microscopic for bacteria, Pandy test for 
globulin, Colloidal Gold tost, Kahn and Vasserman tests. In 
addition, special tests, such as specific gravity, qualitative and 
quantitative sugar, total protein and quantitative chloride tests 
may be run. It is only with the first group that we shall concern 
ourselves. 
I. Cell Counts 
A. Total cell counts this determination is done upon the 
clearest of the three specimens. A special counting 
chamber may be used but a regular heraocytometer will 
suffice. If all cells, both red and white, are to be 
counted; fill a white pipette with spinal fluid (no 
dilution), after first shaking the specimen, then fill 
the counting chamber in the usual manner and count all 
cells in the entire ruled area. That is, count 9 large 
squares and multiply the total by 10/9 which gives the 
total cells per cu. mm. If the leukocytes only are to 
be counted, first rinse out the white pipette with 
Glacial Acetic Aqid and let the acid run out by gravity 
(do not blow it out) then fill the pipette with cerebro- 
spinal fluid and count in the some way, using the same 
factor, 10/9. The, normal count is 1 to 7, 10 is the 
maximum in health. B. Differential Count: the differential count is done in 
the same manner as that on a.blood film. 
An increase in cell count, together with predominence 
of lymphocytes (more than 70$) strongly suggests tuber- 
culosis meningitis or syphilitic disease of the nervous 
system, since it occurs in about 90 to 95$ of cases. 
The number of cells in these cases varies but usually 
runs between 25 and 100 per cu, mm. 
In all forms of acute meningitis, the total cell 
count is high, 100 to several thousand, and neutrophils 
predominate. A notable number of monocytes may also be 
present, especially in acute epidemic meningitis. 
II. Microscopic for Bacteria 
A. This test may be run with the first specimen of fluid. 
In general, the specimen is centrifuged and a smear is 
made from the sediment. The smear is then stained by 
Gram’s method and examined under oil. If tuberculosis 
meningitis is suspected, a tube of fluid is allowed to 
stand overnight. In cases of tuberculosis meningitis, 
if the fluid is allowed to stand for 12 to 2U hours, a 
pellicle or cobweb like veil will form in the spinal 
fluid. The pellicle will entrap the tubercle bacilli, 
which can bo found by placing the pellicle on a slide 
with an applicator stick and then staining the slide for 
tubercle bacilli by the Ziehl-Neelsen Technique. 
Ill, Pandy Test for Globulin 
A. This is a simple sensitive test which should be run on 
the clearest of the three specimens. One small drop 
of spinal fluid is dropped into a small test tube con- 
taining 1 or 2 cc. of Tandy’s reagent (a 10$ aqueous 
solution of phenol crystals) a faint bluish white cloud 
like veil of smoke is a positive test. 
IV. Colloidal Gold Test 
A. Lanf;5s• Colloidal Gold Test, introduced in 1912 and 
now’ very vddoj.y used, consists in mixing cerebrospinal 
fluid in certain proportions with a colloidal solution 
of gold. Normal cerebrospinal fluid causes no change 
in color, , Fluids from cases of syphilis and certain 
pathological conditions of,the nervous system, induce 
changes in color of the gold solution from red to 
purple, deep blue, pale blue, or colorless. Moreover, 
the dilution at which the maximum color change occurs 
is more or less characteristic- of the different 
pathologic conditions. The typical ,fcurves,,are shown in the Figures 
The test gives its most consistent and valuable re- 
sults in cases of general paresis (a late effect of 
syphilis of the brain). In encephalitis lethargica 
and poliomyelitis (infantile paralysis) a typical 
curve in the tabetic zone has been observed. The 
exact explanation of the test is not ye wholly 
clear, but it is undoubtedly dependent 4pon the pre- 
sence of a globulin. 
V, Technic of Test - arrange a series of 12 clean test tubes 
in a rack. Place 1,8 cc, fresh sterile 0,4$ solution of 
sodium chloride in the first tost tube and 1 cc, in each of 
tho others, except the twelfth. In the twelfth tost tube 
place 1,7 cc, of sterile 1% solution of sodium chloride. To 
tho first tube add 0,2 cc, of the spinal fluid, which must 
be free from any trace of blood. Mix well by sucking the 
fluid up into tho pipette and expelling it, and then trans- 
fer 1 cc, to the second tube. Mix and transfer 1 cc, to the 
third tube, repeating this down the row to the tenth tube 
and discard the last 1 cc, portion. This leaves tho eleventh 
and twelfth tubes, with only salt solution to serve as con- 
trols, To each of those test tubes add 5 cc, of Colloidal 
Gold Solution, Let stand at room temperature for an hour 
or longer, at the end of which time, in the case of a posi- 
tive reaction, the solution in some of the tubes will have 
changed from red to purple, deep purple, pale blue, or color- 
less, In the case of normal fluids, no change will occur. 
The fluid in the eleventh and twelfth tubes which serve as 
controls, should be orange-red, and colorless, respectively. 
The results are usually charted, as shown in the figure, in 
which each column represents a tube. For the purpose of bre- 
vity, the colors may be indicated by the corresponding num- 
bers, which are placed in the same order as the tubes. Thus 
the paretic curve may be expressed as 555554-2100, or this 
may be called a first zone curve, A tabetic curve or middle 
zone curve may be expressed as 0123320000, A meningitic 
curve or last zone curve may bo expressed as 0001224.53d, 
(See Pages 92-93) VI, Spinal Fluid Washerman and Kahn Test: these tests may be 
performed on a bloody as well as a clear specimen, if the 
fluid is first centrifuged. As a general rule the Kahn 
Test is done first and if positive, is then checked with 
a Wasserman. As these tod'ts, when performed upon spinal 
fluid, are no different from the same tests as performed 
on blood, they will not be mentioned here. THE URINE 
Color 
1. The color of urine is subject to wide variations but 
possesses some diagnostic importance. 
2. Normally, it is yellow or reddish-yellow (amber) due to 
the presence of several pigments, chiefly urochrome. 
3. The color depends largely upon the concentration of urine. 
Dilute urines are usually pale while concentrated urines are dark. 
Acid urine is usually darker than alkaline urine. 
4. Color may be greatly changed by abnormal pigments and by 
various drugs and poisons, as follows; Bloods red or brown; smoky. 
Bile; yellowish brown, turning green; yello?/ foam. , Chyle: milky. 
Methylene blue; greenish-blue. Phenols; olive-green to brownish 
black, etc. ' 
5. For uniformity in recording color, Vogel’s scale is re- 
commended, the urine being filtered and viewed by transmitted light 
in a glass 3 or J+ inches in diameter; pale yellow, light yellow, 
yellow, reddish - yellow, yellowish-red, red, brownish-red, reddish- 
brown and brownish-black. To these may be added greenish-yellow, 
olive, milky, etc. 
Transparency and Sediments 
1. Freshly passed urine is usually clear or transparent, but 
may be cloudy due to the presence of phosphates or pus. The former 
disappears upon the addition of acid; the latter does not, but may 
become gelatinous (Donne’s Test). A freshly passed urine may also 
be cloudy with bacteria or comparatively clear with numerous shreds 
of mucopurulent material (chronic urethritis). 
2. A record of the transparency is only of value in comparative!; 
fresh specimens. All become cloudy with bacteria and alkaline salts 
upon standing as the result of decomposition. 
3. Upon cooling and standing all specimens develop a faint 
cloud of mucus, leukocytes and epithelial cells which settle to the 
bottom - the so-called ’’nubecula,” This has no significance. 
U. Acid urines may develop a white or pinkish sediment of 
amorphous urates. 
5. Alkaline urines may develop a heavy white sediment of 
amorphous phosphates, 
6. Pus gives a heavy mucoid whitish sediment. 
7. Blood gives a reddish-brown smoky sediment. 
8. Bacteria give a uniform cloudiness which cannot be removed 
by ordinary paper filteration. 
9. The following terminology is recommended? 
a. Clear, slightly cloudy, very cloudy, 
a. Sediment; slight, moderate or heavy; white, pinkish, 
red, brown, reddish-brown, etc., shreds present or absent. Determination of Reaction 
1, Normally fresh voided urine is acid in reaction, the PH rang- 
ing from 4.8 to 7.5 with a general average of 6. Twenty-four 
heur specimens are less acid than freshly passed urine and may 
be neutral or even slightly alkaline as a result of standing. 
2. Freshly passed urine may be neutral or alkaline as the result 
of the administration of alkalis, retention with "ammoniacal 
decomposition, etc.” 
3. Diet influences the reaction. 
Litmus Test 
For ordinary purposes the reaction may be determined with good 
grades of blue and red litmus papers. 
Blue turning red; acid 
Red turning blue; alkaline 
No change in either; neutral 
Changes both red and blue; amphoteric 
Specific Gravity 
Specific weight or specific gravity denotes the weight of a body 
as compared with the weight of an equal bulk or equal volume of 
another substance, which is taken as a standard or unit. This 
standard adopted for all solids and liquids, if not otherwise 
stated, is water at a temperature of 25PC, 
Determination of Specific Gravity 
The normal range is from 1.015 to 1.030. Pathologically it may 
vary from 1.001 to 1,060. If the specimen contains but a small 
or average amount of sediment it makes but little difference . 
whether the urine is mixed up or the specific gravity taken with- 
out mixing in order to, use the sediment later for microscopical 
examination. If, however, there is a large amount of sediment 
the specific gravity is almost always increased by about 0.002 
after thorough mixing. 
For ordinary determination the Squibb urinometer may be used but 
the urinometer used with the immiscible balance is probably the 
best on the market. It settles down quickly after spinning with- 
out bobbing or swaying,, and its special scale makes it much easier 
to read. With the Squibb urinometer the technique is as follows! 
a. Fill the cylinder without producing bubbles. The specific 
gravity may,be taken without mixing the urine. 
b. Float the hydrometer so that it does not touch the bottom 
or sides. ; ' 
c. Make the reading from tne bottom of the meniseua. Qualitative Detection of Albumin 
Principles - Normal urine contains a trace of albumin which is too 
slight to be detected by the simple tests in general use, a large number 
of which have been described. All depend upon its precipitation by 
chemical agents or coagulation by heat. All precipitate both serum 
albumin and serum globulin and do not differentiate between these two 
proteins. Most are subject to some error largely due to the precipitation 
of mucin or other constituents. All require the use of clear specimens, 
preceded by filtration, if necessary, in order to detect small amounts of 
albumin. The methods here given are recommended for ordinary routine work. 
Heat, and Acid Test 
1. Boil about 5 cc. of urine filtered if necessary, in a test tube 
for about a minute. Hold with a clamp or piece of filter paper 
folded around the neck. Boil the upper portion only. 
2. Add one or two drops (no more) of concentrated nitric acid or 
three to five drops of 5% acetic acid solution. 
3. A white cloud now disappearing is due to earthy phosphates. 
Effervescence is generally due to carbonates from the food. 
4. A very faint trace of albumin may appear only upon the addition 
of the acid. Larger traces appear upon boiling and may become 
heavier upon the addition of the acid. The addition of too much 
may dissolve faint traces of albumin and give a falsely negative 
reaction. 
5. For the routine testing of a large number of samples by this 
method, use numbered test tubes and in a boiling water 
bath for at least five minutes. 
Robert’s Test 
1. The test may be carried out bv contact with urine in any of the 
following ways: 
a. Place a few cc. of the reagent in a conical glass or test 
tube. Tilt and run clear urine from a pipet or medicine 
dropper down the side to give a sharp line of contact. 
REAGENT 
. Magnesium sulphate (saturated aqueous solution - 5 : 
Nitric acid (concentrated) -----------1 \ 
b. Place urine in a horismascope and underlay with agent. This 
instrument is too fragile and too expensive for general use 
although good for office work. 
c. Or immerse a pipet in the urine, wipe off the outside, and 
immerse in the reagent. 
2.. If albumin is present, white rings appear at the line of contact, 
best seen against a black background at a distance of a few feet* Methods of Recording Reactions 
- = Negative 
± a Very slight trace. Cloudiness or ring can just be seen 
against a black background. 
+(l)s Slight trace. Cloudiness distinct but not granular; no de- 
finite floculation, Or the cloud or ring is sufficiently 
definite to be seen without a black background. 
+ + (2) = Moderate trace. Cloudy distinct and granular without de- 
finite flocculation. Or the ring is dense but not wholly 
opaque when viewed from above. 
+ ♦ +(3)= Heavy cloud. Cloud is dense with marked flocculation or 
the ring is heavy. 
+ ♦ ♦ +(4)= Very heavy cloud. Heavy precipitate to boiling solid or 
very dense ring. 
Detection of Dextrose (glucose 
Principles - 
1, Dextrose or glucose readily reduces the oxide of copper in alka- 
line solution. When the whitish-blue cupric hydroxide in sus- 
pension in alkaline solution is heated it is converted into in- 
soluble black cupric oxide, but if sugar is present this is re- 
duced to insoluble yellow or red cuprous oxide, 
2, A large number of tests have been devised on this principle for 
the detection of sugar in the urine but that of Benedict is re- 
commended because of its sensitiveness, simplicity and freedom 
from error. The qualitative reagent does not react with the nor- 
mal sugar of the urine but detects incroasos above this lovel as 
low as 0,2 per cent. Furthermore, uric acid, creatinine, chloro- 
form, formalin and other aldehydes do not interfere to such an 
extent as in the case of Fehling’s test, 
3* If albumin is present in large amounts, it may interfere with 
the precipitation of copper and should be removed by acidifying 
with acetic acid, boiling and filtering. Small amounts need not 
be removed. 
BENEDICT’S TEST 
1, Place 5 cc, of Benefict’s qualitative reagent in a clean test 
tube. 
BENEDICT'S QUALITATIVE REAGENT 
Copper suxpnate.. 17,3 gnu 
Sodium Citrate 173*0 gnu { 
Sodium carbonate (anhydrous)• 100,0 gm. i 
Distilled water to make...IOOCwA cc, , Dissolve the citrate and carbonate in about 500 cc. of distilled 
water by boiling. Filter through paper. Dissolve the copper sulfate) 
in ab»ut 100 cc, of water. Add the copper solution slowly to the 
citrate and carbonate solution and stir continuously whiTe adding. 
Measure and add sufficient water to make the total volume' 1000 cc. 
Do not use for quantitative test. 
2. Add 0.5 cc. of urine (8 drops) and mix thoroughly. 
3. Boil thoroughly for two and one-half to 5 minutes; or place 
tubes in a boiling water bath for fiye minutes - a particular! 
convenient method when conducting a large number of tests 
at one time. 
4-. Allow to cool spontaneously. 
5. If no sugar is present the solution will remain clear or show 
only a slight turbidity of a faint bluish color due to urates. 
If sugar is present, a green, red or yellow precipitate will 
form, the color depending upon the amount of sugar present. 
6. Even 0.25$ glucose yields a large bulk of precipitate, 
filling the solution and rendering it opaque so that the 
test may be applied as readily in artificial light as in 
daylight. 
7. The following scheme may be used for reporting (after Todd 
and Sanford). 
+ (1) * Slight trace. No reduction is evident during boiling but 
appears upon cooling (greenish), 
+ + (2) = Trace. Reduction occurs after about one minute’s boiling 
(Yellow) 
+ + + (3) *= Moderate, Reduction occurs after ,ten to fifteen second’s 
boiling. (Orange) 
+ + + + (4.) = Largo amount. Reduction occurs almost immediately 
after adding urine to the boiling Reagent. (Brick red) 
Haine* s Test 
Use three parts of Haine’s Copper Solution and not more than one 
part urine. Boil over flame for about 1 minute. Calculate 
results as with Benedict’s Test, 
(Note) Maine’s Solution, due to the presence of glycerine, seems 
to keep longer without deterioration than most other 
solutions. However, Benedict’s is the more acceptable 
test, for the presence of sugar. 
Principles 
Detection of .Acetone 
The detection of acetone in undistilled urine is based upon a 
color reaction with nitroprusside (Rothera’s test) in which there 
is a formation of ferropentacyanine with the isonitro compound 
of the ketone or the formation of such an ion with the isonitro- 
amine derivative of the ketone. Rothera's Test 
1, To 5 or 10 cc, of urine add 1 gram of ammonium sulphate, 
2, Add 2 or 3 drops of freshly prepared % solution of sodium 
nitroprusside, 
3, Mix thoroughly, 
A# Overlay with ammonium hydroxide, 
$, If acetone is present, a permanganate color will develop at 
the line of contact. 
A modification of this test is as follows: 
1, Place 2 or 3 cc, of urine in a test tube, 
2, Add 2 to 5 drops glacial acetic acid. 
3# Add 1 cc, nitroprusside solution (10$ aqueous solution), (Or 
place 2 or 3 small crystals of nitropursside into the urine) 
A* Overlay with ammonium hydroxide, 
5, Record as above. 
Detection of Diacetic Acid 
Principles 
The detection of diacetic acid depends upon the production of a 
bordeaux red or violet red color with a dilute solution of ferric 
chloride. 
Test 
1, To about a half test tube full of fresh urine add a 10 per 
cent ferric chloride solution drop by drop until the phospha- 
tes are precipitated, 
2, Filter# 
3* To the filtrate add more of the ferric chloride, 
4* If diacetic is present the solution will turn a Bordeaux Red 
color. 
If doubtful apply the following test: 
Lindomannta,Tost 
1# To U cc• of urine in a test tube add 2 or 3 drops of glacial 
acetic acid, 5 drops of Lugol’s solution and 1 cc, of chloro- 
form, 
2. Shake well and allow the chloroform to settle, 
3# If diacetic acid is present, the chloroform does not change 
color, but becomes reddish-violet in its absence, 
K* If the urine contains much uric acid, use double the amount of 
Lugol’s solution. 
Detection of Indican 
Principles 
The detection of indican by the test given below depends upon its 
decomposition and subsequent oxidation of the idoxyl sot free into 
indigo blue and its absorption by chloroform.  Test 
1, Add to about .5 cc. of urine in a test tube an equal 
volume of Obermayer’s reagent and mix thoroughly. 
Reagent 
Ferric chloride 2 grams j 
Hydrochloric acid (cone, sp. gr. 1.19)#. 1000 cc# 1 
until tube is warm# 
3# Add 2 cc# chloroform and mix thoroughly by inverting, but 
avoid violent shaking# 
'4.# Allow chloroform to settle#. 
5# If indican is present, the chloroform will be colored blue, 
depending upon the amount present. The indican in normal 
urine may give a very faint blue. 
6# The urine of patients taking iodides may give a reddish- 
violet color which may obscure an indican reaction# By 
adding a few drops of concentrated sodium hyposulphite 
solution and shaking, the violet color will disappear, lea- 
ving the blue if indican be present. Occasionally, owing 
to slow oxidation, indigo red will form instead of indigo 
blue. This resembles the color given by iodides but does 
not disappear when treated with sodium hyposulphite. 
Detection of Bile Pigments 
Principles 
The test given below depends upon the oxidation of bile pig- 
ments by acids with the formation of a series of colored de- 
rivatives like biliverdin (green) bilicyanine (blue) and cho- 
letelin (yellow). Bilirubin is perhaps the most important pig- 
ment. 
Rosenbachts Modification of Test 
1. Filter 100 cc, or more of a urine through a filter paper# 
2#-' Remove the filter paper from the funnel and allow it to 
partially dry# 
3# Touch the paper with a drop of old or yellow nitric acid# 
4# If bile is present, a most marked spreading ring of rain- 
bow colors with green on the outside will form# 
Detection of Blood 
Principles 
The conditions in which, blood occurs in urine may be classi- 
fied under hematuria and hemoglobinuria. In the former, one 
is able to detect not only hemoglobin, but the unruptured cor- 
puscles as well, whereas in tjie latter the hemoglobin alone is 
present. The detection of blood is usually detected by tho 
color of tho urine but the detection of traces requires micros- 
copical and chemical examination. For the latter tho usual toste 
for w occult blood*1 are required• Benzidine Test 
1. Prepare a saturated solution of benzidine base (Merck*s) 
by dissolving a knife point full in 2 cc. of glacial acetic 
acid in a test tube. Warm if necessary. 
2. Add an equal volume of % hydrogen peroxide. 
3. Add 2 cc. of the urine and mix. 
A* The appearance of a blue color indicates a positive reac- 
tion. 
Orthotolidine Tes 
1. Solutions: a. 1$ Sol of orthotolidine in methyl alcohol. 
b. Glacial Acetic acid 1 part - H2O2 two parts, 
(Sol, a, will keep several months; Sol b, 
one month). 
2. Add two or three drops of sol, a to the sediment to be test- 
ed and then add two or three drops of sol, b, 
3. Positive test is the appearance of a greenish-blue to a 
deep blue color. 
URINARY SEDIMENTS 
Turbidity of the urine is most often due either to bacterial contamina- 
tion, amorphous urates (brick-dust sediment) or phosphates. In case tur- 
bidity is found, due to bacteria contaminating the urine, subsequent to 
its passing, it is best to call for another sample. 
To preserve urinary sediments, formalin is best for casts and epithelial 
cells, while for genera?, use one may employ a piece of camphor or 1 vol- 
ume of saturated borax solution to 4. volumes of urine. 
To take up the sediment for examination, insert a pipette to the bottom 
of the tube with the opposite opening closed by a finger; then holding 
the finger on the open end tightly, withdraw the pipette and deposit the 
sediment on a slide. 
Unorganized Sediments? These are, as a rule, of little clinical inter- 
est, and give no reliable indication of abnormal production or excretion. 
The precipitation of any particular substance is dependent upon many phy- 
sical as well as chemical factors, of which the reaction is very impor- 
tant, In amphoteric urines, one may encounter elements usually associa- 
ted with either an acid or an alkaline reaction. 
In a urine of ACID reaction, we may find the following: 
1, Amorphous sodium or potassium acid-urates. Usually 
yellowish-red. Heat and alkali causes solution. 
2, Uric acid. Yellowish-red crystals,, usually of whet- 
stone shape and in clusters and heaps. Soluable in 
alkalis but not by heat. 
3, Calcium oxalate. Highly refractile octahedral crystals, 
or in dumbbell shapes. Often due to diet (asparagus, 
tomatoes, spinach, rhubarb, etc). Clumping of the cry- 
stals is suggestive when calculus is suspected. 4-* Rare crystals such as cholesterol,cystin, tyrosin, leucin, 
xanthin, haematoidin, indigo, melanin, creatinine, hippuric 
acid, sodium biurate, etc. 
In amphoteric urine, one may encounter dicalcium phosphate, or practical- 
ly any other crystal. 
In urine of ALKALINE reaction we may expect: 
1. Triple phosphates* Usually coffin-lid crystals, or in fern- 
like forms* Easily soluble in acetic acid* 
2* Tri-calcium phosphate, magnesium phosphate, and calcium car- 
bonate* All are amorphous and easily soluble in acetic acid, 
the last with effervescence* 
3* Ammonium biurate. Yellow, thorn-apple structures* 
4# Calcium phosphate. Slender radiating crystals, or flat sheets* 
5* Rarely, magnesium phosphate crystals* 
The presence of ammonium biurate, particularly if with triple phosphates, 
denotes bacterial decomposition within the genito-urinary tract provided 
the urine is freshly passed. Pus colls derived from the site of inflamma- 
tion should be present also. While certain bacteria might possible cause 
chemical changes without giving rise to inflammation, yet such a possibili- 
ty is so rare as to be negligible. If amorphous phosphates are found, one 
should always consider exogenous sources such as vegetable diet, or special 
causes, as withdrawal of protein food. 
Leukocytes: An occasional leukocyte may bo found in the urine of healthy 
people. An abundance of leukocytes indicated inflammation of the genito- 
urinary tract. Some workers count the pus cells in urine by the samo 
technic used for the leukocyte count of the blood. At times, the urine 
of women may contain an abundance of pus cells without pathological signi- 
ficance. 
Erythrocytes: These may retain their biconcave form, be crenated, or only 
show as pale, possibly double-ringed bodies termed ’’shadows,” They are 
however, usually quite uniform in size, a fact that will aid when one 
encounters, as he frequently does, other structures that closely resemble 
them, such as typical calcium oxalate crystals. Vegetable spores are 
often a source of confusion. It is well to always check a positive find- 
ing with a test for occult blood. 
Epithelial Colls; It is almost impossible to state positively the origin 
in the genito-urinary tract of certain cells, A very trustworthy evi- 
dence, however, is finding of epithelial cells in cases of the so-called 
compound-granule cells (fatty degenerated renal epithelium). Sheets of 
more or less round or caudate epithelial colls arc rather significant to 
the clinician. Vaginal epithelium resembles that secured by scraping the 
buccal mucosa. Bladder epithelium resembles vaginal but is of smaller 
size. Urethral is like that from the pelvis of the kidney, but smaller. 
Cells from the region of the prostate are very rofractilo, have a distinct 
nocleus, and are oval rather than round. Cylindruria: This means the presence of cylindroids or casts. It 
will be found that a 2/3 inch objective gives almost all the informa- 
tion required. -Cylindroids are long structures resembling hyaline casts 
but showing tapering ends, irregularly in diameter, and longitudinal 
striations. They have the same significance as hyaline casts. Casts 
are cylindrical structures with rounded ends. One must bear in mind 
their fragility. It is said that prolonged centrifuging will disorganize 
them. They are of a light specific gravity and tend to occur in the 
upper portion of the sediment, either in centrifuge tube or on the slide, 
Todd says, "If the tubule be small and has its usual linging of epithe- 
lium the cast will be narrow; if it be large or entirely denuded of 
epithelium, the cast will be broad". Persistent presence is of graver 
significance, than occasional occurance. 
i 
Hyaline casts are narrow and homogeneous. They follow almost 
any renal distrubance, and are not necessarily indicative of, per- 
manent damage. Finely granular casts may have no greater significance 
than hyaline, and, as a matter of fact., the latter shqw a granular struc- 
ture with dark-ground illimination, iis the granules become coarser, it 
is generally considered that a more severe lesion is present. Fatty 
casts are especially associated with fatty degeneration, Epithelial 
casts are especially to be noted and reported, as the number present 
mean much to the doctor. Blood casts and pus casts mean a serious kid- 
ney condition and also should be noted and the number reported per low 
power field. Waxy casts are highly refractile and show fissuring of the 
margins. They may accompany a severe acute nephritis, and for that mat- 
ter, should be reported. To summarize, always report the presence, kind, 
and size, and any cast noted in a urine, as they mean much to your doc- 
tor , 
Starches and Fibers: In examining urinary sediments it is important to 
be familiar with.the various textile fibers and starch grains which are 
so frequently present, the fibers- coming from the clothing and the starch 
grains from dusting powder. Viool fiber fragments show bark or scale- 
like imbrications and are round. Cotton fibers are flattened and twis- 
ted, while linen ones show a striated, flattened fiber with frayed seg- 
ments as of a cane stalk. Silk shows a glass-like tube with*mashed in 
ends. Corn and rice are the most common of starch grains and their - 
nature is immediately disclosed by their blue color when mounted in-’ 
iodine. Scratches in the glass slide, or dust particles may be present 
in the specimen, but can usually be distinguished. REPARATION OF REAGENTS FOR URINALYSIS 
Dilute acetic acid 
Glacial acetic acid • ••••••• . ... i ..... • 3 cc. 
Distilled water •••••••••* « ... i .... • 100 cc. 
Benedicts Qualitative Solution 
Dissolve, with the aid of heat* 
Sodium citrate 173 grams 
Sodium carbonate (anhydrous) •••••••••••« 100 grams 
Water . * , , , , 700 cc. 
Filter solution if cloudy and then add slowly, with constant 
stirring: 
Copper sulphate ... .......... .17.3 grams 
Water .100 cc. 
Cool and dilute total volume to ...•••••. . 1000 cc. 
Qualitative Solution 
Dissolve in about 300 cc. water: Sodium hydroxide - 32 grams. 
In another container dissolve in about 200 cc. water, with the 
aid of gentle heat: copper sulphate: 12 grams. 
Mix the two solutions in a 1000 cc. graduated flask or cylinder 
and add 90 cc, glycerine. Dilute with water to liter mark and 
mix* 
Ammonia Water 
Regular U.S.P. Sponger ammonia. 
Litmus 
Supplied in both red and blue paper strips at all surgical 
supply or laboratory supply houses. 
Sodium Nitroprusside 
.Sodium nitroprusside crystals •••.••••••• 10 grams 
Water • 50 cc. 
Mix the two and with aid of gentle heat dissolve 
the crystals. 
Glacial acetic acid 
Procurable at any drug store. Robert1s Reagent 
Nitric Acid 1 part 16? cc. 
Magnesium Sulphate Saturated Solution,5 parts.. 833 cc. 
1000 cc,... 
Hydrogen. Peroxide 
Obtain at any drug store. 
Tsuchiya1s Reagent for Quantitative Albumin 
Phosphotungstic acid 1.5 grams 
Hydrochloric acid, concentrated 5 cc. 
Ethyl alcohol, 95$.... * ,. 95. cc. 
Keeps> indefinitely, < ... 
Esbach* s Solution 
Picric ‘ioid (crystals) 10 grams 
G trio arid (crystals 20 grams 
,Distilled water 1000 cc. 
Benedict!s Quantitative Solution 
Crystal 1.23d copper sulphate 10 grams 
Anhydrous sodium carbonate (or double the 
. weight ox the crystalline salt).,.-, 100 grams 
Sodium nitrate., .200 grams 
PoT/assium sulphocyanate 125 grams 
Potass i’lm ferrocyanide (5v> solution).. .5 cc. 
Distilled’vaster, sufficient to make... 1000 cc. 
a. Dissolve the sodium carbonate, sodium citrate and po- 
tassium sulphocyanate in about 700 c-*. water with the 
aid of gentle heat. 
b. Filter. 
c. Dissolve the copper sulphate in about 1000 cc. water, 
d. Pour the copper solution Into the other solution with 
constant stirring, 
e. Add the ferrocyanide solution, 
f. Cool and dilute to 1000 cc. 
Lugol1s Solution 
Iodine, rosublimod crystals ' 5 grams 
Potassium iodide, crystals; 10 grams 
Distilled water 100 cc. 
Benzidine Powder 
Procurable at drug supply house. Specify Merck’s Blue Label, Gram* s Iodine 
Lugol's Solution,*.*,,,, 1 part 
Distilled Water* 14- parts 
Obermayer1s Reagent 
Ferric Chloride ,, 2 grams 
Hydrochloric Acid ..,,1000 cc. 
Buffer* s Solution for Wright1s Stain 
Manobasic Potassium Phosphate 6,63 Gm, 
Dibasic Sodium Phosphate 3.20 Gm. 
H2O ,..,,1000 cc. 
Gram1s Iodine 
Iodine, Resublimed crystals 1 Gm. 
Potassium Iodide Crystals 2 Gms. 
Distilled Water. 300 cc. I. Sputum 
A. Normal sputum may contain: 
1. Small rather dense mucoid masses - may be translucent or 
may be a gray, or may be pigmented. 
2. Lay have pus cells. 
3. May have endothelial cells. 
U. May have bacteria. 
a. Saprohytic bacteria 
(1) Staphylococcus. 
(2) Streptococcus. 
(3) Pneumococcus. 
(A) Micrococus Catarrhalis. 
b. Spirochetes , 
c. Endamebae 
B. Sputum should be brought up from the lungs. Mouth should be 
rinsed out in order that it be free of food particles and 
mouth debris. Be sure the specimen does not come from the 
nose or the naso-pharynx; must be placed in a clean recepti- 
cal and no disinfectants must be added until after examination. 
C. The study of sputum should be divided in Physical Examination, 
Microscopic Examination and Chemical Examination. 
1. Physical Examination comprises: 
a. Quantity which may be in amount as being imperceptible 
to 1000 cc. 
b. Color which may be any shade from clear and transparent 
to colors and their shades through the yellows, greens, 
red and blacks, 
c. Consistency may comprise any degree in character from a 
thin consistency to a very tenacious mass. May be 
serous, mucoid, purulent, a mucopurulent. 
d. May produce layer formation. 
e. Ditrich’s plugs. 
f. Lung stones, 
g. Bronchial casts. 
2, Microscopical Examination consists: 
a. An unstained specimen! should first be examined; this en- 
ables one to choose what particles of the sputum should 
be most productive of organism, or parts to be examined. 
b. In unstained sputum one should find elastic fibers, 
Cuschmann’s spirals, Charcot-Leyden crystals, myelin 
globules, molds, pus corpuscles, mucus and granular 
detritis, also pigmented cells, heart failure cells. 
c. Stained sputum. 
(1) Several smears must be made of each specimen so that 
all stains can be utilized and all organisms, if pre- 
sent, will be stained. 
(2) Technique. 
(a) Proper specimen must be isolated; toothpicks or 
applicators should be used in preference to the 
wire loops. Those used must be disposed of pro- 
perly after their use. . (b) Material when placed on a slide must be allowed to dry. 
(c) Fix in wood alcohol and 1$ sol, corrosive sublimate for 
2 min. 
Or pass through a flame with the film up several times, 
care being taken not to burn the film. 
(d) Stair 
1- Carbol, Fuschin for T.B, 
2- Grams stain for other bacteria and their identifi- 
cation. 
3- Wright's stain for differentiation of white cells. 
3. To stain T.B, 
a. Select the most purulent part, keeping away from the mucoid 
portion. 
b. May use Cabbet's Method or Zienl-Neelson's Method - this lat- 
ter method is used most frequently. 
(1) Steam carbol fuschin on the smear for not less than 3 
minutes; avoiding too much heat, so that it boils or 
allowing it to evaporate. 
(2) Wash until faint pink with either, 
(a) 5$ HNCU Nitric Acid 
(b) Acid alcohol: 3 cc HcL; 97 cc Alcohol (70$) 
(3) Stain lightly with Loffler's blue or equal parts of 
alcohol and saturated solution of picric acid. 
4. Tuberculosis Bacillus or Koch's Bacillus or the Acid Fast Bacillus, 
if present, will be seen as slender small rods in length, half 
the width of a R.B.C, They may be singly or in groups, T.B, Ba- 
cillus is to be differentiated from Smegma B., also edges of deep- 
ly stained material that has not decolorized and looks like ba- 
cilli, and an organism that comes off of Bermuda grass. Failure 
to find T.B. must call for several more examinations of the spu- 
tum and several more specimens of sputum, When definitely sus- 
picious, sputum for T.B. is expected and not found by the usual 
methods. Concentration methods, cultural methods or animal in- 
noculation methods are utilized. 
a, Loffler's method for concentration, 
(1) 10 cc, of sputum. 
(2) 10 cc, of 50$ antiformin (equal parts of 15$ solution of 
caustic soda and 20$ solution of sodium hypochlorite). 
(3) Heat to boiling. 
(4) To each 10 cc. of fluid resulting, add 1*5 cc, of 1 vol. 
chloroform and 9 vol, alcohol, 
(5) Shake vigorously for several minutes, 
(6) Centrifuge for 15 minutes, 
(7) The Bacilli are in the layer over the chloroform which 
is at the bottom. 
(B) Transfer the sediment to slide, 
(9) Add a little original sputum. 
(10) Dry, fix and stain, 
b. Culture method is rarely done due to the difficulty in the 
growth of T.B. on artificial means much as media. c. Animal innoculation of course is the court of last appeal 
and when negative, there are no T.3. present, however, 
there must be 10 to 150 bacilli present in the injected ma- 
terial, depending on the virulence of the organism to show 
positive pathology. 
Other forms of Bacteria cells found in a stained specimen 
of sputum are: Staphylococci, Streptococci, Ivluch’s granules, 
Heart failure cells, dosinophiles, Pneumococci, Friedlanders 
Bacilli, Influenza Bacilli, micrococcus Catarrhalis, Higher 
Bacteria, Spirochetes, fungi and other leucocytes. 
(a-1) An additional concentration method used at the Brooke 
General .Hospital. 
(1) Concentration method: Sodium Hydroxide: mix equal 
parts of sputum and a 3>% sodium hydroxide solution, 
shake well, and incubate at 37° C. for fifteen to 
thirty minutes depending on the consistency of the 
specimen. Neutralize with normal hydrochloric acid, 
checking reaction with litmus. Centrifuge and pre- 
pare films from the sediment, fix, stain and examine 
as above. EXAMINATION OF STOMACH CONTENTS 
(1) Blood is present in the vomitus in a great variety of conditions. 
When found in the fluid removed after a test meal, it commonly 
points toward ulcer of carcinoma. Blood can be detected in 
nearly one half of the cases of gastric cancer. The presence 
of swallowed blood and blood from injury done by the stomach 
tube must be excluded. 
I 
Test for Blood in Stomach Contents; extract with ether to 
remove fat if this be present, which is usually not the case 
after a test meal. 
To 10 cc, of the fat-free fluid, add 3 or 4- cc. of glacial 
acetic acid and shake the mixture thoroughly with about 5 cc, of 
ether. Let stand a short time, remove the ether, which forms a 
layer above the stomach fluid, and use half of it for the guaiac 
or benzidine test. Separation of the ether may be facilitated 
by adding a small amount of alcohol. In the case of a positive 
reaction the remainder of the ether extract may be examined 
spectroscopically after treating so as to develop the bands of 
hemochroraogen. 
When brown particles are present in the fluid the hemin test 
may be applied directly to them. 
(2) Quantitative Tests, 
(a) Total Acidity; the acid-reacting substances which con- 
tribute to the total acidity are free hydrochloric acid, 
combined hydrochloric acid, acid salts, mostly phosphates, 
and, in some pathologic conditions, the organic acids. The 
total acidity is normally about 50 to 100 degrees (see 
method below), or, when estimated as hydrochloric acid, about 
0.2 to 0.3 per cent. 
(b) Topfer's Method for Total Acidity: in an evaporating dish or 
small beaker take 10 cc. filtered stomach contents and add 
3 or 4 drops of the indicator, a 1 per cent alcoholic 
solution of phenolphthalein. When the quantity of stomach 
fluid is small, 5 cc. may be used, but results are less 
accurate than with a larger amount. Add decinormal solution 
of sodium hydroxide drop by drop from a buret, until the 
fluid retains and there remains constant a faint, pink blush. When this point is reached, all the acid has been, 
neutralized. The end reaction will be sharper if the fluid 
be saturated with sodium chloride. A sheet of white paper 
beneath the beaker facilitates recognition of the color change. 
In clinical work the amount of acidity is expressed by 
the number of cubic centimeters of the decinormal sodium 
hydroxide solution which would be required to neutralize 
100 cc. of the gastric juice, each cubic centimeter represent- 
ing one degree of acidity. Hence, multiply the number of 
cubic centimeters of decinormal solution required to neutralize 
the 10 cc. of stomach fluid by 10. This gives the number of 
degrees of acidity. The amount may be expressed in terms of 
hydrochloric acid, if one remembers that each degree is 
equivalent to 0.00365 Gm. of hydrochloric acid. Some one 
suggests that this is the number of days in the year, the 
last figure, 5, indicating the number of decimal places. 
Example: Suppose that 7 cc. of decinormal solution were 
required to bring about the end reaction in 10 cc. gastric 
juice; then 7 x 10 * 70 degrees of acidity; and, expressed 
in terms of hydrochloric acid, 70 x 0.00365 = 0.255 Gm. 
(c) Free Acidity. 
Topfer's Method for Free Hydrochloric Acid: In a beaker 
take 10 cc. filtered stomach fluid and add A drops of 
the indicator, a 0.5 per cent alcoholic solution of 
dimethylamine-razobenzol. A red color instantly appears 
if free hydrochloric acid be present. Add decinormal 
sodium hydroxide solution, drop by drop from a buret, 
until the last trace of red just disappears, and a 
canary-yellow color takes its place. For accuracy it is 
better (Benedict), not to carry the titration quite to 
the canary-yellow stage, although the end point is then 
not so definite. Better still, when all the red has 
disappeared, read off the number of cubic centimeters 
of decinormal solution added, and calculate the degrees 
or percentage of free hydrochloric acid, as in Topfer’s 
method for total acidity. 
When it is impossible to obtain sufficient fluid 
for all the tests, it will be found convenient to esti- 
mate the free hydrochloric acid and total acidity in the 
same portion, and this is frequently adopted as a routine 
regardless of the amount of fluid available. After 
finding the free hydrochloric acid as just described, add 
A drops of phenolphthaiein solution, and continue the 
titration. The total amount of decinormal solution used 
in both the titrations indicates the total acidity. (3) Resume of Results. 
(a) After the Ewald test breakfast, the amount of free hydro- 
chloric acid varies normally between 25 and 50 degrees, or 
about 0.1 to 0.2 Gms. In disease it may go considerably 
higher or may be absent altogether. When the amount of 
free hydrochloric acid is normal, organic disease of the 
stomach probably does not exist, 
(b) , Increase of free hydrochloric acid above 50 degrees 
,• (hyperchlorhydria) generally indicates a neurosis, but also 
occurs in most cases of gastric ulcer and beginning of chronic 
gastritis. It has been found in normal persons. 
(c) Decrease of free hydrochloric acid below 25 degrees (hypo- 
chlorhydria) occurs in some neuroses, chronic gastritis, 
early carcinoma, pellagra, anemias, and most conditions 
associated with general systemic depression. Marked 
variation in the amount at successive examinations strongly 
suggests a neurosis. Too low values are often obtained at 
the. first examination, the patient’s dread of the intro- 
duction of-thel tube probably inhibiting secretion, 
(d) Absence of free hydrochloric acid (achlorhydria) occurs in 
most cases of gastric cancer and far-advanced chronic 
gastritis, in many cases of pellagra, and sometimes in 
hysteria and pulmonary tuberculosis. Achlorhydria is a 
constant and important symptom of pernicious anemia even 
• during remissions. It sometimes appears long before any 
anemia is recognizable, 
(e) The presence of free hydrochloric acid presupposes a normal 
amount of combined hydrochloric acid, hence the combined need 
not be estimated when the free acid has been found. When, 
however, free hydrochloric acid is absent, it is important 
to know whether any acid is secreted, and an estimation of 
. . the combined acid then becomes of great value. The normal 
average after an Ewald breakfast is about 10 to 15 degrees, 
the quantity depending updn the amount of protein in the 
test meal. Somewhat -higher figures are obtained after a 
Riegel test-meal. • Of greater significance than the amount 
of combined acid is -the acid deficit, described later. 
(f) For the determination of combined hydrochloric acid, a 1% 
aqueous solution of sodium alizarin sulfonate is used as 
an indicator. - Titration .is done the same as described 
under free acidity above. The end point is at the time of 
the appearance of purple color. Is also calculated as 
above the amount being determined by subtracting the results 
of this titration from the amount of predetermined total 
acidity.. " ANTIGENS, ANTIBODIES, AND IMMUNITY 
In the worst epidemics in the history of the world, there 
have always been a few survivors, those who recovered from the di- 
sease, and those who did not even contract the disease. These peo- 
ple must have had some natural protection which helped them recover 
or prevented the disease entirely. This is called natural immunity. 
When some disease organisms invade the body, the body develops a 
protective condition which prevents that disease organism from pro- 
ducing disease again. This is called acquired immunity. The sub- 
stance which stimulates the production of this protection is called 
an antigen. Antigen is chemical in nature, seems to be protein, and 
may come from either bacteria or cells. The protecting substance 
which is formed to react against the antigen is called the antibody. 
There are several types of antigens, antibodies, and reactions pro- 
duced by them as shown in the table below. 
ANTIGEN 
ANTIBODY 
REACTION 
Toxin 
Antitoxin 
Neutralization (makes non- 
poisonous) 
Agglutinogen (bacte- 
Agglutinin 
Agglutination (causes clump- 
ria or cells) 
ing) 
Precipitinogen 
Precipitin 
Precipitation (separates 
(soluble -proteins) 
from solution) 
Lysinogen 
Lysin 
Lysis (dissolving) 
(■bacteria or cells) 
> 
3act eri oci dinogen 
(bacteria) 
Bacteriocidin 
Kills bacteria 
Opsoninogen 
Opsonin 
Increases phagocytosis 
(bacteria or cells) 
(ingestion of substance 
by special cells) 
Complement fixing 
Antigen (bacterial, 
Amboceptor 
Complement fixation 
cellular, or protein) 
The a'oove reactions serve as a basis for a number of- tests 
done in the laboratory. The reactions ere specific for both anti- 
gen and antibody. Therefore if a known antigen is added to an un- 
known antibody and the specific reaction is -produced, the specific 
antibody must have been -present. If no reaction is -produced, the 
antibody wee not present. Also if eo unknown antigen is added to a 
known antibody and the s-pecific reaction is produced, the specific 
antigen must be present. If the reaction is not produced, the spe- 
cific antigen is not present, S-oecial procedures and techniques 
are necessary to accomplish these tests. SEROLOGICAL TESTS FOR SYPHILIS 
TESTS PRESCRIBED ' 
For uniformity and for efficiency of supply, Circular Letter 
#10 dated'^February 13, 1941, from the Surgeon Gerieral’.s Office 
makes several provisions for tests for syphilis to be used in the 
Army of the United States. ' . 
The qualitative Kahn flocculation test is to be used for ell 
rout i neatest s’, . - . .. * 
The ¥ksscrmann test with, anti-sheep, hemolytic system^will be 
usjed fo-h' spinal fluid and for confirmatory tests on-new doubtful, 
and positive serums-of patients with no other positive clinical 
symptoms. , - . • 
.Method Of reporting 
Results of tests will be reported ps ’’positive11 , ’’doub.tful’* , 
or “negative.” 
The system ’’ , , and will not be used, .. • •' ’• 
Records 
The first specimen of blood of a patient submitted to any 
Army laboratory will be accompanied with Form 97MD properly ex- 
ecuted, ‘ It will be. kept at the laboratory, as a permanent record 
of serological tests done fpr that particular patient. 
Each'specimen of blood or serum submitted for test for sy- 
philis will be accompanied by Form 55L-3MD properly executed in 
duplicate. The original is returned to the sender with recorded’* 
results. The duplicate is kept by the laboratory as a temporary' 
record and the report entered in the proper space on Form 97 MD 
for permanent record. 
.Each; specimen of1 spinal fluid will bo accompanied by Form 
55L-4MD properly executed in duplicate. Results of1 the'test will 
be reported to the sender oh the original end the duplicate will 
be kept in the laboratory with results recorded in proper place 
on Form 97 MD for permanent record, • : ' 
All the above forms are first.made out or.initiated by. the 
surgeon making the request for the test, ’ u-r ■ 
Every laboratory * doing any test for sydhilis on a patient- 
will have a form'97MD for'that patient, THE STANDARD KAHN TEST 
REAGENTS 
1* Physiological salt solution 
Sodium chloride, CP, 8,50 Crams * 
Distilled water 1000,00 Ml, 
Filter if necessary before usin^,; The solution need 
•not be sterile but must be chemically pure. 
2, Standard Kahn Antigen 
Medical Department Supply Catalog, Class 1, order thus: 
, (Item #) 17030-AlJTIGBtT FOR KAHF TEST, 50 00:(unit)bottle. 
Secured on quarterly reauisition or on emergency requisition from 
the Army Medical Center, Washington, D,C. 
It is prepared by the Army Medical School, Army Medical 
Center, Washington, D.C. It is an alcoholic extract of powdered 
beef heart that has had the fat removed.by ether extraction, and has 
had cholesterol added to Increase its sensitivity. The solution 
has been standardized to a degree of specificity-and sensitivity 
required for the standard Kahn will maintain this spe- 
cificity and sensitivity many years if .the following precautions * 
are taken: 4 ' 
a. Keep stored in the dark. Mailing container satisfactory, 
r b, Store at to on temperature only, 
c. Keep tightly stoppered at all tines, 
, d, Stopper must he adeouately covered with high grade 
tin foil. Rubber- and cork contain alcohol soluble 
substances which affect specificity, 
e. Only dry, chemically clean glass vessels shall be 
used to store the antigen, . 
f. Only dry, chemically clean pipettes shall be used to 
measure the antigen during preparation for the test, 
To prepare antigen suspension for the Kahn tests proceed 
as fellows: * ' ;;4 
Place required amount of antigen in one tube (never 
less than 1 ml,) 
b. Place,required amount of saline in second tube (quan- 
tity indicated by titre stated on label of antigen bottle, as; ,, 
litre 1 1.4 means that for each 1 ml, of antigen u$e 1,4'mi;, ' 
of saline). c. Pour saline into antigen quickly, then pour back and 
forth 12 times, 
d. Allow to age for ten minutes before use, but use before 
thirty minutes for best results. 
3. Unknown Serum 
a. .Eight to ten ml. of blood are collected by venipuncture 
with the usual aseptic technical© from the -patient to be tested, 
then place blood in a clean dry tube. Water causes hemolysis. 
b. Allow blood to stand one hour at room temperature for 
clot to form. Do net agitate because hemolysis may result, 
c. Free the clot from the walls of the tube with a clean 
dry applicator, Use separate, clean applicator for each tube, 
d. Centrifuge at 2000 HPM for ten minutes or -place in a 
refrigerator and allow clot to retract, and -preserve until ready 
to use. 
e. Decant or pipette serum into a clean dry tube. Recen- 
trifuge if cells are -present in serum, 
f. Place tube of serum in a water bath at 56°C for one- 
half hour to inactivate, 
g. Test serum as soon after inactivation as possible. 
Serums not inactivated for five to twenty-four hours should be 
inactivated again for ten minutes. If over twenty-four hours, in- 
activate for fifteen minutes. 
Serums will vary in color because some will have e small 
amount of hemolysis causing a. red color, some will have-chyle 
causing a cloudy serum, and some will have an increased amount of 
bile causing an amber or brownish color. There must be no contami- 
nation with water or other substance. 
4. Known Positive Serum 
Serum previously tested and known to be ’’four ulus”, - i.e., 
strongly positive, It must be inactivated before use which is 
done in the same manner as the unknown serum. 
Blood may be withdrawn by venipuncture from a -person re- 
cently tested and known to be strongly positive. The serum is 
prepared in the same manner as the unknown serum. 
5, Known Negative Serum 
Serum -previously tested and known to be negative* It must 
be inactivated before use in the test. 
Blood may be withdrawn by venipuncture from a person recently tested and known to be negative. The serum is prepared in the 
same manner as the unknown serum, . 
APPARATUS 
1, Blood tubes, 2 for each control serum and fur each blood 
tested (one for whole blood and one for serum), 
2, Kahn tubes,"9 for controls and 3 for*each unknown tested* 
3, Kahn rack, one for each 30 tubes, 
4, Vials or tubes for mixing antigen, 2, 10 to 25 ml, 
5, Pipettes, 1 ml, graduated to 0,01 ml., 3 for controls 
and 1 for each unknown serum. 
6, Pipettes, 0,2 or 0.25 ml. graduated to 0,0125 ml,, 
1 to use for antigen. 
7, Pipettes, 5 or 10 ml. graduated to 0,1 ml, 
8, Centrifuge anc1 balance, 
9, Water bath adjusted to 56^0. 
STANDARD KAHF PROCEDURE - on next page. RESDXiTS; Check: contrcl.s first. If unsatisfactory, reneat entire test, 
negative - Solution remains cloudy, no flocculation present in any 
of the three tubes. 
Positive - Varying degrees of flocculation and loss of cloudiness of fluid. 
Head each tube separately, report average, as front tube 3 plus 
flocculation 1 plus) middle n 3 plus 
50% 2 n } Doubtful rear H 1 M 
75% M 3 « Total 7 
100% 11 . 4 h Positive Average 2 plus 
Report n doubtful,f 
IMETOWU 
. _ Anti gen 
SERUM 
°45 if1- 
ft l» 
1 
Saline 
RESULTS 
See below 
Each Unknown Serum 
Front tube 
Middle ,r 
Rear » 
0.05 ml. 
0.025 « 
0.0125 « 
to 
W 
fcl 
V-' 
1,0 ml, 
0.5 « 
0.5 » 
X 
OGi.'rx’JttOLS; 
Known nositive 
Front tube 
Middle " 
Rear ” 
0.05 ml, 
0.025 »» 
0.0125 « 
nos, serum 
0,15 ml. 
It 11 
11 If 
Mi 
0 
U: 
EH 
0 
fH 
1 
t 
to 
1.0 «1. 
0.5 » 
0.5 H 
ti 
0 
EH 
1 
I 
m 
x 
8 
Marked 
flocculation, 
clear fluid 
Known negative 
Front tube 
Middle n 
Rear " 
0.05 ml, 
0.025 « 
0.0125 " 
Feg, serum 
0.15 ml, 
it ii 
ti it 
1.0 ml, 
0.5 « 
0.5 ,! 
No 
flocculation, 
cloudy fluid 
Saline control 
Front tube 
0,05 ml. 
Saline 
0.15 ml. 
1.0 ml. 
No 
flocculation. 
Middle » 
Rear ,f 
0.025 « 
0.0125 » 
it it 
» it 
0.5 b 
0.5 n 
cloudy fluid 
STANDARD KAHN PROCEDURE (ALL MATERIALS HAVING BBE3T PREPARED) QUANTITATIVE KAHN TEST PROCEDURE 
To be done only on serum showing three or four ulus reaction 
with Kahn test. 
1. Blood collected by venipuncture is allowed to clot, then is 
centrifuged to separate the serum. Serum must be entirely free 
of cells. 
2. Inactive serum in water bath at 56°C for 30 minutes. 
3. Mix Kahn Antigen 
a. Place 1 ml, antigen in one vial or tube. 
b. Place required amount of saline in second vial or tube, 
(quantity indicated by titre on label of antigen bottle) 
c. Pour saline into antigen quickly, then pour back and 
forth twelve times. Allow it to age ten minutes. 
4, Prepare serum dilutions as follows while antigen is aging: 
Tube # Saline Serum or serum dilution Dilution Ratio 
1 0.6 ml + 0.4 ml, serum 1:2,5 
2 0.5 n 4 0,5 M of dilution from tube #1 —1 : 5 
3 n n n n « n n « #2 —1 ; 10 
4 h«, I » h « w tt ti *3 __i ; 20 
5 h « t h ft it it n it #4 __1 • 40 
6 it ti + « tt m it « « #5 .-i . 80 
If necessary the dilutions may be carried higher. 
5, A one-tube Kahn test is then done on each of the above dilu- 
tions as follows: ulace six tubes in a rack and uroceed as 
shown. Start with tube #6, 
Tube # Antigen Serum Saline 
6 - 0.025 nl, 0.15 nl of 1 : 80 dxl.j jo.5 nl.i 
5 - » “ « " » 1 : 40 11 ” " ! ■§ 
4 - " « « » " i : so " L „ " * ; 5 £ 
3 - " " " " " 1 ; 10 • M 
2 - « « " " " 1 ! 5 » §1 " " I % H 
1 - " 0 " " " 1 :2.5 « 5'S| " "|S E 
6. Results: 
a. The highest dilution which shows a three plus or four ulus 
reaction indicates the quantitative dilution. 
Id, Reporting, two methods used, 
(1) By units - Quantitative dilution x 4 : no, of units. 
Example - 1 : 20 dilution is highest dilution 
showing a three ulus reaction, so; 20 x 4 = 80 
units, the quantitative reading. 
(2) By reuorting the reaction of each tube starting 
with tube #1, as: TITRATION OF KAHN AM1! GSM 
' Kahn'antigen is very stable if properly taken care of, It 
occasionally becomes necessary, however, t~ check the titre be- 
cause of possible improper care, 
■’ By determining the titre one determines the amount of Physio- 
logical saline to add to each ml, of antigen when preparing it for 
test. 
To determine this amount various amounts of saline are added 
to 1 ml, amounts of antigen. JL precipitate is formed in each in- 
stance. When a specified amount of saline is added to each, the 
precipitate redissolves in some tubes and does not.in others. The 
antigen-saline mixture having the least amount of saline in which 
the precipitate redissolves in further addition of saline indicates 
the quantity of saline to add to each 1 ml,, of antigen, in other 
words the titre. 
To-accompli sh the titration, set up 25 tubes as shown below. 
PROBLEM: To check AETIGM- TITHE LABELED U 1.4 
- 
Antigen-Saline 
Test 
’each of the Antigen 
-saline 
mixes to "be tried 
•H 
d 
tv!) O P 
•hxi d d 
P P p 
mixtures as follows: 
' Tube 
Tube 
Antigen 
Saline 
. 
. o 
Saline 
la Saline 1,2 
ml. 
Front 
0.05 ml. 
0.15 
ml 
1.0 
ml 
lb Antigen 1.0 
» 
Ph o 
«oa)g 
Middle 
0.025 " 
ft 
ii 
p 
d 
0,5 
tr 
tH-p B3 
O P 
Fear 
0.0125" 
tt 
tt 
d»» 
•P? 
o 
0.5 
it 
2a Saline 1.3 
ml. 
d d p*h 
Front 
0.06 " 
»» 
ti 
1.0 
tt 
2b‘ Antigen 1.0 
n 
•H CU *H 
p. CO 
Middle 
0,025 " 
ii 
it 
COrH 
rH 
0.5 
it 
M 
s—tu 
d o <rtfo 
cs -a) d 
cu.d.c'p 
Rear 
0,0125" 
n 
it 
d O 
' Od 
0.5 
it 
S 
. 3s, Saline 1.4 
ml. 
Front 
0.05 ", 
it 
1.0 
it 
O 
4J> 
3b Antigen 1,0 
if 
• d d to 
Middle 
0.025 " 
li 
« 
>iTO 
rH 
0.5 
it 
si 
9 
P d 
w.O • o 
n.‘ in 
0> 0) -H 
Rear 
0.0125"’ 
' n 
tt 
Wfl) 
dd 
OH 
Phi—1 
0.5 
it 
4a Saline 1.5 
ml. 
Front 
0,05 " 
it 
It 
1.0 
tt 
o 
a 
4b Antigen 1,0 
u' 
d P p 
•H *»H O • 
“ f 
CO 0) to o 
Mi ddle 
Rear 
0.025- " 
010125" 
tf 
ti 
it 
it 
od 
tiOin 
•H 
l>rd 
<xi 
pcd 
M 
0.5 
0.5 
it 
<t 
0) 
<D 
AJ 
s 
XJ 
CO 
5a Saline 1.6 
ml; 
<D f> O .Cl 
HH d W 
Front 
0 L;05‘ " 
r 
tt 
1.0 
ti 
5b Antigen 1.0 
ft 
P d «> 
O j.3 o CO 
Middle 
0.025 " 
If 
it 
jdd 
wS 
0.5 
tt 
p d 
Rear 
0.0125" 
tt 
tt 
0,5 
it 
— 
The saline-anti-gen mixture with the least amount of saline in which 
the precipitate completely redissolves is the mixture or titre to 
'use for'the test. 
When checking antigen of lower titre, use smaller amounts of saline 
for each ml,- of antigen. For example, if antigen is labeled 
1 y 1.1, make mixes 1 t 0.9; 1 + 1.0; 1 ■*. 1.1; 1 + 1.2; 1 * 1.3, 
If higher titre, use larger amounts of saline. SPINAL FLUID KAHN PROCEDURE 
1* Centrifuge spinal fluid until free of cells, 
2. . Place in a centrifuge tube the following: 
a. Clear spinal fluid , , . . ... , . , ,1.5 ml, 
b. Saturated Ammonium sulfate solution, , 1,5 ” , 
3. Place centrifuge tube in water hath 56°C. for 15 minutes, A 
white cloudiness or precipitate indicates the presence of globulin, 
r - * . 
4. Centrifuge 3000 PPM for 10 minutes to pack globulin in bottom of 
tube, 
5. Decant and discard supernatant fluid. Drain remaining super- 
natant fluid by inverting the centrifuge tube on a clean towel 
for several minutes, 
6.,- Dissolve precipitate by adding 0,15 ml, of 0,85$ saline, then 
shaking, 
7. Do a Kahn test on this globulin solution as follows: 
a. Mix Kahn Antigen, 
(1) Place 1 ml, antigen ip one vial or tube, 
(2) Place required amount of saline into sec- 
ond vial or tube, (Quantity indicated by 
titre on label of"antigen bottle,) 
(3) Pour saline into antigen quickly, theri 
pour back and forth twelve times. Allow ; • 
it to age 10 minutes, - ' * 
b. Pipette into bottom of a Kahn tube. 
(1) Antigen, 0.01 ml, 
(2) Globulin ■ solution, " 0.15 11 
Shake.vigorously for 3 minutes, 
(3) Add Saline. 0,5 ml. 
Shake to mix. 
8, Results: 
Positive - Flocculation with loss or clearing of cloudy ap- 
pearance, 
Negative - No flocculation, no change, no loss of cloudiness. 
73 THE COMPLEMENT FIXATION TEST 
history - In 1888 Hut tall demonstrated bacteriolysis, the process 
of dissolving bacteria. He mixed a measured amount of blood serum 
or defibrinated blood with a measured amount of certain bacteria. 
He incubated the mixture for several hours at 37°C, He then poured 
several plates with this mixture in the media. He also poured 
several plates with the bacteria alone without the serum. After 
allowing time for growth, he demonstrated that the plates contain- 
ing the serum had many less colonies, some of them being nearly 
sterile. This showed that there was something ih the serum that 
inhibited the growth of or killed many bacteria. 
Futtall also demonstrated that if the serum was heated to 
55 or 56° C, or was allowed to stand or age, it would no longer 
kill or inhibit the growth of the bacteria. 
In 1894 Pfeiffer demonstrated bacteriolysis to be specific, 
that is, if a specific bacterium is injected into an animal, that 
animal develops a bacteriolysin, the substance producing bacterio- 
lysis, This bacteriolysin will dissolve that particular bacteria 
but no others. This he demonstrated in the following manner. 
Into the peritoneal cavity of a guinea pig which had recovered 
from cholera-was injected a suspension of cholera spirilla. At 
regular intervals after the injection, exudate was withdrawn from 
the peritoneal cavity. The first specimens withdrawn showed swell- 
ing of the spirilla, later specimens showed distortion in shape, 
granulation in appearance, and finally complete disappearance. 
Other bacteria injected into the peritoneal cavity of a 
guinea pig recovered from cholera were not thus affected thus show- 
ing the action to be specific. 
Pfeiffer also observed that guinea pigs that had recovered 
from cholera would tolerate large doses of cholera spirilla in- 
jected' into their peritoneal cavities, whereas other guinea pigs 
died from small doses. 
He also observed that if the peritoneal cavity of a normal 
pig was injected with a mixture of cholera spirilla and the blood 
serum from a guinea pig recovered from cholera, the normal pig 
escaped infection. This proved that a normal guinea pig could be 
protected from the disease, that the blood carried the same bac- 
teriolysins that were present in the peritoneal cavity as shown 
above, 
Pfeiffer also showed that the immunized serum, serum from 
guinea pigs recovered from the disease, as used in the above ex- 
periments, was used just as effectively if heated as if not heated 
when injected in the peritoneal cavity of the guinea pigs. But 
if used in the test tube, the heated serum did not dissolve the 
spirilla* 
Bordet showed that if a small amount of fresh normal serum was 
added to the heated immune serum, bacteriolysis or dissolving of 
the bacteria would also take place in the test tube. Bordet1s experiments showed that unheated immune serum depends 
on three substances - all necessary for bacteriolysis; 
1, A specific bacterium used to produce the immunity. 
2, Blood serum containing the protective substance, 
3, A substance present in all normal serum. 
In 1898 Bordet reported hemolysis, the power to dissolve or 
lake red blood cells. He showed that if a rabbit be injected with 
a man’s red blood cells, a hemolysin was produced that would cause 
laking of the red blood cells in the presence of the serum of this 
rabbit. If the serum was heated to 56°C. for one-half hour, and 
the red blood corpuscles of man added, there would be no laking. 
If normal serum was added to this mixture, a hemolysis occurred. 
This reaction could be carried out for any two species of animals, 
i.e,, rabbit and man, sheep and rabbit, etc. 
It is apparent therefore that three substances are necessary 
here also: (l) Erythrocytes used to sensitize serum, (2) sensi- 
tized' or immune serum, and (3) normal serum. 
The above experiments have given rise to the following terms 
and definitions: 
ANTIGEN - Any substance which when injected into suitable 
animals, will result in the formation of specific antibodies, 
AMBOCEPTOR - The specific antibody produced by the injection 
of an antigen, Amboceptors may be normally present in certain blood 
sera but can be greatly increased by the injection of the suitable 
antigen, Amboceptors are thermostable, that is, retaining their 
activity after heating at 56°0, for one-half hour, 
A BACTERIOLYSIS is a bacteriolytic amboceptor, 
A HEMOLYSIS is a hemolytic amboceptor, The first 
dissolves bacteria and the second dissolves red 
blood cells, 
COMPLEMENT - The substance present in all normal fresh sera, 
which when added to a mixture containing antigen, and amboceptor, 
results in the production of bacteriolysis or hemolysis. Comple- 
ment is rendered inactive by heating for one-half hour at 56° and 
is therefore said to be thermolabile. It also loses strength rapid- 
ly on standing at room temperature. 
BACTERIOLYTIC SYSTEM - The combination of antigen (bacterium), 
amboceptor (immune serum), and complement (normal serum), 
HEMOLYTIC SYSTEM - The combination of antigen (erythrocytes), 
amboceptor (immune serum), and complement (normal serum). 
In the above two systems all three substances must be present 
or no reaction takes place, i.e., antigen, amboceptor, and comple- 
ment , 
Since a bacterial antigen produces a specific antibody or am- 
boceptor, it was reasoned that a test for the presence of this anti- 
body or amboceptor in the blood serum should be possible. With this 
test it should be possible to tell whether a patient has or has had 
an infection from a certain bacterium because of the production of these specific antibodies or amboceutors which would be in the blood. 
Therefore if a. patient’s serum with complement uresent dis- 
solves the cholera suirilla, the patient must have cholera or must 
have had it. If it dissolves the suiracheta -pallida, the causative 
organism of syphilis, the patient must have syphilis* 
Because bacteriolysis is difficult to visualize, practically, 
the test is carried further, 
; The amount of complement necessary to produce complete bacter- 
iolysis can be determined for any measured amount of uatient’s 
serum and antigen. If the uatient’s serum has the specific ambocep- 
tor, the. complement will be used or fixed. 
To determine if any or all of the complement is fixed, a hemo- 
lytic system is added, that is, red blood cells and rabbit serum 
sensitized or immunized to those cells. In order for hemolysis to 
take ula.ee, complement is necessary* So if this hemolytic system 
is added to the bacteriolytic system and there is some complement 
left,, some hemolysis will take place. If no complement is left, 
no hemolysis can take place. Hemolysis is easily visualized and 
readily shows whether any or all the complement is used or fixed, . 
The reaction may be visualized as follows: 
Antigen plus Positive Patient’s serum ulus complement « bacteriolysis 
and no free 
■ complement. 
Antigen plus neg. Pt’s serum ulus complement - no bacteriolysis 
Ho Amboceptor 
'complement free. 
Vhien-hemolytic system is added to the above the following occur: 
Red blood cells plus sensitized serum ulus free complement - hemolysis 
R.B.C, -plus sensitized serum ulus no complement z no hemolysis 
Variation in positivity of serum nay be denonstrated thus: 
Antigen * Pt’s Serum 4- Complement = Fixed* & 4- Amboceptor j. R 2 partial 
partly Free** , B hemolysis 
positive - Comp. C (circle^ THE TWO-TUBE KOLKER TEST 
(Sheep System) 
A. Glassware and Airparatus. 
Piuettes: 0,2 cc, graduated to 0.01 cc 
1,0 cc, graduated to 0.01 cc 
10,0 cc, graduated to 0,1 cc 
Test tubes, 100 x 12 mm,, heavy wall, without lip. 
Test tube ra.cks, carrying two rows of ten tubes ea.ch. 
Centrifuge and centrifuge tubes 
Water baths: Inactivating, set at 56 deg. C, 
Incubating, set at 37 deg. 0. 
Refrigerator, running at 6 to 8 deg. C, 
All glassware should be chemically clean and should be used 
dry or rinsed out with normal saline solution just before using. 
Never use any glassware containing the slightest degree of water. 
B. Reagent s. 
1* Patient*s serum. Blood is collected from the patient by 
venipuncture and'allowed to clot. The serum should be separated 
from the clot and centrifuged until all cells are thrown to the 
bottom of the tube. The serum is poured or pipetted from the top and 
ulaced into a clean tube. The serum must be entirely free of cells. 
Before the test is run, all sera are inactivated in 56 degree water 
bath for 30 minutes. 
2, Salt solution. This is an isotonic solution of sodium 
chloride. Add 0.85 grams of chemically pure sodium chloride 
(Merck’s Blue Label) to 100 cc of distilled water. 
3, Sheen Cell Suspension (indicator Antigen), Collect the 
blood by bleeding the sheep from the external jugular vein into 1 
to 3 percent sodium citra.te solution, or the blood may be received 
into a flask containing a handful of sterile glass beads and shaken 
well to defibrinate it. Either method prevents clotting. The for- 
mer method is preferable, Filter a small amount of blood through 
cotton into graduated centrifuge tube, allowing twice as much blood 
as will be required for the tost to be run. Add 2 or 3 volumes of 
salt solution. Centrifuge at 1500 R.P.M. for ten minutes. Decant 
and add saline. Repeat this washing 5 times. On the last washing 
centrifuge at the 1500 R.P.M. for exactly 15 minutes. Do not vary 
the time or speed, so as to insure the same per cent suspension when 
the cells are finally diluted for use in the test. Read the volute 
of the cells in the centrifuge tube, carefully remove the supernatant 
fluid, and prepare a 2 suspension by washing the cells into a flask 
with 49 volumes of salt solution. Always shake well before using to 
secure an even suspension, as the cells rapidly settle to the bottom 
of the flask on standing. The sheet) cells can he preserved for a period up to three weeks 
hy placing one volume of cells in four volumes of saline and adding 
for each 100 cc, 1 l/4 cc of formalin formaldehyde). When the 
cells are to he used they are washed three or four times with saline 
as above, 
4. Complement. The serum of guinea nigs is used. Animals are 
selected which have not been recently bled, ’The comulementary 
strength will be more uniformly good if the -pooled serum of several 
guinea nigs is used. 
The animal is placed on its back with an ether cone over its 
nose. When the animal has relaxed (care: too much ether causes 
death), blood is withdrawn from the heart with a 10 cc- syringe and . 
18 or 20 gauge short bevel, sham needle, the needle being carefully 
placed through the chest wall into the ventricle. When 8 or 10 cc 
have been secured, withdraw the needle and place the animal aside on 
its back to recover. Remove the needle from the syringe and gently 
run the bloo’d into a centrifuge tube. Do not stir or agitate be- 
cause this causes hemolysis. Set aside for an hour at room temper- 
ature to allow clot to form. Free the clot from the walls of the 
tube with a clean sterile applicator. If it is to be kept over 
night, place in a refrigerator. When it is to be used, centrifuge 
at 1500 R.P.M, for 15 minutes. Decant or pipette off the serum. 
If it contains cells, recentrifuge. The serum should show no 
hemolysis and contain no cells. It must be used when fresh be- 
cause it loses its complementary strength. It can be kept over 
night if the clot is not separated from it. The serum is diluted 
with saline in the: amount determined by titration, 
Lyophilized complement is pooled guinea pig serum which has 
been frozen and dried under vacuum in ampules, each of which con- 
tains a specified amount of serum. In this form, it retains its 
complementary strength for many months. The dried serum is regen- 
erated for use by adding enough distilled water to bring it to its 
original volume. This serum is then diluted and titrated in the 
same manner as is fresh serum. This lyophllized serum is obtained 
from the Army Medical Center on Quarterly or emergency requisition, 
is considered as deteriorating, and is listed as follows: 
Item No, Unit Price 
17034 COMPLEMENT FIXATION TEST, LY0PHILI2ED, 
guinea pig, 5 cc ; bottle $3,00 
It must be stored in refrigerator. 5, Amboceptor, Serum of rabbits sensitized to sheep cells. 
It is diluted to with glycerine to preserve it. Keep in a 
refrigerator. It need not be inactivated. It is prepared at, and 
is secured from the Army Medical Center, Washington, D, 0,, on a 
quarterly or emergency requisition. It is considered as a deterio- 
rating item and is listed as follows: 
Item No, Unit Price 
17000 - AMBOCEPTOR. ANTI SHEEP, HEMOLYSIN, 5 CC Bottle $6.00 
6, Antigen. It is a cholestcrolized and lecithinized alco- 
holic extract of ether extracted powdered beef heart. The same 
precautions should be with this antigen as with the Kahn 
antigen. It is kept at room temperature. It should be titrated 
for antigenic activity about every three weeks. It is prepared 
at and secured from the Army Medical Center, Washington, E, C, on 
a Quarterly or emergency reouisition. It is considered as a deterio- 
rating item and i* listed as follows: 
Item No, Unit Price 
17020 - ANTIGEN, SYPHILIS COMPLEMENT FIXATION 
TESTS, 5CC Bottle $3,60 
7, Known Positive Serum, Serum previously tested and known 
to be strongly positive. It must be free of cells and inactivated. 
8, Known Negative Serum. Serum previously tested and 
known to be negative. It must be free of cells and inactivated. C. TITRATIONS 
1, T it rat ion of Amb o c'ept or , 
. a, -Prepare a dilution Of 1:100 amboceptor as follows; 
’ Glycerolized amboceptor .,(50$) 2 cc 
Salt solution . ■ . 94 cc 
Phenol (5$ in salt solution) 4 cc 
This is to be kept in the refrigerator as a stock 
solution and is good for several weeks, ’ 
b, Dilute this stock solution for the titration as 
follows: * ' 
Stock amboceptor (l;100) • 0.5 cc 
Salt solution.‘ , :■ • 4,5 cc 
This will be 1:1000 in strength,„ 
: c.‘ In a series.of 10 tubes, prepare higher dilutions 
'•» c . •, , 
as f6116ws: • 
# • v , •; • *5 
# 1, Amboceptor 1:1000, 0.5 cc plus no saline 
#2, ” n 0.5 cc plus 0.5 cc saline (1:2000) 
# 3, 11 »■'* « ” ” 1.0 « » (1:3000) 
#4, ” « ■ " « 1.5 " ” (1:4000) 
#5. » ' " . ” " « 2,0 ” " (1:5000) 
4 6. ” 1:3000 « » ” 0.5 ” ” (1:6000) 
4 7. ” 1:4000 ” ” ” 0.5 ”, - ” (l:8000) 
# 8 y ’ J "/j ” 1:5000’ n ” ” 0-.5 ’" ; ” (1:10000) 
#9. ” 1:6000 ” ” ” 0.5 ” « (1:12000) 
#10. v ” 1:8000 • ” ” ” 0.5. (1:16000) 
♦ “ ’ * v - * ' •’ • . . 
Mix the contents of each tube thoroughly. 
d. Prepare a 1:30 dilution of the complement by diluting 
0.2 cc of the regenerated complement or fresh guinea pig serum 
with 5,8 cc of salt solution, 
e. Prepare a 2$ suspension of sheep cells in salt solution, 
f. In a series of 10 tubes set up the amboceptor titration 
as shown in the following table; 
Tube Amboceptor 0,5 cc Complement 1:30 Saline Sheep Cells 
1. 1:1000 O'.3 cc 1,7 cc 0,5 cc 
2. 1:2000 to all to all to all 
3. 1:3000 tubes tubes tubes. 
4. 1:4000 
5. 1:5000 
6. 1:6000 
7. 1;8000 
8. 1:10000 
9. 1:12000 
10. 1:16000 
Mix the contents of each tube thoroughly. g, Incubate in the water bath at 37 deg, C, for 1 hour, 
h. Head the amboceptor unit. The unit is the highest 
dilution of amboceptor that gives complete hemolysis. 
Two units of amboceptor are used in the complement 
and antigen titrations and in the final test. Example: If the 
unit equals 0,5 cc of the 1:6000 dilution, then two units will 
equal 0,5 cc of the 1:3000 dilution. Dilute just enough of the 
stock amboceptor for the titrations and the number of tests to be 
run. 
8. Titration of Complement. 
a. Prepare a 1:30 dilution of the complement. (See para- 
graph d under amboceptor titration) 
b. Dilute the antigen as indicated by the dilution fac- 
tor on the antigen bottle, by placing the required amount of salt 
solution in a small flask and adding the antigen drop by drop, 
shaking the flask continually until the antigen has all been added 
Prepare enough for the complement titration and for the final test 
c. In a series of 10 tubes, set up the titration as 
follows: 
Tube 
Comulenent 
Antigen 
Salt 
Antooceutor 
Sheen Cells 
(1:30) 
Dose 
Solution 
2 Units 
24 
cc 
cc 
cc 
cc 
cc 
1. 
0.1 
0.5 
1.4 Yater 
0,5 
0.5 Yater 
2. 
0.15 
0.5 
1.4 Bath 
0.5 
0.5 Bath 
3. 
0.2 
0.5 
1.3 37°C 
0.5 
0.5 37°0 
4. 
0.25 
0.5 
1.3 for 
0.5 
0.5 for 
5. 
0.3 
0.5' 
1.2 one 
0.5 
0.5 one 
6. 
0.35 
0.5 
1,2 hour 
0.5 
0.5 hour 
7. 
0.4 
0,5 
1.1 
0.5 
0.5 
«. 
0.45 
0.5 
1.1 
0.5 
0.5 
s. 
0.5 
0.5 
1.0 
0.5 
0.5 
10. 
Hone 
None 
2.5 
None 
0.5 
The smallest amount of complement just giving sparkling 
hemolysis is the exact unit. The next higher tube is the full unit, 
which contains 0,05 cc more complement, In the antigen titration 
and in the final test, two full units are used and are so diluted 
as to be contained in 1,0 cc as in the following example: 
Exact unit. 0.3 cc 
Full unit 0,35 cc 
Dose (2 fuj.1 units) 0,7 cc 
To calculate the dilution to employ so that 1,0 cc 
will contain the dose of 2 full units, divide 30 by the dose (0,7) ‘ • •' -K * - 
This equals 43, therefore 1,0 cc of a 1:43 dilution.will contain 
'the required 2 full -units. 
D. Titration of Antigen 
1. Prepare a 1:80 dilution of antigen by adding 0,1 cc, drop 
by drop, with continual shaking* to 7,9 cc of salt solution in a 
large test tube or a small flask, 
2. Higher dilutions are then prepared as follows: 
4 cc of 1:80 A 4 cc of salt- solution ■’ 1:160 dilution 
4 cc of 1:160 * 4 cc of salt solution 1:320 dilution 
4 cc of 1:320 - 4 cc of salt solution 1:640 dilution 
4 cc of 1:640 ± 4 cc of salt solution 1:1280 dilution 
4 cc of 1:1280 4. 4 cc of salt solution 1:2560 dilution 
3. Arrange 5 rows of test tubes- with 6 tubes in each row (30 tubes) 
♦ • ‘ . v 
4. In the first tube of each row place 0,5 cc dilution 1:80. 
r In the second tube of each row place 0,5 cc dilution 1:160, 
In the third tube of each row place 0,5 cc dilution 1:320. 
In the fourth tube of each row place 0,5 cc dilution 1;640. 
In the fifth tube of each row place 0.5 cc dilution 1:1280 
In the sixth tube of each row place 0,5 cc dilution 1:2560 
5. Heat 3 cc of moderately to strongly positive syphilitic 
serum in a water bath at 56 deg. C, for 15 to 20 minutes and pre- 
pare 5 dilutions in large test tubes as follows; 
Tube Serum, cc Spline, cc Resulting Dilution CC of Serum in 
0,5 cc of Dilution 
1“ 1.0 4.0 < 1.5 • 0.1 
2* 0.5 4,5 1:10 * -0.05 - 
3 0.5 9.5 1:20 0.025 
4 2,0 (1:20)2.0 1:40 - 0.0125 4 
5 1.0 (1:20)4,0 1:100’ 0,005 
6, Add 0,5 of 1:5 dilution to each of the 6 tubes of the 1st row. 
Add 0.5 :of 1:10 dilution to each of the 6 tubes of the second row. 
Add 0,5 of 1:20 dilution to each of thd 6 tubes of the third row. 
Add 0.5 of 1:40 dilution to each of the 6 tubes of the fourth row, 
• Add 0.5 of 1:100 dilution to- each of the 6" tubes of the fifth row, 
7'. Add 1.0 cc of complement dilution carrying 2 full units 
to all 30 tubes. 8, In a separate rack, set up a serum' control carrying 0.5 
cc of 1:5 serum and 1,0 cc of complement (2 full units); also a 
hemolytic system control carrying l.Occ of salt solution and 1,0 
cc of complement (2 full units), 
9. Shake the tubes gently and place in the, refrigerator at 
6 to 8 degrees 0. for ,15 to 18 hours, followed by water bath at 
37 deg, C, for 10 minutes. ' "v ■ 
10. Add 0,5 cc of amboceptor (2 units) and 0.5 cc of a 2$ sus- 
pension of sheep cells to all 30 tubes and to the control tubes, 
• * ‘ * f * 
* • f *v t \ 
11. i-ix thoroughly and place in water bath at" 37 deg, C, for 
one hour and make readings. The serum and hemolytic controls should 
show complete hemolysis, *' 1 • ■ ... .. . 
- ' - ; ’ , .1 •, t 
12. Chart the results as per the following example observed 
with a, strongly positive serum: 
Serum 
(in 0.5 cc) 
1:60 
Antigen * 
1:160 
dilutions 
1:320 
(in 0.5 
1:640 
.cc dose) 
1:1280 
1:2560 ) 
.0.005 
4+ 
0.0125 
- 
4 
4-»- 
44 
4 
0.025 
.•f- 
+444 
4444 .. ’ 
4-W-4 
4444 f 
4 
0.05 
• 4-1-4 
44-4+ 
4“>* 
44-«-4 
4444 
-r+ 
0.1 
4444 
444-*- 
4+4+ 
-t*+ + T 
4f 44 
Thf do§e of antigen to employ in the final test'is the largest 
amount' living .a *++■+ reaction with the smallest .amount of serum. 
If three dilutions of antigen give 4-H-+ reactions with the smallest 
amount of serum, the dose is midway between the highest and lowest. 
2, Procedure for the Test 
Kevins ascertained the exact amounts of the reagents to he 
used by the methods,* set un the tvo-tuhe Kclmer test on the 
various hlocd sera for diagnosis as indicated in the table on the 
following page.  
Hemolytic 
System 
Results 
Tube 
No. 
Serum 
cc. 
Antigen 
© 
5 
P 
CD 
Vi 
0) 
t 
© 
P 
6 
o 
o 
Vi 
P 
cd 
© 
P 
2 
d 
*6 
O 
dc 
Complement 
(2 units) 
w 
Vi 
§ 
jd 
00 
I—1 
J> 
l—I 
Vi 
O 
<M 
o 
o 
co 
"© 
u 
o 
p 
cd 
Vi 
© 
ttO 
. 
CO 
© 
P 
2 
d 
•H 
a 
m 
i—i 
1 
o 
i—i 
v+ 
o 
o 
o 
r- 
co 
+5 
cd 
xl 
p 
$ 
u 
© 
p 
cd 
cc 
Amboceptor 
(2 units) 
Sheep 
Cells 
2% susp. 
// 
/ 
/ 
d 
o, 
XJ 
rH 
U 
O 
<H 
O 
o 
r- 
CO 
p 
© 
x!; 
p 
© 
Vi 
© 
P 
© 
£ 
d 
•H 
© 
P 
© 
ihht'+=no hemolysis) Report a.s 
'! Posi ti ye 
>4. •” ) 
4 *75# M ) Doubtful 
-» *100# ” ) Negative 
nSortin'' Control” tube should 
show complete hemolysis. If 
not, report anticomplemen- 
tary serum. 
Unknown 
Serum 
nTes- 
” Serum 
Control” 
1 
2 
0.2 
0.2 
0,5 
~ NONE 
(0.5 cc 
saline) 
1.0 
1.0 
0.5 
0.5 
0.5 
0.5 
Known 
Positive 
Control 
_ 1 . 
2 
0.2 _ 
0.2 
_ 
(8afiSi) 
. _ _ 1.0 _ 
l.o 
.0*5 
0.5 
_ _0j,5__ 
0,5 
No Hemolysis 
Complete Hemolysis. If not 
serum is anticomolementary 
Known 
Negative 
Control 
_ 1 
2 
_ _0 
0.2 
_ 
NONE 
(0.5 cc 
saline) 
_ 
1.0 
0,5 _ 
0.5 
______ 
0.5 # 
-Complete keno lysis 
Comolete hemolysis. If not 
serum is anticomplementary; 
Antigen 
Control 
1 
NONE 
(0,5 cc 
saline^ 
0.5 
Ti 
s 
p 
to 
1.0 
* 
Vi 
d 
0.5 
0.5 
Complete hemolysis 
Ambcceptoi 
NONE 
o 
P 
<D 
d 
Control 
2 
(l.Ooo 
NONE 
% 
1.0 
"© 
•H 
0.5 
0.5 
■§ 
Complete hemolysis 
saline) 
o 
*§ 
d) 
O 
. 
i Si 
ri 
o 
cd 
f-H 
Sheep Cell 
3 
(2,5 cc 
NONE 
NONE 
d 
n 
rH 
p* 
NONE 
0.5 
No hemolysis 
Control 
Saline) 
PROCEDURE FOR TWO-TUBE KOLMER TESP OF BLOOD Spinal Fluids. 
These are usually tested without any preliminary preparation 
as they do not contain enough complement to require inactivation by 
heating at 56°C. If a specimen contains considerable blood which 
has not had time to settle out, it should be centrifuged. 
The following table shows the set-up for complement fixation 
tests on spinal fluids for syphilis: 
Tube 
Spinal 
Antigen 
— 
Conmlement 
■Art# center 
Sheep 
No. 
Fluid 
cc. 
( 2 units ) 
f-H 
[2 units! 
cells 
cc 
O 
V* 6 
cc. 
(2^) 
u 
- o 
o CO 
CW 
, 
O cy 
• 
• 
CD PO 
O 
p 
2 
ct 
o po 
o 
p 
a> 
S 
* 
JN 
1 
0,5 
0.5 
1.0 
CD 
LOP 
0,5 
0.5 
DO 
4> 
CD 
P H ft 
d 
■P 
CC 
P 
O X 
x 
f-t PP 
p 
o 
o d 
d 
o 
P OP 
x 
u 
U, r-t 
M 
n 
w 
© p a 
cu 
V 
r*. p 
p 
-p 
•H X R 
d 
ft -P 
5 
1 
Vh 
Q) •• 
*H • 
d 
•H 
2 
0.5 
None 
(0 
1.0 
•H jd 
0.5 
. 0.5 
© u 
p p 
p 
O 00 
cc o 
,0 XI 
r 
p 
•H 
p p 
g a, 
d 
P c 
d p 
: 
4^ 
O 
p If: 
I—1 r-4 
P o 
Tube number 2 is the control tube and should show complete 
■hemolysis. The antigen, amboceptor and sheep cell control should 
■be- run with each lot, the same as for blood serum. 
A control, consisting of a known positive end a known neg- 
ative fluid should also accompany each lot of fluids tested. EXAMINATION OF CEREBROSPINAL FLUID 
Collection - Collect cerebrospinaTbf luid in two chemically d,ean 
sterile tubes. Label 1 and 2. The first specimen may contain 
blood from the needle puncture, No, 2 is reserved for the cell 
count. Examination should be completed as quickly as possible after 
collection. If 'it is necessary to send the fluid to a distant lab- 
oratory for the Vassernann and colloidal gold tests, the cell count, 
study of smears, and the simpler chemical tests should be done in 
the local laboratory and reported. on a slip accompanying the specimen. 
X. Appearance 
Normally crystal clear, colorless - like clean water. 
Color: 1, Colorless - Normal 
2. Yellow -* Altered hemoglobin (old blood), jaundice, 
3, Red - Hemoglobin or blood. 
Transparency; 1, Crystal clear - Nnv.mal 
2. Slight Haziness - Blood or pus cells, 
3. Turbid - Blood or pus cells. 
Coagulum: 1, No coagulation - Normal, 
2, Pellicles - Forms after standing some hours 
due to meningeal inflaminetion. 
3. "Cobweb" or) 
"Pine Tree") - Tuberculous meningitis, 
Coagulum ) 
II, Cytology - Cell counts must- b£ made with a fresh specimen be- 
cause cells degenerate rapidly, 
A. Material! - 1, Spinal Fluid 
2. Staining solution 
Methyl or Gentian violet 0.2 gas. 
Glacial acetic acid 10,0 cc.. 
Distilled water 90*0 cc. 
Mix well, filter until the solution is clear, 
3. Wright’s stain 3 -carts -1- methyl alcohol 1 part; 
buffer. 
4. Leucocyte pipette, 
5. Fuchs-Rosenthal counting chamber or ordinary 
Kemocytometer, 
6. Microscope, 
7. Glass slides, 
8. Methyl alcohol. B, Procedure for Counting 
1, Shake spinal fluid to get uniform suspension of cells. 
2, Fill pipette to 0.1 mark with stain. 
3, Fill with spinal fluid to 1,1, 
4. Shake well, 
5, Fill counting chamber and allow to stand for 3 minutes, 
6. Count cells in entire ruled area, 
Fuchs-Rosenthal■ - 16 squares 
Hemocytometer - 9 squares 
7. Calculation. 
(a) Fuchs-Ro sent hal - chamber 4x4 mm, x 0.2 mm, 
equals 3.2 cu, mm. The 0.2 being nearly equal 
to the proportion of dilution by stain is dis- 
regarded hence: 
f Total count s Dumber of WBC per'cu, ram, 
„• 3 
(b) Hemo cytometer - chamber is3mmx3mmx0.1 mm, 
equals 0,9 cu, mm. 
Total count x 10 ,, . 
1 g“ a Dumber of WBC per cu. mm. 
Again calculation compensates for small error 
caused by dilution, 
8. Results - Formal is 0 to 8 cells per cu, mm. 
C, Procedure for Differential Count 
1, Centrifuge spinal fluid for 5 minutes, 
2, Prepare thin smears of sediment on glass slides, 
3, Dry in air quickly. 
4, Stain with Wright-methyl alcohol stain.* 
5, Make differential count, polymorphonuclear and 
lymphocytes. 
Results: Dermal - Lymphocytes and few endothelial cells. 
Abnormal - Polymorphonuclears. PREPARATION OF COLLOIDAL GOLD SOLUTION 
METHOD OF PREPARATION AT 
EIGHTH SERVICE COMMAND LABORATORY 
I, Solutions. 
1, Fifteen grains of gold chloride (ampoule by Merck) are made 
up to 100 cc. with double distilled water in a volumetric flask. 
2, Five grams of sodium citrate (Merck, tl.S.P.) are weighed 
out accurately on an analytical balance and made up to 500 cc. with 
double distilled water in a volumetric flask. 
II. Procedure. 
Nine hundred fifty cubic centimeters of double distilled water 
are measured out with a one liter graduate into a three-liter 
Florence flask* Ten cubic centimeters of gold chloride, measured 
with a 10 cc. volumetric pipette, are added to the double distilled 
wafer. This solution is slowly brought up to 94°C; and at that point 
of temperature, 50 cc. of the 1$ citrate measured in a 50 cc, volu- 
metric flask are added all at once to the gold chloride water so- 
lution, Immedicately the flame is turned to full force. As soon 
as the liquid has definite large boiling, bubbles, two and one-half 
minutes are timed with a, time clock. As soon as the' two and one- 
half minutes are over, turn the flame out and set the colloidal 
solution aside to cool. - 
NOTE; Scrupulously clean glassware must be used throughout the 
above procedure. Ne make a practice of using glassware which is 
not used for anything but colloidal gold. All glassware should be 
washed two or three times .double distilled water before using. 
The second distillation of water should be done with a full glass 
still, using tinfoil to cover the bottles after distillation, A 
large gas burner is required for boiling. 
88 Standardization of Colloidal Gold. 
A, Apparatus 
X, 12 chemically clean test tul»es 
2, Test tube rack for tubes 
3, Pipettes: 5 ml,graduated to 0,1 ml, 
1 « " " 0.01 ", 
B. Reagent s 
1, Colloidal gold solution 
2, Saline, 0,4$ to be kent in chemically clean bottle 
Sodium chloride, CP 1 gram 
Rouble distilled water 250 ml. 
3, Synthetic globulin furnished by Army Medical School, 
C, Procedure 
Prepare a 1:60 dilution of the synthetic globulin by 
mixing 0,1 cc of globulin with 5.9 cc of .4$ saline. The diluted 
globulin is good for 1 day’s use only and must be nrepared fresh for 
each day. 
Place 10 chemically clean test tubes in a rack and nlace 
in each tube solutions as indicated: 
Tube No, Saline mix each tube well before nroceeding. 
1 0.5 ml. 4 0.1 ml. Synthetic globulin, mix thoroughly, 
2 0,5 " +0,5 H mixture from tube #1, mix 
3 0.5 " +0.5 « " " « #2, » 
4 0.5 » +0.5 " « " « #3, » 
5 0,5 *•» +0,6 « M " 0 #4, " 
6 0.5 " +0.5 » » n • #5, » 
7 0.5 11 +0.5 " " " " #6, n 
8’ 0.5 « +0.5 « « " »« 47, » 
9 0.5 « +0.5 » " " " #8, " 
10 0.5 ” 4-0.5 » " " « #9, " 
Remove 0.5 ml. from tube #10 and discard. 
Control Tube: 
Saline 0,4$ 1,0 ml, 
Then niece in each tube including the control tubes - 
Colloidal Gold Solution 2.5 ml. 
Mix thoroughly by rotating tubes. Allow to stand at room tempera- 
ture 18 to 24 hours. D, Results ’ 
The readings depend upon the presence or absence of 
color change. For convenience, colors are given numbers as follows; 
#0 - No color change #3,- Blue 
#1 - Slight violet tinge #4 - light - transparent pink or blue 
* #2 Bluish red almost decolorized 
#5 - Complet-e decolorization, 
'Control - Vith saline, no color change, or ”0”, - 
A gold of standard potency should exhibit the following; 
. Zone 1 - Curve, 5,554,310,000 
*' ' A record of the reaction of each gold prepared and Used should be 
kept. More than a slight deviation from the above curve warrants dis- 
carding of the gold and another prepared. 
Colloidal gold should be allowed to stand 24 hours before test- 
ing, Should be stored in sterile bottles using either ground glass 
. stoppers or tin foil protected stoppers,. 
Colloidal Gold Test on Spinal Fluid • 
Same procedure as that of standardization by synthetic globulin, 
except 0,1 cc spinal fluid undiluted is placed in tube # 1* 
Reporting - The color of each tube is reported by number. The 
following are examples of normal and abnormal reports; 
Tubes ;#1 -to #10 inclusive 
Normal - 0,000,000,000 
2,21$,000,000 
•’First Zone” or.’’Paretic” _5,555,542,100 
5,554.310.000 
, , m . 0,123,221,100 
"Mid Zone", "Luetic" or i ■_ ; _ _2 443 100 000 • 
' ”Tabetic” T * ■ >uuu " ' 
iaDeriC 3.455,542,100 
“End Zone” or ’’Meningitic” 0,001,223,31u 
(Showing shift to right) 0,002,455,555 NOTES NOTES LABORATORY TECHNICIANS MANUAL 
PART II 
INDEX 
Chapter 
Page 
1. Glass Handling 98-103 
2. Feeding, Care and Breeding of Laboratory Animals 10-4-10$ 
3. Preparation of Bacteriological Stains and Solutions 106-110 
U. Bacteriology - Introduction 111-11$ 
$. Outline Classification of Bacteriology 116-121 
6. Streptococcus and Staphylococcus 122-123 
7. Diplococcus Pneumoniae and Pneumococcus Typing 124.-128 
8. Neisseria 129-133 
9. Vibro Comma 134-135 
10. Pasteurella 136-139 
11. Genus Brucella 14.0-14-1 
12. Genus Hemophilus 14.2-144 
13. Gram-Negative, Aerobic, Non-Spore-Forming Enteric Bacilli 14$-1$4- 
14.. Cl, Parabotulinum 1$$-1$6 
1$. Corynebacterium Diphtheriae 1$7-160 
16. Mycobacterium Tuberculosis 161-163 
17. Spirochetes I64.-I69 
IB. Fungi 170-174- 
19. Rickettsiae 17$-178 
20. Culture Media and Sterilization of Glassware 179-190 
21. Bacteriological Examination of Water and Milk 191-198 Glass Handling 
1. Cutting of glass tubing and test tubes, 
2. Bending of glass tubing, 
3. Making capillary pipettes from glass tubing, 
U. Preparing an ampule out of a test tube. 
5. Blowing of bulbs in glass tubing or test tubes, 
S. Sealing joints in glass tubing. 
In the average laboratory there is some call for turning 
glass tubing or test tubes into a form for some special purpose, 
and the faculty for doing this may be readily acquired with practice. 
Equipment: only soft glass can be so handled with the 
ordinary laboratory equipment; the hard glasses, such as ,fPyrexH 
having too high a melting point to be handled without special high 
temperature blowpipes. The standard 6, 8 and 10 mm. thick wall 
tubing and the standard test tube will suffice for the average 
requirements, While some simple manipulation may be done on a 
bunsen burner, it is preferable to have a blast lamp, both to provide 
higher temperature and to have smaller controllable heat point. 
Most blast lamps are designed for use with artificial gas; difficulty 
may be encountered in using natural gas, of higher with 
these burners and where natural gas is used special modification of 
the burners may be required. Satisfactory gasoline blast burners 
are also on the market. A small triangular file provides the means 
of cutting the. glass. Cutting of Glass Tubing 
Place the glass tubing on a table, hold it firmly and nick it in one 
spot by firmly drawing across it the edge of a triangular file. 
(It is rarely necessary to extend this file nick around the tubing.) 
Then holding the tubing in both hands with both thumbs opposite the 
nick a quick snap will complete a clean cut break of the tubing. If 
the break is not clean cut it indicates the need of deeper nick or of 
a modified manipulation in effecting the snap, a technic to be acquired 
with practice. If one end of the tubing is too short to so handle, the 
snap may be effected by holding the long end rigidly in one hand and 
hitting the small end by the file held in the other hand. After the 
break any sharp points, frtm failure to attain a satisfactory clean 
break, may be filed down. The surface of the break is finally smoothed 
by melting it in a hot flame; at this time, by overmelting it, uhe bore 
at the top can be reduced to any desired size. 
Of course the hot glass should not be laid down on the table top; an 
asbestos board, the tip of a wire basket or a metal ring stand may 
oe so used. 
Cutting a Test Tube 
Make the file nick as for glass tubing but make it deeper and preferably 
encircle the tube, A thin tube may be broken at this point by a bi- 
manual snap. Thick tubes require additional aids to complete the break? 
at one point make the file nick especially deep, then touch the tubs 
firmly at this point with the red hot file tip - a fracture should then 
result; if the fracture is not complete it may bo traced around the tube 
by keeping the red hot tip just ahead of the fracture line, on a cold 
test tulle. The trim up is the same as for tubing. 
Bending of Glass Tubing 
Holding both ends place the tubing in a hot flame so that at least an 
inch gets hot. Rotate tube while it is heating to make the heat even on 
all sides, When the glass is red hot and soft remove from flame and bend 
to the desired form keeping it in that position until it has hardened. 
If a broad bend is desired, as in making a nU” bend, several inches 
should be so heated. If only a slight bend is to be made, an inch ©f 
glass will suffice. At first you will have a tendency to overheat 
the glass and draw the two ends apart, distorting the shape and caliber. 
Also if you underheat, or put forced pressure on the bending effort, 
an undesirable collapse of the tubing at the bend will occur, A sat- 
isfactory bend retains the same caliber throughout the tubing. If, in working with thin tubing, the collapse at the bend cannot be 
prevented there is a device available for presenting it: having sealed 
one end of the tubing before melting it, the mouth is applied to the 
other end while effecting the bend, and enough air pressure is made into 
the tubing to return the collapsed tubing to its proper form - a procedure 
similar to the blowing of glass bulbs described below. 
Satisfactory Bend 
Unsatisfactory Bend 
glass knotT 
collapsed 
Making Capillary Pipettes from Glass tubing 
Glass tubing is heated, rotated, in flame to softness, removed from 
flame and the two ends drawn apart and held in place until hardened. 
The size of the resultant capillary tubing will depend on the degree 
of heat, the rapidity of drawing out and the extent of the drawing out. 
The tendency is to make the tube too small by too rapid separation of a 
narrow length of heated tubing. There is frequent use in bacteriology 
for such a pipette in this form. 
It is desirable to keep on hand 8 inch lengths of glass tubing, with 
cotton plugs at both ends, the whole sterilized by dry heat for use in 
making into pipettes as desired for special uses. Such pipettes may be 
given a narrowing of lumen about 3/4. inch from the end, to prevent the 
cotton plug from passing deeper into the pipette. 
Preparing an ampoule out of a test tube 
Proceed with a test tube just as above drawing, the two ends only about 
two inches apart resulting in a neck of about 4. mm, diameter. The tube 
may be cut at this point converting the lower end into an ampoule to be 
later sealed, or the test tube may be left intact to be later sealed at 
the constriction. Excessive heat may make tube difficult to holdj this 
may be avoided by placing a large perforated cork over each end and 
holding and rotating these. Blowing bulb in Glass Tubing 
One end of the glass tubing is sealed and sufficient length alltwed 
for holding one end in hand, the other end in mouth while blowing. 
Holding the tube over the flame with both hands rotating it for even 
heat, it is given a red heat to the molting point, then passed to the 
mouth to blow up the bulb to the required size. If the bulb is to be 
of considerable size some concentration of glass must be attained before 
the final blowj this is done by gently approximating the two ends while 
the middle is soft, giving an occasional slight blow to prevent collapse 
of melted glass. Trouble encountered will consist of eccentric bulbs, 
due to uneven heating, or to thin paper shell bulbs due to overblowing 
without glass concentration. A test tube may be similarly handled, A 
terminal bulb may be made at end of glass tubing by a one hand mani- 
pulation and blowing. 
Union of Glass Tubing 
If two straight pieces of glass arc to bo spliced, one piece is first 
given a seal at one end, the two ends to be spliced arc given a slight 
flange by gently rotating within the heated end, the sharp end of a 
triangular file. The two flanged ends should.be - of same size, shape, 
and'1 same grade of glass; they are heated to a red heat and then appro- 
ximated to complete sealing on all sides, there results a union which 
is oversize and too thick; this union may bo trimmed off to even size 
with the tubing by heating over the flame, while rotating evenly, blowing 
slightly into the tubing occasionally as tendency t’o collapse occurs, 
and slightly separating the two ends and blowing slightly if over thick 
glass is noted at the union. 
By similar procedures glass tubing of different size or glass tubing 
and tost tubes may be •united. A hard glass united with a soft glass 
would tend to fracture at the union, A union having thick spots or 
knobs will tend to fracture on change of temperature. Making .joints in glass tubing 
The making of Y of glass tubing, that is, a three way tube, combines 
the process of blowing a bulb and union of two glass tubes. We begin with 
glass tubing of any desired size. Two lengths are prepared, each sealed 
at one end, such as being drawn to capillary length. One of these is 
given a flange at open end, the flange molded into an oval shape by the 
action of end of file on the molten tube. The other piece of glass is 
given a hole in its center to receive the flange; the tubing is held in 
a small hot flame, without rotation, to heat only one-small spot of the 
tubing; when it is melted a lopsided bulb is blown to an extreme thinness 
even to rupture; then the bulb is crushed away with the file leaving the 
hole surrounded by the ragged bulb fragments; heat is applied to the edges 
of this hole to make it evenly, heated and the same size as the flange on 
the other piece of tubing. When both the flange and the hole borders are 
at molten heat the two are brought gently together, being sure of union 
at all points but avoiding collapse. The union is then smoothed off by 
alternate heat and blowing as in the simple union except 'that rotation 
cannot be effected and each side has to bo separately fused. A tendency 
to attain lumps of glass must be prevented and those smoothed out’ if they 
occur, otherwise this will become a weak point in the joint. The three 
ends of the union are then cut to the desired length. Inasmuch as those 
joints arc generally used for holding rubber tubing a desirable refinement 
would consist of giving them, prior to making the joint, a double ferrule 
of glass at the end of each future arm of joint. 
1st Stop 
Two pipettes 
2nd Step 
Ferrule placement 
3rd Stop 
Hole flange Ath Step 
Union and fusion 
5th Step 
Trim to completion 
Exercise in Glass Work: 
1, ’ Prepare a ‘'drinking tube" such as used on wards for bed patients. 
using it yourseix to determine best size and angle.; 
2. Prepare a WUH tube such as used in laboratory bottles. 
3. Prepare a capillary pipette such as will be used for specimen taking. 
A. Prepare a glass bulb about the size of a walnut: Round, smooth, even. 
5. Prepare a union of a piece of 6 to a piece of 10 mm, tubing, 
6. Prepare a "Y" or "T" joint. 
7. As each of the above is completed to your satisfaction, deliver it to 
the instructor on a slip bearing your name and a note that you 
have personally done all parts of the making of the piece. 
Equipment of this work: 
Blast lamp & foot bellows 
Glass tubing: 6 & 10 mm. 
File 
Asbestos board 
Wire basket 
Receptacle for waste glass FEEDING, CAKE AMD BREEDBIG 
OF LABORATORY ANIMALS 
Laboratory Animals: (1) rabbit, (2) Guinea pig, (3) mouse, (Ii) albino rat 
and (5) monkey. 
Reception Quarantine; All animals received from an outside source, should 
be isolated for 10 days to 3 weeks in previously disinfected quarters, and 
found to be free from disease before mixing with regular stock. 
Housing: Animal quarters should be kept clean, dry and completely free from 
vermin. The optimum temperature for most animals is 65° to ?0°F. with 
adequate ventilation. The standard large (10ju) and small (8“) animal Jars 
are suitable for mice and rats; the large Jar also can be used for a small 
guinea pig. The standard galvanized iron animal cage (lit” x ill” x 16”) 
will hold one rabbit or several guinea pigs. For use in breeding rabbits 
or guinea pigs, larger cages or pens, preferably with outside runways, 
should be built. The bottom of the Jar or tray in cage should contain an 
absorbent bed material, such as wood shavings; hay or straw may be used 
in large breeding cages. Clean quarters and renew bedding twice per week. 
Rabbits: 
(1) The diet recommended consists of commercial HRabbit Pellets’* 
supplemented once or twice per week with feeding of green stuff, such as 
carrots, lettuce or celery tops, A diet consisting of equal parts of oats, 
wheat and barley, plus 10% of logume, soybean op linseed meal is suitable. 
Alfalfa or timothy hay will serve both for food and bedding. Always keep 
plenty of water and a small piece of rock salt in the cage. 
(2) Diseases: 
a. "Coccidiosis", an intense and fatal onteritis; is the most 
serious disease. Observe new rabbits for this several days before adding 
to stock. 
b. '‘Ear Mange'1 is caused by a mite; can be cured by local appli- 
cation of a parasiticide. 
c. "Snuffles" is a cold-like disease caused by a filterable 
virus. Isolate infected rabbits until 3 weeks after recovery. 
(3) Breeding; Keep one male (buck) for each 8 to 10 females (does). 
Females are ready for mating at age of 10 months and may be bred every 3 
months thereafter (U litters per year). Keep record of date bred; gestation 
period 31 days; 2 or 3 days before expected arrival of litter place small 
breeding box and ample supply of bedding in cage. 'lean young after 8 weeks 
and separate sexes. 
Guinea Pigs; 
(1) Feeding: Same as for rabbits, except they must have supplementary 
feeding of green stuffs to supply Vitamin "C", at least twice per week. 
(2) Diseases: 
Salmonella infections, chiefly Salmonella typhiraurium and 
S.enteritidis, arc most dangerous of common diseases. Best method of 
control; Kill all potentially infected animals, sterilize room and cages 
and obtain new stock. b. Vitamin MCn deficiency is caused by lack of sufficient green 
stuffs in diet. Characterized by coarse hair and mangy appearance. It is 
transmissible to young through mother. Treatment: - improved diet, 
c, Balantidium coli type of enteritis. 
(3) Breeding: Use colony breeding with U or 5 females in cage with one 
male; duration of pregnancy - 63 days. Wean young and separate sexes when 
U or 5 weeks old. 
Mice; Several different strains used, such as, white mice, Swiss mice (also 
white) and C 57 strain (black), 
(1) Feeding: Commercial dog or fox chow checkers furnish an ample, 
balanced diet for growth and breeding; occasionally add piece of carrot or 
other greenstuff. Must have supply of fresh clean water in cage at all 
times. Mice will do well on simpler diets, such as, (a) the mixed grain 
diet listed above for rabbits, or (b) dry bread with water or skimmed milk, 
with addition of cod liver oil once per week, 
(2) Diseases; 
&, Salmonella infections (mouse typhoid), caused by same organisms 
as for guinea pigs, are common and very dangerous. To control: - destroy 
all infected stock, sterilize room and cages and obtain fresh stock. 
(3) Breeding: Colony breeding, with 4 or 5 females to one male; gestation 
period 21 days; when well advanced pregnancy is observed, place female 
in individual jar. After 21 days, isolate young and return mother to breeding 
jar. Peed young same as adults, but addition of evaporated milk to diet 
hastens growth. 
Albino Hats: 
(1) Feeding: Same as for mice, 
(2) Diseases: If cages are kept clean and ample diet provided, rats are 
very resistant to disease, 
(3) Breeding: Young females are ready for breeding when A months old. Use 
colony method of breeding with J+ females and one male in cage 5 duration of 
pregnancy 22 days5 not necessary to remove pregnant female from breeding 
cage. Wean young and separate sexes when 21 days old. 
Monkeys; 
~(l) Feeding:. Monkeys Trill do very well on ndog-chow checkers" plus canned 
tomatoes, with occasional feeding of fruits and nuts apples, 
bananas, peanuts, sunflower seeds, etc,), 
(2) Diseases: 
a. Pneumonia, usually fatal, 
b, Miliary tuberculosis. 
(3) Breeding; In captivity in small laboratories is not practical. PREPARATION OF BACTERIOLOGICAL STAINS AND SOLUTIONS 
1, Bacteria are stained by the basic aniline dyes. The acid aniline dyes, 
including eosin and acid fuchsin are not suitable for bacterial stain- 
ing. The basic aniline dyes mentioned below, and others, if needed, may 
be conveniently kept as stock solutions of the powder in alcohol to 
saturation, from which are prepared various simple and compound 
staining solutions, 
2. Stock solutions of dyes consist of these dyes in saturated alcoholic 
solution. They are prepared by placing the measured amount for 
solution, or slightly more, into 95% ethyl alcohol at room temperature 
and, after shaking for complete solution, filtering through paper to 
remove surplus dye and debris. Label " , stock solution". 
2zs 
Solubility in 100 cc. of 95% alcohol 
Crystal violet - — - 13.87 gms, 
Fuchsin (basic) 8.16 gms. 
Methylene blue 1.48 gms. 
Safranin ----------------- 3.41 gms. 
Example: To prepare stock solution of safranin; Place slightly in 
excess of 3-4-1 gms. of the dye in 100 cc. of 95% alcohol, shake to 
solution over period of 2 or 3 days, filter through paper, label, 
3. Simple stain solutions: 
General formula: Stock dye solution- 10 cc. 
Distilled water - - 90 cc. 
Uses: Simple stains or as elements of compound stains.. 
Application: Apply stain to a fixed slide for 2-5 minutes, wash 
with water, blot dry, 
4. LoefflerTs Alkaline Methylene Blue: 
Formula: Potassium hydroxide 10% sol, .07 cc. 
Distilled water - - - - — - 70 cc. mix and add 
Methylene blue, stock solution 30 cc. 
Preparation: The K0H is first added to to make 1:10,000 
dilution, then dye added. 
Uses: General bacterial stain. 
5. Carbol-fuchsin, dilute: 
Formula: Carbol-fuchsin (formula elsewhere) 10, cc. 
Distilled water ------- 90. cc. 
Uses; General bacterial stain. 
6. Bismark Brown (not kupt as a stock alcoholic solution). 
Bismark brown powder --------- ,5gm. 
Boiling water - - - — - - — - - - — 100,0 cc. Cool & filter 
7. Gram's Method: This is the most important of all bacteriological 
stains. It includes the application in turn, to a fixed slide, of a 
violet stain, Gram's iodine, a decolorizing agent and a contrast 
counterstain. 
Reagents: (a) Primary stain; Crystal violet - ammonium oxalate solution 
Crystal violet, stock solution ------- 5. cc. 
Alcohol, 95% — 5, cc, mix 
and add 
Ammonium oxalate, 1% aqueous solution - - - 40, cc, 
(b) Gram's iodine: 
Iodine ------------------- 1. gm. 
Potassium iodide -------------- 2, gms. 
Distilled water ---------- 240.) or distilled 
Sodium bicarbonate. 5% aqueous solution-60.) water 300 cc. (c) Deodorizer: 95% ethyl alcohol, or acetone, or alcohol- 
acetone (50-50). 
(d) Counterstain: Any simple contrast stain: Safranin, bis- • 
mark brown, or dilute carbol-fuchsin. 
Technique of Gram-staining: 
(1) Prepare thin even slide spreads, air dry, pass through flame for 
fixation, 
(2) Crystal violet stain is applied for 1 minute, then excess stain is 
poured off, 
(3) Gram’s iodine is then applied for 1 minute. Wash in Water, 
(4) Deodorizer is applied in several washes until no further traces of 
the stain ban be washed out of the preparation (1/2 to 2 minutes). 
Wash in water,- 
(5) Apply counterstain (e.g. safranin) for % minute. Wash in water. 
Blot and air dry. 
Results: 
Gram-positive organisms are stained violet. 
Gram-negative organisms are stained pink (brown or red) 
Gram-ambophile organisms give a variable result. 
General rules of Gram behavior of organisms: 
(1,) Cocci are Gram-positive except gonococcus, meningococcus, and 
catarrhalis group. 
(2.) Bacilli are Gram-negative except the diphtheria, the acid-fast 
group and most spore bearers, 
(3.) Spirilla and spirochetes are Gram-negative, 
(4.) Older cultures tf Gram-positive organisms tend to become Gram- 
ambophile or negative, 
8,Neisser’s Method for Polar Body Staining, 
Formulae: 
(1) Polar body stain (’’Neisser #1”) 
Methylene blue - stock solution - - 10 cc. 
Acetic acid, 5% solution, freshly prepared - 50 cc 
(2) Counterstain: (’’Neisser ”2”) 
Bismark brown - (formula elsewhere) 
(or use the safranin as prepared for Gram’s counter- 
stain) 
Technic of Stain: 
1. Prepare even, thin spreads on slides, air dry and fix by heat. 
2. Apply polar body stain for 1 to 3 minutes, wash. 
3. Apply counterstain for 1 minute. Wash and dry,- 
Result: 
Polar bodies will be stained blue; bacillary bodies take the counter- 
stain. 
Uses: 
Differential stain of the diphtheria bacillus, the plague bacillus 
and others having metachromatic granules, 
9. Acid-fast Stain Method (Ziehl-Neelsen’s carbol-fuchsin). 
Formulae: Includes a primary stain, a deodorizer and a countereitain, 
1, Carbol-fuchsin: Basic fuchsin, stock solution- 10, cc. 
Phenol, 5% solution— 90. cc. 
2. Acid alcohol! Acid, hydrochloric— 3. cc. 
Alcohol, ethyl, 95% ——-97. cc. 
Counterstain: Loeffler’s Methylene blue. Technic: 
1. Prepare Spreads of suspect material on slides, air dry and 
fix by heat, 
2. Apply carbol-fuchsin and heat gently until steam appears over 
the surface. Allow to steam for 5 minutes. Wash in water. 
3# Decolorize with acid alcohol by renewal washer to a faint pink. 
Wash in water. 
U* Counterstain with Methylene blue for § minute, wash in water, 
blot dry. 
Results: 
Acid-fast organisms are stained red. 
Non-acid-fast organisms are stained blue. 
Application: The detection of tubercle bacilli, leprosy bacilli 
and a few other acid-fast organisms. 
10. Hiss’ Method for Capsules. 
Formulae: 
1. Staining solution: Crystal vio3.et, stock solution 10. cc. 
Distilled water 90. cc. 
(Gram’s crystal violet or Ziehl-Neelsen1s carbol-fuchsin may 
be substituted.) ■ * 
2. Mordant: Copper sulphate, 20$ aqueous solution. 
Technique: 
1. a. Exudate: Spread evenly on clean slide. 
b. Cultured organism: Mix with equal parts of animal scrum 
and spread. 
2. Air dry but do not heat fix. 
3* Apply staining solution for 1 minute, heated to steaming. 
U. Wash off the stain with copper sulphate. Do not wash with water* 
5* Blot dry or examine wet under a cover slip. 
Results: Capsule, if present, appears as a faint blue halo about 
a dark purple cell body. 
11. Spore stain: (Corner’s method). 
Formulae: 
Carbol-fuchsin and Methylene blue as in acid-fast stain. 
Technique: 
1. prepare slide spread and stain with carbol-fuchsin as for 
Ziehl-Neelscn method 
2. Cash in hot tap v;ater. 
3. Rinse rapidly with 95$ alcohol. 
H. Apply Loeffler's Methylene blue for 2-5 minutes. Wash, blot dry. 
Results: Spores are red, cell body blue. 12, Nigrosinc method (for spirochetes) 
Formulae: 
Nigrosine 10. gms, * 
Distilled water—— 90. cc. 
Boil in flask for 30 minutes, then add as preservative: 
Formalin (10$) .5 cc. 
Filter twice through double filter paper. Store in small 
sealed test tubes. 
Technique: A loopful of fresh exudate or culture fluid is 
mixed on a slide with a loopful of nigrosine 
solution, then spread over the slide and dried. 
i 
Result: Spirochetes are not stained but are demonstrated nega- 
tively as unstained light areas on a smoky background. , 
13. Van Gieson’s Stain (for Negri bodies). 
Formulae: , 
1. Fixative: Methanol (neutral) — 100. cc. ) freshly 
Picric acid - .1 gm.) prepared, 
2. Stain: Basic fuchsin, stock solution - ,5 to 1 cc.) made fresh 
Methylene blue,stock solution 10. cc, ) just before 
Distilled water —-— 30, cc. ) use. Fuch- 
. sin varied 
to desired 
result. 
Technique: 
1. Make impression or gmear spreads of grey matter of hippocampus 
or cerebellum of brain of suspected rabid animal. 
2. Fix-with a momentary flood of methanol. Wash at once, 
2** Stain is applied for 5 minutes, heated gently to steaming. 
Wash, blot dry. 
Result: Negri bodies are magenta with blue granules. 
Nerve cells are blue. 
Erythrocytes are salmon or bronze color, 
11. Wright’s stain and Gicmsa’s stain: See sections on Hematology 
and Parasitology. 
Miscellaneous Solutions, 
1. Sodium chloride solution ("Saline” or "physiological salt solution") 
Sodium chloride — — 8.5 gms. 
Distilled water — •—r — 1000. cc. 
2. Buffer solution; 
Sodium dihydrogen phosphate (NaH^PO^).—-—-—•- 28.81 gms 
Disodium hydrogen phosphate —-125. "gms. 
Distilled water— to 1000, cc. 
3, Sodium chloride solution, buffered: 
Buffer solution (above) —1— *— 20. cc, . 
iodium chloride —— 8*5 gms* 
Distilled water — —-—-—-t -to 1000, cc* 4, Sodium citrate - sodium chloride solutions: (anticoagulant) 
J./Q <t/b -LU/j 
Sodium citrate c.p. 10. gras. 20. gms, 100, gras.)filtered 
Sodium chloride c.p, 8.5 gms. 8,5 gms. 8.5 gms.) after 
Distilled water 1000. cc. 1000. cc. )solution. 
May be sterilized in autoclave at 15 pounds for 20 minutes. 
When used as anticoagulant of blood,a final sodium citrate concentra- 
tion of over 0,25$ is required; therefore, use to each 10 cc. of 
blood 3.3 cc, of the 1$, 1,4. cc. of the 2$ or ,26 cc. of the 10$ 
solution. 
5. Potassium oxalate solution: (anticoagulant) 
Potassium oxalate 2. gms.) 
Sodium chloride 6. gms.)l cc. of this to 10 
Distilled water 100. cc. )cc, of blood prevents 
coagulation. 
6. Sodium carbonate solution: 2 gms. per liter of water. 
May be used for boiling instruments, as it prevents corrosion. 
7. Disinfectant solution (Desk jar use) sk 
Liquor cresolis comp, U.S.P. 20, 50. cc. 
Tap water to make 1000, 1000. cc. 
8. Preparation of Percentage Solutions by Dilution: 
1. Measure number of cc. of more concentrated solution corresponding 
to the percentage desired for the new solution. 
2. Add distilled water to total volume corresponding to the percen- 
tage numeral of the original solution. For example, to prepare 
a 70$ solution from a 95% solution, measure 70 cc. of the latter 
and add 25 cc. of distilled water, giving therefore 95 cc. of a 
70$ solution. BACTERIOLOGY 
I. Introduction and Bacteriological Techniaue. 
Bacteriology, strictly defined, is that branch of biology 
which treats of the structure and functions of certain minute 
plant forms called bacteria. However, the science of modern 
bacteriology, particularly medical bacteriology, has been 
enlarged to include other microorganisms such as certain fungi, 
plant forms of more complex structure; the spirochaetes, an 
indeterminate group variously classed as plants or animals by 
different investigators; the Rickettsia; and yet unclassified 
group of viruses. 
Bacteria are intimately related to human existence. Without 
them higher animal and plant life would be impossible. Putre- 
faction and decay are dependent upon bacterial activity for the 
breakdown of complex compounds into simple forms which can be 
assimilated by higher plant life. If it were not for bacteria 
we would be lost in a mass of the debris of fallen leaves, dead 
animals, etc., which would not decay and return to the dust 
from which they came in their absence. Bacteria are also 
essential to the synthesis of nitrogen and carbon into compounds 
which can be utilized by plants and in turn by animals and man. 
The dependence of animal life upon plants completes the complex 
cycle of existence, in which bacteria play such a prominent role. 
Unfortunately for us bacteria are not all our friends. Of 
the 1500 or more species which have been described, approximately 
100 are pathogenic (cause disease) to animals and plants, and 
about 50 cause disease in man. It is with the pathogenic 
bacteria and related microorganisms that we are concerned in the 
laboratory. 
Since these organisms are pathogenic. it is necessary to be 
careful in the laboratory when dealing with them for two reasons: 
(l) danger of infection to yourself and others, and (2) danger 
of contamination of the material to be studied. By contamination 
we mean mixing other microorganisms in with those to be studied. 
Since bacteria are everywhere present, in soil, water, air and on 
plants, animals and man, we have to use sterile technique in the 
laboratory to prevent contamination. 
With regard to the danger of infection to yourself and others, 
when dealing with infected material, such as pus, sputum, spinal 
fluid, etc,, do not spill it around on the table and chairs. Keep 
it off of your fingers, if possible, and above all, keep your 
fingers away from your face, mouth and eyes. When you are dealing 
with infected material, always think of your hands and fingers as 
if they had some of the material on them, even though you did not 
see any spill on your fingers; and keep your fingers away from 
your face until you have washed thoroughly with soap 
and water. Frequent washing of the hands with plenty of soap 
and water never hurt anyone, but will save a lot of trouble. 
A, Sterile Technique: 
Sterile technique in bacteriology to prevent contamination 
is essential. In general this is accomplished by using alcohol or gas flame, a platinum wire loop, and sterile glassware, 
and culture media. By sterile we mean the absence of living 
microorganisms. Sterilization is accomplished by holding 
glass or metal objects directly in a flame for.about one 
minute, by boiling in water for 20 minutes or by steam or 
dry heat (see section on culture media for details of 
sterilization.) For the transfer of bacteria from a culture 
to a slide, or from one culture to another, or from the 
specimen to a slide or culture, the platinum wire loop is 
used. (The wire loop consists of a #22 or #24- platinum 
wire 3 or 4 inches long which is fixed to a handle of glass, 
metal or wood. The wire is straight except for a round 
loop at the far end about 4 millimeters in diameter). The 
wire is held in the flame almost perpendicular to the table 
until the entire wire is red hot, then, first allowing it 
to cool for a few seconds, the wire loop is placed in the 
culture or specimen and a small amount of the material is 
smeared on the slide. As soon as this transfer is made 
-the wire loop is again flamed to red hot to remove the 
bacteria. It is important to remember to flame the wire loop 
each time it is used. If the procedure requires several 
transfers, flame the wire loop between each one. 
B. Inoculating Cultures. 
1. Broth Cultures. 
a. Of course the culture media must first be sterile 
and be in a sterile container (be it culture tube or 
flask) with a sterile plug. , 
b. The inside of the container only is considered 
sterile - the outside is contaminated. 
c. Holding the culture container in the left hand, 
grasp the cotton plug with the hand inverted, be- 
tween the fifth and four fingers of the right hand, 
then flame the mouth of the container. Holding the 
handle of the wire loop with the thumb and fore- 
finger of the right hand (after first having flamed 
the wire loop), transfer a loop full of the material 
to the broth culture, being careful not to touch 
the outside of the container. 
d. Then hold the cotton plug in the flame until it burns 
and quickly replace it in the mouth of the container 
to put out the flame - never blow it out. Now flame 
the wire loop again before laying it aside# 
e. If you are to make a blood culture, the blood will be 
collected with a sterile syringe and needle (3 or 
4 cc, of blood is sufficient). Now steps a, b, c, 
and d are followed except that instead of using the 
wire loop for.the transfer, the sterile needle of 
the syringe is placed inside the mouth of the con- 
tainer (being careful not to touch the outside with 
the needle), and the blood is forced into the 
culture by holding the barrel of the syringe between 
the index and middle fingers of the right hand and 
pushing down the plunger with the right thumb. 2. Agar Cultures. 
a. Slant agar the same procedure is followed as for 
broth cultures. The transfer is made with the wire 
loop but instead of dipping the loop into the 
culture it is rubbed lightly over the surface of the 
slant, 
b. Stab agar cultures - the same procedure again except 
instead of a wire loop a straight wire without a 
loop is used and this is stabbed into the agar 
culture. 
3. Plate Cultures: plate cultures are made by placing 
sterile, melted culture media in sterile Petri dishes 
and, after allowing an initial incubation period to 
test their sterility, they may be stored in a refrigerator 
until needed, 
a. Streaked Plate Method: tubes containing 10-15 cc. 
of sterile agar medium are held in boiling water 
until the agar is melted. Then allow it to cool 
until the tube can be handled. Now holding the tube 
in the right hand, remove the cotton plug with the 
inverted left hand, grasping the plug between the 
fifth and fourth fingers. Flame the mouth of the 
tube, then lift one edge of the cover of a sterile 
Petri dish just high enough to pour the melted agar 
into it,: quickly replace the cover of the Petri 
dish and allow the agar to solidify evenly distri- 
buted over the plate. After the plate has hardened 
or set, it is incubated at 37.5° C. for 12 hours to 
check its sterility. A large number of culture 
plates may be prepared in this way and then stored in 
a refrigerator until needed, 
(1) Inoculation cf Streaked Plate: this can be done 
with a sterile swab or a wire loop. One edge 
of the coyer is lifted just high enough to admit 
the swab or wire loop and this , is drawn over 
the surface in parallel rows 4.-6 mm. apart with- 
out recharging the loop, then quickly replace 
the cover. Now invert the Petri dish and mark 
it with a wax pencil. The plate cultures are 
then incubated in an inverted position to 
prevent the spread of the colonies on the sur- 
face by the water cf condensation, 
b. Poured Plate Method: 
(1) Plain agar poured plate - serial dilutions of a 
mixed culture are made in tubes of melted agar. 
Usually three dilutions are sufficient to 
obtain isolated colonies. One or two wire 
loopsful of the mixed culture are put into the 
first tube of melted and cooled agar. The tube 
is rolled between the hands to insure thorough 
mixing without the formation of bubbles. Then 
two loopsful,are transferred from this tube to a 
second cooled agar tube and, after thorough 
mixing, three loopsful from the second to the third tube. The contents of each tube are then 
poured into a sterile Petri dish, on the 
underside of which the date and other necessary 
information has been marked with a wax pencil. 
The work must be carried out rapidly in order 
to be completed before the agar solidifies. 
(2) Blood Agar Pour Plates; by adding sterile 
blood (sheep, rabbit, or human) to the medium, 
blood agar for determining the type of hemolysis 
produced by certain bacteria may be made. 
Hemolysis is best determined by the use of 
poured plates, although for convenience in 
general laboratory work the blood agar is 
allowed to solidify and then inoculated by 
streaking. 
II. Types of Bacteria. 
A, Morphology; by morphology we mean the size, shape and 
general structural form of the bacteria. There are three 
general forms. 
1. Cocci - cocci, when lying free, are approximately 
spherical like a ball. Variations from the strictly 
spherical shape are of common occurrence. The crowded 
conditions of rapid growth often give a variety of 
ellipsoidal, conical and flattened forms, popularly 
designated as bean, kidney and lancet-shaped. Division 
may occur in one direction resulting in the formation of 
diplococci and chains; in two directions in a single 
plane at right angles to each other; in three directions, 
producing the cubical packets of sarcina; and irregularly 
in all three directions, giving the clustered formation 
of the staphylococci. In addition the cocci may have 
capsules. The most important of these encapsulated 
cocci is the pneumococcus, 
2. Bacilli - the Bacilli, which include the greatest number 
of species, are straight, cylindrical, rod-shaped cells, 
with plane, convex, or concave ends. Although they are 
uniformly longer than wide, the ratio of length to width 
varies greatly, some forms being filamentous and others 
approaching the coccal shape. Variations such as ovals, 
spindles and club or dumb-bell shaped forms occur. Mor- 
phological characteristics such as size, ratio of length 
to width,and contour of the ends, serve to identify a 
few species, but final confirmation requires cultural 
and staining methods. Division always occurs at right 
angles to the longitudinal axis. 
3. Spirilla - the spirillar type is characterized by a 
bending or twisting which produces a curved, helical or 
spring-like shape. The spirilla vary from small comma- 
shaped forms (vibrio) with a single turn, representing 
the segment of a helix, to long sinuous forms with 
several curves. In this class the spirochaete of 
syphilis may be included. The organism variously called 
Borellia Vincenti, Spirochaete of Vincent's, can also 
be included here. This organism, which causes Vincent’s 
Angina, is usually found in two forms, one a spindle- 
shaped or cigar-shaped form and the other a wavy 
spirillum. B. Motility? some bacteria, mostly bacilli and the spirilla, 
have the power of motion. This power of motion is used as 
another aid in the classification of the bacteria. The 
bacteria move in their liquid environment by moving their 
flagella. The flagella are very fine projections of the 
bacteria situated at the ends or along the body of the 
organism. The flagella are so small that they cannot be 
seen by ordinary methods, however, in wet preparations, if 
the bacteria are motile, it is assumed that they have 
flagella. By motility of bacteria we do not mean the 
vibration back and forth called Brownian ?tovement, we mean 
active motion about the microscopic field, 
C. Spores: when conditions for bacterial existence become 
unfavorable, some species develop spores, resting forms 
possessing greater powers of resistance than those of 
vegetative bacteria. These spores are known as endospores 
or true spores to differentiate them from the sexual and 
asexual reproductive spores of fungi. They appear in the 
body of the bacteria as highly refractive round or oval 
bodies, which are resistant to ordinary dyes and require 
special staining methods. The mature spore consists of 
condensed nuclear and cytoplasmic material enclosed in a 
relatively impenetrable spore wall. With the completion of 
the fully-formed spore, the parent cell disintegrates. When 
the conditions are favorable the spore is capable of 
developing into the vegetative form of the bacterium. 
The presence or absence of spores is another aid in 
the classification of bacteria. Since the spores are very 
heat resistant, high temperatures are necessary to destroy 
them. 
D. Cultural Characteristics: the kind of culture media that 
the organism grows best on; (some will grow only on special 
media) the color, size, shape, and elevation of the 
colonies; whether they will grow in the presence of oxygen 
(aerobic) or will grow only in the absence of oxygen 
(anaerobic) all are aids in the classification of the kind 
of bacteria. 
E. Fermentation of Sugars: certain of the bacteria will 
ferment or produce gas in various kinds of carbohydrates 
such as: indol, dextrose, maltose, mannite, sucrose and 
lactose. Some organisms will produce acids when grown in 
the above carbohydrates and, by the addition of an 
indicator, this can be determined by the color change. 
All of this is important in the identification of the various 
types of bacteria. (See the Bacteriology Reaction Chart). ORGANISM 
//$• 
7 1 
y AGAR 
/ SLANT 
-1 
BROTH 
PEPTONE 
HABITAT 
REMARKS AND 
DIAGNOSTIC CHARACT. 
Staph, Albas 
Spherical 
4 
— 
Abundant 
Shiny.ffhite 
Turbld,pel 11- 
cie,Sediment 
Sediment 
Skin,air,etc. 
White,Grape-1Ike Clusters 
Stach. Aureus 
Spherical 
4 
- 
— 
— 
Ab. Sh. 
Oranae 
Tur, Sad, 
Sediment 
Skin.air,etc. 
Orange,Grape-1 Ike Clusters 
Strep. Pyogenes 
Pairs or 
Chains 
4 
- 
— 
— 
Scant 
Colonies 
Scant 
Scant 
Skin 
Pathogenic 
Hemol, on bl, agar 
Strep, Viridans 
Pairs or 
Chains 
4 
- 
- 
Scant 
Colonies 
Scant 
Scant 
Mouth 
intestine 
Green lone on bl. agar 
Diplo. pneumoniae 
Pairs or 
Chains 
4 
- 
- 
- 
Scant 
Colonies 
Scant 
Scant 
Mouth, Nose 
Capsules. Bile Soluble 
Meningococcus 
Pairs 
— 
— 
— 
Tiny or nom 
Scant 
Scant 
Meningitis 
Nosg 
Growth best on Blood Media 
Gonococcus 
Pairs 
- 
- 
— 
- 
No Growth 
No Growth 
No Growth 
Gonorrhea 
Growth only on Special Media 
Esch. Coli 
Short Rods 
— 
f 
t 
Moi st,opaqu< 
Fecai Odor 
Turbid Sed- 
Turbid Sed. 
Intestine 
Ferment Suaars. Methv! Red 
Salmonella 
paratyphi 
Rods 
— 
— 
4 
Moist,Gray 
Turbid Sed. 
Turbid Sec- 
Paratyphoid 
Fermented Suqafs.KjS formed 
Salmonella . 
scnottmu. .leri 
Rods 
— 
— 
4 
- 
Moist.Gray 
Turbid Sed. 
Turbid Sed- 
Food poison- 
ing.paratvDh, 
Ferment sugars Formed 
Shegella 
dvsenteriae 
Short Rods 
- 
- 
- 
Mo 1st,Gray 
Si. Turbid 
SI- Turbid 
Dysentery 
Ferment sugars,exotoxAn produce* 
Short Rods 
— 
— 
— 
f 
Moist.Gray 
Turbid 
Turbid 
Dysentery 
Ferment Sugars. Indol 1 
B. anthracis 
Square 
End Rods 
4- 
4 
- 
— 
Gray 
Spreading 
Pel 1icie 
Sed- 
Pei IIcle 
Sed, 
Causes 
Anthrax 
In tissue as encapsulated chains 
Clos. tetani 
Larqe Rods 
4 
4 
4 
Small.Gray 
SI. Turbid 
SI- Turbid 
So iI.Tetanus 
Anaerobic Toxin produced 
Clos, welchii 
Large Rods 
4 
4 
— 
— 
Moist,Gray 
Turbid 
Turbid 
Soil, Gas 
Gangrene 
Anaerobic Toxin Produced 
Clos.botulinum 
Large Rods 
4 
r 
4 
Flat,Gray 
Turbid 
Turbid 
Soil. 
BotulIsm 
Anaerobic Tn*ln Produced 
Tubercle-bacillus - 
Slender 
Rods 
4 
- 
- 
No Growth 
No Growth 
No Growth 
Human 
Pathogen 
Acid Fast.qrows In guinea pig 
Diphtheria 
■haMITrif? 
Rods 
4 
- 
- 
- 
scant gray, 
granular 
Si. Turbid 
Si, Turbid 
Nose & throat 
Pjph.thecia— 
Grows on Loeffler*s bl, serum, 
Club shaped, beaded 
Eberthella 
Ttrohosa 
Short Rods 
— 
- 
4 
— 
Moist,gray 
Turbid 
Turbid 
Tvohoid fever 
Ferment Sugars. Motile 
. B. Vincent! 
Fusiform 
Spirillum 
Sta 
dll 
Ins 
ute 
wli 
car 
h Crlght*s or , 
•bol fuschln 
Mouth & throal 
V, Angina 
Cigar—shaped 4-8a* 
Wavy threads IQ-2Q Outline Classification of Bacteria of Greatest Importance in 
Medical Bacteriology, due to their Pathogenicity or to their 
Frequent presence in Cultures as Contaminants or Saprophytes, 
I. COCCI. Cells spherical or somewhat elliptical. Aerobic. 
A. Gram Positive 
1. Streptococcus forms. Cells in short or long chains, never 
in packets. Genus Streptococcus. 
a. Pyogenic group. Generally (beta) hemolytic. Four species 
several serological types. Most important species - 
S.pyogenes. 
b. Viridans group. Not beta hemolytic but may show varying 
degrees of greening (alpha) of blood. Most important 
species - S.salivarius 
c. Saprophytic group. No hemolysis or only indistinct zone 
(alpha prime type). Usually not pathogenic. Example - 
S.faocalis 
2. Diplococcus forms. Cells usually in pairs. No hemolysis. 
Colonies greenish on blood agar. Diplococcus pneumoniae. 
3. Micrococcus forms. Cells in plates, groups or irregular 
packets or masses. Never in chains. Usually non-pathogenic. 
Genus Micrococcus. 
A. Staphylococcus forms. Cells as a rule in irregular groups. 
Usually pathogenic. Genus Staphylococcus. 
a. Orange pigment. Staph, aureus. 
b. Lemon-yellow pigment. Staph, citreus. 
c. White or colorless growth on solid media. Staph, albus. 
5, Tetragcna forms. Occurs in pairs and tetrads, 
6. Sarcina forms. Division occurs in three planes, producing 
regular packets. Example - Sarcina lutca. 
B. Gram Negative 
1, Cells normally in pairs, with adjacent sides usually flattened. 
Genus Neisseria. 
a. Grow best on special culture media at 37°C. 
(1) Acid from dextrose, not from maltose. N,gonorrhoea. 
(2) Acid from dextrose and maltose. H.intracollularis. 
b. Grow well on ordinary culture media at 22°C. Usually not 
pathogenic. Several species. 
Examples: (1) Non-chromogcnic - N. catarrhalis. 
(2) Chromogcnic - N.flava. 
II. CURVED FORMS. Cells elongate, more or less spirally curved. Gram- 
negative . Vibrio comma. Ill* NON-SPORULATING BACILLI* Elongated rod-shaped cells, without 
endospores. Gram-positive or Gram-negative* Aerobic 
A. Gram-positive* 
T• Long slender, non-motile rods, occurring singly in pairs 
and in chains* Genus Lactobacillus. 
B* Gram negative* 
Small, motile or non-motile rods* Not active in fermenta- 
tion of carbohydrates. Usually parasitic on warm blooded 
animals. Frequently require body fluids for growth. 
(Family Parvobacteriaceae)• 
a. Majority grow on ordinary media. Show bi-polar staining. 
Majority ferment carbohydrates. 
Tribe Pasteurelleae - Genus pasteurella* 
(1) Grow on ordinary media* Indol' anci H2S produced. No 
growth in bile. Sorbitol fermented. Animal pasteur- 
ellas (3 species). 
(2) Grow on ordinary media. Neither indol nor pro- 
duced. Growth in bile. Sorbitol not fermented. 
Pasteurella pestis. 
(3) No growth on ordinary media. Pasteurella'iularensis* 
b. • Majority grow on ordinary media. >t)o not show bi-polar 
staining. None ferment carbohydrates. Non-motile. Three 
species of genus Brucella: 
Growth in media containing: ; • 
Thionin Basic fuchsin 
A ■■■■ ... ..I.. 
Br.melitensis /// . /// 
f . * . • ‘ , - ... 
Br.abortus - . /// 
Br.suis /// 
c. On first isolation require some factor or factors con- 
tained in blood or plant tissues. Usually do not show 
bi-polar staining. Genus Hemophilus. 
(1) X and V factors required - H.influenzae. 
(2) Neither X nor V factors required - H.pertussis and 
H.ducreyi. 
2. Motile or non-motile rods widely distributed in nature. 
Majority of species attack carbohydrates forming acid, 
or acid and gas. Grow well on artificial media, 
a. Do not produce acid in media containing carbohydrates. 
(1) Rounded colony, no pigment produced. 
Alcaligejnes facealis. 
(2) Large spreading colony, yellowish-green pigment 
produced. Pseudomonas aeruginosa* b', “Ferment dextrose and sucrose but not laptose with forma- 
tion of acid and small amount of gas. Produce characteris- 
tic spreading, amoeboid colonies. Liquefy gelatin. 
Proteus vulgaris. 
c. Ferment dextrose and lactose with formation of acid -and gas 
So called coli-aerogenes group. 
Species 
Methyl' 
red 
•V.P. 
Citrate 
utilization 
Indol 
Gelatin li- 
quefaction 
Escherichia coli 
/ 
- 
- 
/ 
- 
Escherichia freundii 
/ 
(?) 
/ 
? 
7 
Aerobacter aerogenes 
- 
? 
/ 
? 
- 
Aerobacter cloacae 
• 
/ 
/ 
- 
/ 
Klebsiella pneumoniae 
/ 
- 
/ 
- ' 
- 
d. Ferment dextrose with formation of acid, or acid and gas. 
A few species of genus Shigella ferment lactose with form- 
ation of acid, but never visible gas. Tribe Sftlmonelleae. 
(1) Ferment dextrose with formation of acid ahd gas . 
Genus Salmonella. 
(2) Ferment dextrose with the formation of acid but no gas. 
(a) Motile - Eberthella typhosa, 
(b) Non-morile - Genus Shigella. > Salmonella - typhoid-dysentery group 
Dextrose 
L 
1 
“1 
O) 
CO 
0 
P 
n 
• 
. 
,C0 
& 
X 
J 
H 
S 
•H 
| Lactose 
Sacchrose 
rH 
o 
■p 
•H 
CO 
Q> 
a 
M 
rH 
O 
'H 
M 
' 
c 
o 
•H 
-P 
O 
S 
T3 
O 
b 
Ph 
to 
CM 
, X 
Citrate 
utilization 
■PC 
co 
P-P 
Pcd 
«3N| 
-P*H 
1 rH 
rOH 
+3 
Motility 
Salmonella choleraesuis 
AG 
AG 
AG 
AG 
(AG) 
- 
- 
- 
- 
/ 
/ 
/ 
Salmonella pullorum 
AG 
AG 
. 
(AG) 
- 
r 
- 
- 
/ 
- 
- 
Salmonella paratyphi 
AG 
AG 
AG 
- 
(AG) 
- 
- 
- 
- 1 
:/) 
- 
- 
/ 
Salmonella enteritidis 
AG 
AG 
AG 
AG 
r‘rrr'1 n 
AG 
- 
- 
- 
/ 
/ 
/ 
/ 
Salmonella schottmuelleri 
AG 
AG 
AG 
(AG) 
(AG) 
- 
(AG) 
- 
/ 
/ 
- 
/ 
Salmonella typhimurium 
AG 
AG 
AG 
AG 
AG 
V - 
(AG) 
- 
/ 
r 
/ 
/ 
Eberthella typhosq 
A 
A 
a' 
(A) 
(A) 
. 
- 
- 
/ 
- 
/ 
/ 
Shigella dysenteriae 
A 
, r* 
- 
- 
- 
- 
• 
- 
Shigella paradysenteriae 
A 
A 
(A) 
- 
- 
- 
(A) 
/ 
- 
- 
- 
Shigella alkalescene 
A 
'.A 
A 
A | A 
- 
(A) 
/ 
- 
Shigella sonnei 
A 
A 
A 
“ 
- 
A 
A 
~ 
- 
- 
Shigella madampensis 
ii 
A 
A. 
A | A 
I-— 
A j “ 
/ 
- 
Note: Brackets () abound symbol denotes variable or delayed reaction. 
IV. SPQRULATING BAOIXJJ,. Rods producing endospores, usually Gran-positive. 
Often decompose protein media" actively. 
A. Grov; aerobically. Mostly saprophytes. Genus Bacillus. 
1. Pathogenic forms. Non-motile rods with square cut to concave 
ends, occurring in long chains, central spores. 
Bacillus anthracis. 
2. Non-pathbgenic forms. Usually motile, having' central or excen- 
tric spores. B.subtilis group (lU5 species) B. Grow only anaerobically. Often parasitic. Genus Clostridium. 
1. Non-motile rods. Rods nvt swollen at sporulation. Spores 
central or excentric. , Cl.perfringens. 
2. Motile; Rods swollen at sporulation. 
a. Spores terminal or subterminal. Spherical or nearly so. 
Cl.tctani. 
b. Spores oval, central or excentric. 
(1) Pathogenic to man - due to preformed toxin. 
(a) Cl.parabotulinun. 
(b) Cl.botulinum. 
(2) Pathogenic to man - associated with gas gangrene. 
(a) Cl.novyi 
(b) Cl.septicum 
(c) Cl.blfermentans 
(d) Cl.histolyticum 
(e) Cl.fallax 
(3) Not pathogenic to man - many species. Examples: 
(a) Cl.sporogenes 
(b) Cl.terbium 
V. BACILLI HAVING BRANCHING CHARACTERISTICS. Show parallelism, slight 
branching, curving forms, V-shapes, clubbing at ends, and segmental 
staining. Gram-positive. 
1. Not acid fast. Colonies more flat and moist like other bacteria. 
Rods frequently club shaped. Genus Corynebacterium. 
a. True diphtheria organism. Slender rods, curved or straight, 
of variable lengths; granular or segmented; generally club 
shaped, Metachromatic granules large except in gravis type. 
Moderate growth on ordinary media, G.diphtheriae. 
b. The diphtheroid group of bacteria - 20 species. 
(1) Short, thick, straight rods. Stain uniformally. Luxuriant 
growth on ordinary media, C.pseudodiphthericum 
(2) Medium sized rods showing solid and barred forms, 
Metachromatic granules small. Scanty and slow growth 
on ordinary media, C.xerose. - 
| C.segmentosum 
C.xerose 
o 
• 
TJ 
Co 
CD 
R 
a 
H- 
tr 
c+ 
f3* 
CD 
•i 
H- 
O 
P 
B 
A'AI'III ssdXi „ 
H Type II 
C.diphtherias Type I 
u 
Species 
1 
X^ 
x 
X 
Dextrose 
« 
x^ 
i 
X. 
Maltose 
■ 
. 
l 
X. 
» 
X 
Dextrin 
x. I i 
- _ .• - . 
I 
X 
• 
1 
Glycerol 
• !> 
1 
X 
i 
1 
Galactose 
f » 
| rv, | "k. 
« 
1 
i 
• 
Saccharose 
1 
1 
t 
« 
1 
Litmus 
milk 
» 
i 
1 
X 
X 
’ 
X 
1 
Production 
of an 
exotoxin 
i 
.j 
i 
t 
X 
X. 
Production 
of - 
hemolysis 
4 
2, Acid fast. Colonies more or less wrinkled and dry, more like 
moulds. Slender rods, seldom filaments, which are stained with 
difficulty, but when once stained are acid fast. Cells some- 
times show swollen, clavate or cunoate forms and"sometimes - J 
'even branched forms. ■ /•* Genus Mycobacterium.' 
a. Saprophytes, or parasites on,cold blooded animals; grow 
rapidly on most media at room temperature, (B species). 
Examples: (1) M.lacticola.' 
- ' (2) M.phlel 
, b. Parasites on warm blooded animals; grow slowly on all media. 
(1) Pathogenic for man, 
(a) M.tuberculosis var. hominis. . • , ; • 
(b) M.tuberculosis var. bovis. . ; " ,, • - 
(2) Not pathogenic for man, M.avium. 
c. Pathogenic for man. Will not grow on usual culture media. 
U* leprae. GENUS STREPTOCOCCUS 
Habitat: Common pathogenic forms; a.lso frequently on skin and body 
orifices without invasive tendency. Some species are the specific causes 
of infectious diseases. A number of saprophytic species are commonly 
present in dairy products and elsewhere. 
Characteristics: Gram positive cocci of medium size, in pairs or short 
chains, never in packets; grow best on blood or serum agar, aerobically, 
at 37°G., the 2h hour colony being small, circular, slightly raised, 
surrounded at times by zone of haemolysis. Killed at 5£°C., in 30 
minutes. 
Haemolytic Group (Seta type) have clear zone of haemolysis around 
colony on blood agar. 
Viridans Group (Alpha type) have greenish zone around colony on blood 
agar. 
Non-haemolytic Group (Gamma type) have no area of haemolysis or green ' 
zone around colony. 
Streptococcus pyogenes: Colonies have Beta zone of haemolysis 2 to 3 mm. 
wide. Grow in long chains. Found in man in acute inflammations including 
septicaemia, cellulitis, wound infections, middle ear or sinus disease 
or elsewhere. Tend to be more severe and generalized than Staph, aureus 
infections. 
Streptococcus salivarious: (S. viridans) (S. mitior) is a parasite of the 
normal nose and throat, also encountered in dental accesses, in endo- 
carditis and in some blood cultures. Grow in short chains. This colony 
readily recognized on a blood agar plate by its greenish zone of haemolysis* 
Usually not pathogenic for small animals. Distinguished from Diplococcus 
pneumoniae by inability to ferment inulin and by not being bile soluble. 
Streptococcus lactis: Is non-pathogenic, occurs in milk and milk products 
and in mouth and intestinal tract of man. Colonies on blood plates pro- 
duce no haemolysis or only trace of green. 
Streptococcus faocalis: Is feebly pathogenic, found in feces of man and 
other animals. Sometimes found in inflammatory exudates and subacute 
endocarditis. No haemolysis on blood agar. 
Identification: 
1. Microscopies Gram stain of direct or culture spreads will show 
Gram / cocci, singly, in pairs or in chains of varving length. The chain 
form is best seen in spreads made from liquid culture, or in liquid b#ody 
fluids. 2, Culture: Blood agar plates at 37° C. for 24 hours will give 
the small colony type and form of haemolysis classifying roughly the 
species. For routine clinical wohk the examination is usually limited 
to the study of colonies on blood agar and the results reported as the 
case may be: 
Streptococcus, haemolytic 
" non-haemolytic 
M viridans 
GENUS STAPHYLOCOCCUS 
Habitat: Common,.potential or actual parasites, occurring on normal 
Fkin and body orifices, and in feces, therefore in dust, soils and as 
culture contaminants? frequently the cause of suppurative lesions in 
man. 
Characteristics: Moderate size cocci, are in pairs or grape-like clus- 
ters; gram-positive; grow freely, aerobically, on common culture media, 
giving in 24 hrs. at 37° c. medium size, low, convex, smooth, glistening 
colonies with an even edge; color of colony variable with species; some 
strains produce haemolysis on blood agar. 
Staphylococcus aureus: Golden yellow colonyq usually haemolytic; 
frequently found in boils, carbuncles and other skin lesions; sometimes 
in blood cultures in the event of septicaemia. 
Staphylococcus albus: Porcelain-white colony; feebly pathogenic. 
Staphylococcus citreus: Lemon-yellow colony. A non-pathogenic saprophyte. 
Identification; 
1, Microscopic: Gram / staphylococci on direct or culture stained 
spread, 
2. Culture: blood agar plate, 24 hrs. at 37° C, gives colony 
features of staphylococcus, species determined by color of colony. Note 
also presence or absence of haemolysis, ■ PIPLOCCCCUS PNEUMONIAE (Pneumococcus) 
Characteristics: Large lancet shaped cocci, usually occurring in 
pairs; sometimes found singly or in short chains. When in pairs, 
the adjacent ends of the cocci are usually bluntly rounded, the 
opposite ends are acutely pointed. In films from sputum, blood and 
cultures on serum containing media, a definite capsule can be seen. 
Gram-positive; stains well with aniline stains and special capsule 
stains. Poor growth on plain agar; grows best on blood or serum 
agar with pH 7.6 to 7.F; colonies on blood agar plate, surface flat 
and smooth with edge sharply raised from the medium, surrounded by 
a narrow zone of alpha-haemolysis (green discoloration); some 
strains (Types III and VIII) give characteristic mucoid colonies. 
Killed in 20 minutes or less at 55°C, Bile soluble; ferments 
inulin, 31 distinct serological types have been identified; called 
p. pneumoniae Type I, II, etc. to XXXIII; however, types 26 and 30 
apparently are identical with types 6 and 1$, respectively. 
f 
Habitat? The principal cause of lobar pneumonia (over 90$); also 
may cause bronchitis; bronchopneumonia, conjunctivitis, otitis 
media, brain abscess, meningitis, endocarditis and arthritis. Fre- 
quently present in normal mouths. Highly pathogenic for mice and 
slightly less so for rabbits. 
Identification? 
1. Direct microscopy: make spreads of specimen on slide, fix 
and stain by Gram's method and/or Hiss's capsule stain. Examine 
for diplococci showing typical morphology; if present confirm by 
procedures below, 
2. Typing by Neufeld Reaction: This is the rapid method of 
choice for identification of type on materials direct from the 
patient, giving the type within 30 minutes. It is less applicable 
to typing of cultures or to detection of type in patients who have 
received one of the sulphanilamido compounds. Only ofter pneumococci 
have been shown by stained spread to be present in appreciable 
numbers, is this typing effort to be attempted. 
Identification? LABORATORY DIAGNOSIS OF PNEUMOCOCCUS 
PNEUMONIA 
The treatment of penumonia with type specific therapeutic serum has 
increased in recent years. Coincident with this, the Neufeld reaction 
has assumed a greater significance, since specific treatment cannot be 
administered without knowledge of the type of pneumococcus causing the 
infection. The more_recent‘introduction- of sulfapyridlne, and other 
sulfone derivativesr as a chemical means of treating'pneumonia has not 
abolished the desirability of the bacterial method of control. In order 
to have a comprehensive treatment, it is important to know the kind of 
organism. , Even if chemical therapy alone is to be used, it is necessary 
to know the type of the causative pneumococcus since certain types require 
different dospges of the drug. Furthermore, for statistical purposes in 
order t,o develop rational-methods for the prevention and.control of 
pneumonia, it is necessary to determineJthe infective-agent in every case. 
f * Sputum •” ... 
Although pneumococci from any source can be typed, for usual 
diagnostic purposes in pneumonia, sputum is most satisfactory. It should 
be collected in clean, dry and preferably sterile wide-mouthed containers. 
Since the determination of the type- may depend on- t,he presence of living 
organisms, no antiseptic should be added. If the specimen is to be 
transported any distance requiring more than 5 hours, and cannot be kept 
cold, formalin (0.5%) may be added as a preservative. This will not 
interfere with the test. Generally the sputum obtained from pneumonia 
patients contains some blood and tends to be ttyfcck and sticky, or may be 
the color and consistency of prune juice. It is not necessary to have a 
large amount of sputum? whenever possible, at least*one or two teaspoonsfull 
should be obtained. • 
The sputum should bp fresh and should be examined soon after ex- 
pectoration by the patient; If there Is a delay.in the examination, the 
specimen .should be in. a refrigerator to retard the overgrowth of 
other organisms. nneumococcus typing should be considered-as an emergency, 
and the report should be*submit ted as sooh as the results will,permit. 
>' • ' • /.* • , ,, _ . 
Blood Cultures ‘ '' ’ • ‘ iV? 
Blood cultures are mode bv mixing,-, two or three cubic centimeters 
of blood with 50- cc. or more of brolh, and observed after eight to twelve 
hours of incubation at 37.5°, If bacterial growth is not evident,,,, 
incubation is continued and the culture observed at intervals, 
for at least ,72 hours, .q,r>d preferably 7 days. In positive broth ! u 
blood cultures-, the supernatant rfuid shows a diffuse turbidity 
end on agitation the mixture appears brownish-red to chocolate in 
color. Usually a sufficient number of organisms is present, as shown 
♦ •• by a gran-stained smear of the culture, so that the Neufeld technic 
may be carried out directly. If growth is sparse, it is advisable to 
place 10 be of the culture aseptically into a sterile tube and centri- 
fuge at Ipw speed to remove the blood cells. The supernatant fluid is 
poured into a second tube and centrifuged dt a high speed to throw down 
the organisms. The sediment of organisms is suspended in a small amount 
of broth tor typing. 
Serums 
In the Neufeld test, highly type-specific antiserum should be used 
which are prepared in rabbits especially for this reaction. Neither 
therapeutic nor diagnostic agglutinating horse antiserums are satis- 
factory because of occasional non-specific reactions. 
There are now some 3L different recognized types of pneumococci. 
In order to shorten the procedure varying numbers of these are mixed to- 
gether into 6 different combinations of from 3 to 6 types in each. These 
6 combinations are lettered from A to F inclusive. The specimen is first 
tested against each of the 6 combinations. If a positive result is 
obtained in one of them, then the specimen is tested against each of the 
component types of that combination* These noyt include: 
Group A - - Type 1, 2, 7 
» fi - - " 3, A, 5, 6, 8 
**•€--» 9, 12, U, 15, 17, 33 
" IV - - « 10, 11, 13, 20, 22, 2A 
" E - - ” 16, 18, 19, 21, 28 
« F?- " 23, 25, 27, 29, 31, 32 
It is not necessary to cry to memorize these combinations since they 
are furnished with each diagnostic set and the various component types are 
on the label of the vials. When not in use the serums should be kept in 
the refrigerators* Care also should be exercised to prevent their con- 
tamination* 
Neufeld Reaction - Procedure 
The Neufeld "quelling” reaction is based on the interaction between 
the encapsulated organism and the specific homologous antibody in the 
antiserum, resulting in the apparent swelling of the capsule without 
obvious change in size of the organ!«m within* 
Wet Preparation Method 
1* Areas are marked off and labeled on clean glass slides for the 
group typing serums. 
4 2* By means of a small loop measuring approximately 1 mm. in 
diameter, a small amount of the specimen ia placed on each area of the 
slides* 3* With a large loop measuring i; mm. in diameter* approximately 
ten times the amount of undilated serum is mixed with the material 
according to the relative number of organisms present; a different 
type serum or group of serums is used for each area. The loop 
should be cooled and flamed between each transfer. 
U- Loeffler’s methylene blue is next added with the 1 m.m. 
loop and mixed with the specimen and serum. Some typing serums 
already may contain this dye* but it is often insufficient for 
staining. 
3. A cover-slip is placed over each preparation# The slides 
are allowed to stand for a few minutes before examination. 
Examination 
A good microsc-ope with an oil immersion lens is essential and 
a powerful microscope light* subdued with blue glass* is a great aid. 
The entire preparation should be examined with an oil immersion 
lens. This test is highly specific when satisfactory antiserums are 
used. If no reaction occurs immediately* the preparation should be 
examined again in about 30 minutes. 
In a preparation containing the homologous antiserum, a positive 
reaction is indicated by the appearance of a definitely outlined but 
colorless halo surrounding the blue-stained pneumococci. This zone 
is swollen capsular material* having a distinct "ground glass" 
appearance. It is not the size* but the typical appearance of the 
swollen capsule which determines the positive reaction. The degree 
of swelling is usually equivalent to the width of the pneumococcus* 
although that of Type III. is much greater. In most types* the sharp- 
ness of outline is most significant. In some negative reactions* a 
thin halo without definite outline may confuse the inexperienced ob- 
server. Focusing just above and below' the pneumococcus itself, reveals 
nothing in such preparations; whereas* in positive reactions the swell- 
ing is easily visible at these focal planes. Tiihcn a positive reaction 
is found in one of the 6 groups* then the same process is repeated 
for each of the specific types composing that group. 
Sources of Error Involved in the 
Noufeld, Test 
1. Proportion of serum to sputum.-This is one of the most 
common causes of failure to obtain a reaction. Satisfactory ratio of 
serum to sputum should be more than 5 to 1. If the specimen contains 
very large numbers of pneumococci* it should be diluted with broth or 
saline. 
2. Influence of thick tenacious sputum. - Thick tenacious sputum 
will retard or prevent the reaction entirely. In addition, there is 
great difficulty in picking up a small portion of sputum. To avoid this thick sputum should be mixed with a small quantity of saline or 
broth and beaten with a wooden applicator, thus releasing sufficient num- 
burs of the organisms. 
3. Specimens containing two or more types. - This may occur and for 
this reason it is necessary to examine all six of the groups. When such a 
case is found, it is. necessary to make a note as to which specific type 
is the most numerous; since the type which is most numerous is probably 
the causative organism. Types III, VI, XIX, and XXIII, are relatively 
more common in normal throats than any other types, and may be found along 
with the causative organism. Types I and II are rarely found in normal 
throats. 
Mouse Inoculation 
Specimens containing pneumocooci which fail to type by the Neufeld 
reaction should be examined further by inoculation into a blood agar 
plate and by intraperitoneal injection into a mouse. This will demonstrate 
the presence of pneumococci which were overlooked by the direct examination. 
About 0,5 cc. of the material is injected into the peritoneum of a mouse, 
and the animal observed at frequent intervals for signs of illness. 
Symptoms may occur after 4 or 5 hours. At that time, or after 18 hours, 
a puncture of the peritoneum is made. This is done with a capillary 
pipette (75 mm..long by 1 mm. in diameter) or with a tuberculin syringe 
and a 25 gauge needle. The material thus obtained is examined by the 
Neufeld tost, care being taken that only small amounts of the exudate is 
used. If the mouse dies the abdomen is opened and the exudate examined in 
the usual manner* 
Bile Solubility Test 
The solubility of the pneumococcus in bile affords one of the most 
satisfactory tests for distinguishing pneumococci from streptococci. Broth 
cultures 18 to 24 hours old are used for this test. Into a small precipi- 
tin tube 0.2 cc. of the culture is placed, to which is added 0.1 cc. of 
ox-bile, A control tube is set up, using a similar amount of culture and 
0.1 cc. of saline. Another control is generally sot up for clarity and 
color to be expected, mixing 0.2 cc. of plain broth with O.l't**, of ox- 
bile. The tubes are incubated in a water bath at 45° C., for 1 liQur or at 
37° C. for 2 hours and then examined. Those cultures which lyse (ail 
bacteria dissolved) completely in bile are soluble, and are 
pneumococci, 
Bile for this purpose must of necessity be crystal clear. Centri- 
fugation may clarify it, although filtration is sometimes necessary. A 
10$ solution of bile salts (sodium desoxycholate or taurocholate) may bo 
substituted for bile. Each new lot of bile or bile salts should be first 
tested with known pneumococcus cultures. NEISSERIA 
Characteristics: Gram-negative cocci of variable growth vigor and 
variable pathogenicity. All members give a positive oxydase reaction. 
Habitat! N. gonorrhoeae (gonococcus) is the cause of gonorrhea; N. intra- 
cellularis (meningococcus) is the cause of a specific meningitis; both 
organisms may be readily demonstrated in the exudates from involved 
tissues; M. catarrhalis and several other species, which are found in the 
nose and throat of normal individuals, are sometimes associated with 
certain .epidemics of respiratory or eye infections* 
NEISSERIA GONORRHOEAE (gonococcus): A strict parasite of man; found in 
discharges from the genito-urinary system in acute or chronic gonorrhea, 
in the, pus .from gonorrhoeal conjunctivitis, rarely in tke blood stream. 
Characteristics: Oval or spherical cocci of moderate size, frequently • 
arranged in pairs with adjacent sides flattened or slightly, concave, 
resembling a pair of kidney beans side by side; in exudates the cocci are 
fairly regular in size and shape and are usually inside the pus cells; - 
in cultures the cocci will not grow on plain agar, enrichment of media is 
needed; grow on moist chocolate agar at 37° C. in 24- hours to small, 
round, convex, greyish-white colonies; growth is aerobic, favored by 
atmosphere of 10% CCU. Highly susceptible to inimical agencies: when 
dried the cocci die In 2 hours; moist heat at 55°C. kills in 5 minutes; 
quickly killed by 1:4.000 silver nitrate; cultures kept at room temperature 
die in a few .days, but at 37°C, they may survive several weeks. 
Identification: Microscopic examination only is generally done, cultural 
confirmation done only under special conditions. 
1, Microscopic? Make direct spreads of the infected urethral, cervical 
or conjunctival discharges bn glass slides, fix with heat and stain by 
Gram's method. Examine the stained preparation for gram-negative, coffee 
bean shaped, intrrcellular or extracellular diplococci having the typical 
morphology of gonococci. Report whether diplococci are intra or extra 
cellular, or both. Also report any other bacterial forms present, 
noting for each whether Gram-negative or Gram-positive and whether coccus 
or bacillus; also the relative numbers and kinds of tissue cells present. 
2, Ordinary culture methods, especially in chronic urethral or cervical 
infections, will reveal only the secondary organisms'which may occur. A 
special culture program is needed for growing N. gonorrhoeae. 
3, Special culture program: The cultural demonstration of the gonococcus 
is superior to direct spread examinations In cases of chronic gonorrhoeae 
in both sexes and in all cases in the female, especially when material 
for examination is taken from the cervix. a. The cultivation of the gonococcus, mixed with freer growing micro- 
organisms, requires observance of the following special procedures: 
(1) Take specimens of representative material and apply directly to 
plate media, 
(2) Use a medium such as moist chocolate agar, which will readily 
grow the gonococcus in mixed culture. 
(3) Grow in 10% CO2» 
(/+) Identify the gonococcus-meningococcus group by colony form and 
oxydase reaction, 
(5) Confirm the identification carbohydrate fermentation tests. 
b. Specimen taking and transmission: (Optional methods listed in order of 
preference.) 
(1) Platinum loop is touched to drop of pus, to urethra or to cleansed 
cervical os, and is immediately stroked broadly over a warm culture plate 
at the bedside or clinic chair, 
(2) Sterile swab is similarly contaminated with the suspected material 
at the bedside or clinic chair, immediately placed in a tube containing 
1 cc. of nutrient broth for prompt transmission to laboratory and inocu- 
lation of warm culture plate (broad spread of .1 cc. of this broth). 
(3) For delayed inoculation (up to 8 hours), the swab-broth tube #2 
is stored in icebox until culture plate inoculation is made. 
c. Culture media: (1) chocolate agar, soft, moist, warm. 
(2) The media of McLeod is elsewhere described. 
(3) Difco "Proteose #3 Agar" and "Bacto Hemoglobin" may be combined. 
d. Incubation: 37° C. in 10$ CO2 in closed jar, 24 - 48 hours. 
e. Examination of Culture: Observation made of two features: 
(1) Colony form: convex, slightly opague colonies, 1-3 mm, in 
diameter, with undulated margins. Their slight opacity and characteristic 
undulated margins serve to differentiate them from colonies of strepto- 
cocci and of diphtheroids, 
(2) Oxydase reaction: Flood a segment of the plate with 1 cc. of 1% 
aqueous solution of dimethyl paraphenylene diamine hydrochloride (Eastman 
Kodak Co.). (The McLeod program similarly uses 1% tetramethjtl parapheny- 
lene diamine hydrochloride, giving the colonies a bright purple color, is 
more expensive but has the advantage of a more rapid reaction and not 
killing the cocci in 30 minutes as does the dimethyl). Gonococcus colonies 
develop a pink color which on further oxidation becomes maroon and finally 
black, streptococcus and diphtheroid colonies fail to undergo this color 
change. Caution is indicated not to be mislead by a mere darkening of 
the surrounding media. Spreads made and stained from the oxydase-positive 
colonies must verify the tinctorial and morphological properties of the 
micro-organisms as this stain is not entirely specific for the Neisseria 
group, Medium sized, convex and translucent colonies which give the 
oxydase reaction may be accepted as gonococci if they consist of Gram- 
negative diplococci; in cases of doubt, i.e,, if appearance of colonies 
is not entirely characteristic or when the complete identification is of 
special importance, subcultures are made and the fermentation reactions 
and ability to grow on ordinary agar are determined (The dye does not 
interfere with the staining properties of the gonococcus though it does 
interfere with its cultivation if it has proceeded beyond the pink stage). NEISSERIA INTRACELLULAR!S 
(Meningococcus) 
Characteristicst Similar to the gonococcus, but found in different lo- 
cations and possessed with different invasiveness; distinguishable by 
serological tests. Divided into five types by serological behavior, 
types I and II and less cofamonly types III, IV and V; types I- and III,. .and 
II and IV, respectively, are very closely related,' Responsible for endemic 
and epidemic cerebrospinal meningitis in man; may be found in and isolated 
from infected spinal fluid, blood or nasopharyngeal Secretions of patients 
suffering with cerebrospinal meningitis and from the nasopharyneal secre- 
tions of carriers. Highly susceptible to inimical agencies; cocci die 
in less than 3 hours when dried and kept at room temperature; killed bj 
moist heat at 55° C. in less than five minutes; cultures die in a few 
days when kept at room temperature. 
'Identification: 
1. Macroscopic appearance of the spinal fluid is to be noted and reported, 
formal fluid is water-clear and colorless. Meningitis fluid is more or 
less turbid. Color, turbidity, blood and clot are to be noted. Blood, 
if fresh, may have come from the spinal puncture and make examination of 
the fluid difficult, 
2. Microscopic: An immediate presumptive diagnosis of meningococcic 
meningitis may be made by direct study of cerebrospinal fluid. 
a. Stained films of suspected final fluid: centrifuge the fluid, prepare 
spreads of the sediment on glass slides, fix and stain by Gram’s method. 
Examine for typical Gram-negative, coffee-bean shaped, intracellular 
diplococci. If present, they ’should be considered as meningococci and 
tentatively reported as such,- to be confirmed by culture and agglu- 
tination tests. ‘The presence of other organisms a»d ..the relative number 
'arid kind 65 tissue cells are also reported, • • • >. 1 
Cell counts of spinal fluid: make total and differential counts, , 
comparable tfb the counting of blood calls. The relative number of poly- 
morphonuclear and mononuclear leucocytes are to be noted - the former are 
usually enormously increased in cerebrospinal meningitis, , 
3. Culture of Sediment of Spinal Fluid. , 
a. Plant several loopfuls of sediment-on warm blood agar plate, 
b. Inoculate tube of warm serum dextrose broth with 1 cc< 
c. Incubate cultures at 37°C. for IB-24- hours and observe for the typical 
Gram-negative, coffee-bean shaped diplococci. Cultures are; generally . 
pure; if mixed, pure growth is to be obtained by subcultures on solid 
media (as for the gonococcus). Pure cultures are used for fermentation 
tests to rule out N. gonorrheeae. and for tube-agglutination tests. 
4. Culture of blood: This is not a routine procedure; the meningococcus 
may be recovered fr»m the blood by routine methods, in anomalous 
infections with septicaemia, with or without meningitis, 
5. Culture of nasopharynx: This is done for the detection of carriers 
only. The nasopharynx of convalescents and of potential carriers are 
touched with a sterile applicator or inoculating needle, and this inoculum 
is spread diffusely onto warm blood agar or chocolate agar plates; after 
incubation at 37° C,, suspect colonies are fished to warm serum dextrose 
broth for confirmation of identity. 6. Agglutination tests of pure cultures: A presumptive slide agglu- 
tination may hasten the procedure and cast out atypical organisms. A 
macroscopic tube-agglutination test with polyvalent meningococcic anti- 
serum is used for final proof of identity. Occasionally type determina- 
tion will be indicated. Most of the saprophytic Neisseria are salt or 
serum sensitive; to rule out non-specific clumping it is necessary, in all 
agglutination tests for meningococci, to run controls using normal horse 
serum (diluted 1:10) and saline. 
a. Presumptive test: Place a drop each of polyvalent antimeningococcic 
serum (1:10), normal horse serum (1:10) and sterile saline on separate 
areas of a slide; emulsify bacteria (portion of suspected colony) in each 
drop; observe for clumping of organisms. 
b. Macroscopic test tube agglutination test: Add 0,5 cc, amounts of each 
sera, diluted to -£■ of titer shown on vial, into labeled tubes; use separate 
tube for polyvalent and for each type antimeningococci serum (usually I & 
II only) to be tested; another tube receives 0,5 cc. of normal horse serum 
(diluted 1:10) and last tube receives 0,5 cc, saline; to each tube add 
0. cc, of suspension of cocci being tested; incubate overnight at 45- 
55°C. or for 2 hours at 37°C, end overnight in icebox. 
c. If the organism is a meningococcus, it should agglutinate in tube con- 
taining polyvalent and homologous type serum and not in other tubes. 
If clumping occurs in either control tube, the test is "unsatisfactory". 
7, Fermentation reactions; with material from a pure culture, inoculate 
tubes of serum water media containing 4 pivotal sugars (see chart) and 
incubate at 37°C, 
Neisseria catarrhalis 
Characteristics: Gram-negative diplococci; in sputum, the organisms are 
shaped like coffee-beans and may be both intra and extracellular; in 
cultures they are generally larger and are found in pairs and tetrads; 
grow freely, forming large colonies in 24 hours. They are normally found 
in the nose and throat; have meager pathogenicity; may be found, inci- 
dentally, in inflammatory secretions especially of respiratory area. A 
number of closely related Neisseria, clso found in respiratory area, are 
included on differentiation chart. 
Identification; 
1, Microscopic: Make gram-stained spreads of the infected secretions and 
examine for Gram-negative cocci. These organisms are larger than meningo- 
cocci * may not be arranged in pairs, may be intracellular, 
2, Culture: 
a. Inoculate plain agar, incubate at 22° C,; N, catarrhalis will grow, 
gonococcus and meningococcus will not, 
b. Pure culture is inoculated into sugar series in serum water media, 
(See chart for results) Differentiation of Various Species of Neisseria. 
Dextrose 
Maltose 
Levulose 
Sucrose 
r* 
i£ 
O 
u 
a 
u 
C3 
M 
22°C. Growth 
•H 
c § 
o 3 
r, 
■“ <3 
XJ w 
-p 
•H'H 
fS O 
N « 
• O 
MO 
MO 
bi 
, Special colony feature 
N. 
gonorrhoeas 
A 
- 
- 
- 
- 
- 
- 
Small, round, convex. 
N. 
intracellularis 
A 
A 
- 
- 
L 
k> 
- 
/ 
Small, round, bluish-grey 
K. 
catarrhal!s 
- 
- 
- 
- 
/ 
/ 
- 
Large, greyish-white. 
Hju 
sicca 
A 
A 
A 
A 
/ 
/ 
- 
Large, wrinkled, impossible to 
emulsify. 
I*. 
nerflava 
A 
A 
A 
A 
/ 
- 
Greenish yellow, adherent to 
medium. 
flava 
A 
A 
A 
- 
- 
- 
- 
Yellow 
1L. 
subflava 
A 
A 
- 
- 
i 
i 
- 
Greenish-yellow, adherent to 
medium. 
flavescens 
- 
- 
- 
- 
? 
? 
- 
Golden-yellow 
A indicates formation of acid. VIBRIO COMA 
Description* Slightly curved rods with rounded ends often resembling 
a comma; occur singly, in S-shaped pairs, short chains or spirals; ac- 
tively motile; grow readily aerobically on simple media at 37°C.; agar 
plate colony; 1-2 mm. diameter, greyish yellow, -translucent, low con- 
vex, with smooth or finely granular glistening surface and an entire 
edge, butyrous consistency; broth growth: abundant, with powdry deposit, 
thick surface pellicle. 
Identifying Characteristics; 
(a) Their power to grow on solid media which are so alkaline (pH 8.0 to 
8.1) that other organisms cannot develop. : 
(b) Their initial growth at surface of liquid media, while accompanying 
organisms grow throughout the liquid. . ; 
(c) Cholera red reaction / (also given by two saprophytic species) 
(d) Indol /, M.R. -, V.P, acid, no gas in glucose, levulose, galac- 
tose, maltose, mannite, and sucrose; lactose may become acid after 11 
days; litmus milk alkaline at top, slightly acid at bottom, not coagu- 
lated, slowly peptonized; nitrites produced from nitrates. 
(e) Gelatin stab growth: good filiform growth, confluent at top, dis- 
crete below, funnel-shaped liquefaction, with thick yellowish-brown 
pellicle on surface. 
(f) Agglutination with cholera'immune serum. 
Cholera-like VibriosThere are several classified and probably many 
unclassified vibrios isolated from feces or water arid differentiated on 
serological and biochemical characteristics. . „ , . 
Examination of Clinical Material; Vibrio comma may be isolated from the 
stools or intestinal contents of cases or carriers, from contaminated 
water .or foods and identified by microscopic, cultural and serological 
methods. 1 
Specimen Collection; 
(a) The ’’rice water” stool of cases or the feces of carriers are trans- 
mitted without the addition of glycerol or.other preservative, 
(b) Surface'water transmitted in a sterile liter flas.k. 
Microscopic: A presumptive diagnosis of suspected cases, not of carriers, 
may be quickly made by examining stained spreads of flakes of mucus from 
the ’’rice water” stool; stain by Gram’s method and with dilute-carbol- 
fuchsin; if Gran negative, comma-shaped organisms are present, examine 
a hanging drop preparation. Presumptive positive report may be made if 
large numbers of typical, actively motile, vibrios are found. This finding 
must then bo confirmed by cultural and serological examination. 
Cultural; Specimens of feces from suspected cases or carriers should be 
planted, using two or more loopfulls of intestinal mucus or liquid 
feces, with the least possible delay and incubated at 37°C. 
(a) Alkaline peptone water pH 8-8.1 (several tubes) 
(b) Alkaline nutrient agar pH 6-8.1 
(c) Dicudonne’s agar. Water under test is placed in 100 cc, amounts in sterile flasks, to each 
flask is added 10 cc, of peptone water. After 6-12 hours incubation 
at 37°C,, transfer a portion of the surface growth to the three media 
above. 
Rapid Presumptive Test 
After 6-8 hours at 37°C, examine hanging drop and stained film prepara- 
tions made from the surface growth of peptone water. If Gram-negative, 
motile vibrios in large numbers are noted, test as follows; 
(a) Microscopic Agglutination Test: Deposit near one end of a slide a 
drop of agglutinating serum of a dilution of 1:200 (titre not less than 
1:4-000) and near the other end a drop of saline; also place a third drop 
consisting of normal serum (diluted 1:10) near center of the slide as a 
control; then touch the suspected surface growth with point of the inoc- 
ulating needle, rub up in the drop of saline solution; flame the point, 
again touch the surface pellicle with the point and rub it in the drop 
of serum dilution; flame the point of the platinum needle again and add 
bacteria to the serum control in the same manner, agglutination will al- 
most instantly appear in the anticholera serum (if cholera); the drops 
may be allowed to dry, then fix and stain; if agglutination has taken 
place, it will be evident in the stained specimen to the naked eye or 
on slight magnification with the hand lens, 
(b) Cholera Red Test; A few drops of concentrated sulphuric acid are 
added to a 24- hour peptone water culture; a resulting red color depends 
upon the nitroso-indol reaction from the production of indol and the re- 
duction of nitrite in the peptone. 
Confirmatory Tests: If either of the presumptive tests are positive, 
obtain pure cultures for confirmation by selecting isolated colonies 
from plate media and transferring to: 
(a) Gelatin tube; to note characteristic type of liquefaction, 
(b) Alkaline peptone water: for cholera red test, 
(c) Agar slant for macroscopic tube-agglutination test, 
Pfeiffer1s Phenomenon: Small loop full from colony in 1 cc sterile saline 
mix with 1 cc cholira immun serum 1:1000 dil, inject I.P, into G,P. after 
20 min, withdraw peritoneal fluid 0 cap pipettes—if pos. non motile 
disintigrating vibrios can bo seen microscopically. Pasteurella 
(Hemorrhagic septicaemia group) 
Characteristics: Small Gram-negative rods showing bipolar staining. Aerobe, 
facultative anaerobe. Non-motile or motile. Frequently pathogenic, produc- 
ing characteristic hemorrhagic infections in man and animals. Includes: 
P. pestis - causing plague in man and rodents. 
P. tularensis - causing tularaemia in man 
and rodents. 
P. aviclda ) 
P. muricida )associated with fowl cholera or 
P. cuniculicida)hemorrhagic septicaemia of birds 
or lower animals. 
Pasteurella pestis 
Habitat: A parasite of rats and other rodents, causes plague in man. 
Transmitted by the bite of infected rat flea, or by contact or contamination 
with rodent, or human case or carrier. 
Characteristics: Short, thick bacillus; pleomorphic, especially in % salt 
agar; bipolar staining; grows readily on agar at 37°C. with raised, trans- 
lucent, grayish-yellow, glistening, viscid growth. 
May live for months in bodies of dead animals. Agglutinated by plague anti- 
serum. Infectious by inoculation for small animals; subcutaneous injection 
into guinea pigs provokes local oedema followed by inflammatory swelling 
of regional lymph nodes, and a generalized infection to death in 2 - 5 days; 
postmortem appearence: glands enlarged, surrounded by hemorrhagic exudate; 
small grayish, necrotic areas in liver and spleen; bacilli found in local 
lesions, bubo, internal organs, especially spleen, and blood. 
Collection of Specimens: 
II' Pus or gland fluid from bubos,aspirated by syringe or collected after 
incision (may be forwarded to distant laboratories on agar slants). 
2. Portions of affected tissues, removed at operation, to be forwarded in 
sterile bottles, 
3. Blood specimens, taken during period of sopticcmia. 
A. Autopsy materials, preferably bubo, lung, liver and spleen, 
5. Sputum, in .cases of pneumonic plague, 
6. Rodent: The whole rodent, shipped in fruit preserving jar, scaled. 
Microscopic Examination: 
1, Stain films from suspect materials by Gram’s method and methylene blue 
or dilute carbol-fuchsin (for bipolar staining), 
2, The presence of typical Gram-negative, short, ovoid, polar-staining 
bacilli, including many degenerated and poorly stained forms, is suggestive 
but not conclusive evidence of P. pestis infection. 
Culture: 
1, Inoculate surface of blood agar, glycerol agar and 3% NaCl agar plates. 
2, Plant blood specimen into nutrient broth and incubate before plating, 
3, Incubate cultures at 30 to 35°C. for 36 to AS hours. 
A. Observe growth and transfer to agar, broth, litmus milk, gelatin, tryp- 
tone broth, lead acetate medium and sorbitol broth for further study. (See 
chapter Classification of Bacteria”), Agglutination: (Macroscopic method preferred, to avoid the spontaneous 
clumping confusing the microscopic test) 
1. Make suspension of young agar culture in normal saline, using only 
the fine supernatant emulsion remaining after period of settling, 
2. High-titre agglutinating serum (horse) is generally used, 
3. Test is of greatest value in identifying suspect cultures, positive 
titre being interpreted in comparison with the titre of same serum tested 
with a known plague antigen, 
i, Test is of little value as applied to patient’s serum, for agglutinins 
do not appear in patients suffering from plague until about 9th day. 
5. Salt solution controls are necessary in all tests to detect auto-agglu- 
tination. 
Animal Inoculation: 
1. Caution: Animals should be freed of all ecto-parasites, prior to use, 
by dipping in an antiseptic solution. Then place in glass jars covered 
with fine mesh gauze to prevent access or escape of any parasites. When 
handling animals, living or dead, protect the hands and arms by wearing 
rubber gloves and long sleeved gown, 
2. Inoculate guinea pigs or mice subcutaneously with small amount of the 
original specimen or with a loepful of suspected culture. Putrefied mate- 
rials may be applied to the freshly shaved abdomen of a guinea pig (plague 
bacilli penetrate the abraded skin, contaminants do not). 
3. If P. pestis is present, the animals will develop characteristic lesions, 
die in 2 - 5 days with characteristic postmortem appearance; cultures of 
P. pestis may be isolated from the lesions. 
Diagnosis of Plague in Rodents: 
1. Postmortem appear .nee will usually evidence the natural infection in ro- 
dents. 
a. Bubo, with hemorrhagic spots and areas of gray necrosis. 
b. Subcutaneous and general congestion. 
c. Granular liver, with punctate hemorrhage and grey-yellow spots. 
d. Congested spleen. 
e. Pleural effusion. 
2. Bacilli may be found in bubo, liver, spleen and blood, and isolated from 
thence for study in pure culture by methods used for clinical materials. 
3. Shipment to a distant laboratory for examination: The entire carcass is 
placed, without any preservative, in a tightly sealed container, which is 
packed in a second container to avoid breakage and escape of contents. 
The package must bo shipped by express; federal laws prohibit the shipment 
of' plague-infected materials by mail. Decomposition may be avoided by 
surrounding the inner container with ice or "dry ice”. Label package 
"Perishable - for Bacteriological Examination - Please Expedite," 
Pastourella tularensis 
Characteristics: Small, Gram-negative, non-motile rods; pleomorphic, bacil- 
lary and cccpoid forms; stained best with carbol-fuchsin and crystal violet, 
show bipolar staining; fail to grow on ordinary media; aerobic; require 
specially enriched media for growth; an organism which grows on plain agar or in broth is not P. tularensis; growth on serum-glucose-cystine agar, 
2 to 5 days at 37°C.: minute, greyish-white colonies. Fairly susceptible 
to inimical agencies; killed by moist heat at 56 C. in 10 minutes. Ag- 
glutination tests of great value in diagnosis of disease by serum study, 
or in identification of cultures: agglutinins may persist for 20 years 
after recovery and a positive serum agglutination does not necessarily 
mean active infection. P. tularensis antiserum also agglutinates Brucella 
antigens to about of its titre. P. tularensis is the cause of "tulare- 
mia17, a plague-like infection of rodents, especially rabbits, and oc- 
casionally in man. Generally transmitted from rodents to man by infected 
blood-sucking insects, such as flies, ticks, lice, fleas and bedbug, or 
by direct handling of infected rabbits or squirrels. Accidental labora- 
tory infections occur due to its ability to invade unbroken skin. 
Microscopic Examination; 
Of value (l) to study morphology of organisms and (2) to rule out M. tu- 
berculosis by observing acid-fast stain of spreads made from pathological 
materials. 
Culture: 
1. Piece of infected tissue, pus, fluid or blood is planted on slants of 
glucose-cystine agar or blood cystine agar. Incubate at 37°C. for 3 to 
5 days. 
2. Blood agar plates also are planted to detect other organisms. 
3. Observe cystine slants for characteristic colonies. If negative, con- 
tinue observation for 21 days; if growth occurs, identify organism by 
stained spread, pure culture transplants and macroscopic agglutination 
tests with high titre immune serum. 
A. Cultures made from blood and lesions of man are usually unsatisfactory. 
Cultures should be made from heart’s blood, spleen, lymph nodes and liver 
of guinea pigs following inoculation with material from patient. 
Animal Inoculation: 
1. Inoculate guinea pigs, rabbits, or mice with suspected materials from 
glands, ulcers or blood: (a) subcutaneously and (b) rubbed on the recently 
shaven, abraded abdomen if other bacteria are present. 
2, Result: Death in 5 to 1C days (generally) with characteristic lesions': 
a. At site of inoculation, hemorrhagic oedema, no pus. 
b. Bubos, cervical, axillary or inguinal. 
c. Glands enlarged and filled with dry, yellow, caseous material. 
d. Spleen enlarged dark. 
e. Liver contains discrete, white caseous granules. 
f. Organisms can bo seen in spreads and be cultured from spleen, liver 
bubo and blood. Agglutination Reaction: Macroscopic tube method preferred, 
1, Set up agglutination tests of patient’s serum against P.tularensis 
and Brucella (abortus or melitensis) antigens. Incubate in water bath 
at 45 - 55°C. for 12 to 18 hours, 
2, Agglutination of P, tularensis by serum in dilutions of 1 to 80 or 
higher is considered diagnostic of tularemia, provided there is no cross 
agglutination with Brucella. Agglutinins appear in the patient’s blood 
after the first week of the disease and usually increase rapidly, 
3, Identity of a suspect culture may be established by a similar test 
using a suspension of the organisms and serial dilutions of a P.tularensis 
antiserum of known titre. The resultant agglutination to be significant, 
must be present in dilutions approaching the known titre of the serum. Genus Brucella 
Description: Minute rods vdth many coccid cells; 0.5 by 0.5 to 2.0 mic- 
rons; Gram-negative; do not show bipolar staining; all species patho- 
genic to man are non-motile; do not liquefy gelatin; and fail to ferment 
any carbohydrates. 
Habitat; Strict parasites, invading animal tissue, producing infection 
of the genital tract, the mammary gland or the lymphatic tissues and the 
intestinal tract. Br. melitensis, Br. abortus and Br. suis, primarily in- 
fect goats, cows and hogs, respectively, causing abortion and systemic 
infection; infectious to other domesticated animals; may infect man 
causing undulant fever (brucellosis). The motile species, Br. bronchi- 
septica causes distemper in dogs; also causes acute infection in other 
animals; and rarely infects man. 
Br. melitensis, Br* abortus and Br. suis. 
Description; Gram-negative, non-motile coccobacilli as for genus; Br. 
melitensis and Br. suis grow aerobically, Br. abortus requires 
for initial isolation and early culture transplants;growth on all media 
is slow, grows best on glucose liver infusion agar with pH 6.6: U8 hour 
colonies on plate are small, circular, convex, amorphous, smooth, glis- 
tening and entire; agar cultures turn media brownish after 7 days. The 
three species are very closely related; may be separated with difficulty 
on basis of (l) CO? requirement for growth, (2) growth on media contain- 
ing certain dyes, (3) H2S production and (It) agglutinin absorption tests. 
Habitat: Found in blood, urine, feces, exudates and occasionally sputum 
and nasal drainage of human cases; also in milk, cheese and other dairy 
products from unpasteurized milk from infected animals. 
Laboratory examination of clinical material; 
1. Microscopic.- Indistinguishable morphologically. However, Gram-stained 
smears from pathological lesions should be examined for the small Gram- 
negative rods described above. 
2. Cultural.-While the organisms may be found in the blood early in the 
disease and during febrile periods and in urine and milk specimens at 
irregular intervals, the percentage of positive cultures, even from 
proved cases, is low. 
a. Obtain specimen consisting of 10-12 cc. of blood or £0 cc. of 
urine or milk. Other body foci such as contents of ovarian cyst, synovial 
fluid, or excised glands may also be subjected to cultural study. 
b. Inoculate two flasks containing 100 cc. of veal infusion broth 
pH E.6 with 5 cc. of blood, several loopfulls of sediment from cath- 
eterized urine specimen, or several loopfulls of sediment and of cream 
layer from milk. Also streak specimen on two infusion agar plates, 
c. Incubate one set of media in incubator 37°C. for growth of Br. meli- 
tensis and Br. suis; place other set of media in jar containing 10$ CO2 
and incubate at Br. abortus. d. Examine plates and Gram-stained films from broth after 24-48 hours 
and at frequent intervals thereafter for growth. Streak new plates from 
broth at least once per week, even if no evidence of growth is discernible. 
Observe cultures for at least 4 weeks before reporting as negative. 
e. Identify any positive culture as belonging to this group by agglutina- 
tion with antisera prepared against either Br. abortus. Br. melitensis or 
Br. suis. 
Note; Although not usually required the species of young cultures can be 
determine by (1) agglutinin absorption tests, (2) tests for production, 
and (3) ability of the organism to grow on media containing certain dyes 
(basic fuchsin and thionin). 
Table. - Differential characters of the three related species of 
Genus Brucella, 
• 
• 
: 
10$ CC0. required 
• 
• 
HpS * 
Growth 
on media 
• 
• 
i 
for Primary 
: 
formation1 
containing: 
♦ 
• 
• 
t 
isolation 
• 
(days). 5 
Thionin : 
Basic fuchsin 
• 
! Br. 
melitensis 
♦ 
• 
• 
• 
0 
• 
• 
: 
n : 
/// : 
/// 
I Br. 
abortus 
: 
; 
// 
• 
• 
♦ 
t 
• 
• 
2 . 
o ; 
/// 
! Br. 
suis 
• 
t 
0 
t 
• 
• 
k : 
• 
/// I 
* 0 
• 
• 
• 
• 
3* animal inoculations. - Br. melitensis and Br. suis, and less constantly 
Br. abortus, may be isolateTTr materially subcutaneous inocu- 
lation into guinea pigs (preferably males); after 4 weeks, kill the animal; 
examine Gram-stained smears from the lymph glands, spleen and liver; and 
make cultures from the liver, spleen, blood and lymph.nodes. This test is 
seldom used because of the groat danger of laboratory infection* 
4. Serological. - ... V' ‘ 
a. Identification of pure cultures: 
There is complete cross agglutination to titer between an antigen pre- 
pared with either species and antisera prepared against any other species. 
However, Br. abortus and Br. suis can be differentiated from Br. melitensis. 
but not from each other, by agglutinin absorption tests, 
b. Scrum from a patient taken after the fifth day of disease will usually 
contain agglutins. Set up macroscopic agglutination tests in dilution of 
l/20 to 1/640 or higher against a Brucella antigen (abortus, melitensis or 
suis) and against Pastcurclla tularonsis antigen. Agglutination of Brucella 
antigen in dilution of l/lCO or higher is considered to be significant. 
Cross agglutination in serum from patients with Brucellosis or tularemia is 
frequently present, but is loss marked with the heterologous antigen. Ag- 
glutinins may persist for years after recovery. This is the most valuable 
test for diagnosing Brucella infections and is the only one routinely used. Genus Hemophilus 
Characteristics: Minute rods, sometimes almost,coccoid, sometimes thread- 
like and pleomorphic; Gram-negative; not acid-fast; non-motile, non-spor- 
ing, non-encapsulated. Strict parasites, do not grow on common media, 
require for their cultivation accessory substances present in the blood 
and fresh vegetable tissue. H. influenzae. H. ducreyi and H. pertussis 
are the three most important species. 
Hemophilus influenzae 
Characteristics: Very small, short rod; stains faintly, best by dilute 
carbol-fuchsin or Giemsa stain; grows best on media containing hemoglobin, 
subcultures, on plain or serum agar fail- to grow; chocolate agar plate I 
colony, 24 hours at 37°C,: small, pinpoint, transparent, smooth, raised; 
tendency to grow best near colony of other aerobic organism, i.e., "Sat- 
ellite” colonies; not subject to agglutination test; does not have 
conspicuous biochemical activities. 
Habitat: Commonly found in cultures of upper respiratory tract, their sig- 
nificance there questionable; probably not as name implies, related to the 
disease influenza. Occasionally found in pathological spinal fluids. 
”Koch-Weeks” bacillus, formerly called H. con.iunctivitidis. found in eye 
fluids in acute infectious conjunctivitis ("Pink eye"), is now classified 
as H. influenzae. 
Identification; 
1. Koch-Weeks bacillus; 
(a) Make slide spread from conjunctiva, stain by Gram’s method and with 
dilute carbol-fuchsin, 
(b) Observe for minute Gram-negative bacilli; often intracellular, 
(c) Culture is not informative except to reveal other organisms. 
2, Spinal fluid, respiratory tract and other suspect materials: 
(a) Make culture ©n chocolate agar, incubate at 37°C, for 2 days. 
(b) Suspect colonies are identified on colony appearance, microscopic 
morphology of organisms and failure of subcultures to grow on plain agar. 
Colony may be, confused with a streptococcus colony, 
(c) Specific identification considers source of the specimen, hemolytic 
properties and requirements of accessory growth factors. 
Hemophilus pertussis 
Characteristics: Like H. influenzae except bacilli are more unifotm in^size, 
with less pleomorphisra, ferment no carbohydrates and do not require 
accessory factors for growth, but cannot be distinguished on.morphology 
alone; colony on potato-glycerin-blood medium (pH 5.0) at 37dC, barely 
visible in 24 hours, plainly visible after 48-72 hpprs as small, .greyish; 
raised, pearl-like growth; after several generations growth is freer, 
glistening, becoming in a few days heavy, almost like the growth of typhoid 
bacilli; then transplants will grow on plain agar. 
Habitat: Constantly present in the respiratory secretions of whooping cough. 
-1L2- Identification: Cough plate method for isolating H. pertussis is preferable 
to sputum cultures; Open Petri dish, containing potato-glycerin-blood 
medium, is held in front of the mouth during a cough paroxysm* The 
organisms, sprayed on the plate with droplets of secretion appear in' 
colonies after 37°C. for /S' hours. Colonies are larger, more opaque and 
whiter than those of H. influenza. 
Hemophilus duplex 
(Morax-Axerfeld bacillus) 
Characteristics: Short stumpy, moderate size bacillus, often in diplo- 
form and chains. Cultivated only on media containing blood, serum or 
ascitic fluid. On Loeffler's blood slant colonies appear after 24-36 
hours at 37°c. as small identations which indicate a liquefaction of the 
medium. 
Habitat: Found in eye in subacute infectious conjunctivitis. Not patho- 
genic for animals. 
Identification; 
1. Prepare slide spreads from conjunctival sac, stain with dilute carbol- 
fuchsin or Gram stain. 
2. Short, stumpy bacilli in direct spreads are presumptively Morax-Axenfeld 
bacilli. 
3. Culture on Loeffler's blood slant or other special media may confirm. 
Hemophilus ducreyi 
Characteristics: Very small ovoid rod, non-motile, tendency to be in 
short chains and parallel rows; Gram-negative; tendency to be more deeply 
stained at the poles. In pus, the bacilli are often found within leu- 
cocytes. Difficult to cultivate; coagulated blood which has been kept for 
several days in sterile tubes (fresh blood will not do unless heated to 55°C 
for 15 minutes) has been found to be a favorable medium. 
Habitat: The cause of chancroid, the soft chancre, are found in the pus 
of ulcerating chancroidal ulcers, mixed with secondary infection, and In f 
purer state in the chancroidal bubo. Not inoculable to lower animals. 
Identification: 
1, Examination of spreads or cultures for H. ducreyi is seldom practiced 
because of the technical difficulties of identification and the fact that 
chancroid lesions are usually distinguishable as such without laboratory 
confirmation. 
2. Direct diagnostic cultivation from chancroidal lesion: 
(a) Media: 1 cc. of sterile rabbit blood (freshly drawn) is placed in 
each of several small tubes, allowed to clot, then heated to 55°C. for 15 
minutes and kept in icebox until used. 
(b) Thoroughly cleanse lesion with sterile water or salt solution. 
(c) Scrape material from bottom of ulcer or from beneath its edges, 
with a stiff platinum loop and plant in a tube of clotted blood by passing 
the wire around the clot. 
143 (d) After 37°C, for 24- hours, the serum around the clot is stirred 
with the platinum loop and a spread is made and examined by Gram method. 
(e) Characteristic chains of Gram-negative bacilli, sometimes in 
pure, sometimes in mixed culture, will sufficiently identify the organism. 
(f) Transfer onto soft moist blood agar of pH 7.2 may give in 4-8 hours 
pinhead size, transparent, grey colonies with a firm, finely granular 
consistency, 
3. Culture from unruptured bubo; pus is withdrawn by aspiration with a 
sterile hypodermic syringe and needle; cultured as above. 
144 GRAM-KEGATIVE. AEROBIC; KGN-SP03E-F0RKING ENTERIC BACILLI 
(FAMILY ENTERG3ACTEPJ ACEAE) 
Characteristics: Gran-negative rods, widely distributed in nature. Grow 
aerobically. Many species are parasitic for man, several of which cause 
typical diseasej other species are saprophytes, or parasites on plants 
and animals. Grow well on ordinary culture media. All species attack cer 
tain carbohydrates forming acid, or acid and visible gas. May be motile 
or non-motile. Non-spore forming. Has been divided into five tribes, 
only three of which (Bschericheae, Proteae and Salmonelleae) contain 
species of interest in Medical Bacteriology, All of these bacteria are 
morphologically similar. They have many other characteristics in common, 
and serological as well as cultural methods may be required to definitely 
identify a member of the group. 
THE COLI-AEROGSMES GROUP 
*XTribe Eschorichecie) 
Characteristics? Motile or non-motile rods, commonly occurring in the 
intestinal canal of normal animals, in the respiratory tract of man, or 
widely distributed in nature. All ferment dextrose and lactose with the 
formation of acid and visible gas. Do not liquefy gelatin except slowly 
by one species (Aerbacter cloacae), Separated into throe genera on 
basis of results of methyl red test, Voges-Proskauer test, and ability 
to utilize citric acid as sole source of carbon. See table below. 
Genus and species 
Methyl 
red 
test 
Vogcs- i sCitrate 
Proskauer sIndol:utili- 
test stest szation 
sGelatin 
slique- 
:faction 
:h2s 
formed 
Escherichia coli 
/ 
. 
/ 
mm 
• 
« 
• 
E. freundii 
/ 
(/) 
/ 
• 
• _ 
• 
/ 
Aerobacter aerogencs 
- 
/ 
(-) 
/ 
• 
• 
t • 
5 
J / 
(-) 
A. cloacae 
- 
/ 
- 
/ 
(-) 
Klebsiella pneumoniae 
(/) 
(-) 
- 
(/). 
• 
• 
• 
- 
Notes Some species give variable results; 
reaction, 
* Page 570 Simmons Manual. 
(/) or (-) indicates 
usual 
Escherichia Coli 
Characteristics: 'Cdccoid to long rods, occurring singly, in pairs and 
long chains. Gram-negative, Motile or non-motilc. Not usually capsu- 
latcd. Ferments many carbohydrates, including dextrose and lactose, with 
formation of acid and gas. The large number of species formerly identi- 
fied on basis of motility and carbohydrate fermentation are now Included 
within this species as varieties. 
145 Habitat: Occurs in normal intestinal tract of animals; frequently found 
in soil and water, as a result of fecal contamination. Sometimes ac- 
quires pathogenic power and may cause local or general infectionsi fre- 
quently causes infections of the genito-urinary tract; invades the cir- 
culation in agonal stages of diseases. . 
Identification; 1. For isolating E. coli from water and sewage see section L 
on "Bacteriological Examination of Water," ’ 
2. For E. coli in feces, urine, etc,, follow the procedure 
outlined under examination of feces for E. typhosa and identify according 
to the reactions in chart above, 
Aerbacter aerogenes 
Characteristics; Short rods with rounded ends, usually shorter and plumper 
than E. coli. They are aerobic, Gram-negative, non-spore-forming and fre- 
quently capsulated. Ferment many carbohydrates, including dextrose, lac- 
tose and glycerol, with formation of acid and gas. Do not liquefy gelatin. 
Colonies on solid media are large and very viscid. 
Habitat: Widely distributed in nature; normally found on grains and plants, 
sometimes found in the intestinal canal of man and animals. It has been 
reported as the cause of cystitis. v , ■ • 
Identification: 1, See section on "Bacteriological Examination of Water" 
for method of isolating A. aerogenes from water. 
' 2. Isolate organism from feces, food, and soil by plating 
on eosin methylene blue agar or other media asVdescribcd under E. typhosa 
and identify by characteristic biochemical reactions shown in. above table, 
Klebsiella pneumonia j . 
- v — 
Characteristics: Short, plump, ncn-motilc. Gram-negative-rods; aerobic, 
growing well on ordinary media; produces a large, mucoid colony on solid 
media. It has a large‘capsule’which can be demonstrated readily in spreads 
from sputum, animal exudates and other pathological material. Ferments 
dextrose, lovulose, galactose,"saccharose and usually lactose with pro-. . 
duction of-acid and gas. 
Habitat: Common commensal in respiratory tract; occasionally found in soil, 
dust and water. Associated with pneumonia and other inflammations of the 
respiratory tract. Occasionally found in various suppurative lesions of 
the body, and may give rise to septicemia. 
Identification? 1. Examine stained spreads from pus,„ sputum, or fluid from 
lesions for Gram-negative encapsulated,bacilli, 
- v 2. Inoculate' eosin methylene blue agar plates or other me- 
dia, Examine for mucoid colonies consisting of bacilli, with typical morpho- 
logy. Identify suspected colonies through cultural and biochemical tests. 
146 3. Blood culture ma3r be made by usual methods; iden- 
tify any suspect colonies as above. ' 
Genus Proteus. : 
Characteristics: (The only genus in tribe Froteae.) 
pleomorphic, Gram-negative rods; filamentous and curved rods, and invo- 
lution forms are common. Generally actively notile. Characteristically 
produce ameboid colonies on moist media and decompose proteins; gelatin 
is rapidly liquefied by most species. Ferment dextrose and generally 
sucrose, but not lactose, with formation of acid and small amount of gas. 
Usually Voges-Proskauer test is negative. Genus consists of 8 species; 
type species is Proteus vulgaris. : 
Habitat: Putrefying animal and vegetable materials; found in feces, soil 
and gunshot wounds. Certain Proteus strains, identified as X19, X2 and X 
Kingsbury, originally isolated from typhus fever cases, are used as anti- 
gens in the Weil-Felix test (sec section on Rickettsiae). One species, 
P. morgani. has been reported as the cause of mild enteritis. 
Identification: 1. Most laboratories roughly identify ary Gram-negative, 
motile bacillus that produces an ameboid colony on moist agar at 37CC. 
as belonging to the Proteus group and do not classify further. 
2. However, one species of the genus, Proteus morgani, 
produces the typical ameboid colony only when grown on agar at 21-2S°C. 
Isolate pure cultures of this organism as described under E. typhosa, 
and identify on basis of fermentation of dextrose and other hexoses only, 
with formation of acid and slight amount of gas. 
TYPHOID-DYSENTERY AND PARATYPHOID-ENTERITIS GROUPS. 
TtrIBE" SAlMOiIEYIjEAE') " 
Characteristics; Motile or non-motile, Gram-negative rods; grow aerobical- 
ly; non-spore-forming; Vogos-Proskaucr test negative; gelatin not lique- 
fied; and no spreading growth. Attack many carbohydrates with formation 
of acid, or acid and gas. Certain species of genus Shigella and .genus 
Eberthella attack lactose with gas formation only. Tribe consists of 
three genera; genus Salmonella organisms ferment dextrose with the forma- 
tion of acid and usually gas; genus Eberthella and genus Shigella or- 
ganisms ferment dextrose with formation of acid, but no gas, Eberthella 
being motile and Shigella non-motile, 
GENUS SALMONELLA 
The organisms of this genus are defined as: usually motile, but 
non-motile forms occur. Attack numerous carbohydrates with the formation 
of acid and usually gas; lactose, saccharose and salicin am never at- 
tacked. Do not form indol or liquefy gelatin. Differ from coli-aerogenes 
group in failing to ferment lactose; and from typhoid-dysentery group in 
forming gas from dextrose. Can bo separated into 37 species, several of 
which arc pathogenic for man, causing a typhoid-like fever, food poisoning, 
or an acute gastro-onteritis. All species pathogenic for man are motile. 
147 Important Species: 1, S. paratyphi. the cause of paratyphoid 
A fever in man. Characteristic reactions: never ferments xylose, rare- 
ly able to produce H2S, and fails to utilize citrate and d-tartrate. 
2. S» schottmuellcri, the cause of paratyphoid B fever in man. 
Characteristic reactions: ferments xylose and usually attacks inositol; 
formed; citrate /; and tartrate usually -. 
3. S. typhimurium, a natural pathogen of rodents, especially mice, and 
many other animals; causes food poisoning in man. 
Characteristic reactions: very diffucult to distinguish from S.schott- 
muellcri. by means of either biochemical or serological reactions; most 
reliable tests for separating them being, (a) S. typhimurium is usually 
tartrate /, and (b) agglutination reactions with ,fHn antigens of organ- 
isms in the specific phase, 
A. S. enteritidis, and its varieties, are widely distributed among 
animals; sometimes the cause of food poisoning in man. 
Characteristic reactions: ferments xylose, but never attacks inositol; 
H2S /; citrate / and tartrate /. 
5. S. hirschfeldii. the cause of a typhoid-like fever in man, sometimes 
referred to as paratyphoid C, bacillus; found principally in Europe. 
Characteristic reactions: biochemical reactions similar to those of S. 
enteritidis; serologically, closely related to S. choleraesuis. 
6. S. choleraesuis. two varieties, causing American and European hog 
cholera, respectively; occasionally infect man. 
Characteristic reaction? fails to ferment arabinose, a carbohydrate at- 
tacked by other Salmonella. 
Identifications 1. Isolate paratyphoid.fever group from feces, urine or 
blood as described under E. typhosa. 
2. Food poisoning group. Isolate pure cultures from 
fcccs or food. (See section on Food Poisoning), 
3. Identify pure cultures by means of carbohydrate 
fermentations and other biochemical tests (see section- on Classification 
of Bacteria) and by agglutination reactions. 
A. The Salmonella group, including E. typhosa is very 
complex, serologically. Each species possesses from one to three dis- 
tinct antigenic components in the body of the bacillus (MCH antigens) 
and other distinct components in the flagella (MHlf antigens), the lat- 
ter occurring in many species in two alternate phases, the specific 
phase and the group phase, each possessing different antigens,, The 
same antigenic components may be found in several different species, 
in various combinations. However, most strains of the pathogenic species 
listed above can be definitely classified on basis of (a) source of speci- 
men, (b) biochemical reactions and (c) series of agglutination tests. 
148 GE|TJ_Sj:3ERTIISLU 
The organisms of this genus are defined as Gram-negative, mo- 
tile rods, generally occurring in the intestinal canal of man, usually 
in different forms of enteric inflammation. Attack dextrose and several 
other carbohydrates with the formation of acid, but no gas; certain non- 
pathogenic species may attack lactose, saccharose and/or salicin with 
formation of acid, but no gas, E. typhosa is the only species regularly 
pathogenic for man, 
Eberthella typhosa. 
Characteristics: Actively motile, Gram-negative rods, possessing the 
general features of the tribe and genus. Never attack lactose, saccha- 
rose or salicin. The normal smooth motile form has one somatic and one 
flagellar antigen, thus producing both H and 0 agglutinins; non-motile 
variants are rare; the somatic antigens are related to those of 
Salmonella enteritidis and a number of other species of Salmonella, 
Colonies on plain agar, after 2U hours incubation at 37°C., are smooth, 
round, domed, greyish in color, transparent to opaque, with entire edge; 
after cultivation on artificial media, rough type variants may develop. 
See table in section on ’’Classification of Bacteria” for biochemical 
characteristics and table in this section for reaction on Russell’s 
double sugar tubes, and for type colonies on differential plate media. 
Habitat: Found in feces and blood, and occasionally in bile and urine, 
of patients ill with typhoid fever of which it is the causative agent; 
also present in feces, urine and bile of carriers. 
Laboratory Examination of Specimen 
The Specimens to be examined will usually consist of blood, 
feces, urine or .bile of suspected cases of typhoid or paratyphoid fever 
and of bile, feces and urine of carriers for cultural study; also, of 
serum from patients for agglutination (’.Tidal) test. 
1. Microscopic examination. - This is of no value, 
2, Culture - a. Feces, urine, bile, etc.; 
(1) Spread the material, suspended in broth or saline if solid feces, 
over the dry surface of eosin-mothylene blue agar, Lcifson’s dexoxycho- 
late-citrate or other special differential media in Petri dishes, in 
such a manner as to insure the grov/th of well isoldatod colonics. Also, 
inoculate specimen into tube of selinito broth or bile broth, 
(2) Incubate IS-24 hours at 37°C, 
(3) Study the plate cultures carefully,.select several well isolated 
colonies of the type desired (see table) and from each inoculate 
Russell's double sugar (RDS) tube and plain agar slant, 
'.kA) After 2U hours incubation examine cultures for typo reaction on R.D.S. 
motility and Gram-staining properties. (5) Identify any suspected pure culture (a) by inoculating various car- 
bohydrate media and media for the other biochemical tests, and (b) by 
setting up macroscopic agglutination tests against known type antisera 
(E, typhosa. S. paratyphi. and S. schottmuelleri; other antisera may be 
used, if indicated), 
(6) If at end of 2U hours plate cultures show no colonies of the type 
produced by pathogenic organisms, streak new set of plates from the 
broth culture and reincubate old plates for an additional 2L> hours be- 
fore discarding as negative. 
b. Bloods Blood for culturing should be taken early in the di- 
sease, preferably during the first week. 
(1) Obtain 10-15 cc. of citrated or defibrinated blood; whole blood can 
be used for immediate inoculation of media at bedside. 
(2) Inoculate (a) flask containing 100 cc. of bile broth, 1$ dextrose in- 
fusion broth or brilliant green broth with 2 to 5 cc. of blood (b) two 
agar pour plates with 1,0 cc. of blood each and (c) streak 2 or 3 loop- 
fuls of blood on eosin methylene blue agar plate. 
(3) Incubate at 37°C. and make daily transfers to blood agar and E.H.B. 
agar plates. If colonies develop, transfer to Russell’s double sugar 
and identify by the procedure outlined above, 
3. Serological examination. - a. A macroscopic tube-agglutina- 
tion test, as indicated above, should be used to confirm the identity of 
an organism isolated from cultures; use suspension of suspected culture as 
antigen along with known type antisera, 
b. Widal test: After the first or second week, demonstrable anti- 
bodies including agglutinins, develop in the blood of patients with an 
enteric fever. These may be demonstrated by Widal test. This test con- 
sists of a macroscopic (preferred) or microscopic agglutination test, 
Using the patients’ serum, and stock E. typhoaa ”Hn, E. typhosa ”0”, 
S. paratyphi and S. schottmuelleri antigens. 
Genus Shigella 
Characteristics: Snail, Gram-negative, non-motile rods. Attack a num- 
ber of carbohydrates with formation of acid but no gas. 
Habitat: Several species are pathogenic for man, causing bacillary dy- 
sentery; other species nay be found in the ncrnr.l .human intestinal tract; 
several species are pathogens of fowls and ether small animals. 
The Dysentery Group 
1, Shigella dysenteries (Shiga): A cause of dysentery in man and mon- 
keys* Produces acid but no gas from dextrose, levulose and a few other 
carbohydrates. Never attacks mannitol, maltose, lactose or sucrose, In- 
dol not formed. Serologically homogenous and different from the other 
species of Shigella. 2. Shigella sp. (Newcastle type): A cause of human dysentery. In 
peptone water solution, dextrose, maltose and occasionally dulcitol are 
fermented with acid production; lactose, mannitol and saccharose usually 
not fermented. Peculiarities of the organism are (1) occasionally a 
slight bubble of gas is produced from dextrose and dulcitol, (2) when 
dissolved in beef extract broth, dextrose, dulcitol and maltose are al- 
ways fermented to acid and gas. Indol not formed. Serologically homo- 
genous and not agglutinated by antisera prepared against S. dyseriteriae 
or S. paradysenteriae. 
3. Shigella paradysenteriae s' A cause of dysentery in man, and of summer 
diarrhoea in children. Produces acid but no gas from dextrose and manni- 
tol; Some strains attack maltose or saccharose; dulcitol and lactose never 
fermented. , Indol formation is variable. Has been divided into five races 
(l!Vl!, ”Wn, nIn, nY” and !,ZM) by agglutination tests based upon the pre- 
ponderance of one or another of four antigenic components, V, W, X and 
Z; considerable cross agglutination between races; seroligically dis- 
tinct from 3. dysenteriae and New castle*s bacillus; slight cross agglu-, 
tination with S. sonnei. S. alkalescens and S. madampensis. 
U. Shigella alkalescens: * Isolated from human feces and intestines; 
pathogenicity doubtful. Ferments dextrose, mannitol, maltose, dulci- 
tol and sometimes saccharose; never attacks lactose. The most character- 
istic reaction is an initial and lasting, intense alkalinity produced 
in litmus milk. Serologically homogenous and distinct except minor 
cross agglutination with S. sonnei, S. paradysenteriae and S. madam- 
pensis. 
5. Shigella sonnei: A cause of mild,dysentery in man, or of summer 
diarrhoea in children. Ferments dextrose, mannitol, maltose, lactose, 
saccharose and several other carbohydrates with formation of acid, but 
no gas; dulcitol is never, and'xylose seldom, attacked; fermentation • 
of substances other than the monosaccharides may require days or weeks. 
Indol not formed. Serologically divisible into two types; some cross 
agglutination with S. .paradysenteriae, S. alkalescens and S. madamponsis. 
6. Shigella madampensis (S. dispar): Isolated from human feces, apparent- 
ly not pathogenic. Fermentation reactions similar to those of S. sonnei 
Indol is formed, Antigenically heterogeneous; nay show slight cross ag- 
glutination with S. paradysenteriae. S. alkalescens and S. sonnei. 
Laboratory Examination of Specimens 
1. Microscopic examination: In bacillary dysentery, especially 
in infections with S. dysenteriae (Shiga), an early presumptive diagnosis 
can usually be made by direct microscopic examination of fresh fecal dis- 
charges. a. Select portions of a very fresh specimen' containing 
bits of mucus, bloody feces or shreds of the exudate. Prepare (1) 
thin films on slide and (2) cover slip preparations, both unstained 
and stained with Loeffler’s methylene blue or 1% aqueous solution 
of brilliant cresyl blue, in order to study the cells present. 
b. If the disease is the bacillary type of dysentery, 
microscopic examination will show blood in varying amounts, but 
usually abundant early in the disease; polymorphonuclear neutro- 
philes form about 90* of the exudate, and many of these show nuclear 
dogeneration (ringing), while the cytoplasm frequently contains fat; 
endothelial macrophages, which are present in varying numbers are ac- 
tively phagocytic and frequently contain engulfed bacteria, erythro- 
cytes and leukocytes; these under go degernation and form ’’ghost 
cells”; plasma colls are present and are more abundant early in 
the disease; bacterial content is scanty. 
c. For characteristic findings in amoebic dysentery 
stools, see section on Protozoology. 
2. Cultural examination: Shigella may be isolated from the feces of 
patieiits and carriers by the methods Indicated under Eborthella. How- 
ever, both eosin methylene blue agar plates and desoxycholate-citrus 
agar plates should be inoculated,'routinely, since the latter is an 
especially favorable culture medium for Shigella. 
i . 
3. Serological examinationi a. The suspected organisms may be 
identified by agglutination tests using polyvalent and species specific 
antisera; S. dysenteriac. S* S. sonnej and polyvalent 
(Shiga, Sonne and paradysentery) antidysenteric antisera are generally 
used. 
b. Agglutination tests, using serum from patient 
against known antigens, are of limited value. Genus Shigella 
(Dysentery) 
GENERAL CHARACTER IST1C1 GRAM NEG,, NONSPCREFORMING, AEROBIC, FOUND IN INTESTINAL TRACT, All FERMENT CERTAIN CARBOHYDRATES V'lTH FORMATION OF 
ACID AND SOME \?1TH GAS. MAY BE MOTILE OR NON MOTILE, 
! 
IS-1.6ESALMQNEJLLEA 
Gelatin not IIquified 
Lactose not fermented 
Intestinal pathogen group* 
Colonies colorless on 
E*M*Bo Plates 
Genus Eberthella 
(Typhoid Fever) 
Genus Salmonella 
(paratyphoid Fever) 
TRIBE ESCHERICHEAE 
Gelatin not liquified (one exception) 
Alt fermeilt Dextrose and Lactose 
with formation of Acid and Gas* 
Mo Ro or V0 Po positive 
Motile or non mot{le 
Generally not pathogenic to 
intestinal tract* 
Colonies colored on E©M0Be Plates 
I 
Genus Kjebs 11 a 
Ko pneumoniia_ 
M.R +■ 
V oPa ° 
Indol " 
Citrate + 
Gelatin ° 
H2S =» 
Genus Aerobacter 
A* acrog^enes 
M.R, ~ 
V*P, + 
Indol « 
C i trate f 
Gelatin ° 
h2s ° 
Aq chloacae 
M*R rs =* 
V,Po 4 
Indol ■= 
Citrate + 
Gelatin* 
h2s - 
TRIBE PROTEAE (only one 
Gelatin liquified 
Ferment dextrose and gen* Sucrose, 
but not Lactose, with formation Ac 
and small amt* gas* 
Actively motile, amoeboid colonies 
on mo|st media- 
Generally not pathogenic, one species 
P0 raorganl causes mild enteritis 
Colonies colorless on E-M*6* Plates 
Type Species «=> Proteus vulgaris 
Genu s _E sc he r ec {a 
E» coU 
M.R. + 
V,P* ° 
Jndol + 
Citrate «=■ 
Gelatin « 
h2s - 
£n fruendj 
f. - -o + 
V*P* = 
Indol -r 
CItrate + 
Gelatin ° 
h2s + Dulcitol Ac 
S h c ey I Is 
fndol f 
Sh Ijj, ffiadampcns i s 
I 
Lactose Ac 
(slowly) 
I 
Genus.ShigelU 
(Dextrose A no G, Motility 
JV,P„ - 
h2s ~ 
Mannitol Ac 
1 
Dextrin Ac 
Sh ijg.a£c I Una rum 
Xylose Ac 
Lactose- 
> 
Dulcitol 
Sucrose Ac 
ft 
Sh I g,paradysent-» 
erlae (r|.-xner) 
Indol * 
Dextrin <=> 
Sh i g j a 1 k a see n a 
Dulcitol Ac 
Indol 4 
» 
Indol «■ 
sonnei 
Maltose H* 
Sucrose Ac 
E , oedemations 
dcTeTi n iTqu i f a 
Lactose Ac 
Xyfose- 
Milk coagulated 
Shi o.,roi nut i ssima 
S.U.Tt'4.T -j-.r_-. -a 
TRIBE SALMONELLEAE 
(Qen~Charact,^Graraa neg, rods non spore forming, 
Lactose not fermented. 
Gelatin not liquified) 
LJLsj: 
Mannitol =» 
! 
Shj tjiua 
S hi _ jsep ticaeffl i a c 
I 
Sucrose ~ 
h2s r 
jEo tjypjiosa 
Ac from; 
Dextrose 
Xylose 
Maltose 
Raff loose 
Dextrin 
Mannitol 
Sorb!to! 
♦- 
HgS", Rhamnose •*= 
E Sends} 
fndol + 
f 
Genus Eberthel la 
(Dextrose A no G, Motility *) 
f 
Milk net coagulated 
I 
fndol •-* 
Shi dysep l- 
Sh<g.* "astjje 
Maltose « 
£-.pui loruai 
Lit.MlIk 
Ac * A Ik 
H^S 
AG from: 
Dextrose 
Arablnose 
Xylose 
siann \ to I 
Ir.osltol o 
f„enter|t}dis A S^hirschfcldil 
c fFfTtT** -*=~~— “ 
Tartrate ««• 
Qengs Sajmg.nelta 
(Dextrose AG, do not 
ferment Lactose, Sucrose and Salicin) 
Xylose 
?| Ac 
H2S sg 
AG from: 
Dextrose 
Arab!nose 
tfattese 
Dextrin 
hiann I to I 
Dulcltol 
Gorbi tel 
Clirata - 
Tartrate* 
! ' 
Arabir.ose*. 
h2s * 
%c-!i°J.e^a.su i,s 
FpS « 
Sa tvpntsuls 
S.Sp. Kewcasile 
r^smsmsrA=t-.- r-xr** * .-S 
Maltose AG 
Inositol A3 
S_«schottiiiut lefi 
CitTate V 
Tartrate Z 
S«typhimu r {jum 
Ci tr *tT~+' 
Tartrate t 
I 
Xylose AG 
Arabinose AG 
i 
h2s t BACTERIOLOGY - CASE WORK SHEET 
PATIENT- 
DATE 
SPECIMEN 
RECfD FROM. 
ADDITIONAL. DATA. 
CRAM STAIN: 
MOTILITY; 
Hours 
jJL | 72- 
Daysj 
Hours 
24 i 48 I 72 
' - ' --.-r TT f * '* 
I 
Day a 
R.D.S. 
LACTOSE 
MALTOSE 
DEXTROSE 
--SlftCBROSB. 
XYLOSE 
..JBAffEINQSE ., 
.... AEABXNQSE ,, . , 
BHAMNQSE 
UW MILK 
HoS PRODUCTION 
amCTs cimTE. 
ymmoi 
DULCITOL 
SORBITOL 
INOSITOL 
DEXTRIN 
SALICIN 
imiN 
. smsxs 
d~TAHTRAT5. 
INDOL 
JLJL 
M,R, 
# - doubtful 
>£uacid & gae 
■/ - acid, no gas 
Aik - alkaline 
Remarks 
(if necessary, use other side) ......BACTERIOLOGY - CASE worn SHEET 
PAT I MI— 
DATE 
SPECIMEN 
REC»D FROM— 
ADDITIONAL DATA- 
GRAM STAIN; 
MOTILITY; 
i 
j Hours j Days 
24 I 48 ‘ [ 72 j 
Hours 
24 i 48 i 72 
■ • r' "rm ■ 
i 
Days 
H-P-S. 
lactose 
MALTOSE 
DEXTROSE 
SACCHROSE 
moss 
HATEIHOSB 
arabie-ose 
Rhamhose 
..LITMUS MILK 
HoS FHODUCTIOH 
SIMMOMS (!TTO ATE 
HAMITOL 
# - doubtful 
DULCITOL 
SORBITOL 
INOSITOL 
DEXTRIN 
SALICIN 
INULIN 
-MUMig 
d-TARTRATE 
jmsk 
M.R. 
(r>- acid & gas 
/ - acid, no gas 
Aik - alkaline 
|EK UK S 
(if necessary, use other side) BACTBRIOLOGY - CASE.WORK SHEET .. 
PATIENT- 
DATE 
SPECIMEN 
REC'D FROM- 
ADDITIONAL DATA- 
MOTILITY; 
GRAM STAIN: 
' Hours | Days 
24’ i 48 I 72 ~ 
Hours 
24 ! 48 i 78 
Days 
-SA.s. 
LACTOSE 
MALTOSE 
DEXTROSE 
-JAg.ggR.OSE 
XYLOSE 
....RmiNQSE . 
.■■JffiADIUQ.SE , . 
BHAMOSE 
HoS PRODUCTION 
.JSXMMDNS Cl CATE 
MANNITOL 
DULCITOL 
SORBITOL 
INOSITOL 
DEXTRIN 
SALICIN 
ITOIN 
ArMa 
_IN2CL__ 
.-LlA 
MtR. 
# - doubtful 
acid & gas 
/ - acid, no gas 
Aik - alkaline 
REMARKS 
(if necessary, use other side) BACTERIOLOGY - CASE WORK SHEET 
PATIENT— 
DATE 
SPECIMEN 
RECfD FROR 
ADDITIONAL DATA. 
GRAM STAIN: 
MOTILITY; 
Hours 
24 I 46 | 72 
Days 
Hours 
24 i 48 I 72 
■« , B1 •* 
Days 
i .1 
LACTOSE 
KALTOSE 
DEXTROSE 
. 
XYLOSE 
-■■SmCifOSE . 
....ABABINQSE 
■-EHAMNOSa 
-MEWS. HIM .. . 
HoS production 
■■SXUMDgS.CIgUTS 
MANNITOL 
DULCITOL 
SORBITOL 
INOSITOL 
DEXTRIN 
SALT CUT 
IFJIIN 
d-TAHTRATE 
JJQQL 
M.R. 
# - doubtful 
(£*■» acid & gas 
/ - acid, no gas 
Aik - alkaline 
REMARKS 
(if necessary, use other side) BACTERIOLOGT - CASE TOBK SHEET 
PATIENT- 
_ DATE 
SPECIMEN 
RECfD EROE. 
ADDITIONAL DATA 
GRAM STAIN: 
MOTILITY: 
Hours 
_24~_ I 46 [ 72" 
Daysj 
Hours 
24 i 48 S 72 
1 ,r \ 'L 11' "r' —; 
Days 
i 
JLZul, f 
LACTOSE 
MALTOSE 
DEXTROSE 
- SACmOSB 
XYLOSE 
...Mmoss. 
...ABADXNQSB , , , 
-JSE&MQSB 
LITW?,MILK . 
HsS PRODUCTION 
jiiouim. 
mmim 
DDLCITOL 
SORBITOL 
INOSITOL 
DEXTRIN 
SALICIN 
INULIN 
, wct 
d-TARTRATE 
INDOL 
-LJL 
M.R. 
# - doubtful 
add & gae 
</ - acid, no gas 
Aik - alkaline 
REMARKS 
(if necessary, use other side) , .^CTmo^QOY, 
CASE WORK SHEET 
PATIENT- 
DATE 
SPECIMEN 
EDG’D FROM- 
ADDITIONAL DATA- 
gram STAIN: 
MOTILITY; 
Hours 
J24 _ } 48 72 
Day b[ 
Hours 
24 i 48 72 
Days 
LMi 
LACTOSE 
MALTOSE 
DEXTROSE 
3&Qmom 
XYLOSE 
.SAEmOSE,,. , 
. ababxnq.se , 
jrhamqse 
.MSitM 
HoS PRODUCTION 
SIMMONS,01SEATS 
...MANNITOL 
DULCITOL 
SORBITOL 
INOSITOL 
DEXTRIN 
SALT GIN 
INULIN 
, 9-BLAXm 
d-TAHTHATS 
INDOL 
Lu.P, 
M.R. 
# - doubtful 
acid & gas 
/ - acid, no gas 
Aik - alkaline 
REMARKS 
(if necessary, use other side) BACTERIOLOGY ~ CASE WORK SHEET 
PATIENT- 
DATE 
SPECIMEN 
REC*D FRO&_ 
ADDITIONAL DATA— 
GRAM STAIN: 
MOTILITY; 
! i 
i 
I 
Hours | Days 
24 I 46 ~| 72 ~i ~ 
Hours 
84 I 48 j 72 
Days 
JL.k.§lt 
LACTOSE 
MALTOSE 
DEXTROSE 
_5ACCHBQ5S__ 
JGBiOa 
..JAEPINQSS . 
-ABAB1SOS3S , 
...SSAMQSS 
H?S PRODUCTION 
JSXMMDSS CITRATE 
-JmiTQL 
DULCITOL 
SORBITOL 
INOSITOL 
DEXTRIN 
SALICIN 
INULIN 
„ . 
d-TARTRATB 
...Yx JE, 
M.R. 
# - doubtful 
acid & gas 
/ - acid, no gas 
Aik - alkaline 
REMARKS 
(if necessary, use other side) BACTERIOLOGY ~ CASE WORK SHEET 
P AT I ENT- 
DATE 
SPECIMEN 
RBCfD FROM- 
ADDITIONAL DATA. 
GRAM STAIN: 
MOTILITY; 
Hours | j 
>48 j 72 | 
Days; 
Hours 
24 i 48 i 72 
Days 
JkSA, 
LACTOSE 
MALTOSE 
DEXTROSE 
.,gAS.gSRQS.S, . . 
XYLOSE 
.■■RAHXIIQSE . 
ARABXIIQSE,,, , 
■RHAMNQSE 
jLiMs.Mm..., 
HsS PRODUCTION 
SIHCTS CITRATE 
-.jmiTQL 
DULCITOL 
n 
SORBITOL 
INOSITOL 
| DEXTRIN 
j SALICIN 
! INULIN 
I 
j, Mg 
j d-TARTRATE 
1-jmk 
i 
.j. 
! M.R. ' 
# - doubtful 
\£>~ acid & gas 
/ - acid, no gas 
Aik ~ alkaline 
remarks 
(If necessary, use other side) Characteristic Reactions of Gram-negative Intestinal Bacilli 
on Russell’s Double Sugar Medium, - Phenol red Indicator (Alkaline 
is red, Acid is yellowish). 
• 
• 
Slant i Butt 
Mechanism 
• 
The small amount of 
acid produced by dex- 
trose (O.ljS) is dif- 
fused, leaving alka- 
line slant. 
The larger amount of 
acid from lactose (1$) 
gives acid slant. 
Organisms producing 
acid from either dex- 
trose or lactose give 
acid butt. Salmonella 
give acid and gas (bub- 
bles in medium). Ty- 
phoid-dysentery group 
produce acid only from 
dextrose. 
Genus Escherichia 
Acid 
Acid and gas (////)• 
Genus Aerobacter 
and 
Genus Klebsiella 
Acid, returning to 
neutral or alkaline 
after several days. 
Acid and gas (////). 
Genus Salmonella 
Alkaline 
Acid and gas (//), 
Shigella dysenteriae 
and' Shigella 
paradysenteriae 
Alkaline 
Acid 
Shigella sonnei 
and 
Shigella madampensis 
Alkaline, Small acid 
producing daughter 
colonies may be formed 
several days. 
Acid 
Eberthella typhosa 
Alkaline 
Acid 
Genus Proteus 
Alkaline 
Acid and gas (/). .Colony Characteristics of Gram-negative Intestinal Bacilli 
_ _ on Differential Plate Media, „ 
Medium 
Eosin methylene blue agar 
a. agar. 
b. Dosexycholato-citrate 
agar. 
Mechanism 
Coli-aerogenes group 
ferment lactose and grow 
into large, opaque colo- 
nies; also absorb dye to 
give color to colony. 
The non-lactose-fermen- 
ting pathogenic species 
develop as small, color- 
less, translucent colonies 
Non-lactose-fermenting 
organisms grow into small, 
clear,colorless,translucent 
colonies. 
Cn a lactose fermenting 
organisms produce large, 
reddish colonies. Growth 
on b is same except great- 
er inhibition cf coli- 
aerogenes group. 
Genus Escherichia 
- 
Large colonies with 
large, dark, almost black 
centers, and with green- 
ish metallic sheen. 
a. Largo opaque,red- 
dish colonies; occasion- 
ally have colorless rim, 
b. Much inhibited; if 
present, growth is pink 
and opaque, opacity 
spreading to surrounding 
medium. 
Genus 
and 
Genus Klebsiella 
Large pinkish mucoid 
colonies with small, 
dark brown or black cen- 
ters; rarely show metal- 
lic sheen. 
a. Similar to Escheri- 
chia colony except lar- 
ger and mucoid, 
b. Much inhibited; same 
as Escherichia shown in 
b above. 
Genus Salmonella 
Translucent, colorless 
or pinkish colonies, 
■usually slightly larger 
than S. typhosa? later 
"have bluish tint. 
Large translucent co- 
lonics, domed, shiny, 
smooth and colorless. 
Shigella dysenteriac 
Small translucent, 
colorless colonies. 
Same as Salmonella co- 
lonies except smaller. 
and Shigella 
paradysenteriae 
Shigella sonnoi and 
nadampensis 
Small, translucent, 
colorless colonies. Later 
may ferment lactose. 
Same as S. dysenteriac 
colonics during first 2A 
hours, later may show red- 
dish daughter colonies, cr 
entire colony may become 
red. 
Eberthella typhosa 
Translucent,colorless 
colonies. 
Translucent colonics; 
domed, shiny, smooth and 
colorless, . 
Alcaligenes 
facealls 
Translucent,colorless 
colonies. 
Similar to E. typhosa 
Genus Proteus 
Translucent colorless 
colonies; slight 
spreading. 
Sane as Salmonella; 
spreading inhibited. Cl.parabctulinum (Types A & B) and Cl.botulinum(Type C) 
Habitat: These are primarily saprophytes of the soil, may 
occasionally be found in the intestinal tract of domesticated 
animals and on various foods contaminated by soil or dust. They 
are not infective' to man or animals but do produce disease by means 
of the violent poison,; toxin, it may produce in foods which act as 
culture media fo.r its saprophytic growth. This toxin is not formed 
within the body. This poisonous toxin, variously applied, produces 
’’botulism” or food poisoning in man, ’’forage poisoning’Lin-animals 
pr ’’limber heck”, in poultry. The living organism may be sought for 
in the infected food but not in the poisoned man or animal for it is 
not an infection. Botulism may be associated with meat or meat.pro- 
ducts, fruits, vegetables, canned goods and various pickled and pre- 
served foodstuffs. Broth cultures injected subcutaneously in mice, 
g.pigs, rabbits, cats,• monkeys prove fatal in 1 to 4 days. 
Morphology, and Staining: Large sporulatlng rods with parallel 
sides and rounded ends, occurring singly or in chains.. Slightly mo- 
tile; not-capsulated. Gram-positive. Spores are oval, larger than 
the bacilli and usually situated at or near the end. Spores form 
best in sugar free media at a temperature of 20 to 25°. 
Metabolism:- Strict anaerobe growingwell in ordinary media 
with neutral or slightly alkaline reaction. Optimum temperature 35° 
(growth poor at 37°). Hemolysis produced on erythrocytes (human and 
horse). Types A & B are generally proteolytic; type C only slightly 
proteolytic. Optimum pH 7.4 to 8. Above 37° toxin formation is im- 
peded, below 20° toxin formation stops, . v •' 
Cultivation: . 
Agar: 4 day growth: flat irregular, greyish yellow, filamentous 
colonies with alternately smooth and granular surface, and in* 
definite fringed periphery. 
Deep glucose agar shake: 4 days growth: colonies thin ’ semi-*®paque 
discs with bi-convex brownish centers, translucent edges. 
Abundant gas formation. 
Blood agar plate (Horse): 3 days: Irregular, round, 2-3 mm, colonies 
with smooth center fimbriate periphery. Alpha type hemolysis. 
Cooked Meat Mediums (Brain): 4 days: Abundant growth, turbidity, gas 
formation, brain digested and blackened. Butyric acid odor. 
Broth: 4 days: Dense turbidity, rancid odor. Bio-chemical Reactions: 
nAH 
"Q" 
"C « 
Glucose 
AG 
AG 
AG 
Maltose 
AG 
AG 
AG 
Salicin 
AG 
AG - 
-, 
Glycerol 
AG 7... 
AG - 
Lactose 
- 
- 
- 
Inositol 
- 
AG 
Indol 
- 
- 
- 
Nitrates 
- 
- 
- ■ 
NHq 
7 
7 
/ 
fbS 
/ 
/ 
/ 
Meth.Red 
V.P. 
- 
- 
- 
Litmus milk Reduced and alkaline, no. coagulation, 
Serology: Types A & B are identifiable by agg. and toxicity tests. 
Their toxin is specific only for type, the antitoxin of one 
is not neutralised by the toxin of another. Type C, forming another -- 
separate specific toxin is distinguished chiefly by lack of proteo- 
lytic powers. 
Resistence: The bacilli without spores are readily killed by heat 
and chemicals. Spores withstand drv heat of 180° for 15 - 30 min- 
1 o v 
utes, moist heat at 100 for 3-5 hours. Toxin is destroyed by 
80° C in 5 - 15 minutes. 
Identification: (see ’’Bacterial Food Poisoning”) CORYNEBACTERIUM DIPKTHERIAE 
(Diphtheria bacillus’) 
Description: Slender rods,' straight or slightly curved, of medium 
size; often lie,at various angles to one another 
forming V or Y shapes, or clumped as Chinese letters; generally not 
uniform in thickness, exhibiting rounded, pointed or swollen ends or 
enlargements along the length of the cell; usually stain unevenly, 
showing barred and granular large forms, .solid staining short forms; 
gram-positive, nonmotile; grow readily at 37°, preferably on Loeffler’s 
serum or blood agar as small, circular, smooth,, moist, grayish to 
creamy-white colonies, some strains giving narrow zone of haemolysis 
on blood agar; pathogenic to man and,to guinea pigs* 
Habitat: The cause of diphtheria, usually found in the mucous mem- 
branes of the nose,.throat and larynx of cases and carriers, oc- 
casionally found as cause of. conjunctivitis, wound infection, middle 
ear infection and broncho-pneumonia. It is not, found in the blood 
stream, the general symptoms being caused by the powerful toxin formed 
at the local site of infection. 
Identifying Characteristics: (l) Shape, size, irregular staining, V. 
or Y arrangements as seen in a direct 
spread or spread from Loeffler’s medium and stained by Loeffler’s 
methylene blue or Reisser’s- stain. 
(2) Growth freer on Leoffler’s serum medium than that of other 
organisms. 
(3) Colony form on blood agar. Colony on tellurite medium be- 
comes black. 
, (4) Pathogenicity for guinea pig (see virulence test). 
(5) Carbohydrate fermentation (see chart below). 
Collection and Transmission of Specimen for Examination: Cotton swab maj 
be applied to 
the involved area (throat,,nose, wound) or to the membrane or exudate 
from that area with care to gather a considerable amount, of the exudate 
on the swab, with caution not to'contaminate the swab by it touching 
the tongue or other ncninvolvod areas. Use this swab for: 
(a) Immediate inoculation of Locfflcr’s scrum slant 
for 1S-2/+ hours, incubation at 37CG., or for shipment to distant 
laboratory. •' | 
(bf Immediate inoculation of blood agar plate, for in- 
cubation at 37°, 2U hours. ' -| I 
(e) Spread on slide for direct Noisscr stain examina- 
tion. 
(d) (Optional) plant on ’’tellurite media”. 
Microscopic Examination: (Direct spread, Rcissor’s or Loofflor’s 
stain), An immediate presumptive diagnosis 
can sometimes bo made on the basis of morphology and staining features of. what few diphtheria bacilli may be observed in the direct smear, 
but here they will be confusedly mixed with the many other microor- 
ganisms of mouth or wound flora. Vincent's organisms and diphtheroids 
may give confusion, should be noted on report if found. Negative find- 
ing by direct method cannot be given value. Presumptive positive 
finding should be confirmed by cultural and virulence tests. 
Cultural Examination: (a) Loeffler’s tube, after 18-24 hours incu- 
bation at 37SC is examined by broad needle drag along its surface, 
this then spread on a slide and planted on blood agar and tellurite 
media for later pure colony isolation. The -slide spread is stained by 
Neisser method and observed for diphtheria bacilli; the irregularity 
of staining and shape, the metAchromatic granules may be noted. If 
typical diphtheria bacilli are found and the culture is from a sus- 
pected case, a presumptive diagnosis should be made at once. If the 
culture is from a suspected carrier, diphtheria-like bacilli should 
be further identified by fermentation and virulence test. 
(b) Blood agar plate will provide information on general bac- 
terial flora, particularly streptococci, and will give opportunity for 
notation of colony form and single colony isolation of diphtheria-like 
bacilli. 
(c) Tellurite media will point out the diphtheria-like co- 
lonies by black color. 
(d) (Optional - rapid) Apply a sterile serum-swab to in- 
volved area, return to serum-tube -and incubate for a few hours; trans- 
fer to other media and examine slide made by gently rolling swab out 
into thin film. Sterile serum swabs are prepared by placing sterile 
swabs into sterile tubes containing a few cc. of serum. Some such 
swabs are made to contain 2% potassium tellurite to attain blackening 
from growth of diphtheria bacilli. 
Fermentation Reactions: G. diphthcriae can be differentiated from re- 
lated organisms by their fermentation reaction 
in dextrose, saccharose and dextrin. The absence of power of a parti- 
cular organism to ferment glucose and its ability to ferment saccharose 
is usually sufficient to exclude the organism from being a diphtheria 
bacillus. 
Virulence Test; This is the only certain method by which the identity 
and virulence of C. diphthcriae can be confirmed or 
distinguished from nonvirulent varients. No other known species of 
this genus occurring in man produces a fatal toxaemia in guinea pigs. 
Pure cultures are preferred for this test, but for speedy test the 
suspension of a heavily positive Loefflcr’s tube may be substituted. 
(a) Subcutaneous Method: Inject 2 cc. of a pure culture grown 
for 48 hours in infusion broth or 4 
cc. of a Loefflor’s slant suspension in 10 cc, of saline, subcutaneously 
into a 250 gram guinea pig. At the same time a similar injection of the 
culture is made into a control guinea pig which had been given 250 units 
of diphtheria antitoxin, intraperitoncally, 24 hours previously. If the organism is a virulent diphtheria bacillus the unprotected animal 
will die within 3 to 5 days and on post mortem will show a gelatin- 
ous aedema around point of injection and enlarged hemorrhagic ad- 
renals. 
(b) Intracutaneous Method: Two guinea pigs of 250 grams 
are used, one of which has 
been injected intraperitoneally with 250 units of diphtheria anti- 
toxin 2 k hours previously. The growth from a 2k hour Loeffler’s 
slant is suspended in 20 cc. of normal saline and .15 cc. is in- 
jected intracutaneously at corresponding site of each pig. Six 
cultures may be tested at the same time on two animals. Virulent 
strains of diphtheria bacilli produce a definitely circumscribed 
local infiltrated lesion which shows superficial necrosis in 2-3 
days. In the control pig the skin remains normal. If a mixed cul- 
ture was used for test, and contained streptococci or staphlococci 
with sufficient virulence, local lesions will occur in both animals; 
the test would then be considered inconclusive and repeated using a 
pure culture. 
Schick Test: This is an intracutaneous skin test to evidence the 
presence or absence of immunity to C. diphtherias. 
The injection consists of .1 cc, of diphtheria toxin (l/50 m.l.d,), 
A control test uses the same material which has been made inert by 
heat (75° for 5 minutes). Results are noted daily for k days and 
recorded as Positive, Negative, Positive Combined or Negative Combined 
reactions. 
C. pseudodiphthericum bacillus): This organism is shorter 
and thicker than 
C. diphtherias» usually straight and clubbed at one end, rarely at 
both; when Loefflcr stained it occasionally shows unstained trans- 
verse bands which, unlike these in C. diphtherias, hardly ever ex- 
ceed one or two. Sometimes the transverse band gives the bacillus 
a diplococcoid appearance; no polar bodies arc demonstrable by 
Ncisser's stain; it grows moro luxuriantly, colonics larger, less 
transparent and whiter than are those of true diphtheria bacilli; 
a positive means of distinction is its inability to form acid on 
sugar media; it is not pathogenic to guinea pigs or to man. It is 
a common mouth commensal, may be found in k2% of normal throat cul- 
tures; diphtheria-like bacilli which prove to be avirulent generally 
are found to be C. pseudodiphthericum. 
C. xcroso (Xerosis bacillus): This is a harmless saprophyte, common- 
ly found in the normal or inflamed con- 
junctiva of the eye. It closely resembles C.. diphtherias, and is in- 
distinguishable morphologically and culturally though generally shor- 
ter; polar bodies may occasionally be seen; it differs in its acid- 
ifying action on sugar media and its nonpathogenicity to guinea 
pigs. ' 
*.' : l 
Hiss Serum Water plus 1% 
Virulence 
Dextrose 
Saccharose 
Dextrin 
C. diphtherias 
+- 
— 
Virulent 
C. pseudodiphtheriae 
— 
— 
— 
Nonvirulent 
C. Xerose 
4 
4- 
— 
Nonvirulent 
Diphtheroid Bacilli: There is a large group of ill defined or- 
ganisms given this general name because of 
their morphological resemblance to the diphtheria bacillus; often 
show metachromatic granules; are not virulent when tested by the 
guinea pig virulence test. They are common saprophytes of the 
• throat, skin and other body areas, are so ubiquitous that any as- 
sociation of them with specific disease must be avoided; they must 
be distinguished from virulent and therefore significant diphtheria 
bacilli. MYCOBACTERIUM TUBERCULOSIS 
(Tubercle bacillusT" 
Descriptions Slender rods, straight or slightly curved with rounded 
ends; occur singly, in threads or in clumps; may stain 
evenly or irregularly showing granular, beaded or banded forms; stain 
with difficulty but when once stained are acid fast; growth on media 
show, aerobic, aided by glycerine or other enrichments; growth on gly- 
cerine agar in 4- weeks at 37°C; colonies minute, crumb-like, irregular, 
whitish-yellow, later brownish, ridged, becoming dry; pathogenic to 
guinea pig. 
Habitat: A strict parasite, causing tuberculosis of man, cattle and 
other animals; human and bovine varieties are distinguishable, 
both infectible to man; there are other species of this genus causing 
avian tuberculosis, infecting fish, snakes, turtles and other cold 
blooded animals. There are a number of acid fast bacilli which are 
strictly saprophytic but confuslble with M. tuberculosis. 
Identifying Characteristics: (a) Acid fast bacillus, when stained 
by Ziehl-Keelsen method. 
(b) Pathogenic to guinea pigs, producing 
tuberculosis in 6 weeks. 
(c) Growth on special media slow, wrink- 
ly and contains acid fast bacilli. 
- \ 
Collection and Transmission of Specimens, for Examination: Sputum, 
exudates, 
urine, spinal fluid and tissues may be examined for tubercle bacilli. 
They should be collected under as sterile precautions as feasible 
(not possible with sputum) and transmitted in suitable container to 
laboratory. Sputum collection should be so guided as to provide 
bronchial material rather than the fluids from the mouth or nose. 
Microscopical Examination (directly applied to specimen): A presump- 
tive diag- 
nosis can be made by applying an acid fast stain, such as Ziehl-Neel- 
son carbo-fuchsin to a slide spread of selected (caseous) fragments 
of the specimen. The red acid fast bacilli will be readily noted in 
contrast to the value of the counterstain of all other bacteria cells 
and debris. Stained spreads may be made from the centrifuged sediment 
of urine or spinal fluid, using small film of sterile egg albumin on 
the slide to prevent the sediment being washed off during the staining 
process. 
Concentration Method: If tubercle bacilli are too few to be found by 
above method they may be concentrated by di- 
gesting mucous with sodium hydroxide or antiformin and examining the 
centrifuged sediment by direct spread> by culture or by guinea pig 
inoculation. Sodium Hydroxide Method: Mix equal parts of the specimen and a 3% MaCH 
solution, shake well, incubate at 37°C for 
l/2 hour, add HC1 to neutrality to litmus, centrifuge and use sediment 
for test. 
Animal Inoculation; This is the most certain method of establishing the 
specific diagnosis of ’tuberculosis. Centrifuge the 
NaOH digested sputum or the urine or spinal fluid, suspend the sediment 
in sterile saline and inject this subcutaneously or intramuscularly 
into the thigh of a young guinea pig (250 gm.). Autopsy of animal at 
its death several weeks later, or if it lives at 6 weeks, will reveal 
generalized tuberculosis, apparent particularly by caseation of glands, 
spotted liver and large spotted spleen, which may be confirmed by 
finding acid fast bacilli by direct spread or special culture of these 
tissues. 
Cultural Examination; This is not employed as a routine procedure. 
Several loopsfull of the sediment in the sodium 
hydroxide concentrate or tissue fragment from guineapig tissue, are 
planted on the surface of tubes of Pctroff (or other suitable) medium. 
Incubate the cultures for two days, then seal - use cotton plugs by 
dipping them in melted paraffin. Incubate at 37°C. for 6 weeks and 
examine for colonies of M. tuberculosis. SPUTUM EXAMINATION FOR TUBERCLE BACILLI 
(Sodium Hydroxide-alum flocculation Method) 
Reagents: 
1. Digester: Sodium Hydroxide 40. (4$) 
Potassium Alum 2, (.2$) 
Bromthymol blue .02 (.002%) 
Water to 1000. 
Yellow -> Blue 
Note: Range of indicator 6.0 —- 7.6 
pH 7.0 • Light bluish green 
2* Acid: Hydrochloric Acid, Concentration 250. (25$) 
Water to 1000. (about 2,5 N) 
Test: 
(1) Mix sputum (5cc.) with 1 to 4 parts of digestor. 
Shake well. 
Incubate at 37°, 3,0 minutes for culture on animals. 
Incubate at 37° to homogeneous mass for concentration. 
(2) Adjust pH to pH 7 with Acid-digestor adjustment. 
(3) Centrifuge at top speed for 5 minutes, 
(4) Remove supernatant fluids. 
(5) a. Spread on slides - heat fix, or 
b. Culture or 
c. Inject animals with saline suspension of sediment. 
Result: the sodium hydroxide digests the organic matter. Flocculation 
occurs when acid is added. This flocculation carries into sediment 
the organisms, including Tubercle bacilli, not killed or dissolved 
by the alkali. NOTES SPIROCHETES 
Characteristics: Slender, undulating, corkscrew-like, flexible, 
filamentous organisms. They have short or long spirals 
with the twists in three dimensions. The number, depth 
and relative length, and sharpness of angle of spirals 
have diagnostic importance, though somewhat variable. 
Motile by sinuous, rotating movement of the body, not by 
flagella as in the case of bacteria. Stain with diffi- 
culty by ordinary stains though some (genus Borrelia) 
stain readily; the polychrome methylene blue stains of 
Wright and Giemsa are most used; silver impregnation 
method is applicable to the more resistant forms. Fontana 
stain for spreads, Levaditi stain for tissues. Most rea- 
dily demonstrable, to reveal their characteristic motility, 
in the fresh state by dark ground illumination,- Cultiva- 
tion difficult and generally not practical. Animal inocu- 
lation significant in a few pathogenic species. 
Habitat: Ubiquitous, occurring in nature in soil, water, decaying 
organic materials and on and in the bodies of man, animals 
and plants. Some are saprophytes, others are commensals, 
a few are pathogenic, causing such severe diseases as: 
syphilis, yaws, relapsing fever and infectious jaundice. 
BORRELIA RECURREUTIS 
(Relapsing fever spirochete) 
Characteristics: Spirochetes having large wavy, inconstant spirals, 
usually about 5; when seen under darkfield illumination, 
the organisms are very active, in length several times the 
diameter of an erythrocyte, rapidly progress in either di- 
rection, disturbing the red cells by their motion; stain 
readily and uniformly by polychrome stains (Wright*s or 
Giemsa’s) and by simple stains; difficult to cultivate; 
inoculable into mice and rats, there causing periodicity 
of spirochetemia but no demonstrable clinical symptoms or 
tissue lesions. 
Habitat: The cause of Relapsing fever; found in blood and tissues 
of patients suffering from relapsing fever and in the body 
and intestinal contents of the infector vectors, ticks and 
lice. The name applies to the spirochete of European re- 
lapsing fever; a number of other species—names have been 
given for the spirochetes of the United States and Mexico 
(3. turicata), Central and South America (B. vonezuclensis) 
and others, differentiation of which is based only on 
specific immunological reactions. Some lower animals may 
servo as reservoirs of infection, in the United States, 
the armadillo and the opposum. Identification: Fresh or citrated blood, taken during febrile para- 
xysro, is examined: 
(1) Darkfield illumination of fresh, thin slide- 
cover glass preparation, for the characteristic motility 
and morphology. 
(2) Slide spread, stained by Giemsa’s method or 
by dilute carbol-fuchsin for morphology. Here the forms 
are much distorted, the spirals often obliterated, so that 
the characteristic morphology cannot be found. These spi- 
rochetes may sometimes be detected and the diagnosis sug- 
gested, in a routine Wright’s stain for differential blood 
count. 
(3) White mouse or rat inoculation, intraperitoneal, 
of ,2 to ,$ cc.of blood; examine fresh tail blood from the 
2nd to 14.th day for spirochetes. 
I 1 
FUSOSPIROCHETAL DISEASE 
(Vincent’s Angina) 
Definition: Vincent’s angina is an inflammatory lesion in the mouth, 
pharynx or throat, most often affecting gum margins and ton- 
sils. An acute inflammation may lead to the formation of a 
pseudomembrane, suggesting that of diphtheria; later there 
are punched out ulcers, suggestive of those of syphilis. 
The disease is localized, generally mild with minimal sy- 
stemic disturbances. Two microorganisms are almost always 
found together, in great numbers, in films from these le- 
sions; the two forms apparently living in symbiosis. They 
are rarely present alone, being usually accompanied by other 
microorganisms, such as staphylococci, streptococci, even 
diphtheria bacilli; the latter finding being more significant 
than the Vincent organisms alone. 
Fusobacterium plauti-vincenti (fusiform bacillus of Vincent): Large 
bacilli, thick in middle, tapering toward ends to blunt or : 
sharp points. Readily stained by Loeffler’s methylene blue, 
carbol-fuchsin or Giemsa stain, with characteristic unequa- 
lity in the intensity of the stain, being more deeply stained 
near the end; banded alteration of stained or unstained areas 
in the central body, not unlike the metachromatic granules 
of diphtheria bacilli. 
vincentii: Spirochetes somewhat like those of relapsing fever, 
longer than the fusiform bacilli; made up of variable numbers 
of undulations, shallow and irregular in their curvatures, 
unlike the more regularly steep waves of Treponema pallidum. 
They stain more evenly and less distinctly than the fusiform 
bacilli. Identification: 
1. Make slide spreads from the ulcerative lesions, fix in 
flame and stain deeply with dilute carbol-fuchsin, cry- 
stal violet or Wright’s stain and examine for fusiform 
bacilli and spirochetes. 
2. Positive results will be evidenced by finding great num- 
bers of- both fusiform bacilli and spirochetes, A few 
forms of either type is not significant* ' 
TREPOKm PALLIDUM 
Characteristics; Delicate spirochete coiled in S to 14- regular, ri- 
gid, sharp spirals; spirals equal or greater in depth than 
in length, with acute-rather than obtuse angles. As seen 
under darkfield illumination, it appears as a highly refrac- 
tilp*, long, slender, spiral, silvery form with serpentine, 
corkscrewiike movement; motile, but.does not progress rapid- 
. ly or far, motion rotational with undulations. 
Made visible most effectively by darkfield illumina- 
tion. Difficult to stain with analine dyes other than the 
Qiemsa stain; body.stained pink by Giemsa stain or black by 
silver impregnation method, Fontana stain in spreads, Levaditi 
in tissues. 
May be cultured by special methods and inoculated in- 
to some lower animals, neither procedure being practical for 
diagnostic purposes. 
Habitat: Strict parasite of humans, causing the infectious disease of 
syphilis, with protean manifestations; transmitted only by 
direct contact, generally through sexual intercourse, occas- 
ionally through intimate contact of other mucous membrane or 
skin sites. Syphilis, one of the most prevalent and impor- 
tant of all infectious diseases, usually progresses through 
a number of states, irregular and varied: 
1. Incubation period of 4- to 6 weeks. Spirochetes 
then cannot be demonstrated. 
- 2. Primary stage: ’’Hard chancre” at site of inocu- 
lation. Starts as a papule, enlarges, becomes hardened and 
then ulcerates, forming,an ulcer, with a firm base and hard 
■ edge in typical form, but atypical lesions frequently occur, 
especially, if mixed with secondary infection or coexistant 
with chancroid. Spirochetes can be found in fluid expressed 
■ from this chancre. The spirochetes will not necessarily bo 
' on the surface, rather in the deeper tissues and in the se- 
rum exuding from scarified lesion; at this stage they have 
already become disseminated to a general infection, can be 
. demonstrated in fluid aspirated from satellite lymph gland, 
• but cannot readily be found in the blood or other areas 
though potentially there, 
3. Secondary stage; Characterized by mucous 
patches, skin rashe-s and a variety of superficial lesions. 
Treponema pallidum can usually be found in material from 
those lesions. A* Tertiary stage with lesions of viscera, bones, 
central nervous and cardiovascular systems; tendency to deep 
rather than superficial lesions. Spirochetes are usually 
scanty, not readily demonstrated in these lesions. 
Identification: (Different procedures applicable to different le- 
sions and stages). 
1. Darkfield Examination: 
ft! ~Lc sions arc cleansed of surface crust, de- 
tritus, pus and surface organisms by gauze, or cotton 
applicator. If lesion has received any germicidal 
agent, examination is deferred until all germicide 
has been removed and the lesion has had applied to it, 
only a saline pack for a day or two, 
(b) Primary lesions are then given some trauma, 
to provoke exudation of scrum, by gently rolling the 
lesions between the gloved finger and thumb or by rub- 
bing its surface with a dry cotton applicator; avoid 
hemorrhage (though a few erythrocytes or pus cells are 
desirable to aid in obtaining proper illumination), 
(c) Secondary lesions are merely cleansed and 
abraded. 
(d) Slide-cover glass, fresh preparation may be 
made from accessible lesions by merely touching the 
slide to tissue juice and immediately placing the cover 
glass over this moist drop. Vaseline placed around edge • 
will prevent drying. If lesion is less accessible, the 
fluid may bo collected in a capillary pipette and placed 
on slide from this. * - 
(e) Examine immediately on darkfield microscope 
for characteristic morphology and motion of Treponema 
pallidum. Exercise caution not to misinterpret obser- 
vation. There are many saprophytic spirochetes which 
are easily distinguished;, there are a few spirochetes, 
especially in the mouth, which are more difficult to 
distinguish. 
(f) ”Artifact spirochetes” provoke mistakes to 
those unfamiliar with the appearance of blood, pus and 
cultures under darkfield illumination. Wavy filamen- 
tous structures may occur there which give a false im- 
pression of spirochetes; forms given off by red cor- 
puscles in a drop under a cover glass, may falsely 
suggest spirochetes. 
(g) Report findings with qualifying data, sueh 
as notation of location of lesion, examined, the oc- 
currence of conditions making examination unrepresen- 
tative, etc. 
2. Delayed darkfield method: This is a scheme of forwarding 
lesion fluids to a distant laboratory for darkfield exami- 
nation; employable when facilities for darkfield examina- 
tion are not locally available, or when local examiners 
desire confirmation of their own findings by a consul- 
tant, A tissue fluid from a suspected lesion is allowed to flow into a capillary tube 8 cm, long by 1 mm. diameter; the 
two ends of this tube are sealed by pressing into a soft paraffin- 
vaseline mixture {50% of each) and these tubes forwarded for the 
darkfield examination. At examining laboratory the serum may be 
transferred to a slide by pressing one end of the capillary tube 
into a paraffin-vaseline mixture until the opposite end plug is 
forced out, 
3. Nigrosine method; This is not strictly a staining method, for it 
leaves the unstained spirochete in a black field, A loopful of the 
fresh tissue fluid is mixed with a loopful of 5% aqueous solution 
of nigrosine (plus *5% formalin as a preservative); this mixture 
is spread on a glass slide, dried and examined by.ordinary illumina- 
tion with oil immersion objective, A remote- examination may be made 
by forwarding an air dried drop, of the exudate on a slide; the labor- 
atory adds a loopful of water to this to dissolve the exudate and 
proceeds with the nigrosine preparation. Results are far inferior 
to the darkfield method, for the characteristic motility is absent 
and the spirochetes, by distortion, have lost much of their charac- 
teristic morphology, 
A, India ink method; Like the nigrosine method, a drop of material is 
mixed with a drop of drawing ink and the mixture spread on a slide. 
When dry, examine for white spirals against a dark background. 
5. Stained spread examination: By Giemsa or Fontana methods. 
6. Local Wassormann; Considerable amount of serum is collected from the 
local lesion and used for complement fixation tost, 
7. Blood serum and spinal fluid serology> Applicable to later stages. 
It is customary to subject all venereal patients, even after repeated 
negative darkfields, to follow-up blood tests for several months. 
LEPTOSPIRA ICTEROHAEMORRHAGIAE 
(Weil’s Disease - Infectious Jaundice) 
Characteristics: Spirochetes of many fine coils, so fine as to be dif- 
ficult to distinguish; one or both ends may be bent into a hook. 
Rapid spinning notion with intermittent active lashings. Difficult 
to stain; stained reddish by Giemsa method. Cultivated only by 
special method's, Inoculable into guinea pig with distinctive lesions, 
Habitat: The blood and kidneys of infected wild rats. The blood, urine, 
kidney, biliary tract of patients with infectious jaundice (Weil’s 
Disease). Identification; 
1. Guinea pig inoculation: Inoculate 3 to 5 cc. 
of fresh blood, fresh urine sediment or tissue suspension, 
intraperitonoally into white guinea pig; observe it daily 
for fever, .jaundice in the ears, eyes and about genitalia 
and for leptospira in the blood (usually found after the 
4-th day). After the animal dies, large numbers of lep- 
tospira can bo demonstrated in emulsions of the liver, 
kidneys and adrenals. 
2. DarkfieId examination of tissue emulsions, oc- 
casionally of urine or biliary sediment, for the motile 
leptospira, 
3. Stained spreads and cultures have limited appli- 
cation. FUNGI (MOULDS AND YEASTS) 
Introduction: The fungi are complex plant organisms, devoid of chlorophyll. 
The single cell types, as the common budding yeast (Saccharomyces 
cerevisiae grow and multiply much as do bacteria, except as to their 
method of multiplication (by budding, not by fission). Each individual 
cell combines ohe functions of nutrition and reproduction. Other fungi, 
the moulds, are made up of many cells, usually cylindrical (hyphae), 
joined into filaments (mycelia) from which spores (small round cells) 
develop, the structure built up by the filaments and spores being 
characteristic for each species. 
Habitat; Fungi occur widespread in nature and to a less extent in disease. 
Saprophytic fungi obtain their food from dead plants or decaying materials. 
Parasitic fungi obtain their food from living animal or plant life. A 
few species of fungi are pathogenic and capable of producing minor or major 
skin infections (dermatomycosis), hair infections (trichophytosis), 
bronchial infections (bronchomycosis) and certain specific generalized 
infections (blastomycosis,’ actinomycosis and others). Some fungi have 
commercial importance, such as those that give flavor to cheese and cause 
broad to rise (yeast). Many fungi attain laboratory attention because of 
their ubiquitousness in dust and their contamination of laboratory media. 
Descriptive Terms: Fungi occurim many forms, often variable within species 
under different conditions of growth. Those that are pathogenic tend to 
grow differently in the tissues than on culture media. Their classifi- 
cation is too complex to be given here, A few terms used in describing ' 
and classifying fungi are given below. 
Budding fungus: Yeast like; grow by budding; in the tissues and in culture 
appear as round or oval budding cells; may, but generally do not, develop 
rudimentary mycelia. 
Filamentous fungi; Mould like; develop long filamentous threads with or 
without apparent spore formation. 
Hypha: the single thread-like portion. 
Mycelium: a group or matted mass of branching hyphae. 
Septa: Divisions of hypha formed by transverse partitions. 
Spores: Cells developed for the propagation of the species. 
Thallus: The actively growing, vegetative organism as distinguished.from 
spores. 
Apcospores: Group of spores, U or B, enclosed in a sac or ascus. 
Endospore: A spore formed within an outer envelope. 
Conidiophore; Hypha bearing a spore or group of spores. Blastospore? A spore formed by budding. 
Arthrospore; A spore formed of segments of a hypha and released by dis- 
articulation. 
Chlamydospore? A large spore with tough and frequently double contoured 
wall, undergoing encystment. 
Sterigma? A short stalk bearing chains of conidia (as in Aspergillus). 
Sporangium: A sac containing an indefinite number of spores at the end of 
a hypha (as in Mucor). 
Fuseaux? Fusiform septate spores, produced by certain fungi (Trichophyton). 
Spirals? Terminal coils seen in some species. 
Pectinate Bodies: Comb-like structures formed by some fungi. 
Cryptococcust A genus of budding fungi devoid of ascospores and mycelia 
\e.g., Cryptococcus gilchristi. the causative organism of one form of 
blastomycosis). 
Saccharomyces? A genus of budding fungi having ascospores but no mycelia 
Je,g,, Saccharomyces cerevisiae. brewers yeast, oval or spherical cells, 
cause alcoholic fermentation). • 
Monilia? A genus of budding fungi having no ascospores, mycelia of rudi- 
mentary type and capable of fermenting certain sugars with the production 
of acid and gas. (e.g., Monilia psilosis. formerly thought to cause sprue). 
Endomvces: A genus of .budding fungi, having ascospores and segmented 
mycelia (e.g,, Endomyces albicans, found in thrush). 
Mndurella: A genus of filamentous .fungi characterized by septate, branching 
hyphae and chlamydospores; they are contained in black granules of in- 
fected tissue (e.g., "Madura Foot") (e.g., Madurella mvcetomi in "Madura 
Foot".) 
Nocardia (Actinomyces): A genus of filamentous fungi characterized by very 
fine, non-segmented mycelial filaments and no spores (e.g,, Nocardia 
bovis. the "ray fungus" of "lumpy jaw" of cattle, actinomycosis of man), 
Sporotrichum; A genus of filamentous fungi which appear in the tissues as 
oval spores -nd develop in cultures as mycelium with characteristic grouped 
spores or conidia. (e.g., Sporotrichum schenki. the causative agent of 
sporotrichosis, appearing in fresh spreads of the pus or tissue as oval 
or cigar shaped cells, and when examined by hanging drop culture appear 
as fine interlacing septate hyphae with oval or pear shaped spores, attached 
to the hyphae.) 
Aspergillus? A genus of filamentous fungi characterized by its spore organ. 
They are 'Common and troublesome laboratory contaminants, appearing in culture plates as cottony masses dotted with minute colored spots, be- 
coming in older cultures profusely black, yellow or green, according 
to the species. Microscopically, the colored spots are seen to be the 
spore organs, the spores (conidia) borne on aerial hyphae which ter- 
minate in a large rounded head with rows of spores projecting in all 
directions. The main cottony mass is a network of septated mycelial 
filaments, (e.g,, Aspergillus niger. the black mould.) 
Penicillium; This genus differs in its spore organ in that the fertile 
hyphae show numerous branches, rather than a rounded head, bearing rows 
of spores, a structure somewhat resembling a broom. The color of the 
colony varies with the species - green, yellow, etc. A common variety is 
Penicillium brcvicaule and its strains, it causes spoilage of cheese 
and other dairy products. 
Phycomycetes: A group of genera having, in addition to the mycelium, 
spores contained in a spherical, case-like structure (a sporangium) at the 
end of a hypha. Species of this group frequently contaminate laboratory 
media and food products, and occur in soil, dust and water, (e.g., Mucor 
mucedo. the blue-black mould.) 
I . ' 
Pleomorphism; This term refers to the great variation in characteristics 
of morphology and culture which many fungi, undergo under different con- 
ditions of life. The ringworm group are particularly likely to undergo 
these degenerative changes, and once a culture has so changed, it cannot 
easily be restored to its original condition. Prolonged growth on 
sugar-containing media leads to this permanent change, hence the use 
of "conservation agar1' for stock culture maintenance. 
Ringworm Group of Fungi; These filamentous fungi produce superficial 
skin infections, generally growing as leathery masses of closely inter- 
woven hyphae, growing slowly with development of bumps and ridges, and 
covered by a powdery or velvety "duvet" of aerial hyphae. The next four 
genera belong in this group. The so-called "Athlete's foot” may be 
caused by the same group of fungi. 
Microsporum: The small spored ringworm fungus: in the diseased epider- 
mis they appear as a fine mycelium, one to*- five microns in diameter, com- 
posed ©f rectangular elements; they penetrate into the haira and grow up 
and down in the hair. When the infected hairs are examined, they are 
found to be encased with an irregular mosaic of small round spores about 
2 micrens in diameter, (e.g., Microsporum audouini. causing the common 
ringworm of the scalp.) 
Trichophyton? The large spored ringworm fungus: the mycelium consists 
of chains of oval or rectangular spore-like bodies 5 to 8 microns in 
diameter, in regular alignment. Various species commonly produce ringworm 
of the scalp, beard, skin and nails, (e.g,, Trichophyton tonsurans, 
which is found only within the medulla of the hair.) 
Epldermophyton: A genus of skin-invading fungi, appearing as long inter- 
lacing filaments, never invading the hair as do trichophytons. May be 
readily recognized by direct microscopic examination of skin scraping, 
(e.g., jEpidermophyton cruris, causing the common "Dhobie Itch" or ring- 
worm of the groin and other areas.) Achorint A genus of filamentous fungi of which a species causes favus of 
scalp TAchorion schoenleini,) 
Materials for Examination: 
1, Hairs, ski* scales or scrapings from lesions, 
2. Tissue masses or scrapings from internal lesions. 
3, Secretions or excretions from infected areas, 
4. Incidental cultures (contaminants, etc,). 
Methods of Examination 
1. Choice of Method; Varies with expectancy of findings. Skin scrapings 
and hair may yield informative data by immediate direct methods only and 
be less informative on culture or animal inoculation, being difficult 
to grew, to infect or to exactly identify. Some species give the desired 
information only on cultivation or animal inoculation, especially in the 
case of tissue invaders. A few pathogens may be detected by histo- 
pathological examination. 
2, Microscopic examination; 
a. Collection of specimen? (preferably in laboratory by a medical 
After cleansing the affected part with alcohol, such 
materials as hairs, nails, scales or bits of tissue may be 
scraped into a sterile Petri dish. Moist specimens should be 
prepared and examined without delay. Biopsy specimens should be 
divided, one-half for direct examination, the other half for 
fixation and histological examination. All specimens should be 
obtained from an active infected area and not fijpm dried or 
inactive lesions. Collection of sputum for this examination 
requires especial care to avoid mouth contaminants by previously 
rinsing mouth with sterile saline solution, the expectoration then 
to be placed in a sterile Petri dish and examined within a few 
h*urs, 
b. Fresh preparation for direct study: outline a vaseline circle 
on a slide, place the material under examination within this cir- 
cle, add. a few drops of 1055' sodium hydroxide, cover with a cover 
glass -and examine after a period of digestion - a few minutes to 
twelve hours. Fungi resist the digestive action of the hydroxide 
and retain their form, whereas tissue elements disappear. Avoid 
mistaking artifacts, resembling yeast-like organisms and hyphae, 
c. Stained spreads: Moist materials may be spread oa a slide as for 
bacteriological study, for bacterial stains and for a polychrome 
(Wright or Giemsa) stain. The former stains will reveal bacterial 
content, the latter will assist in the study of any fungi present 
but will also bring out cellular detail and may lead to the 
discovery of a protozoan infection as leishmaniasis. 3. Cultivation! Seventy per cent alcohol may be used to cleanse scales 
or hair of contaminating bacteria, by allowing the specimens to soak 
therein for about one hour prior to placing them on culture media (control 
culture to be made of the alcohol to detect contaminating spores in it). 
Materials, with or without above preparation, should be placed upon 
media having a pH of 5 to 6, unfavorable for bacterial growth. Inoculate 
several Sabouraud’s maltose agar tubes, planting the inoculum into a 
slightly broken surface of the slant. Incubate at room temperature 
(22°C,) and at 37°C. Many fungi show cultural differences when grown at 
22°C and at 37°C, Retain the cultures for at least four weeks before 
considering them negative, 
Observe the cultures daily, but do not open the tubes unless definite 
growth is observed, then make a subculture as soon as the tube is opened# 
are made on Sabouraud's maltose agar or on his conservation 
agar; the Tormer is best for primary isolation, the latter for storage and 
the study of characteristics. In studying the yeast-like organisms, 
corn meal agar plates are useful for isolation, purification and low- 
power study under the microscope. These are best inoculated by so streak- 
ing the plates that the wire produces a slit in the medium. 
Cultures may be studied by observing colony characteristics by naked 
eye, under low power magnification and by slide preparations, fresh or 
stained, for microscopic examination. Instead of removing, as for 
bacterial spread examination, a surface loopful of the colony, it is best 
to remove, with a stiff wire, a fragment of the culture supported intact 
in a fragment of the culture media; this is placed in a drop of lacto- 
phenol (equal parts of lactic acid and phenol) or of water, on a slide, 
and covered with a cover glass. In studying these slides the microscope 
light is modified to provide a subdued light (by lowering of substage 
and control of diaphragm). Study hyphae, branching, budding, sporulation, 
septation, etc., for descriptive report of the cultured fungus. 
Hanging drop cultures in maltose broth, afford another method of 
study, 
4. Animal Inoculation: Most of ttie fungi are not pathogenic for labora- 
tory animals. A few species have animal pathogenicity and animals may 
therefore be used in determining the characteristics of only those few 
species. The form of the fungi seen in tissues tends to be quite different 
from the form seen in cultures. Some of the yeast-like fungi, producing 
as a group, blastomycosis, are pathogenic for animals; diagnostic in- 
formation is, in'these obtainable by the subcutaneous, intramuscular, 
intraperitoneal or intravenous inoculation of the suspected material, or 
preferably, a pure culture of the isolated fungus. The mouse, rat, 
guinea pig, or rabbit may be so used, and observed for a prolonged period 
for local or general evidences of infection. THE RICKETTSIAE 
Description; Extremely small bacterium-like organisms, varying from just- 
visible coccobacilli to filaments 1,5 to 2.0 microns long; found in the 
alimentary tract of certain Arthropods, and in the tissues of man and 
animals during a variety of diseases of which they are the causative agents; 
usually non-motile; Gram-negative, but stain poorly with usual dyes; 
Giemsa stain applied for 10-24 hours is preferred stain for Rickettsiae in 
infected tissues, with which stain they appear as purple dots, or bipolar 
staining bacilli, packed within tissue cells, or extracellularly; fail to 
grow on ordinary culture media, but may be grown in tissue cultures and in 
living chick-embryo cultures. 
Habitat: Many species, all of which are strictly parasitic on Arthropods, 
animals and/or man; the cause of five more or less clearly defined groups 
of diseases in man, many of which are primarily diseases of rodents, or 
other animals, being transmitted to man by the bite of infected lice, ticks, 
mites, or fleas; no or only slight cross-immunity produced between groups. 
They are found in the blood and all the organs of man or other infected 
animal, but show a preference for mesothelial cells, especially those cells 
of the tunica vaginalis, peritoneal cavity and intima of small blood 
vessels. 
1. Typhus fever group; Consists of two types of disease which immunize 
one against the other; the rickettsiae always develop within the cytoplasm 
of infected cells; transmitted by lice and fleas; 
a. Epidemic Typhus is transmitted from man to man by the body 
louse (Pedicuius hurnanus var. corporis), Infective agent - 
Rickettsia prowazeki. 
b. Endemic typhus is normally a pathogen of rats and other rodents 
and is transmitted from rat to rat and rat to man by rat 
fleas (X. cheopis and C. fasciatus)" however, during epidemics 
in man, it may be transmitted from man to man by the body 
louse. Infective agent - Rickettsia mooseri. 
2. Rocky Mountain Spotted Fever Group: Consists of two types of 
disease found in the United States, ’’Western type” and ’’Eastern 
type” R.M.S.F. and several identical or closely related diseases 
under different names in other parts of the world, which immunize 
one against the others; the rickettsia often are found packed 
within the nucleus of infected cells; transmitted by ticks, which, 
when once infected, are capable of transmitting the virus through 
the egg to their offspring: vector for ’’Western type” is 
Dermacentor andersoni and for ’’Eastern type” is D, variabilis. 
Each type is caused by strains of the same virus (Rickettsia 
rickettsi). 
3. Other groups: 
a, Tsutsugamushi fever group: Found in Japan and Qther oriental 
countries (Tsutsugamushi in Japan, Rural or Scrub typhus in 
Malaya, and ’’Mite fever” in Sumatra); causative agent - R. 
nipponica; transmitted by bite of infected larval mite 
’[genus Trombicula), b. Trench fever: Epidemic amongst troops on all fronts in Europe 
during World War I; causative, agent -r- R. quintana; transmitted 
by body louse. 
c. tfQft feverr Found in Australia and western U. S,; causative 
agent - R, burneti; vectors not yet determined, but the tick 
(D. andersoni) has been incriminated in the U, S.' 
Collection and of Specimens for Examination: 
1. Clear, sterile serum of patient for Weil-Felix agglutination test, 
2. Sterile defibrinated or citrated blood for animal inoculations. 
3. Autopsy material, fresh and fixed in formalin, such as portions 
for brain, spleen and tunica vaginalis, for animal inoculation 
and for sectioning. 
Weil-Felix Reaction: This is the most reliable diagnostic test for identi- 
• fying fevers due to rickettsiae. It is based upon the fact that, in 
patients with this group of fevers, agglutinins which react with certain 
varieties of Proteus organisms develop in the patient’s serum after the 
fourth day; in the case of typhus fever, agglutinations as high as 1/4-0,000 
have been reported; a titer of l/lOO or higher is usually considered as 
diagnostic;, see table for interpretation of results. The reaction is based 
upon a macroscopic test-tube agglutination test, using a series of dilutions 
of the patient’s serum c.gainst Proteus antigens. 
The Proteus strains, used,were originally isolated from typhus fever 
patients, but apparently had no connection with the disease. The X19, X2, 
and ’’Kingsbury" strains have been found to be of the greatest value. 
Since the antigens common to the Proteus strains, and the rickettsiae are 
found in the body of the bacilli, the antigens, used, should be prepared 
from non-motile "0" variants, preferably heat killed or alcohol treated. 
The antigens are,then designated as 0X19, 0X2 and 0XK. 
Animal Inoculations: Guinea pigs, rabbits and monkeys are susceptible to 
many of these viruses-when inoculated intraperitoneally with 3 to 5 cc. 
of blood, or of suspension of brain or other tissues. The animal of choice 
for inoculation is a nearly grown, male guinea pig to attain the 
characteristic scrotal lesions. Cross immunity tests by Inoculating several 
guinea bigs, each protected against a specific virus, are of great differ- 
ential diagnostic value. In addition to reactions given below, proof of 
type disease depends upon (1) transfer of infection to second guinea pig 
and ..(2) sterility of ordinary bacterial cultures from the organs. 
Reactions in the guinea pig: 
1. Epidemic typhus; Incubation period, 5 to 14- days; temperature of 
103.5 to 106°F, for 3 to 14 days; no gross, scrotal or testicular 
lesions; brain shows small, proliferative nodules, with perivascular 
infiltration; guinea pig usually recovers; a solid immunity is 
produced, also protecting against endemic typhus. 2. Endemic typhus* Incubation period, Ly to 6 days; temperature of 
103 to 105°F. lasting for 3 to 5 days, showing a saddle back 
curve, with a drop during the second day of fever; scrotal 
lesions, erythema and swelling of the scrotal skin, the tunica 
vaginalis is adherent and Giesma-stained scrapings will show 
the presence of numerous intracytoplasmic rickettsiae; brain 
lesions, similar to but less marked than epidemic typhus; the 
guinea pig never diesc. immunity, same as for epidemic typhus. 
3. Rocky Mountain Spotted Fever. Western type: Incubation time, 2 
to U days; temperature rises rapidly to 106°F.; scrotal lesions, 
erythema and edema, commonly accompanied by necrosis; brain 
lesions, same as for epidemic typhus; 90 to 100% of guinea pigs 
die within week to 10 days; recovered animals are immune to 
both types of R.M.S.F. but not to typhus or other groups. 
R»cky Mountain Spotted Fever. Eastern type: Incubation time, 3 
to 5 days; temperature, 104- to 106°F.; scrotal lesions, erythema 
and edema, no necrosis; brain lesions present, but sparse; 
mortality 25 to 30% immunity, same as for Western type. 
5. nQ.n fever? Incubation time, U to 6 days; temperature, 103 to 
106°F.; no scrotal or brain lesions, the chief post mortem 
finding being an enlarged spleen; frequently kills animal; 
specific immunity produced. 
6, Tsutsugamushi and Trench Fever Groups: Not infective, or produce 
only slight rise in temperature. 
Cultural Examination? This procedure is not routinely employed. The 
rickettsiae will not grow on usual media, or in the absence of sus- 
ceptible tissues, where they develop within the cells; after passage 
through guinea pigs, they may be cultured (1) in a modified Maitland’s 
medium containing 20% guinea pig or horse serum and sterile minced tissue, 
such as the tunica vaginalis of guinea pig, or mouse embryo, or (2) 
in living chick-embryo tissues by direct inoculation into the yolk of a 
sterile egg after 5 to 10 days incubation. 
Microscopical Examination?\ Not usually made; rickettsiae may be found 
in small numbers in the blood and various tissues but their demonstration 
is of no practical differential value. Table - INTERPRETATION OF LABORATORY TESTS FOR RICKETTSIAL DISEASES 
Disease 
Weil-Felix Reaction 
Results of Guinea 
pig inoculations 
0X19 
0X2 
' OXK 
Scrotal lesions 
Brain lesions 
Epidemic typhus 
/// 
/ 
// 
Endemic typhus 
/// 
/ 
Erythema, edema 
i 
Western Spotted Fever 
i 
/ 
/ 
Erythema, edema 
Necrosis 
// 
Eastern Spotted Fever 
/ 
/ 
Erythema, edema 
Sparse 
”0.” Fever 
— 
— 
— 
— 
Trooical Scrub Svphus 
— 
— 
/// 
Not infective 
Not infective 
..Isulaugamiiclii- 
/// 
Mot infective 
Not infective 
Trench Fever 
? 
7 
7 
Not infective 
Not infective CULTURE MEDIA 
I. Preparation and Sterilization of Glassware. 
A. Cleaning: one of the most important steps in the preparation 
of culture media is clean glassware. There are three kinds 
of clean glassware, 
1. Physically cleaned glassware: this is done by washing 
the glassware with soap and water. New glassware may 
be soaked in warm soap water for an hour or two, rinsed 
in tap water several times, and finally rinsed in dis- 
tilled water. Most used glassware may be washed in the 
same manner. If there is anything on the tubes or flasks 
that won’t come off by simple washing, then it is 
necessary to put it in dichromate cleaning solution. 
The glassware should be placed in a soap solution. 
The soap solution is prepared in a sink by taking a 
rather large can and punching holes with a knife in the 
bottom and lower 1/3, Yellow GI soap is sliced into 
small pieces and hot water is run through the can of 
soap. After the desired amount of water is drawn, the 
solution can be made as soapy as desired. Before putting 
dirty glassware into this soap water, all agar, blood, etc, 
should be rinsed out with tap water. This glassware is 
then placed in the soap water for an hour, then cleaned 
with a brush. There is a variety of brushes available, 
and one can be found to clean any kind of glassware. 
Heavy wax pencil marking may be removed with scouring 
soap or powders. Scorched agar and dye stains are 
removed by the bichromate cleaning fluid. Paraffin may 
be removed by xylol or placed in the cresol solution. 
Adhesive tape can be removed by ether or xylol, 
A very small amount of glacial acetic acid may be 
added to the distilled water used for rinsing the glass- 
ware. After the glassware has been run through the 
acidulated distilled it is placed in wire baskets 
or small packing boxes and dried in the hot air sterilizer 
or allowed to drain dry. Physically cleaned glassware 
will answer most purposes of culture media. 
2, Chemically Cleaned Glassware; this is glassware especially 
prepared for use where the presence of a foreign chemical 
may be detrimental to a test, 
/41 spinal fluid tubes (tubes which are to be used 
to draw spinal fluid specimens); pipettes (especially 
those used for titration media) must be cleaned chemically. 
This is done by leaving them in the dichromate cleaning 
solution overnight, or longer. The glassware is removed 
from this solution, rinsed in tap water; distilled water, 
then boiled for one hour in distilled water. The tubes 
or pipettes are then dried. 
Bichromate cleaning solution; potassium (or sodium) 
bichromate, technical - 100 gms,; concentrated sulfuric 
acid (either technical or commercial) - 250 cc,; distilled 
water - 750 cc. Dissolve the potassium bichromate in the 
water and add the acid very slowly. Extreme care must be exercised due to the heat 
generated by the addition of the acid. It is best to 
make the solution in a 2000 m, pyrex flask. 
3. Bacteriologically Cleaned Glassware? this is glassware 
that has had all cultures or contaminated materials 
destroyed. This is done by placing them in a large 
container which has a 2-5% solution of saponated cresol 
for 21+ hours, or placing in an autoclave and sterilizing 
for thirty minutes at a 15 lb, pressure. 
Rubber gloves should be worn while removing the 
glassware from the cresol, as it will burn the hands 
after a few minutes unprotected. Care must be taken in 
handling the glassware, because it is very slippery 
when it comes out of the cresol. 
After the glassware has been left in the cresol long 
enough, place it all into the sink, rinse the cresol off 
thoroughly and remove all the agar in tubes by means of 
a small test tube brush, which has been used for some time 
and is partially worn out. All traces of agar must be 
removed from tubos. Petri dishes and flasks. 
B. Sterilisation of Glassware, 
Gter.ile glassware is essential for accurate work in 
Bacteriology, since it would be difficult to identify an 
organism in the mixed contamination caused by unsterile 
glassware, 
Whereas, in the vegetative forra> many spore-bearing 
organisms are easily destroyed; some of the spores are very 
resistant, so this must be taken into consideration. 
Heat, both dry and steam, is used for sterilizing. Hot 
air sterilizing is preferable. Glassware may be sterilized 
with steam under pressure (that is, in the autoclave) at a 
3/5 or 20 pound pressure for from 15 to 30 minutes, then 
placing in the hot air sterilizer to dry. 
The Hot Air Sterilizer 
This typo of sterilizer is similar to a detached oven of 
a gasoline or oil stove. It is double-walled and usually gas is the 
source of heat, A sterilizer with some sort of adjustment for heat 
should be used. One or two holes should be provided for thermometer, 
and a constant vigilance maintained until the proper temperature is 
obtained. Two or three metal shelves should be used in the sterilizer. 
The temperature may run from 180° C. - 200° C,, 185°C, being 
an ideal temperature. Following is a general time for sterilizing 
and drying various things; 170° C.- 
180° C. 
• 
Item ; 
Minimum Time 
Maximum Time 
Test tubes, flasks, vaccine 
bottles, needles, glass stoppered 
2 1/2 hrs. 
3 1/2 hrs. 
bottles, Petri dishes 
4 hrs. 
mm mm 
syringes and wrapped pipettes 
3 hrs. 
K hr a* 
For drying: about the same length of time, except Petri dishes, 
which will dry in about 30 to 45 minutes, and pipettes, which should 
be left dry at least 4 hours or more. 
; iso0 c. 
- 190° C. 
Test tubes, flasks, vaccine 
bottles, etc* 
1 hr..45 min*. 
2 hrs* 15 min. 
Petri dishes 
. 31/2 hrs. 
- - 
Syringes and pipettes (wrapped) 
2 1/2 hrs* 
3 hrs* 
For drying: 
1 1/2 hrs. 
Tubes and flasks 
1 hr. 
Bottles 
2 hrs. 
3 hrs. 
Petri dishes 
30 min. 
- - 
Pipettes 
3 hrs. 
- - 
190° C, 
- 200° C. 
Caution: Cotton and paper and cloth disintegrate at temperatures 
above 190° C,, so when sterilizing-, watch your glassware carefully. 
Test tubes, flasks, needles, etc. 
1 hr. 
1 1/2 hrs. 
Petri dishes * 
21/2 hrs. 
- - 
Syringes and Pipettes (wrapped) 
2 hrs. 
2 1/2 hrs. 
For drying: 
Tubes and flasks 
45 min. 
Bottles 
1 hr. 
1 1/2 hrs. 
Petri dishes 
15-20 min. 
- - 
Pipettes 
2 hrs. 
3 hrs. 
Cotton plugs in test tubes should be a cream or light tan color when 
sterile. Cloth should be light tan to a light brown, . 
Petri dishes may be placed on the top shelf of the sterilizer. 
Avoid putting dripping glassware into the sterilizer in such a manner 
as to allow water to drop on hot -glassware. If a wooden or cardboard 
box is used as a container for drying. glass?/are, it should be placed 
on the bottom away from the sides of the sterilizer, if the temperature 
exceeds 190° C, Tubes containing cork stoppers should also be placed on the 
lower shelf of the sterilizer when temperature is 190°C. C, Preparation of Glassware for Sterilization. 
1* Syringes. 
a. 30 cos 30 cc syringes are sterilized for the purpose of 
drawing blood for blood agar, ’'chocolate agar”, and 
blood for typing serum. 
The syringe and needle are prepared together. After 
the needles have been well sharpened, put them on the 
syringe tightly with pliers, being sure the bevel of the 
needle is facing up with the graduations on the barrel of 
the syringe. Then the stylet is inserted and a Wasserman 
tube is put over the needle and stylet for protection. 
Next a piece of unbleached muslin about 1 foot square 
is cut and laid down on a table. Place the syringe 
diagonally and roll up in the muslin securely, tucking 
in the corners. Then tie a string around it, and it is 
ready for sterilizing, 
b. 10 cc: the 10 cc syringe is prepared the same as a 30 
cc, only, instead of wrapping in muslin, the syringe may 
be placed in a large test tube and muslin (about 4 inches 
square) tied over the oop of the syringe and tube. 
The syringe and needle may be sterilized separately. 
Then, wrap the syringe in muslin, and put the needle with 
a stylet point down in a Wasserman tube, plug with cotton 
and sterilize. Needles used for taking Fassennans, blood 
chemistry, etc., are prepared in the same manner. 
2, Pipettes: pipettes may be sterilized in metal cans made for 
that purpose, or they may be wrapped in paper individually 
and sterilized. Toilet tissue is an excellent paper to wrap 
pipettes in, 
3. Flasks: Erlenmeyer flasks of all sizes may be sterilized by 
taking a piece of cotton and making it into a roll, according 
to the size of the flask. Place this cotton roll in a piece 
of gauze from 3 inches to 6 inches square, then insert into 
the mouth of the flask, 
4* Test Tubes: test tubes are plugged with Grade A cotton. 
There are several methods of doing this but I personally have 
found that the best method is to roll the plugs. This is 
. done by taking a piece of cotton about 2 l/2 inches wide and 
about 3 inches long;'fold the cotton so that it is about 
1 1/4 inches wide and 1/4 inch thick. Then roll the cotton 
rather tightly.and insert into tube. This method is somewhat 
difficult at first but with practice, a good durable plug can 
be made. All test tubes, and vacinne bottles are plugged in 
this manner, 
5* Bottles. 
a. Wide-mouthed specimen bottles are plugged either with 
gauze and cotton or with a #20 cork stopper, and a piece 
of muslin over the top. These bottles are used for 
stomach contents, sputum, and for shipping specimens from 
one station to another. b. 6 oz. glass-stoppered bottles are provided for the 
shipment of milk and water specimens for 
bacteriological examination, A piece of muslin 
about 4- inches square is tied securely over the 
top of the bottle. 
c. Small vials are prepared for feces by inserting 
a small metal paddle in a cork and putting it in 
the vial and sterilizing. 
d. 24.0 cc. glass-stoppered bottles are sometimes 
used for the shipment of food for examination. 
These are sterilized in the same manner as the 6 
oz. bottle. All test tubes, flasks, pipettes, 
syringes, etc., must be absolutely dry before being 
sterilized, 
II. Preparation of Culture Media, 
A. General Considerations; almost all the work done in 
bacteriology depends entirely upon culture media. 
1, What is Culture Media? 
Culture media is artificial food for bacteria. 
It is composed of organic substances and inorganic 
or mineralsOf the organic we have chiefly a 
nitrogenous substance called peptone, meat extract, 
blood, serum, eggs, agar and many others. Minerals used 
are various salts, such as sodium chloride, potassium 
phosphate, sodium citrate and others. 
Now bacteria are very exacting in their food 
requirements, more so than human beings, because if a 
person is hungry, he will eat almost any food set before 
him. But bacteria are different, some will grow on 
one thing in which another family of bacteria can’t 
possibly live. 
So, if the bacteriologist is to do accurate work, 
he must have a wide choice of well prepared media to 
work with. 
B. Determination of the pH of Media Titration, 
1. pH is the log reciprocal of the hydrogen ion con- 
centration, or to put it simply, it means the acidity 
or alkalinity of a substance. The pH scale is zero 
to fourteen. Seven being neutral. Above seven is 
alkaline or basic. Below seven is acid. Bacteria have what is called their ’’optimum 
pH.” If the pH of the medium deviates from the 
optimum pH of the bacteria to too great an extent, 
the growth of the bacteria will be inhibited 
or stopped altogether. 
Most bacteria grow best in a slightly alkaline 
medium. It is my belief that pH is the most important 
consideration of Culture Media, 
The technic most commonly used in the adjustment 
of the pH of media is the colorimetric method. 
2, Reagents, equipment and apparatus required. 
a. Pipettes. 
(1) 10 cc. graduated in 0,1*s; two or more; 
1 cc. graduated in 0.01’s. 
b. One 25 cc, graduate,, graduated in cc’s, 
c. Culture tubes, holding approximately 20 cc, 
d. pH standards: Phenol - red standards are 
used. pH 6.8; pH 7.0; pH 7.2; pH 7.4; pH 
7.6; pH 7.8; pH 8,0; pH 8.2; and pH 8.4 
e. Phenol-red indicator. 0.02$ aqueous solution. 
An indicator is a substance, usually a 
dye, which has the ability to change its color 
progressively as the pH changes. For example, 
phenol red, when in an acid solution is lemon 
yellow. At pH 6.8, it has a slight pink 
color. At pH 7.0, it has a definite pink 
color, and as the pH rises to pH 8.4, it 
becomes pinker until it is red. Indicators 
have what is called their ’’range,” .that is, 
where the color change begins and ends. The 
range of phenol-red, is pH 6.8 - pH 8.4. 
The phenol-red standards may be obtained 
from the Army Medical School at Washington, 
f. Normal NaOH. 
g. Distilled water. 
h. A wooden block painted black, 3 inches long, 
2 inches wide and 2 inches high, with four 
holes large enough to accomodate your 
standard and three culture tubes,, and four 
holes in the side about 3/4 to 1 inch apart, 
two on either side of the block permitting 
one to see through the block. 3, Procedures 
a. Take the 2$ cc- graduate and get exactly 19 cc of 
distilled water. Add one cc of normal NaOH, Mix 
thoroughly by drawing the distilled water into the 1 
cc pipette and blowing it out,. Repeat this several 
times. This gives you N/20 (1/20 normal) NaOH, 
b. In the back row of two holes in the block, place the 
followings on the left, a tube with 10 cc of distilled 
water; on the right, your pH standard, for example; 
pH 7.6 standard, 
c. Place two empty tubes in front of the distilled water 
and standard. In the tube in front of the pH standard, 
put 10 cc of the media that is being tested. 
In the other tube put 9,5 cc of media and 0,5 cc 
phenol rod indicator. Mix by inverting one time. If 
the tip of the thumb is dipped into the medium, it will 
have the same reaction and the contents of the tube will 
bo unaffected, 
d. Compare the color of the medium and the indicator in the 
one tube with the color of the standard and other tube 
together, 
e. If the colors do not match, the medium is too acid. 
Slowly add N/20 NaOH, inverting from time to time to 
mix. When the color on the left compares with right, 
then, you are ready for the next step; 
lit us assume that we have 8000 cc of media. If it 
takes 1,3 cc of N/20 NaOH for 10 cc of media, then for 
8000 cc it would take; 
10 cc - - 1,3 cc 
100 cc - - 13 cc 
1000 cc - - 130 cc 
8000 cc - - 1040 cc of N/20 NaOH 
Obviously we cannot add 1040 cc because it would 
dilute the media, so we calculate the amount of normal 
NaOH. That is done by dividing 1040 cc by 20s 
52 cc 
20/IO4O cc 
Hence, 52 cc N NaOH is added to the medium and thoroughly 
mixed. Then the pH is checked. If the reaction isnft 
satisfactory, repeat the titration until it is. The pH 
is usually adjusted at or near the boiling of the medium. 
Usually, during sterilization of media, the reaction 
changes somewhat, so about 0,2 pH should be allowed# C. Clarification: Media must be clarified. This is done by 
centrifuging, by filtration, by coagulants, and by decanting. 
All broths may be filtered through paper. All agar is 
filtered through cotton and gauze. 
D. Distribution, 
1. The stock media is kept in Erlenmeyer flasks. The 1 
liter flasks containing 600 cc; the 2 liter flasks 
1500 cc; and 500 cc. flasks 300 cc. 
2* Tubing of media is done by placing in a large container 
or funnel to which is attached a rubber hose and controll- 
ed by a pinchcock. 
3. For agar slants, about 5 cc; for unslanted agar, 10 to 
15 cc; and for fluid media 10 cc. Agar media must not 
be allowed to cool too much while tubing. 
E. Storage; after sterilization, the culture media should be 
kept in a refrigerator. Paper should be tied over the neck 
of flasks or lead foil may be used. This prevents the 
entrance of fungi and contaminating of the contents. 
F. Sterilization of Culture Media: all media must be 
sterilized; this is usually done in an autoclave, or with 
steam as in the Arnold Sterilizer, by filtration through a 
Berkfeld Filter, in an inspissator, or by boiling water 
bath. All the stock agars and broths are sterilized at 15 
lb, pressure for 15 minutes. Most sugar broths are steri- 
lized at 10 lb. pressure for 10-15 minutes. 
1. Fractional or intermittent sterilization: 
a. Media is s-terilized for 3 successive days either by 
boiling or in the Arnold sterilizer, for from 30 
to 45 minutes, 
b. On the first day of sterilizing, the vegetative 
forms of bacteria are killed, and some spore forms 
are activated. On the second day, those spores 
activated from the first day have been changed to the 
vegetative forms. These are killed by the second 
sterilizing. The most resistant spores are activated 
and on the third day, the vegetative forms of the 
most resistant organisms are destroyed. This may be 
illustrated in the following chart; 
j Vegetative Form 
Spore Form 
1st Day 
- * t s J 
S 
2nd Day 
; y ; s 
re- 
3rd Day 
s y s ; 
sistant spores) 
• • J 
G, Pour-Plates; are media distributed in Petri dishes, such as 
blood agar plates, EMB plates, etc. All the plates are 
poured aseptically. 10-20 cc. of agar is poured. Enough, so 
that the media will be about l/8 inch thick. This is done by 
first flaming the neck of the flask containing the agar. 
Raise the top of the Petri dish in such a way that one edge 
is touching the table top, then pour the desired amount. While the agar is still warm, flame the entire surface of 
the plate to destroy what bubbles may be present, 
H. During the process of cooking media we figure 25% of the volume to 
be lost through evaporation. There are two ways of taking care of 
this loss. 
1. Either make a mark on the side of the pot or measure with a 
stick and add distilled water to the original volume, 
2, The other method is to take 25% of the volume and add to 
the original volume before you start cooking. For instance, 
if the volume of media being prepared is 8000 cc» then 25$ 
of this would be 2000 cc. This 2000 cc is added to the 
8000 cc. About 100-125 cc will boil off in one minute, so 
you would boil vigorously for about 20 minutes. 
I. Differential Media: Many groups of organisms are very' similar 
and can only be identified by certain reactions on various 
media. This media is called “differential media” and includes 
the following: 
1. Sugar Broths. 
2. Russell’s Double Sugar. 
3. Simmon’s Citrate Agar. 
4. EMB agar, 
5. Clark and Lub’s Medium. 
6. Jordan’s tartrate agar and others. 
These various media are especially useful in 
differentiating organisms of the colon - Typhoid - Dysentery 
Groups. Usually all of the above media is used with the 
same organis., and identification is clinched, 
J. Solid Media: solid media is prepared with agar, gelatin, or 
coagulated eggs or serum. Almost every broth made up, there is 
a corresponding agar, for instance, beef extract broth, and 
beef extract agar. Agar is the word used both for the medium 
and for the substance which makes the medium solid. Agar is 
made from a certain species of Asiatic seaweed; it is a glue- 
like substance. It may be dissolved in hot water, and when 
cooled forms a solid. From ly to 5% is usually used. 
K. Liquid Media: Liquid media is the various broths prepared, 
L. Semis*lid media: This is prepared by adding a small amount of 
agar or gelatin, (0,5# agar) 
Now I will give you some formulae commonly 
used in routine bccteriological work: 
III. Formulae. 
A, Broths: Beef extract broth (Meat) (For routine use), 
1. Beef extract 3 gms, 
2. Peptone 10 gms, 
3. Sodium Chloride 5 gms. 
A. Distilled water 1000 cc. Add the weighed ingredients to the distilled water 
and dissolve manually or by heat. Adjust the reaction so 
that the final pH will be between 7.4 - 7.6 filter 
through paper and distribute in tubes or flasks. Sterilize 
16 lbs. pressure for 15 minutes. 
Nutrient Broth 
(Sta 
ndard for water analysis) 
Beef extract 
3 gms. 
Peptone 
5 gms. 
Distilled 1^0 
1000 cc.. 
Prepare as directed above. Adjust reaction between 
pH 6.4 and 7. 
Meat Infusion Broth 
Beef or veal round, free from fat, 
ground 500 gms. 
Distilled water 1000 cc. 
Mix the meat and water and infuse in the icebox for 
18-24- hours. Heat in e. boiling water bath over a low 
flame for about 1 hour. Filter through a cotton and gauze 
filter. Add 5 gms. of sodium chloride and 10 gms. peptone 
to 1 liter of the broth. Adjust the reaction to pH 7.8, 
and prepare in the same manner as beef extract broth. 
Lactose Broth 
(Standard for water analysis) 
To 1000 cc of nutrient broth, add 5 gms. lactose (0.5%); 
dissolve tube in a fermentation tube, and sterilize in 
autoclave 15 lbs. for 15 minutes. 
Clark and Lub’s 
Medium 
(For Voges-Proskauer 
and Methyl-Red 
Tests) 
1. Peptone (Difco Proteose 
5 gms. 
2. Dextrose (C?) 
5 gms. 
3. Potassium Phosphate K HPo 
4-. Distilled water 
5 gip. 
1000 cc. 
Dissolve 1, 2 and 3 in 4- 
with heat. Filter and tube 
in Loeffler tubes. Sterilize 
by boiling in a water bath 
30 minutes on three successive days. 
Brain Broth or Blood Culture 
Medium 
(for blood cultures). 
1 calf brain 
Dextrose 10 
gnu 
Sodium Citrate 10 
gnu 
Infusion broth (pH l.U - 7.6) 1000 
cc. Dissolve the dextrose and sodium citrate in the 
infusion broth. Filter, wash and remove all membrane 
and blood from the brain and chop it into pieces as large 
as the end of your thumb. Wash a number of marble chips 
the size of the end of a finger. Place the marble chip 
and piece of brain in a large tube and the filtered broth 
with the dextrose and sodium citrate. Sterilize 15 
lbs. for 15 minutes. 
1. Dextrose 
2. Maltose 
3. Mannit 
h» Xylose 
5. Arabinose 
Sugar Broths 
6. Lactose 
7. Saccharose 
8. Dulcitol 
9. Inositol 
10. Inulin 
To 100 cc. of beef extract broth pH 7.U - 7.6, add 0.5 
gm. of the desired sugar, and 0.1 cc. of Brom-Cresol-Purple 
indicator 1.6$ alcoholic solution. Tube in small fermentation 
tubes 5-10 cc. Sterilize by boiling in water bath '30 
minutes for 3 days. Or sterilize in the autoclave at a 
pressure not exceeding 7 lbs. for 10 minutes. 
B. Agars: Sterilize all agars 15 lbs. for 15 minutes, unless 
otherwise stated. 
Beef Extract Agar 
(For routine use) 
To 1000 cc. of Beef Extract Broth, add 20 gm. of agar, 
Heat the broth to about 80°C., before adding the agar. Stir 
constantly while the agar is being added to prevent lumping 
and scorching. When the media begins to boil, titrate. 
Adjust the reaction to pH 7.6. Filter through a cotton and 
gauze filter. This is stock medium and is used for plain 
agar plates and slants, and as bases of blood agar and 
Russell’s double sugar. After filtering, the agar should be 
distributed in f>00 cc, and 1 liter flasks. Put 300 cc, of 
agar in the 5>00 cc, flask, and 600 cc. in the one liter 
flask. 
Levine’s Eosin - Methylene - Blue Agar (EMB) 
(Standard for water analysis) 
1. 
Pepton (Difco) 
10 
gm. 
2. 
KoHPO) (Pot. phosphate, dibasis) 
2 
gm. 
3. 
Agar 
15 
gm. 
lu 
Distilled water 
1000 
cc. 
Prepare in the usual manner, adjust pH to 
7.1*- 
7.5, distribute in 1 liter flasks 600 cc. 
This 
is best base agar. 
5. 
Lactose 10 gm. of 20$ sterile sol. 
50 
cc. 
6. 
Eosin, yellowish, 2$ aqueous sol. 
20 
cc. 
7; 
Methylene blue, 0.325$ aqueous sol. 
20 
cc. 
Just before use, to each 100 cc. of the base 
agar. 
Lactose, 20$ sol., sterile 
5 
cc. 
Eosin, yellowish, 2$ 
2 
cc. 
Methylene blue 
2 
cc. Mix well and pour into Petri dishes. 
Use? For the routine determination of organisms of the 
colonaerogenes groups in water. 
Note? For the isolation of pathogenic organisms from 
feces, it is necessary to reduce the dye content one half. 
Sterilize the lactose 10 lbs. pressure ten minutes. 
Russell’s Double Sugar 
To 100 cc. of beef extract agar pH 7.3 - 7.4, add; 1 
gm. of lactose and 0.1 gm. glucose, ""his may be dissolved 
in a minimum of water, 5 cc. 0.02% aqueous solution phenol 
red. Stepilize in the autoclave at a pressure of P lbs. 
for 25 minutes. Slant with a deep butt. Upon solidifying 
check the reaction with known cultures of E, Coli, Para 
"B" and E. Typhosa; the stab and streak method being 
employed. The reactions are as follows? 
E, Coli - - acid and gas throughout the whole tube. 
Para ’'A,f or nB” - acid and gas butt. Alkaline slant. 
E. Typhosa * acid butt alkaline slant. 
Sabourand’s Medium 
(for fungi) 
Peptone 10 gm. 
Maltose, crude 40 gm. 
Agar 20 gm. 
Distilled water 1000 cc. 
Adjust reaction to pH 5.2; tube and slant; autoclave 
at P bis. for 30,minutes. 
Blood Agar 
To 100 cc's of Beef Extract agar (pH 7.2 - 7.4), 
melted and cooled to about 40°C. add: 5 cc’s of sterile 
citrated blood. Mix and pour Into either sterile test tubes 
(agar slants) or sterile petri dishes (blood plates). 
Incubate at 37°C. for 24 hours to insure sterility. BACTERIOLOGICAL EXAMINATION OF WATER 
AND*"MILK 
I, Water: 
It is customary to submit specimens of drinking water from 
army stations for bacteriological examination at frequent 
intervals. If laboratory facilities are available, this is 
done locally; otherwise, it is shipped to the nearest Service 
Command Laboratory, or to the Army Medical School. 
The purpose of a bacteriological examination of milk and 
water is to determine the potability. Generally, two 
different tests are done (l) to determine the total number of 
bacteria by means of the standard plate count; (2) to ascertain 
if there has been fecal contamination by demonstration of 
organisms of the Colon-Aerogenes Group. 
A. Apparatus: all apparatus must be sterile. 
1. Glass stoppered 120 cc. sample bottles, protected by 
muslin cap. 
2. Pipettes 10 cc, 1 cc, with cotton plugs in the mouth 
ends, 
3. Glass test tubes for making dilutions. 
4. Petri dishes, 
B, Culture Media. 
1, Final reaction for broth and Agar Media should be 
between pH- 6.4, and 7. 
2. Following is a list of media used: 
a. Lactose broth. 
b. Nutrient Agar. 
c. Clark and Lub’s Medium, 
d. Simmon’s citrate agar slants, 
e. E.M.E. plates. 
f. Brilliant Green Bile Lactose 5$ and 2$, 
g. Dulcitol, 
C, Reagents. 
1. Sterile distilled or tap water, 
2. 0,04$ solution of methyl red in 60$ alcohol, 
3. 10$ aqueous solution K0H. 
4-. Xylol. 
$. 90$ alcohol. 
6. Loeffler’s Methylene Blue. 
7. Phenolphthalein indicator, 1$ in 50$ E, alcohol. 
8. Concentrated HpSO,, 
9. NaOH, N/10. 
D. Special Apparatus. 
1. Babcock pipettes 17,6 cc. 
2. Babcock fat bottles. 
3. Lactometer with Quevenne scale, 
4., Wooden racks with holes for 5 large fermentation tubes 
and 2 small fermentation tubes. 
5. Lens magnifying 21/2 times for plate counts. 
6. Incubator with temperature set at 37.5°0. 
7. 56° Water Bath. 
- 8. - Centrifuge, E. Collection of Specimen. 
1. Must be done under as sterile conditions as possible. 
2. Should be representative. 
3. Collected in a sterile glass-stoppered bottle. 
4-. If some time elapses between receipt of specimen and 
time tests are made, it should be placed in a 
refrigerator. 
F. The Total Bacterial Count. 
1. Dilutions. 
a. Undiluted: 1:10; 1:100; 1:1000, etc. 
b. Set up two test tubes containing 9 cc. of sterile 
water: in the first tube and 1 cc. of undiluted 
water and in the second transfer 1 cc. of water 
from the first tube. 
2. Plating. 
a. Transfer 1 cc, amounts from the 1:10 dilution and 
the 1:100 dilution to two sterile Petri dishes, 
b. Add about 10 cc. of melted nutrient agar, cooled 
clown to 4-0°C. to each Petri dish. 
c. Mix by tilting and rotation. 
d. Allow to solidify and incubate. 
e. Pour a plate of nothing but agar, and 
f. Pour a plate using 1 cc, of water which was used 
for the dilutions, 
g. These plates are controls. 
3. Incubation: plates should be incubated for 24- hours. 
4-. Counting. 
a. Use a lens with a magnification of 2 l/2 times. A 
special ruled apparatus is made for plate counting. 
b. Try to prepare dilutions so that at least 2 plates 
will give from 30 to 300 colonies. 
c. Count the total number of colonies and multiply 
by the dilution factor. For example, if 140 
colonies are counted on the plate 'where the 1:10 
dilution was made, then 14-0 x 10 *= 14-00 colonies, 
which is the plate count, 
d. Under 500 colonies per cc. is considered potable 
raw water. Over 500 colonies, unpotable. 200 
colonies per cc, or less is potable for treated 
water, 
G. Tests for the presence of Coli-Aerogenes. 
1. Introduction and definition, 
a. Gram-Negative non-spore forming bacilli which 
ferment lactose with gas formation and grow aero- 
bically on standard solid media. 
b. Formation of 10$ gas or more in a standard lactose 
broth fermentation tubes within 24- hours at 37°C. is 
presumptive evidence of the presence of members of 
this group, 
c. Appearance of lactose-splitting colonies on E.M.B. 
plates made from a fermentation tube with gas 
confirms considerably the presumption that gas 
formation was due to the presence of members of 
Coli-Aerogenes Group, d, To complete the demonstration of the presence of 
members of this group, it is necessary to show that 
one or more of the aerobic plate colonies are gram- 
negative non-spore-bearing bacilli, which, when 
inoculated into a lactose broth, fermentation tube, 
form gas. 
2. Presumptive Test, 
a. Inoculation 
(1) At least twice as much media as water. 
(2) Inoculate 5 large fermentation tubes with 10 
cc’s water, two small fermentation tubes, 1 
with 1 cc, and the other with 1 cc, of the 1:10 
dilution mentioned before. 
b. Incubation and reading. 
(1) Incubate the tubes for 48 hours. Examine at 
24- hours and 48' hours and record gas formation. 
Records should be such as to distinguish 
between; 
(a) Absence of gas formation. 
(b) Formation of less than 10$ gas in the 
inverted tubes. 
(c) Formation of more than 10$ in the small 
tubes, 
c. Positive Presumptive tests formation of 10$ or more 
gas within 24 hours constitutes a positive pre- 
sumptive test. 
d. Doubtful Test. 
(1) No gas or less than 10$ within 24 hours. 
(2) The presence of gas in any amount at 48 hours, 
e. Negative Test; absence of gas formation after 48 
hours, • 
3. Partially Confirmed Test, 
a. At the end of 48 hours, if gas has formed in tubes 
containing less of the water specimen, then plate, 
(For example, if water has been tested in amounts of 
10 cc, 1 cc and 0.1 cc, and gas is formed in 10 cc 
and 1 cc, not' in 0.1 cc, the test need be confirmed 
only in the 1 cc amount). 
b. Make transfers to plates as soon as possible after 
gas formation. If gas occurs at the end of 24 hours, 
then transfers may be made, 
c. Make one or more Endo or E.M.B, plates from the tube 
which shows gas formation from the smallest amount of 
water tested. 
d. Incubation of plates: incubate the plates for 18 to 
24 hours. 
e. Results, typical and atypical: 
.. (l) If typical colonies have developed during the 
time of incubation, the. partially confirmed may 
be considered positive. 
(2) If no typical colonies develop within 24 hours, 
incubate- for another 24 hours. 
(3) If no typical colonies develop after 48 hours, 
then two or three colonies most likely to be 
Coli-Aerogenes, are chosen. 4-. Completed Test, 
a. After the colonies are selected, either typical or 
atypical, they are inoculated into the following. 
(1) 2 tubes of Clark and Lub’s. 
(2) 1 Simmon's Citrate Agar. 
(3) 1 dulcitol sugar fermentation tube. 
(4) Brilliant Green Bile Lactose, 5%, small tube. 
b. All incubated 24. hours to 36 hours, except Clark and 
Lub* s. 
c. Following is the reaction obtained on the above 
mentioned media; 
(1) For Colon Group; 
(a) Voges-Proskauer Test-Neg. 
(b) Methyl Red Positive 
(c) Citrate Negative 
(d) Dulcitol Acid and Gas 
(e) Brilliant Green . Gas 
(2) Aerogenes Group. 
(a) V.P Positive 
(b) M.R. Negative 
(c) Citrate Positive 
(a) Dulcitol Negative 
(e) Brilliant Green Gas 
Brilliant green bile lactose is used to 
rule out all organisms that ferment lactose, 
but are not of the Coli-Aerogenes Group. 
d. Procedure for Voges-Proskauer (V.P,)’ and Methyl Red 
(M.R.) Tests; 
(1) V.P. - to a Clark and tub's tube, inoculated and 
incubated for 36 to 48 hours, add 5 cc. of 10% KOH 
(or the same amount of KOH as broth). The color 
develops slowly and a long time should elapse 
before reading, A pink fluorescence indicates a 
positive, no color, a negative reaction, 
(2) M.R. - add 5 drops of an 0.04-% solution of Methyl 
Red in 60% alcohol to a Clark & Lub's tube, which 
has been inoculated and incubated 4-8 to 92 hours. 
A red color is positive and a yellow color 
negative, 
e. Reaction of Simmon’s Citrate; Simmon's Citrate Agar 
slants are dark green and if positive, there is a dark 
blue slant. 
5. Swimming Pool Water - water in swimming pools should be of 
the same standards as drinking water. 
6. If colonies are found on the E.M.B, plate resembling 
organisms of the Typhoid Dysentery Group, Russell’s Double 
Sugar tube should be inoculated, 
7. Sewage; sewage and grossly polluted water may be examined 
the same as other water, but higher dilutions must be used. II. Milk. 
A, Enumeration of Bacteria 
1. Standard Plate Count, 
a. Dilutions: dilutions are made as with water, 1:100, 
1:1000 are usually used. The milk specimen should 
be shaken 25 times with an up and down motion of 
about 1 foot. 
b. Transfer 1 cc. amounts from each tube of diluted 
milk (sterile water is used), to a sterile Petri 
dish. 
c. Nutrient agar is used as in water; it should be 
cooled to 4.0 to 45°C. 
d. Incubate the plates at 37°C. for 48 hours. 
e. Colonies are counted and the number determined as 
in water analysis. 
f. Standard Plate counts may be made on buttermilk, 
cream, chocolate milk, raw milk, ice cream, etc. 
2. Test for the presence of organisms of the Coli-Aerogenes 
Group. 
a. Inoculate three small fermentation tubes containing 
2% Brilliant Green Bile lactose with the following: 
Standard Plate (1:10 dil.Jj 2nd tube, 1 cc. of the 
1:100 dilution; and the 3rd tube, 1 cc, of the 
1:1000 dilution. 
b. Incubate for 4$ hours. 
c. If gas is present, proceed with the partially con- 
firmed and completed test as described under WATER 
G3 and 4-). 
3. Microscopic Count of Bacteria (Breed Method). 
a. Exactly 0.01 cc. of milk is drawn up into a special 
capillary pipette (M.S. item No. 43540) and is 
spread over an area of 1 sq. cm. on a microscopic 
slide. 
b. The uniformly spread film is dried in a warm place 
for not more than 5 to 10 minutes. 
c. (1) Immerse in Xylol one minute to remove fat. 
Drain and, allow to dry, 
(2) Fix for one minute in 90% alcohol. 
(3) Stain with Loeffler's methylene-blue solution. 
(4) Rinse with water, 
(5) Decolorize with alcohol until only a faint 
blue tint is left, 
(6) Dry and examine microscopically, 
d. The number of bacteria per cc. of milk is estimated 
by counting all the organisms within a given area 
in a microscopic field, this area having been 
carefully measured and its ratio to a square centi- 
meter determined. At least 1/100,000 part of a cc. 
of milk is to bs examined. The microscope should 
be so adjusted that each field covers a certain 
known fraction of the area of a square centimeter. e. Advantages. 
(1) Makes possible the counting of all 
bacteria living and dead, and can there- 
fore be used on specimens preserved with 
formalin or other antiseptics, 
(2) It is more economical and can be carried 
out rapidly in the field. 
(3) It gives information concerning the 
sanitary condition of the dairy and the 
contaminated milk before pasteurization, 
f. Disadvantages, 
(l) The small amounts of milk used lead to 
inaccuracy and the large factors used 
in estimating the bacterial count introduce 
a large factor of error, 
(2 Much time must be spent on the counts, 
in order to reduce this factor of error, 
especially when examining very good milk, 
(3) The individual technic of the counter 
may be responsible for a greater variation 
in results than when the plate method is 
used. 
g. The ratio used in comparing the standard plate 
count with the Breed Count is estimated at 1 to 
4* 
h. For preserving milk for shipment, use 1 cc. of 
formalin (37$) full strength, to 120 cc, of 
milk, 
4. Methylene Blue Reduction Method: known as the 
reductive test. It is useful where laboratory 
facilities are limited, or making a rapid inspection 
of a large number or samples. It is based on the 
fact that when methylene blue is added to milk, 
the color may be reduced or lost, depending on the 
oxygen consumption of the bacteria present, 
a. Methylene Blue Reagent. 
(1) Standard tablet available manufactured by 
National Aniline Company. 
(2) Dissolve 1 tablet in $0 cc. of boiling 
distilled water and add 150 cc. of cold 
distilled water. b. Technique. 
(1) Measure 10 cc. of milk in a thick-walled 
test tube fitted with a rubber or cork stopper, 
(2) Add 1 cc. of the certified methylene blue 
solution. 
(3) If the blue color is not evenly distributed, 
invert and mix uniformly. 
(4) Place in a water bath and heat to 37°C, This 
temperature is maintained until test is 
completed. 
(5) Tubes should be observed at 15 minute intervals 
and the end point recorded (disappearance of 
the blue color). 
c. Interpretation of Results. 
Glass 1 - Good milk, not decolorized in 5 1/2 hours. 
Glass 2 - Milk of fair average quality, decolorized 
in less than 5 l/2 hours, but not less than 2 hours. 
Class 3 - Unsatisfactory milk, decolorized in less 
than two hours, but not less than 20 minutes. 
Glass 4- - very unsatisfactory milk, decolorized in 
20 minutes or less. 
B. Tests other than Bacteriological. 
1, Specific Gravity. 
a. Ranges between 1.027 and 1,035. 
b. May be determined by means of a hydrometer. The 
Quevenne lactometer with a range of 1.015 - 1.04-0 
is usually used, however, the hydrometer designed 
for taking the Sp.Gr, of urine may be used, 
c. Scale of Quevenne lactometer reads in two figures, 
which are the second and third decimals of the full 
sp. gr, reading, e.g,, a milk of sp, gr. 1.030 
would have a Quevenne reading of 30. 
d. Temperature should be 15*6 C. - corrections for 
readings close to this temperature are made by 
adding 0,0002 to the observed sp.gr. for each 
degree above and subtracting 0,0002 from the sp.gr, 
observed for each degree below 15.6° C, Such 
corrections should be made within a range of 13 
to IB0 C. 
2. Percentage of Fat - may run from 2.00$ to 4.4-5$. 
a. Babcock Method. 
(1) Apparatus and Reagents. 
(a) Test bottles (Babcock) 
(b) Centrifuge - speed 600 - 1200 r.p.m, 
(c) Pipettes 17.6 cc and 17.5 cc graduations 
only, 
(d) Sulfuric Acid in commercial or technical, 
with specific gravity 1.82 to 1.83 at 
20° G. 
(2) Procedure, 
(a) Transfer 17.6 cc of milk to the test 
bottle and 17.5 cc, of sulfuric acid, 
preferably not all at one time, pouring 
it down to bottleneck so as to wash down 
all traces of milk. Temperature of the 
add should be between 15 and 20°C, Shake 
until all trades of curd disappear; then (b) Transfer bottle to centrifuge, counter- 
balance it and whirl for 5 minutes 
after the proper speed is attained. 
(c) Add soft or distilled water at 60°C,, 
or above, until the bulk of the bottle 
is filled, 
(d) Centrifuge 2 minutes. 
(e) Add hot water until the liquid approaches 
the top graduation on the scale. 
(f) Centrifuge one minute. 
(g) Place bottle in a 55 to 60°C, water 
bath. Immerse it to the level of the top 
of the fat column. Leave in water bath 
10-15 minutes. 
(h) Remove bottle and with the aid of 
calipers or dividers, measure the column 
of fat from the lowest surface to the 
highest point of the meniscus. 
(i) Fat column at time of measurement should 
be translucent, golden yellow or amber 
color, and' free from visible suspended 
particles. LABORATORY TECHNICIANS MANUAL 
PART III 
INDEX 
Chapter Page 
1. General Chemistry ............ 1-1*3 
2, Blood Chemistry . .i*l*-5l 
I Protein Free Filters 1*1* 
II Blood Sugar 1*1* 
III Blood Sugar: Lewis and Benedict Modified ...... 1*6 
IF Marshall Test 1*7 
V Hall Test 1*9 
Tables and Equivalents, 52 
VI Non-Protein Nitrogen . 52 bl 
VII Blood Urea 52 b2 
VIII Creatinine 52 b2 
IX Chlorides , * 52 b3 
X Plasma Proteins 52 bi* 
XI Calcium 52 bl* 
XII Icterus Index 52 bl* CH5MIS TRY 
1. Scope and definitions - Chemistry is a branch of 
natural science. It is, in fact, a branch of physics, which is 
the science dealing with changes in matter, with the composition 
of matter, and with the properties of matter, 
* / 
a. Physics deals with changes in the state of matter, 
or with the properties of matter in large masses or general as- 
pect, It is also concerned with changes in matter which do not 
involve molecular rearrangement or composition, 
b. Chemistry deals with two main groups of 
phenomena, namely; 
(1) The composition of substances, especially the 
structure of their molecules, and the properties which are 
dependent upon this structure. 
(2) Changes in the composition of matter or of 
molecular structure and the effects attending these changes. 
c. For example, suppose we consider a block of wood 
which is placed in an oven and heated. After a period, the block 
of wood becomes hot or heated. Thus far the change is a physical 
one. When, however, the heat is increased and prolonged the 
block of wood burns and finally no wood is left. What we call 
"ash" and ”smoke” have been produced from the wood. Chemistry 
is hers involved and the change, burning, is a chemical change. 
d. General Chemistry (Theoretical Chemistry or 
Physical Chemistry) treats with principles which generalize the 
facts relating to the composition of substances and to changes 
in composition. In other words, it deals with the fundamentals 
underlying all chemistry end chemical action. It is the branch 
of chemistry we shall study first. 
2. Matter and its subdivisions - In the study of any 
scientific subject, it is essential to define orle's terms in 
order that all who work with them are "speaking the same 
language.” Scientific definitions will be found, usually, t« be 
limitations up#n the meanings of words, making them more 
specific than the ordinary *r general meaning of the w#rd used in 
everyday speech or writing. We will proceed to define matter 
and some of the terms used to describe it, its divisions and 
its properties. 
a. Matter is anything which has weight or occupies , 
space. b. A substance in chemistry, is a particular kind of 
matter. 
(1) A pure substance is a substance which when 
divided yields particles of identical properties with each other 
and with the original substance. 
c. A property of matter is a means or any means by which 
matter makes itself evident to our senses. All of our knowledge 
of matter has been gained by observation of"the properties of 
matter. Examples are given below under general and specific 
properties, 
(1) General properties of matter are those possessed 
by all matter regardless of kind. Most of these are well-known 
to us, so well known that we take*them for granted and do not 
often think about them. For instance, we know that matter cannot 
be destroyed, we know that two bodies cannot occupy the same 
space at the same time. In other words, matter has the general 
properties; indestructibility (it cannot be destroyed) and 
impenetrability (no two bodies can occupy the same space at the 
same time). 
(2) Specific .properties of matter are peculiar to each 
kind of matter and are usually described by the degree or extent 
to which they are possessed by a substance. It is upon these 
properties we base our work in chemistry. In fact, we recognize 
substances by means of properties which they possess to some 
marked degree. For some examples: solids have the properties of 
hardness, form, solubility, melting point,, density and others. 
Liquids have density, boiling points, color, odor, taste and other 
properties. Gases have color, odor, taste, critical temperature, 
density and many other properties. 
(a) Specific properties of any substance are 
uaually measurable; that is we have some means of determining 
the degree to ■-which a-substance'has a property. 
(b) Specific properties of any substance are always 
possessed by that substance to the same degree, if other 
conditions are held constant. In other v/ords, ice always melts 
at 0°C,; boric acid always dissolves in water in the, proportion 
of 1 Gnu of boric acid to 1? cc. of water: mercury always weighs 
13.6 Gm. per cc. under the same conditions. 
d. Mass or weight - Mass or weight will be treated in our 
work as identical terms. They refer to the quantity of matter. 
Weight is defined as the measure of tfye earth’s attraction for a 
body. In actual practice, the attraction of the earth f#r a body 
is compared with the same attraction for a standard body (a 
weight), - a gram, or a pound, etc. (1) A body is any particle of matter which can make 
its presence known to any or all of the five senses of man, 
(2) A molecule is the smallest particle of matter 
which is capable of independent existence. It is, for each 
substance, definite in size, ?;eight and composition. These sizes 
and weights will be considered at a later date. The whole of 
our course will be concerned in a large part with the composition 
of molecules. 
(3) An atom is a particle which with one or more other 
atoms goes to make up a molecule. It is then a division of a 
molecule but it is not capable of independent existence. All 
atoms of a given element are alike and are uniform in composition 
and weight. 
(4-) Ions are atoms endowed with a charge of electricity 
and temporarily combined with a solvent (usually water). They are 
not existing independently, although they have become dissociated 
from the molecule to which they previously (before being dissolved) 
belonged. They will be discussed in some detail at a later date, 
(5) Smaller divisions of matter, electrons, protons, 
etc,, will not be considered in this course. 
The condition or state of matter refers to matter as it is 
affected by the circumstances whifth surround it. For instance, 
we say that a body is hot or cold, or under pressure. Here 
frequently, however, we refer to the states of matter known as 
solid,.liquid and gaseous. 
(1) Solid Matter or matter in the solid state, means 
above and beyond our ordinary conception of the term, that the 
molecules of solid matter are fixed in relation to one another; 
that is, they cannot move around or away from one another. This, 
of course, explains why solids have their fixed form. 
(2) Liquid matter, or matter in the liquid state, 
is matter whose molecules have sufficient freedom cf motion to 
move around each other, but yet have sufficient attraction one 
for another to maintain a relatively constant bulk or volume. 
This makes liquids capable of assuming the shapes of the vessels 
used to contain them. 
(3) Gases or matter in the gaseous state is matter 
whose molecules have complete freedom of motion and in addition 
a certain repulsion for each other. This is what causes the 
gases to tend to expand or exert pressure on their containers. 3. Energy - Modern day physics treats energy separately 
from matter. We will continue under the older idea of considering 
energy to be a property of matter; namely, its capacity to 
do work. This is much simpler and for all the use we will make 
of it, as adequate as the modern conception of energy as something 
fundamentally similar to matter and ultimately interchangeable 
with it. 
4. The Laws of Nature or Natural Laws - One of the first 
ideas that the beginner in any science must learn is that natural 
lav/s were made after the lawmakers had observed the acts of nature. 
There is no way to make nature obey these "laws.11 YLLth this fact 
as a background, we can define a Law of Nature as a statement of 
someone's experience; and the collection of assembled fact 
commonly referred to as Natural Laws may be defined as the combined 
experience of the years which have been spent studying the various 
sciences. They merely state that, given such and such a state of 
conditions, this and that action will result. Most natural laws 
are experiences which have been repeatedly undergone, without ever 
having been disproven. In many cases they are capable of 
application as mathematical formulas. Not all of these "laws” 
apply with such accuracy, however, and according to their accuracy, 
laws are classified as EXACT LAWS, APPROXIMATE LAWS, AND EMPIRIC 
LAWS. 
a. Most of the "laws of nature" began as theories or 
hypotheses in the mind of some investigator. These theories are 
merely olausible exolanatians or suppositions advanced in an 
attempt to explain certain observed facts. In other words, the 
investigator says to himself, "This is how and why it might have 
happened." He then has a theory. Next he says, "If that is how 
and why what I saw happened, then if I do thus and so, then this 
is what is going to happen." He tries it, and if what he expected 
does happen, the investigator has a hypothesis. These theories and 
hypotheses are used as guides to show the direction further 
experiment should take, or perhrps, to point the way to direct 
proof in some manner or other. If such proof is accomplished, 
then the theory or hypothesis becomes a "law." 
b. Exact Laws - Exact Laws are those to which no 
exceptions are known; and any minor deviations in the results of 
application of such laws which occur, become increasingly small 
as the skill of the manipulator increases. 
(l) The Law of the Conservation of Matter is such 
a law. It states that matter cannot be destroyed. In other words, 
regardless of what changes matter may be made to undergo, its 
weight remains the same, none being destroyed, none'being created. 
This is one of the oldest as well as one of the most exact of all 
natural laws. It has been expressed in many ways, down through 
all time. We are all familiar with the old proverb, "From nothing, nothing comes.n In fhemistry, a favorite statement 
of the law is, '’You can’t get anything out of a test tube that 
you didn’t put into it.” As a corollary of this law, we see 
that the total of all the matter in the universe is and will 
always be a constant. 
c. Approximate. Laws - Approximate laws of nature are 
those which are true for most conditions, but vary under some 
few conditions (usually at some extreme). Minor variations in 
the results of application of approximate laws usually remain 
the same regardless of the skill of the investigator who is 
applying them. 
(l) Boyle’s Law is an example of an approximate 
law of nature. It states that the volume of a gas varies in 
inverse proportion to the pressure exerted on the gas. This 
works out according to p ? p' ?! v' ? v, with mathematical 
precision for ordinary temperatures and pressures. Near the point 
of liquefaction of gases, that is to say, at very low tem- 
peratures, and very high pressures, it fails to describe what 
happens. Most approximate laws are eventually corrected by further 
study which discovers some minor feet previously not taken into 
consideration. Approximate laws also have usually some easily 
seen basis in fact, that is, they explain what happens. 
dr Bnroiric Laws - Oneiric laws are merely statements 
of observed facts with no attempt whatever to explain anything. 
They are frequently only gross approximations and may even 
vary widely from f-ct. On the other hand, they are often 
convenient for arriving at some result not easily seen or under- 
stood. Unlike either exact or-approximate laws or even well 
founded theory or hypothesis, they seldom can be made of use as 
a guide to further study. 
(1) Trouton’s Rule is an example of an empiric 
law. With no attempt at an explanation, it states that it takes 
22 times the number of calories expressed by the boiling point 
of a liquid to boil cr volatilize a gram-molecular weight of 
the liquid. 
5. Atomic and Kinetic Theory - We are now ready to begin 
the study cf chemistry. Our approach is perhaps not the usual 
•ne for we will omit historical and other considerations and 
begin with the ultimate particle cr piece cf matter and build up 
our picture of what happens from there. Moreover, we have not 
time in the short course allotted us to attempt a great deal of 
proof either in the, laboratory cr mathematically and theoretically 
on paper. We will have to accept as fact, and it is such, the 
existence and behavior of these particles or units of matter. a. Almost ever since men fcegan to try to puzzle out 
why things happened under certain circumstances, it has seemed 
logical that matter was composed of an enormous number of 
small particles. As investigators became more skillful, it 
became more and more evident that this was the case, until the 
present day, and then only very recently has this been 
substantiated, the particles being observed with the new 
electronic microscope. 
b. Atomic theory - in 1804- John Dalton in England 
formulated and advanced a theory built upon the lines suggested 
by the idea of such a structure of matter. From it he and 
others were able to reason ways in which the theory could be 
tested out. Not one of these deductions based on sound 
reasoning from Dalton’s theory has yet failed to prove true. 
This atomic theory states that? 
(1) Elements are made up of inconceivably (?) small 
particles which are indivisible in chemical actions and which 
are called atoms. 
(2) The atoms not only have definite weights, but 
the atoms of any one element have the same weight, which is 
different from the weight of the atoms of some other element. 
(3) When elements unite chemically, the action 
takes place between the atoms. 
These three statements are really one of the basic 
principles of chemistry, and if the student will learn them well 
and think of them in connection with later work he will see that 
much useless rote learning will be avoided. For our purposes 
the statements may be regarded as undisputed facts and the 
student is required to learn them verbatim as facts. 
c. Kinetic theory - Before and since the time that 
chemists were elaborating and proving experimentally that 
assumptions made upon the atomic theory were correct, physicists 
were developing a somewhat similar theory to explain certain 
phases of physics. They, of course, were interested in the larger 
particles which atoms made up when they united. Finally a sort 
of theory grew from their efforts, and it is still growing. 
Unlike the atomic theory, the molecular or kinetic theory was 
not the work of any one person and it is perhaps not so clearly 
stated; in fact, the theory cannot yet be completely stated be- 
cause we are still building up proofs of it, and developing still 
further applications of its postulates. For our use, we will 
accept as established fact the following as the kinetic theory? (1) Matter is composed of infinitesimally small 
particles called molecules* 
(2) These molecules of any particular substance 
are indivisible by physical means, but can be divided into 
their constituent atoms by chemical means. 
(3) The molecules of any particular substance are 
all alike and have a definite weight, this v/eight being known as 
the molecular weight of the substance. 
(4) Each molecule of each substance is endowed 
with a certain measurable quota or quantum of energy. This 
quantity of energy is the same for all molecules regardless of 
the size or kind, being dependent only upon the temperature. 
(5) 3y means of its endowed energy each molecule 
is in a state of rapid motion or momentum (depending on the 
temperature and state of the substance), 
(6) A gram-molecular weight of any substance 
contains a definite number of molecules, and this number is the 
same for all substances. This number of molecules is 606,000, 
000,000,000,000,000,000, or better 6.06 x and is called 
Avogadre’s number because he was the first to use, not the 
number, but the basic principle. 
Upon these six postulates which are the more fundamental 
principles of the kinetic theory, it is possible to explain or 
to predict so many of the laws and discoveries of science that 
chemists and physicists universally no?/ accept the theory as 
proven. 
It is easy to explain the behavior of matter in all 
of its forms by kinetic theory, and to deduce many facts that 
were well known before the theory assumed such large stature, 
as well as many facts that have since come to light as a,result 
of deductions made from the theory. ' Let us consider the three 
states of matter previously discussed in paragraph 2 above. 
Solids are definite in volume, external conditions 
being kept the same, and molecular motion is confined to rapid 
vibration of the molecules within the solid, without exerting 
enough pressure to change the external shell. Evidences of this 
vibration are the conductance of heat and sound through solids, 
electrical charge, and if it is a magnetic substance, the ability 
to maintain its magnetic polarity during division. 
Let us now suppose this solid to be heated, thus 
adding to the energy of its molecules, for heat is r form of 
energy. When sufficient heat has been added, the substances lose its form because the molecules have acquired sufficient 
energy to move around one another. We say that the substance 
has melted. Note that the molecules of the substance attract 
each other for the most part and make no attempt to repel each 
other; a few molecules, however, do acquire enough energy to 
escape through the surface shell of molecules, and the higher 
the temperature the more the energy and the greater the number 
that will so escape. We say that the substance has evaporated 
and we know that evaporation takes place to a greater extent on 
a warm than on a cold day. 
Suppose, now, we continue to heat the substance until 
the molecules become very violent, and break up the surface shell 
becoming gaseous or vapor molecules. Due to the great number, 
the great speed, and the loss of the power to cohere, the 
molecules are in a state of chaotic collisions, the molecules 
striking each other and the sides of the container and bouncing 
off in every conceivable direction. We say that the gas is 
exerting pressure on the sides of the container, just as a very 
hard shower of rain seems to be pressing on all points of a 
tent at one time. 
The student is referred to pages IB to -48 in Simons’ 
Manual of Chemistry, for examples of phenomena dependent upon 
molecular motion and energy, and to pages 110 to 113 for a fuller 
discussion of atomic theory. These nine postulates are very 
important to a quick understanding of chemistry, which is the 
reason they have been introduced early in the course and they 
must be learned. 
6. Elements - the word element means, fundamentally, 
something which cannot be changed. We use the term element in 
chemistry to mean a substance whose molecule cannot be broken 
down into more than one kind of atom. 
a. The number of elements - eighty-nine elements are 
today well known to chemistry, and there is reason to believe 
that the total number of elements is limited to ninety-two. 
Of these 89 elements, not all are of importance to medicine and 
so to pharmacy, although such is the progress of medical science 
that they may become so at any time. For instance, helium once 
believed to be one of the most inert substances known is now 
being used in the treatment of asthma. There are, nevertheless, 
comparatively few of the elements that we need study in any 
detail. The elements are listed below more or less in the order 
in which we will study them. The underlined ones are those 
important to us in medicinet As of 194-3 
(1) Gaseous elements - (of which there are eleven) 
Hydrogen 
H 
1,008 
1 
Oxygen 
0 
16.000 
2, 
5 
Nitrogen 
N 
14.008 
3, 
5 
Fluorine 
F 
19,000 
1 
Chlorine 
Cl 
35.457 
1 
Helium 
He 
4*002 
d> 
) 
Neon 
Ne 
20.2 
0 
) 
These are the so-call- 
Argon 
A 
39.9 
0 
) 
ed "noble gases," 
Krypton 
Kr 
82.9 
0 
) 
they do not enter into 
Xenon 
Xe 
130.2 
0 
) 
combination with any 
Radon 
Rn 
222. 
0 
) 
other substance, so we 
are not concerned with 
them, 
-i . \ 
(2) Liquid elements - (of which there are only two) 
Bromine 
Br 
79.916 
1,3,5,7 
Mercury 
Hg 
200.61 
1,2 
(3) Solid elements (of which there are 76). The 
following list is for reference and is inserted here only for 
convenience. 
Lithium 
Li 
6.9A9 
1 
) 
Sodium 
Ka 
22.9*7 
i) 
The "ALKALI METALS"; 
Potassium 
K 
39.096 
1 
) 
All are metallic 
Rubidium 
Rb 
95. U 
1 
) 
elements 
Cesium 
Cs 
132.91 
1 
) 
Cooper 
Cu 
63.57 
1, 
2 
) 
Silver 
Ag 
107.BBO 
1 
) All are metals 
Gold 
Au 
19?. 2 
1, 
2 
) 
Calcium 
Ca 
Z.0.07 
2 
) 
Strontium 
Sr 
W.63 
2 
) Metals 
Barium 
Ba 
137.37 
2 
) 
Radium 
Ra 
225.95 
2 
.) 
Beryllium 
Be 
9.02 
2 
) 
Magnesium 
Mg 
24.32 
2 
) Metals 
Zino 
Zn 
65.39 
2 
) 
Cadmium 
Cd 
112.41 
2 
) 
Mercury 
Kg 
200.61 
h. 
2_ 
J 
Platinum 
PI 
195.23 Scandium 
Sc 
45.10 
3 ) 
Metals 
Yttrium 
Y 
SB.9 
3 ) 
3 ) 
... 
lanthanum 
La 
138.92 
Actinium 
Boron 
Aluminum 
Ac 
227. 
B 
A1 
10.82 
26.97 
3 ) 
3 ) 
3 ) 
3 ) 
3 ) 
All metals except 
boron 
Gallium 
Ga 
69.72 
Indium 
In 
114.80 
Thallium 
T1 
204.39 
Carbon 
C 
12.00 
4 ) 
Carbon and silicon 
Silicon 
Si 
28.06 
4 ) 
are non-metals; the 
Germai ium 
Ge 
72.60 
4 ) 
others are metals. 
Tin 
Sn 
118.70 
2,4 ) 
Lead 
Titanium * 
Pb 
Ti 
207.2 
48.10 
zaJ 
3,4 ) 
Metals 
Zirconium 
Zr 
91.00 
4 ) 
Cerium 
Ce 
140.13 
3,4 ) 
Thorium 
Th 
232.15 
f±_) 
Nitrogen 
N 
14.008 
3,5 ) 
Nitrogen and Phos- 
Phosphorus 
P 
31.02 
3,5 ) 
phorus are non- 
Arsenic 
As 
74. 96 
3,5 ) metals?, others are 
Antimony 
Sb 
121,77 
3.5 ) metals. 
Bismuth 
Bi 
209.00 
3.5 ) 
Vanadium 
V 
.hu , 50.96 " 
3.5 ) Metals- 
Columbium 
Cb 
93.10 
3,5 ) 
5 ) 
) 
Tantalum 
Ta 
181.50 
Prfttoactiniura 
Pa 
- 231.00 
0 
16.00 
2 ) 
Oxygen and sulfur 
■Sulfur 
s 
32,064 
2,4,6 ) 
are non-metals; 
Selenium 
Se 
79.20 
2,4,6 ) 
others are metals 
Tellurium 
Polonium 
Chromium 
Te 
Po 
. 127.51 
210.0 
2,4,6 ) 
) 
Cr 
52.01 " 
2,3,6 ) 
Metals 
Molybdenum 
Mo 
v« 96.00 
3,4,6 ) 
Tungsten 
W 
184.00 
'6 j 
Uranium 
U 
238.17 
4,6 ) 
Fluorine 
F 
19.00 
1 ) 
Nen-metals 
Chlorine 
Cl 
35.4-57 1,3,5,7 ) 
The Halogens 
Bromine 
Br 
* 79.916 1.3.5.7 ) 
Iodine 
I 
126.932 
1,3,5,7 ) 
Manganese 
Mn 
54.93 
-2,3,4,6,71 
) Metals 
Masurium 
Ma 
98.00 
Rhenium 
Re 
186.31 Iren 
Fe 
55.84- 
2,3 ) Metals 
Cobalt 
Co 
58.94 
2,3 ) 
Nickel 
Ni 
58,69 
2.3 ) 
Ruthenium 
Ru 
101 ,'7 
3,4-,6,8 ) Metals 
Rhodium 
Rh 
102.91 
- 3 ) 
Palladium 
Pd 
106.7 
2,4 ) 
Osmium 
Cs 
190.8 
2,3,4,8 ) 
Iridium 
Ir 
• 193.1 
3,4 ) 
Platinum 
Pt 
195.23 
2.4 ) 
Hafnium 
Hf 
180.8 
) 
It will have been noted that the list above contains 
metals, -non-metals, gases, liquid, and solid elements. 
b. Elementary molecules - Most elementary molecules 
consist of two identical atoms which are united chemically to 
form the molecule. Thus most of the molecules of elements 
weigh twice as much, proportionately, as their respective atoms 
A few monatomic elements are known, and a few whose molecules 
contain more than one atom. In every elementary molecule, 
however, all of the molecules are alike. 
v 
c. Families of elements - The student will find in 
his reading, references to ’’families of elements.” It is not 
intended to go deeply into a study of elementary families at 
this time; and it is sufficient to point out that the elements 
are grouped into families according to the similarity of their 
more important properties. In the list of elements given under 
(3) above, the elements are grouped according to families, and 
reference will be made to the similarity of these properties 
from time to time. 
7. Compounds - (Text Reference, Simons, page 56). We 
defined an element, in the previous paragraph as a substance wh 
molecules contain only one kind of atom. In contradistinction, 
a compound is a substance whose molecules contain more than one 
kind of atom; and a compound may be broken down chemically into 
two or more different kinds of atoms. The idea of the term 
compound also implies that the two or more atoms are combined 
chemically, which is to say, they are held together by a force, 
which is called chemical affinity, or chemism. 
a. Examples? 
' Salt or sodium chloride contains in each of its 
molecules; 
1 atom of chlorine 
1 atom of sodium 
Calcium Carbonate or chalk contains in each of its 
molecules; 
1 atom of calcium 
1 atom of carbon 
3 atoms of.oxygen b. LAW OF CONSTANCY OF COMPOSITION and LAW OF MULTIPLE 
PROPORTIONS - Several of the basic laws of chemistry are intended 
to help one to understand the formation of compounds. Two which 
have a direct bearing on the subject are the laws of (l) 
constancy of composition, and (2) multiple proportions. 
(1) The LAW OF CONSTANCY OF COMPOSITION states that: 
A definite chemical compound always contains the same elements 
in the same proportion. 
(2) The LAW OF MULTIPLE PROPORTIONS states that: 
If two elements, MArt and "S11 are capable of uniting chemically 
in more than one proportion, then the quantities of ’’B” which can 
combine with a fixed quantity of MAn bear a simple ratio to one 
another. (By a simple ratio, here, we mean a ratio between small 
whole numbers like 1 to 2 or 2 to 3). 
(a) Examples: there are two chlorides of iron 
as follows: 
Ferrous chloride contains? 
1 atom of iron 
2 atoms of chlorine 
Ferric chloride contains: 
1 atom of iron 
3 atoms of chlorine 
Likewise, there are two oxides of hydrogen: 
r * "■ .iv i . 
Hydrogen monoxide contains: 
1 atom of oxygen 
2 atoms of hydrogen 
Hydrogen dioxide contains: 
2 atoms of oxygen 
2 atoms of hydrogen 
8. Symbols - In writing about chemistry and chemicals, it 
was early found that the names we give substances, and the manner 
of stating what they contained, were cumbersome to handle. The 
examples shown in the two previous paragraphs are ample proof of 
this difficulty. A method of indicating more easily what was 
meant was needed to save words and soace and to indicate the 
intention of the writer more clearly. Even the ancients recognized 
the need and their writings contain crude symbols or sign writing. 
Accordingly, a system of shorthand writing was developed. 
Symbols were developed for each element, and out of these symbols, 
it is possible to write formulas for each compound. Later, ways 
were learned to write the manner in which compounds and element 
entered chemical reactions, and these are called chemical equations. a. What a symbol is: 
(l) A symbol usually consists of the initial 
letter of the Latin name of the element. Where two or more 
elements begin with the same initial letter, some other 
distinctive letter from the name of the element is written 
beside the initial letter. The initial letter is written as 
a capital, the second letter as a lower case letter. 
(a) Examples of symbols - 
Single letter symbols? 
Two letter symbols: 
Argon 
A 
Aluminum 
A1 
Arsenic 
As 
Boron 
B 
Bismuth 
Bi 
Bromine 
Br 
Carbon 
C 
Calcium 
Ca 
Chromium 
Cr 
(2) A symbol means one atom of an element - In 
addition to being merely an abbreviation for the name of an 
element, a symbol refers to a single atom of an element. Thus, 
A above means 1 atom of Argon; A1 means 1 atom of aluminum, Ca 
means 1 atom of calcium, etc. 
(3) A symbol means a definite relative weight of 
an element. In addition to the above meaning, we may read into 
each symbol, in addition to the name and number of atoms, a 
certain definite relative weight of an element, as contemplated 
in the statement of atomic theory. This, then, is a complete 
statement of what a symbol tells us: 
A means an atom of argon having a definite weight; 
As means 1 atom of arsenic having a definite weight; 
Cr means 1 atom of chromium having a certain weight,etc. 
In the list of elements in paragraph 6 above, 
the symbols are written as the second column, directly following 
the name of each element. 
(A) Subscripts - where it is desired to write a 
symbol which is to indicate more than one atom of an element, 
a subscript is used. A subscript is a number written to the 
right of a symbol to indicate the number of atoms or atomic 
weights desired. Examples follow: 
AS2 means two atoms of arsenic cr two atomic weights of i 
C means an atom of carbon or 1 atomic weight. 
PL*, means 35 atoms of hydrogen or 35 atomic portions 
of it by weight. 9. Atomic Weight - the atomic weight of an element is the 
relation between the weight of 1 of its atoms and an atom of 
oxygen. It is necessary to elaborate a little on what the above 
statement means. 
An atom of any element is by definition, "an inconceivably 
small" particle. If it is inconceivable, then it is certainly 
too small to weigh. Recently we have learned the number of 
atoms into which a certain weight of substance can be divided, 
and we can calculate the weight of an atom. Such a weight, how- 
ever, runs to upwards of 20 decimal places and is hard to apply 
to any practical use. Rather early in the study of chemistry 
it was learned that the relative weights of elements could be 
determined. We could determine that oxygen weighed 16 times 
as much as hydrogen per unit volume, and Avogadro early proved 
that, other things being equal, equal volumes of elementary gases 
contain the same number of molecules. From that, it was con- 
cluded that an atom of oxygen weighed 16 times as much as an 
atom of hydrogen. And in a similar manner, these relative 
weights were obtained for other elements. Instead of using the 
long statement that "oxygen weighs 16 times as much per atom 
as hydrogen," we say that the atomic weight of oxygen is 16, 
or simpler still, 0 s 16. 
In the list of elements in paragraph 6a above, the 
figures in the third column are the atomic weights corresponding 
to the elements who-se names and symbols occupy the first two 
columns. It will be noted that no units of weight are given. 
This, of course, is because the weights shown are relative weights, 
or ratios between the elements nsmed and a standard or reference 
element. 
Hydrogen, because it is the lightest known element, was, 
at first, chosen for the standard by which the weights of other 
elements were compared. The weights of other elements were 
determined by weighing the amount of another element which had 
combined with hydrogen. If it was found that, for instance, 35 
parts by weight of chlorine, had combined with 1 part by weight 
of hydrogen, then the atomic weight of chlorine was called 35. 
In similar manner, the weights, of other elements were, determined 
and, in addition, other ways of comparing the weights were 
devised. TTith progress the atomic weights became more and more 
accurate, the elements under study became more and more numerous, 
and the number of known atomic weights increased. It was found 
that relatively few of the atoms combined directly with hydrogen, 
and that in a great many cases, awkward decimal fractions were 
obtained. Some investigators began to compare the other elements 
to oxygen because more elements combine directly with that 
element, and found that the weights so obtained were nearer to 
whole numbers. Gradually the use of oxygen as a standard became 
established and is universal today. SO THE STANDARD FOR ATOMIC 
HEIGHTS IS THE RELATION 0-16. instead of the older H = 1. It will be seen that for rough work there is very little 
difference (if 0 = 16, then H = 1,008); but for fine work the 
oxygen standard is preferred, and all tables of atomic weight 
are so made. 
Use of symbols end atomic weights - the student might 
well ask at this time, "How many atomic weights and symbols am 
I expected to learn?" The answer is, "None," It is common 
practice to use tables in order to obtain this data, and in our 
work access to tables will be allowed or necessary data will be 
furnished in all work. It will be found as a matter of practice, 
that symbols are so handy and so useful thcat they teach 
themselves to the student, and he very quickly begins to use 
them to save time. It is not necessary to spend time actually 
memorizing a set of symbols. 
10. Formulas - (Text reference page 113) - (Simons). 
Formulas bear the same relationship to compounds that symbols 
bear to elements. They are shorthand representations of what 
a compound contains or is composed of. They are simply a 
concise statement of the number of and kind of atoms in a 
molecule of the compound. Also, in an analogous manner to the 
writing and reading of symbols, we mean by the formula for a 
compound, 1 molecule of the compound, weighing a certain 
definite relative amount. (This molecular weight is the sum 
of the weights of the atoms which the molecule contains). 
a. Examples of formulas - we have used in our dis- 
cussion thus far, several compounds as examples in various 
instances. These are tabulated below, with their formulas, for 
comparison to the cumbersom method previously used to write 
their composition? 
Sodium Chloride «- NaCl 
Calcium Carbonate - CaCO^ 
Ferrous Chloride - FeCli 
Ferric Chloride - FeCl~ 
Hydrogen Monoxide - HpO 
Hydrogen Dioxide - 
b. The writing of formulas - Formulas are written 
according to certain customs which have grown up, and are well 
understood by chemists everywhere. Some of these follows 
(1) Write formulas with the metallic elements on 
the left and other elements following. 
(2) Formulas are read from left to right. 
(3) Subscripts are used to indicate the number 
of at»ms of each element in the compound. (4.) Coefficients (numbers placed before the whole 
formula and on a line with it) are used to indicate the number 
of molecules intended; e.g., 2 NaCI means two molecules of sodium 
chloride, and therefore, two atoms of sodium and two atoms of 
chlorine, 3 CaCCL means three molecules of calciuto carbonate and, 
therefore, 3 atoms of calcium, 3 atoms of carbon, and 9 atoms 
of oxygen. This is important-in many respects. From a standpoint 
of weight alone, there would be no difference between 2 NaCI and 
Na CI2. From the'standpoint of molecular make-up, however, 2 NaCI 
means two molecules each of which contains two atoms, whereas 
means one molecule which contains four atoms. This must 
be kept in mind. It is also necessary to remember that a 
coefficient multiplies every atom in a formula which it precedes, 
while a subscript multiplies only that atom to which it is 
attached (follows). 
c. It is necessary that a compound have a definite 
composition before a formula can be written for it; but this 
follows from the law of constancy of composition and our definition 
of a compound. 
11, Combining power of the elements - we have already stated 
that a force, chemical affinity, holds compounds together, and some 
of you may have begun to wonder why one molecule has only two 
atoms, another three, a third fifty-six, etc. The reason is that 
ndt all elements have the same amount of combining power. Some 
unite with others in a one to one ratio, while the same element 
will react with another different .element in a 1 to 2 ratio, or 
some different ratio altogether. That phase of an element’s 
combining power which determines its power to hold 1 or 2 or 3 
other atoms in combination is called valency or atomicity, 
valency is the commoner term. 
Valence a measure of combining power - Valency is one 
of the measures of combining power; it is not the force itself. 
Valence tells us how many atoms of another kind an atom will combine 
with. Naturally, some basic measure is necessary, some unit of 
measure, by which we may express the “size1’ or amount of combining 
power. Again hydrogen is taken as a basis; and ,we say that any 
element which combines with hydrogen in the proportion of 1 atom 
per atom of hydrogen has a valence of one, and that any element 
which combines with hydrogen in the proportion of 2 atoms of hydrogen 
atom of itself has a valence of two, and so on. Thus chlorine 
combines with hydrogen in a 1 to 1 ratio, forming hydrogen 
chloride - HC1, so we say that chlorine has a valence of one. 
Likewise, sulfur combines with hydrogen in a 1 to 2 ratio, forming 
hydrogen sulfide - H?S, so we say that sulfur has a valence of two. 
Nitrogen combines with hydrogen in a 1 to 3 ratio, forming ammonia - 
so we say that nitrogen has a valence of three, etc. 12, Structural Formulas - By means of the valence bonds 
mentioned in the previous paragraph, we can draw a diagram of a 
molecule of a compound instead of merely writing its formula. 
Such diagrams are called structural formulas. They are merely 
an indication of how we believe the elements of a compound to be 
united. They are not to be thought of as actual pictures of 
compounds, although they are almost as useful as such pictures 
would be if we had such things. Examples of such structural 
formulas are shown below, along with the name and molecular 
formulas of the compounds: 
Hydrochloric Acid 
Potassium Iodide 
Carbon Monoxide 
Carbon Dioxide 
HC1 
KI 
CO 
C0o 
2 
H - Cl 
K* - I 
o 
it 
o 
o 
it 
o 
H 
o 
Ferrous Chloride 
Ferric Chloride 
Calcium Carbonate 
Water 
FeCl 
2 
FeCl3 
CaCC3 
H2° 
Cl Fe - Cl 
Cl - Fe - Cl 
o 
ti 
o 
1 
o 
H - 0 - H 
Cl 
Ca- 0 
13, Radicals - A great many chemical compounds contain 
groups of elements which have a character of their own and which, 
in reactions, move from compound to compound as a group, seemingly 
without internal change. Such groups of elements are called 
radicals. These radicals are well known in organic reactions, so 
well known that many of them have acquired names: we speak of 
the hydroxyl radical, the nitrate radical, the sulfate radical, 
the phosphate radical, etc. Radicals act as though they were 
single elements in reactions, moving intact from salt to salt. 
Radicals also are said to have valence, which is probably a 
misnomer; but if we draw structural formulas of various radicals 
we see that there must of necessity remain certain free bonds 
attached to some of the elements of the radical. It is these free 
bonds which give radicals their valence. Some typical radicals 
are shown below: 
Hydroxyl 
Sulfate 
Nitrate 
Phosphate 
Ammonium 
Radical 
Radical 
Radical 
Radical 
Radical 
-OH 
SO, 
N0~ 
PC 
NK. 
A 
3 
A 
A 
0 
0 
0 
H 
0 S 0 
0 N 
o 
P-i 
o 
H N H 
0 
0 
0 
H 14.. Acids - Inorganic chemicals may be divided into several 
groups, each of which has characteristics common to all of the 
chemicals in the groups. One of these groups is the one 
containing what we call acids. We call all of them acids 
because they have certain properties in common and we call these 
properties ’’acid properties.” 
a. Common properties of all acids; 
(1) Structure - all acids are compounds of 
hydrogen with some other element, a non-metal. We say that they 
have the general formula, HR or H+R~, where H or H+ represents 
hydrogen, and R or R~ represents another element or radical 
containing another element. The plus and minus signs are added 
to show that in the compound the hydrogen is electropositive, and 
the radical is electronegative. 
(2) Physical state of acids - acids may be gases 
as HC1 or or liquids as or solids as 
(3) Chemical properties of acids; 
(a) All acids have a sour taste when in 
solution. 
(b) The hydrogen of acids is liberated by 
treatment with a metal; 
HR + Me « MeR + H 
(The symbol, Me, represents any metal, MeR 
means a salt of the acid with the same 
metal) 
(c) Acids react with bases characteristically; 
HR + MeOH « MeR + H20 
(The formula MeOH represents the hydroxide 
of any metal). 
(d) Acids have characteristic reactions with 
indicators. Acids change blue litmus paper 
to red. Acids change methyl orange from 
yellow to pink, etc. 
b. Hydrogen ion ~ The H+ of an acid is called a hydrogen 
ion; and it is the hydrogen ion that gives acids their chemical 
properties. We see from the symbol that hydrogen ion is an 
atom of hydrogen carrying a positive charge of electricity. We 
will learn later that it is the H+ and only the H+ which gives 
acids all of their properties, that the ’’strength” of acids is 
measured by the amount of hydrogen ion they are able to produce in 
solution, and that any compound that is able to liberate hydrogen 
ion is acidic whether or not its actual formula conforms to the 
above general one. For the present, however, we will hold to the 
general formula HR, as our definition of an acid, 
15. 15. Bases - Another large group of chemicals is the one 
called ’’bases'1, and the properties which they posses in common 
are called basic properties. This group is the one commonly 
referred to as alkalies, or caustic alkalies, or simply 
hydroxides. 
a. Common properties of basest 
(1) Structure - all bases are hydroxides of metals. 
They have the general formula MeOH, in which "Me" represents any 
metal. To show the nature of the compounds the general formula 
is written Me+ OH’", in which Me+ represents any metallic ion and 
0H“ represents hydroxyl ion. The group - 0 - H, commonly called 
hydroxyl ion is very interesting. It is this ion that gives 
bases all of their properties, and as we are soon to learn it is 
the exact complement of hydrogen ion, and in many ways may be 
considered as its opposite. As in the case of hydrogen ion, we 
also find that any substance which is capable of releasing 
hydroxyl ion when it is dissolved in water, will have basic 
properties, whether or not the substance actually is a base. 
(2) Chemical properties of bases? 
All bases have bitter taste when dissolved. 
Bases react characteristically with acids. (MeOH + HR - MeR + 
H2O. Metals are, in general, unaffected by treatment with bases, 
unless some other agent is present. Bases give characteristic 
reactions with indicators. Bases turn red litmus paper blue. 
Bases turn methyl orange yellow. Bases turn phenolphthalein red. 
16. Neutralization - we have seen that acids have certain 
specific properties, while bases have a different set of properties, 
just as specific, and in some ways opposite. When substances 
having acidic properties are mixed with substances having basic 
properties in the proper proportion (molecule for molecule), both 
the acidic properties and the basic properties disappear, the 
solution is no longer sour.or bitter, and if an indicator has 
been added to the solution it will have neither the color it has 
in acidic solutions nor that which it has in basic or alkaline 
solutions. 
This reaction, neutralization, is a very common and a 
very important one. It is shown above under the paragraphs on 
acids and bases. It is repeated below, with the electrical 
charges shown, both in general and in a specific neutralizations 
Me+OH** + H+FT = MeR + 
Na+OH- V Htr* = Nal t % 6 
oir + h+ « h2o 
It will be noticed that in every neutralization reaction water 
is formed, and moreover, as the third reaction emphasizes, that 
the water is formed by the combination of hydrogen ion and hydroxyl ion. Some of each in a free state exist in water, 
but as they are present in equal amounts, their presence is 
now shown by the appearance of either basic or acidic properties. 
Similarly we call any other substance neutral which has 
every hydrogen ion counterbalanced by a hydroxyl ion. In fact, 
this balance of hydrogen ion and hydroxyl ion is neutrality. 
In the second equation shown above? if molecular 
proportions of each chemical have been used, the solution will 
be neutral, all of the positive charges will have become balanced 
by negative charges. But in addition, a new substance is 
formed, namely, Nal, or sodium iodide. This is the other 
characteristic of neutralization reactions, - salt formation. 
We shall further discuss salts in the next paragraph. 
To summarize, in a neutralization, four things always 
happen: 
a. A substance having acidic properties reacts with 
one having basic properties, 
b. Both acidic and basic properties disappear, 
c. Water is formed. 
d. A salt is formed. 
17. Salts - salts comprise another large group of chemicals. 
Salts are composed of one or more atoms of a metal combined with 
one or more atoms of non-metals. They have the general formula 
MeR. 
The properties of metals are too many and varied to 
attempt to describe, beyond saying that they are neutral in 
comparison to acids and bases, and relatively stable. This is 
to say, they do not react with as many other substances or 
react as readily as acids or bases. Most of the ordinary 
chemicals used in medicine are salts. 
a. Normal salts are salts which are formed by the 
complete neutralization of acids and bases. They may be re- 
cognized at once from their formulas. If the formula contains 
neither hydrogen nor oxygen (apart from the oxygen of the 
electronegative radical) the salt is a normal one or a neutral 
one, as they are sometimes called. All of the following are 
normal salts. They contain none of the hydrogen from the acid 
and none of the hydroxyl from the base. NaCI 
Sodium Chloride 
A12(S° ) 
BiClo 
Aluminum Sulfate 
KBr 
Potassium Bromide 
Bismuth Trichloride 
Lil 
Lithium Iodide 
SnCl/ 
Stannic Chloride 
NHiF 
CaC12 
Ammonium Fluoride 
MgSOT 
Nagbesuyn Chloride 
Calcium Chloride 
AgN0j 
ca(K03)2 
Silver Nitrate 
BaSO, 
Cu3(P°4)2 
Barium Sulfate 
Calcium Nitrate 
Cupric Phosphate 
U(NO376 
Uranium Nitrate 
b. Acid Salts - some acids have more than one hydrogen 
ion per e.g., sulfuric acid H2S0y, and phosphoric 
acid HoPO., It is possible to replace only one of these hydrogen 
ions withra metal; or in other words it is possible to only 
partially neutralize these acids. A salt formed by such a 
partial neutralization is termed an "acid salt." Any acid salt, 
shows in its formula, part of the hydrogen of the acid. Sodium 
Acid Sulfate is NaHSO/j Potassium Acid Sulfide is KHS; Sodium 
Acid Carbonate is NaHC03; the following equations show the 
formation of these three acid salts, along with the formation 
of neutral salts of the same bases and acids; 
2NaOH + H2 * NagSO + 21^0 
NaOH ♦ * + H20 
2K0H + H2S = K2S + H20 
KCH + H2S « KHS H20 
2NaOH + H2C03 = Na2C03 + 2H20 
NaOH + H2C03 » NaHC03 ♦ H20 
Acid salts are either so named, or the syllable 
bi - inserted in the name to indicate that the salt is an acid 
one. In each of the following, the name in the two columns refers 
to the same salt: 
Sodium Acid Sulfate Sodium Bisulfate 
Potassium Acid Sulfide Potassium Bisulfide 
Sodium Acid Carbonate Sodium Bicarbonate 
Ammonium Acid Silicate Ammonium Bisilicate 
(1) Acids which form acid salts; we have not so 
far referred to the basicity of acids. By basicity is meant the 
number of basic valence bonds the acid is capable of satisfying. 
This is exactly saying the same thing as that basicity is the 
number of H+ atoms the acid contains. We say that any acid which 
has one positive H atom is monobasic, or that it will satisfy 
one molecule of a base containing a monovalent basic element. 
Monobasic acids having only one positive hydrogen ion can form 
only normal salts, but as the number of hydrogen atoms increases, 
the number of possible acid salts increases. Thus, dibasic acids. like sulfuric or carbonic acids have a series of normal and 
one series of acid salts. Tribasic acids, like phosphoric and 
boric acids, have a series of normal, and two series of acid 
salts. This is illustrated as follows: 
3NaOH + H3PC4 = + 3H20 
2NaOH + H3P04 “ Na2 + 2H20 
NaOH + H3PO4 » + H20 
With three salts so similar, one immediately 
wonders how they are named to differentiate them. It is a 
problem that is solved only by completely stating what the salt 
contains. This is not always done, and when it is not, 
confusion remains. In some cases a logical definition clarifies 
the matter somewhat, as is the case with the three sodium 
phosphates. Several possibilities are shown below. Only those 
in the first column are self-evident: 
U.S.P. Names 
Trisodium Phosphate Tribasic Sodium Phosphate 
Disodium Hydrogen Phosphate Dibasic Sodium Phosphate Sodium Biphosphate 
Sodium Dihydrogen Phosphate Monobasic Sodium Phosphate Sodium Phosphate 
c. Basic Salts - basic salts while they are just the 
opposite of acid salts, are not so easily explained from a stand- 
point of neutralization. In general, a basic salt is one 
containing more base than is necessary for the formation of 
normal salts. 
The base which remains in the salt is not so easily 
identified as the H+ of an acid salt, due chiefly to the tendency 
of two -OH radicals to decompose to a molecule of H20 and an = 0 
■ • We will, therefore, find either hydroxyl or oxygen 
from the base in the formula of a basic salt. 
Note in the examples given below that basic salts 
may have their formulas written in two ways: 
Basic salts of triatomatic bases; 
Basic Ferric Chloride Fe20Cl^ 
Basic Aluminum Chloride ALpOCl/ 
Basic Bismuth Nitrate Bi(0H)tN03)2 
Basic Ferric Chloride pe 
Basic Aluminum Chloride Al20?Clp 
Basic Bismuth Nitrate BitoH)2N03 « + H^O Basic salts of diatomic bases: 
Basic Lead nitrate Pb(OH)NO or Pb(N0o)p.Fb(0H)p 
Basic Mercuric Sulfate or HgSO^.(HgO^) 
Basic Magnesium Carbonate or MgCO^.MgO 
(1) It may be seen that the naming of basic 
compounds is difficult, if one is to differentiate the compounds 
by name only. From the few examples above, whose names are 
given only in a general way, several ambiguities may be noted. 
We will not, however, go further into the nomenclature of these 
compounds at this time, for we will see later that the basic 
compounds which are used in medicine are mixtures of the several 
possible basic compounds in almost every case, 
d. Double salts are salts formed when two different 
bases go to neutralize a dibasic acid. They are comparatively 
simple, and fairly common; e.g., Potassium and Sodium Sulfate, 
KNaSO/, Sodium and Potassium Carbonate, NaKGO-3, Silver and 
Sodium Sulfate, AgNaSO^. 
18. Chemical change - Chemical changes are of fundamental 
interest to us. We outlined the scope of chemistry as the study 
of composition and of changes in the composition of substances. 
But the composition of substances cannot be studied without 
studying changes in composition; all analyses involve chemical 
change; all syntheses involve chemical change. In fact, we have 
not been able to discuss the little matter we have thus far 
undertaken without bringing chemical change into the picture to 
explcain ourselves, 
a. Types of Chemical Change - there are six different 
ways in which chemical reactions take place: 
(l) Direct Combination or Addition Reactions - 
In addition reactions, two or more elements unite to form a 
compound, without the formation of any by-product. It may be 
illustrated by the following reactions; 
Na + I = Nal 
Mg + 0 s MgO 
2Fe + O3 * Fe^O^ 
(a) General formula for direct combination 
or addition reactions - 
A ♦ 3 * AB (2) Simple Decomposition - Simple decomposition 
isn’t always as simple as the name would seem to indicate. The 
term -simple is used to differentiate this type of change from 
double.decomposition, with which you now have more than a 
passing knowledge in the laboratory. What is meant is that 
one compound breaks down into two or more simpler compounds. 
It will be seen to be the exact opposite of addition reaction. 
The following examples illustrate various types of simple 
decomposition! 
Any oxide of a "noble metal” breaks down 
when heated as follows: Ag20 * Ag2 * 0, 
Carbonyl chloride, when heated, decomposes into a simpler 
compound and the element, chlorine: COClp * CO + Clp. 
Calcium Carbonate, or chalk, when heated, breaks down into two 
simpler compounds: CaC03 * CaO + CO^* 
(a) General formulas for simple decomposition: 
the three reactions above are merely specific applications of 
the three general reactions below: 
AB = A + 3 
ABC* AB + C 
ABC* AB + BC 
(3) Displacement ~ a third kind of reaction, 
already familiar from our study of acids, is the kind of reaction 
in which an element replaces some other element in a compound. 
Any metal which reacts with an acid, displaces hydrogen from 
the acid and takes the place of the hydrogen in the compound: 
2 Hg ♦ 2 HN03 = 2, HgNC'3 ♦ H2 
(a) General formula for displacement 
reactions: AB + C * AC + B, 
(A) Double Decomposition (metathesis) - double 
decomposition is the type of reaction we have been carrying out 
in the laboratory, where the elementary atoms ’’change partners”, 
e.g,, 
KgClp + 2K.I * Hgl2 + 2KC1 
2FeCl3 + 3H2S= Fe2B3+ 6HC1 
(a) General Reaction for Double 
Decomposition: AB + CD * AC + BD. 
(b) Neutralization which, we have already 
discussed, is a kind of double decomposition. ($) Oxidation - a fifth type of reaction may 
possibly be better discussed at a later time. We will, however, 
mention here: Oxidation literally means adding oxygen to a 
compound; and in this sense, the reaction Mg + 0 * MgO, which 
we discussed as an addition reaction* is an example of an 
oxidation as well. The term is used also in a larger sense. 
Any reaction in which the oxygen content of a compound is 
increased, or in which the valence of an atom is raised, is 
an oxidation reaction. These will be discussed in greater 
detail at later dates. 
(6) Reduction - reduction is the opposite of 
oxidation. It is a type of reaction in which oxygen is 
abstracted from compounds or in which the valence of an atom is 
lowered by chemical means. Reduction is carried out by 
reducing agents, many of which are important drugs and will 
be discussed as we meet them. It should be noted even at a 
first glance at reduction reactions that they frequently 
involve hydrogen in a similar manner to the way oxidation 
reactions involve oxygen. 
b. Conduct of Chemical Reactions - in conducting 
chemical reactions there are many things to be considered. 
Perhaps chief among these is why we are carrying out the reaction, 
which should always be clearly in mind. More material 
considerations are (1) what substances take part in the reaction; 
(2) how much of them is to be used; and (3) under what physical 
conditions the reaction is to be carried out. 
(1) Reagents - Reagents are the substances which . 
enter the reaction and if we are to have a reaction, we must, 
of course, have reagents that will produce the change that we 
are aiming for in carrying out the reaction. Along the same 
lines, a further consideration is that we choose reagents which 
will produce the desired change in a way that we can use the 
result. To use a more specific example, if we are aiming to 
manufacture a chemical, we must choose reagents which will give 
us our compound in a way in which we can recover it. If we 
are analyzing a substance, we must choose a reagent which will 
cause a change which we can note, and, if possible, measure 
in some way. It is only by experience with similar reactions, 
or by exoeriraent with a particular one, that we can tell what 
will be the best reagent in a specific case. Let us further 
illustrate by means of an example, and suppose we are striving 
to make lead nitrate. In casting about for various reactions 
that might produce the result, a chemist would certainly 
consider the following: 
Pb + 2HNO3 « Pb(N03)2 + H2 
PbO + 2HN03 * Pb(N03)2 ♦ HO 
PbC03 + 2HN03 * Pb(N03)2 + HO + C02 All of these reactions result in the desired salt, lead nitrate. 
In the first, the by-product is a gas, which removes itself 
from Jthe solution. In the second, the by-product is only 
water. ' In the third, water and a gas are produced. In any 
of these reactions, the by-product may be easily and completely 
removed. If we actually try, we will find that the first 
reaction is carried out only with difficulty, and consumes a 
great deal of heat, while either the second or third goes on 
spontaneously at room temperature. So we eliminate the first. 
To choose between the second and third, we need consider only 
which of the substances are at hand or are easily to be obtained. 
Price may also be a factor, in which case we find PbO the 
cheaper. It is also more readily available, particularly in 
the Army. 
It is sometimes more difficult to choose 
reagents, but in any case, the reasoning or experiment must 
follow some such course as that above, 
(2) Quantities of Reagents - how much of the 
reagents we are to use in a specific case may be calculated 
if we can write an equation for the reaction. This involves a 
use for chemical equations which we have not yet touched on 
and we can best discuss it by considering some specific reaction. 
Suppose our purpose is carrying out a reaction to produce 100 Gm. 
of mercurous iodide. To choose reagents with which we are 
familiar, let us prepare the iodide from mercurous nitrate and 
potassium iodide according to: 
HgN03 + KI * Hgl + KNCL 
Now the thing we have not considered about chemical equations 
is that they represent relative weights of the substances 
involved, being composed of the formulas of the compounds 
involved. These relative weights total the same on the two 
sides of the quality sign in just the same way that the various 
atoms must reach the same total. In the following the equation 
is expanded by showing the relative weights involved: 
HgN03 
♦ KI 
- Hgl + 
KNC 
J3 
200.6 
39. 
1 
200. 
6 
39.1 
u.o 
126. 
92 
126. 
92 
u.o 
AB.O 
48.0 
262.6 
166. 
02 
•327. 
52 
+101.1 By simple arithmetic, it may be seen that the equation is 
mathematically true, each side totaling 428,62. (Does this 
satisfy the Law of the Conservation of Mass?) 
Our problem now is to change the relative 
weights to actual weights which we can measure. This we can 
easily do by the use of proportion, for the actual weights 
involved are certainly proportional to the relative weights. 
Then; » ... 
HgN03 » Hgl 262,6 t 327.52 
and since we want 100 Gnu of Hgl, we substitute that figure and 
solve; 
HgN03 ; 100 Gm. ;? 262.6 ; 327.52 
HgNOo = 262.6 x 100 Gm. c go.18 Gm. 
327.52 
Similarly, for potassium iodide; 
KI ; Hgl 166.02 ; 327.52 
KI ; 100 Gm.;* 166.02 ; 327.52 
KI * 166.02 x 100 Gm. * 50.69 Gm, 
327.52 
So we use 80.18 Gm. of mercurous nitrate ahd 50.69 Gm. of 
potassium iodide to make 100 Gm. of mercurous iodide, and we 
have as a by-product (80,18 + 50.69) - 100 Gm. or 30,87 Gm, of 
potassium nitrate in solution. 
(3) Physical Conditions Surrounding Reactions - 
having decided what reagents are to be used, and in what 
quantity they are to be used, there yet remains to decide the 
conditions under which the reaction is to be carried out, that 
is to say, how the reagents are to be used. Here we have 
considerably more leeway than in the previous steps; but we cannot 
hope to throw the dry reagents into a beaker and take out dry 
mercurous iodide. In order for a reaction to occur, we must 
bring the reagents into intimate contact, for chemical reactions 
take place between atoms, not between lumps, or crystals, or 
bottlefulls. Since both the salts are soluble, we dissolve 
them ad mix the solutions. The mercurous iodied precipitates and 
may be washed until free of potassium nitrate and then dried. 
Not every reaction goes along so simply, 
however, and there are often many other things to be considered. 
Even in the fairly simple reaction we have chosen as an 
illustration we can vary the nature of our precipitate somewhat 
by altering the conditions under which-the reaction takes place. 
If hot, concentrated solutions of the salts are made and mixed, 
a heavy, dense precipitate will result; while if dilute, cold 
solutions are to be used, a lighter powder and a more finely 
divided one results. Some of the factors which, in general, are 
used to control or modify the speed of reaction, or the degree 
of completeness to which a reaction will take place are shown 
below: 
(a) State of Aggregation 
Gases react most rapidly due to their 
molecules being in a state of rapid motion which results in an 
intimacy of contact throughout the mass of the gas, 
liquids react less actively than gases, 
but since they are readily mixed intimately, reactions betv/een 
liquids take place fairly rapidly. 
Solids, since they are rigid, react only 
at the surface, and often very slowly and incompletely. Again 
this is due to the fact that there is not intimate contact 
between the particles. 
(b) Temperature - Temperature has a large 
effect both on the speed of the reaction and upon the degree of 
completeness to which the reaction takes place. Both the speed 
of reaction and the degree of completeness of reaction are, 
in general, increased by increasing the temperature. This also 
follows from the kinetic theory, the heat increasing the energy 
of the molecules. 
(c) Concentration - by concentration is meant 
the actual number of molecules in a definite amount of space or 
volume. Increasing the concentration, naturally increases the 
molecular contact and speeds up reaction. 
Concentration has another effect, which is 
used in some reactions which do not ordinarily proceed to 100% 
completion. In these cases, a complete reaction is often 
obtained if one of the reagents is present in excess, i.e,, in 
greater concentration than the other. 
(d) Removal of End-Products - Frequently the 
speed of reactions is increased if the products of the reaction 
are removed as they are formed. Some reactions may be carried 
to completion only by this means. 
(e) Catalysts - Catalysts are substances, which 
by their presence cause reactions to proceed at a greater 
speed or to a greater extent than they would if the catalyst 
were not present. At first the action of catalysts was surrounded 
by much mystery and guess-work, but their use can nearly always 
be explained by sound physical or chemical reasoning. There are, 
nevertheless, no general rules one can learn regarding the action of catalysts except in comparatively isolated reactions or types 
of reactions. 
(f) Pressure - the'effect of pressure is 
similar to that of heat, in increasing the sphere of contact of 
the molecules. It has little effect, however, on reactions 
between solids and liquids, in which gases are not involved, 
(g) Electricity - Electricity has many and 
varied uses in connection with chemical reactions, too many for 
consideration here. It can be used to begin, to speed, or to 
complete reactions, and it acts quantitatively. A given amount 
of electricity will produce a given amount of chemical change. In 
fact, one of the measures of electricity, is the amount of 
chemical change it will produce. 
c. Reversible Reactions and Chemical Equilibrium - 
from many of the statemets above, it will have been seen that 
not all chemical reactions proceed until all of the reagents are 
completely changed to new compounds, as the chemical equation 
shows. Reactions which do not go to completion are known as 
reversible reactions, and the point at which they seemingly stop 
is known as* chemical equilibrium. These are more fully explained 
below, and as well in Simons, page 129. 
All of the reactions carried out in the laboratory 
have gone to completion, as do nearly ali of the reactions we have 
discussed in class. For example, the reaction, ♦ 2K0H * 
HgO + ♦ 2KC1, goes on until all of the mercury bichloride is 
used up, or all of the ootassium hydroxide, or both, depending on 
the concentration of the two reagents. T7hen either or both of the 
reagents is used up, there can be more HgO formed because there is 
no more material left to make it, and the reaction stops. To use 
a different example, one with' which we are all familiars C + ® 
representing the burning of coke (carbon) in a stove or 
furnace, the coke continues to burn (react) until it is all used 
up, or until the air (oxygen) supply is cut off. If either happens, 
the fire goes out (the reaction stops). 
In reversible reactions, however, not all of the 
reagents are permanently changed to new products; but after a 
certain proportion of the reagents has reacted, an apparently 
stable mixture results. As an example of a reversible reaction let 
us consider; 2 NaCl ♦ 2 HC1 ♦ If sulfuric acid 
is added to salt, some of the salt is converted to hydrochloric 
acid, but not all of it is. In other words, if we used molecular 
quantities of the reagents, we should expect to find only the two 
products, sodium sulfate and hydrochloric acid, in the solution. 
Instead we find fours sodium chloride, sodium sulfate, hydrochloric 
acid, and sulfuric acid. Moreover, if repeated analyses of this solution are made at various times but at the same temperature, 
they show that the four substances are present each in the same 
concentration, at each of the analyses. We say that a state of 
chemical equilibrium (balance) has been reached. 
We know, as a result of the kinetic theory, that the 
reaction is not just at a standstill, as it seems to be, but that 
it has reached a point at which just as many atoms are changing 
one way as there are changing back again. In other words, the 
reaction is proceeding in both directions at the same time. That 
is. the reason the reaction is called reversible, and also the 
reason that the equality sign is modified to become an arrow 
pointing in each direction. It is also the reason for the state 
of balance or equilibrium. It is equally true that if we start 
with the substances on the right of the equality sign, instead 
of those on the left, we get the same equilibrium mixture, if the 
temperature is the same. 
There are several ways to complete these reactions, 
as has been intimated in discussing conditions under which 
substances react. If the concentration of one of the reagents 
is considerably more than the other, the reaction proceeds to 
a greater extent; and, if one of the end-products is taken out of 
the solution, the same thing happens. 
19. Physical Properties and their Importance in Chemistry - 
Properties have already been described to some extent as the 
means by which substances make themselves evident to our senses. 
They have already been divided into general and specific proper- 
ties, according to whether or not they are possessed by all 
substances to the same degree. Another useful division of 
properties is according to whether or not they are measured by 
physical or chemical means. As chemical properties are our main 
interest in this course, we shall not attempt to discuss them in 
general here, beyond stating that frequently we are unable to 
measure a chemical property except by physical means. For this 
reason we are compelled to first learn something about physical 
properties, 
a, Physical Properties Possessed by all Substances - 
In this paragraph, an attempt is made to define and describe in 
general those properties, often called "PHYSICAL CONSTANTS11 which 
are possessed by practically all substances, regardless of their 
state of aggregation or chemical nature. No absolute separation 
of this sort can be made since many of these properties are 
carried over from one state to the other; but a little later the 
properties possessed only by gases, those by liquids, and those 
by solids, will be treated separately. (1) Specific Gravity - Specific gravity is merely 
a comparison of the weight of two substances. Let us not 
surround it with any mystery, and attempt to make it difficult. 
All of us are familiar with the statement that lead is heavier 
than feathers. This is a statement of specific gravity, and 
other statements of specific gravity are just as simple, if 
we really understand this one. All the scientific definition 
of specific gravity does is to make the statemehts uniform for 
large groups of substances. It does this by reducing the 
statement of specific gravity to a mathematical ratio, a 
fraction. This ratio or fraction ist 
Weight of some volume of substance HAM 
Weight of an equal volume of substance MBH 
Let us examine this for lead and feathers, in order to go back 
to our illustration. Suppose we take a bucket and fill it with 
lead and weigh it, finding the weight, say 256 pounds. If we 
repeat the same procedure with feathers, we find the weight is 
7 pounds. But this weight includes the bucket. If we weigh 
the bucket empty and find it weighs 6 pounds, we can subtract 
this amount from the other two weights and find how much the 
lead alone and the feathers alone weighed. The specific gravity 
then is the ratios 
2^0 
1 
Or we say that lead is 250 times as heavy as feathers. 
(2) Specific gravity compared to water - the use 
of water as a basis of comparison for specific gravity has 
become so common that when the term specific gravity is used 
without stating the basis of comparison, it is understood that 
comparison to water is intended. There are many reasons for this; 
the figures obtained are in the neighborhood of 1; water is 
easily obtainable; water does not change as much with temperature 
as some other substances; figures obtained with it are inter- 
changeable, in the metric system, with density, and many others. 
For ordinary or common 
specific gravities, then our fraction always has as a denominator 
the weight of an equal volume of water, ors 
Specific Gravity * Weight of some volume of substance tfA>l 
Weight of an equal volume of water 
Water is used as a basis of comparison for all 
liquids and solids and may be used for gases as well. (2) Density - Density is frequently confused with 
specific gravity. It is the relation between the weight of a 
substance and the volume it occupies. To state the same thing 
mathematically! weight 
Density = 
volume 
The reason for the confusion between density 
and specific gravity is the peculiar set-up of the metric system 
of weight and measure, by which volumes are measured by the 
amount of space occupied by 1 Gm. of water. In the metric 
system then, the denominator of the fraction will be the same 
whether we are computing density or specific gravity. Let us 
illustrate this by determining both the specific gravity and 
the density of alcohol. 
Let us suppose we weigh the contents of a glass 
of water, then the contents of the same glass of alcohol, then 
measure the contents by pouring them into a graduates 
Weight of glassful of water 200.0 Gm. 
Weight of glassful of alcohol 163.2 Gm. 
Volume of glass 200.0 cc. 
Specific gravity of alcohol = 163.2 * q 
200.0 
Density of alcohol - 163.2 Gm. - q,816 
200.0 cc. Gm/cc 
In specific gravity the relation is between 
weights alone and it makes no difference what system we use, the 
specific gravity will be the same. In density we are dividing 
weight by volume, and we cannot logically drop the units of 
measure. They remain in the result. Densities calculated in , 
some other system will be different from those calculated in 
the metric system. An engineer will tell us that the density of 
water is 6-4.4- pounds/cubic foot; some pharmacists say that it 
is 4-54-«6 grains/fluid ounce; while we ordinarily say it is 1 
Gm./cc. The only difference is in the system of weight and 
measure used. It is, however, important to express the units of 
measure whenever confusion might result, 
(3) Solubility - Solubility is the extent to which 
a substance dissolves in a solvent. Like specific gravity, we 
speak of the solubility of a substance as the extent to which it 
is dissolved by water, unless we specifically designate some 
other solvent. There are two methods of stating solubilities: 
(l) as the number of cc. of solvent required to dissolve a fixed 
amount of a substance, or (2) as the amount of a substance which 
can be dissolved by a fixed amount of a solvent. The U.S.P. uses 
the first method, and we shall prefer its use, although there is 
little difference. b, Specific physical properties of gases - There are 
several properties almost always discussed when any gas is 
being considered. Although these are of little direct 
application to laboratory work, we may find at some time 
that we wish we knew at least what these properties mean. Then, 
in addition, there are more and more gases being used in 
medicine. 
(1) Specific gravity - Specific gravity of gases 
is the same as other specific gravities, except the specific 
gravity of a gas may be compared to another gas. The gases used 
for comparison are air and hydrogen. When these other gases 
are used as standards for the specific gravity, a statement to 
that effect usually accompanies the figure. These statements 
are usually in the form HA = l,r meaning compared to air, or 
UH * 1!I meaning compared to hydrogen, 
(2) Density - the density of gases is frequently 
given as Gm. per liter, because the figures obtained when Gm./cc 
are used run to several decimal places. Again, there is no; 
confusion if the statement is complete. 
(3) Solubility-due to the nature of gases, one 
sees solubility of gases expressed in parts by volume. 
(4) Critical Data - Critical date of gases refer 
to the ease with which gases may be liquefied. The critical 
temperature of a gas is the temperature above which a gas 
cannot be liquefied no matter how much pressure is applied to it. 
And the critical pressure is the pressure at which the liquefactior 
begins when the gas is maintained at its critical temperature. 
Generally speaking, the higher the critical temperature and the 
lower the critical pressure of a gas is, the easire it is to 
liquefy. 
c. Specific properties of liquids; 
(1) Specific gravity 
(2) Density 
(3) Solubility - solubility of liquids is often 
given by volume. 
(4) Boiling Point - the boiling point of a liquid 
is the temperature at which the liquid and vapor phases of a 
liquid remain in equilibrium at 1 atmosphere of pressure. (5) Freezing Point - the freezing point or congeals 
ing point of a liquid- is the temperature at which the liquid and 
its solid form remain in a state of equilibrium. Needless to 
say, this is the same as the melting point of a solid. 
(6) Viscosity - this is the property of liquids by 
virtue of which they stick to surfaces. It is measured by 
the length of time it takes the liquid to drain from a fine tube. 
(7) Surface Tension - surface tension is the 
property of forming a film of closely packed molecules at the 
surface of liquids, 
d. Specific properties of solids: 
(1) Specific Gravity 
(2) Density 
(3) Solubility 
(4) Crystalline Form - the crystalline form of a 
solid is a description of the shape taken by its crystals when 
it is allowed to crystallize. These forms are very definite and 
always the same for the same solid under the same conditions. 
The. study of them is a science in itself, and for our purposes 
the mere statement that the substance, is crystalline or amorphous 
.(not crystalline) is usually sufficient. 
(5) Melting Point -see freezing point above, 
20. Ionization - Dissociation of compounds upon solution, 
or as the phenomenon is generally called, ionization, is of the 
utmost importance to all concerned with medicine and laboratory 
and as only a brief treatment of the subject is given here, the 
student is expected-to master all of it and to gradually become 
able to apply all of the matter to extemporaneous problems of 
compounding as they come up. This is no small task, and in the 
limited time available will require.the best efforts of all 
concerned, 
a. General - Dissociation, it will be remembered, was 
one of the general types of chemical•reaction; the following 
general reactions were given as examplesK simple decomposition) 
AB = A- + B 
ABC - AB + C 
ABD * AB + BG It has been found that many substances on being dissolved in 
liquids, especially on being dissolved in water, undergo 
dissociation and remain dissociated as long as they are in 
solution* Such substances, which dissociate on being dissolved 
are called electrolytes because they are the same substances which 
increase the capacity of water to conduct electricity. 
It was long ago discovered that these same 
substances deviated from non-electrolytes in the degree to which 
they changed the properties of solvents used to dissolve them. 
Electrolytes produce a greater depression of the freezing point, 
a greater elevation of the boiling point, and a higher osmotic 
pressure in solutions of them. Since it is known that these 
properties are proportional to the number of particles dissolved 
in a given amount of liquid, there was nothing left to believe 
except that electrolytes split into smaller particles and a 
greater number of them. This gave rise to the theory of 
electrolytic dissociation, which states that MOLECULES OF 
ELECTROLYTES WHEN DISSOLVED IN WATER, BREAK UP TO A VARYING 
DEGREE INTO INDEPENDENT PARTICLES CHARGED WITH ELECTRICITY AND 
THE NATURE AND NUMBER OF THESE PARTICLES DETERMINE, TO A LARGE 
DEGREE, CERTAIN PHYSICAL AND CHEMICAL PROPERTIES OF THE SOLUTIONS. 
These independent charged particles are called IONS. 
b. Properties of ions as compared to atoms - 
(1) Kinds of Ions - Ions are of two kinds, different 
as regards the nature of the electrical charge on them; 
CATIONS - or electro-positive ions have a 
positive charge on them, and when an electrical current is applied 
to a solution, the cations move toward the cathode or negative 
pole, 
ANIONS - or electro-negative ions have a charge 
of negative electricity on them and upon application of a current, 
move toward the anode or positive pole. 
(2) Relation between anions and cations - neutrality 
of solutions - in every solution, anions and cations are present 
in equal numbers - for each anion. one cation; for each positive 
charge, one negative charge. It is for this reason, that the 
solution as a whole is electrically neutral. Moreover, while the 
statement of the electrolytic theory describes ions as 
"independent particles," they are never present without the 
corresponding oppositely charged ion, so that they should not 
really be said to be independent particles. (3) Ions are atoms plus a charge of electricity - 
Ions react according to electrical laws rather than to chemical 
laws: they unite with other ions only if the other ions are 
oppositely charged. Thus, while two chlorine atems will react 
to form a molecule, two chlorine ions will not, unless they are 
first removed from solution. In other words, before ions can 
react like atoms, they must become atoms. Moreover, in the case 
of many ions, there would be a reaction between the ion and 
water, if the ion were an atom. For instance, take the 
dissociation of sodium chloride in solution. If the sodium ion 
were the same as the sodium atom, it would react with the water 
to form a molecule of NaOH and hydrogen would be given off. 
Sodium chloride solutions do not do this unless sufficient 
electrical force is applied to remove the ion from solution. In 
the sane solution, if the chlorine were in the form of atoms, 
it would color the solution yellow, which it does not. 
c. Symbols for ions - the symbol for an ion is the 
symbol for the corresponding atom with the proper electrical 
charge added, as a plus or minus sign. Thus the symbol for the 
chlorine ion is Cl", for the sulfate ion it is , for the 
sodium ion Na+, for the ferrous ion Fe++, and for the ferric 
ion Fe+++. From these symbols it may beeseen that the charge 
on an ion is proportional to its valence. This has already been 
applied practically in the laboratory in balancing chemical 
equations. 
d. Dissociation reactions - it was stated at the be- 
ginning of this discussion that dissociation in solution was a 
chemical reaction. It is, moreover, a reversible reaction, which 
means that it does not, in all cases, go to completion, but 
reaches a state in which there are as many molecules 
into ions as there are ions reuniting to form molecules , chemical 
equilibrium, in other words. As with all reversible reactions 
there are physical conditions which influence the composition 
of the ’’equilibrium mixture,” The condition which has the most 
influence is concentration, i.e., the relation between the 
number of solute and of .solvent molecules. Ordinarily, dilute 
solutions ionize to a greater extent than more concentrated ones 
of the same solute and solvent. 
(1) Equations - Ionic equations are written as 
other reversible reactions, using the symbols for ions. Usually 
esch ion is shown separately? HC1 H* + Cl~ 
H2 S04 ?=* + H+ + SO — 
Ca(0H)2 Ca++ + OH“ + OH~~ 
It should be noted that radicals do not ionize within themselves 
to liberate the ions of each of the elements of which the 
radical is composed, but that the charge is on the radical as a 
whole, thus 0H~ and SO—. 
4 
To interpret the above a little more fully? the 
first reaction tells one that hydrochloric acid dissociates into 
hydrogen ion and chloride ion until a certain point is reached 
depending upon the concentration. At this point of equilibrium 
there are as many hydrogen and chloride ions reacting to form 
hydrochloric acid molecules as there are hydrochloric acid 
molecules dissociating into hydrogen ion and chloride ion. At 
this point there are three kinds of solute present: (l) 
hydrogen ion; (2) chloride ion: and (3) hydrochloric acid 
molecules. 
e. Ionization constants - the ionization constant is 
a measure of the degree of completion of a dissociation reaction. 
It varies with concentration and with temperature; and it is 
too cumbersome a figure for use in ordinary labora- 
tory work. In fact, ionization data are of little use to the 
practical pharmacist, since very little is yet known about the 
ionization of concentrated solutions. The student is referred 
to the electromotive series, and from the standing of an ion 
in the general series and experience with similar salts, he can 
usually estimate the degree of ionization closely enough; see 
paragraph on hydrolysis below. 
f. Chemical reactions in the light of electrolytic 
theory - chemical actions in aqueous solutions are practically 
always reactions between ions. Some workers claim that reactions 
are always between ions. There remain, however, reactions 
between substances that ionize only slightly, if at all, so that 
theory cannot be altogether correct, 
g. “Strength” of acids and bases - an attempt has been 
made throughout this course to use the term “concentration'1 to 
describe the weight of solute per unit of weight or volume of 
solvent in contrast to the term “strength." The term strength 
is used to refer to the intensity of acid or basic properties, 
which is a direct consequence of ionization. Acids which ionize 
to a large extent are called STRONG ACIDS; those which ionize only slightly are called WEAK ACIDS, etc. To put the same 
statement a little differently, the acids which ionize most 
have the greatest concentration of hydrogen ion per unit of 
concentration, and acid properties are the result of hydrogen 
ion. Those bases which ionize to the greatest degree have 
the greatest concentration of hydroxyl ion and are therefore 
called the STRONG BASES. 
21. Hydrolysis ~ hydrolysis means literally: splitting 
by water, and it has been defined as double decomposition between 
another substance and water. Like most phenomena, hydrolysis 
was known long before it was explained. Chemists early found 
that many substances, which of themselves are neutral, impart 
acid or alkaline reactions to water when they are dissolved. 
From early observations it was noticed that certain acids nearly 
always produced salts that gave solutions an acid reaction, 
and they called these acids strong acids. Similarly, it was 
noted that bases of certain metals produced salts with a tendency 
to impart basic reactions to solutions. They called these strong 
bases. Ionization gave these studies a new meaning, and it 
is in terms of ions that we will discuss hydrolysis. 
It has already been pointed out that strong acids and 
bases are those which ionize largely, and that weak acids and 
bases are those which ionize only slightly. We note further 
that metals high in the electromotive series have strong bases, 
while metals lower in the series produce weak bases. As 
regards acids, most of the inorganic acids are relatively strong, 
most of the organic acids are weak acids. For-the most part, 
the binary acids are stronger than ternary acids of the same 
element (sulfur is a notable exception). 
22. Hydrogen ion concentration of water; in our discussion 
of chemistry we have seen how inorganic compounds break up or 
ionize in water. We are now going to take up a special 
ionization that has to do with the production of hydrogen ions. 
Before doing this it will be well to review three 
things that we will use in our discussion. These are the 
definition of an acid, base and neutralization. The first is 
the definition of an acid which we might define by saying that 
it is any substance capable of liberating hydrogen ions, i.e., 
HC1 + H20=H+ + 01"“, similarly a base might he defined as any 
substance capable of liberating hydroxyl ions, i.e,, NaOH + 
H2O *Na+ ♦ GH”, Neutralization is the process in which an 
acid reacts with a base to form a salt and HgO. For our purposes 
a consideration of the formation of water is the main point for 
we may consider our acid as H* and Cl“, and the base as Na+ and 
OH". When these react we have water (*L0) and NaCl which in 
solution is Na+ and Cl~. Hence we may write our reaction as 
H* + ci~ + Na+ + 0H~ = H20 + Na+ + Cl~ or more simply 
H* ♦ OH* H20. Keeping this in mind we can continue on our discussion 
with a treatment of the difference between total acidity and 
hydrogen ion concentration. 
For an example, let us take some N/lO acid. Here we 
say that the total acidity is tenth normal for it will take 
exactly an equivalent amount of N/lO base to neutralize it. 
Now let us consider the hydrogen ion concentration. 
In any ionization, the process is not totally complete. This 
is usually written as HC1 01““ + H* and called a 
“reversible process." This phenomenon is especially marked in 
weak acids such as acetic acid. In this case ionization occurs 
only to the extent of about 1%. This would mean that for every 
100 molecules of acetic acid dissolved in water, only one would 
ionize to form hydrogen ions. Thus we could say that in N/lO 
acetic acid the total acidity was tenth normal and the hydrogen 
ion concentration was .1 x .Olor.OOlN. or N/1000, 
We will now boldly define pH and then define the words 
used in the definition, 
£H is the log of the reciprocal of the hydrogen ion 
concentration. From our discussion we already know what 
hydrogen ion concentration is, but to repeat it is merely the 
concentration of actual hydrogen ions in solution. 
In order to define the term reciprocal we will merely 
have to recall a little of our grammar school arithmetic. To 
take the reciprocal of a number we invert that number or turn 
it upside down. For a fraction this is easy, for here we merely 
interchange denominator and numerator, i.e., 3/4; the reciprdcal 
of it would be 4/3. To go on a little further, we will consider 
decimals. These of course are fractions in Y*hich the denominator 
is some power of ten, i.e., ,1 can be written as 1/10 or .02 can 
be written as 2/100. Therefore, the reciprocal of .02 or 2/100 
would be 100/2 or 50. To complete this discussion of inversion 
we will have to talk of whole numbers. We commonly write down 
a whole number, i.e,, 50. If we now divide this whole number 
by one, it will not be changed. Therefore, we could say 50 » 
50/1. If we do this, it is easy to take the reciprocal of it 
by taking 50/l, and turning it upside down, giving us 1/50, 
We now have only one more term left to define. The 
definition of a log is just as simple as those we have just 
discussed, but not quite so familiar to you. Before we make our 
definition, though, let’s have a little more arithmetic. We all know that 100 * 10 x 10 or Similarly, 
1000 = 10 x 10 x 10 or ICp. Another way of saying this is 
that ten to the power two equals one hundred and ten to the 
power three equals one thousand. Now let us consider 10^*303^ 
We know this will be greater than 102, or 100 and less than 
or 1000. By the use of suitable tables we can find that 
1q2.303 200. You are no doubt now beginning to suspect that 
any number can be written as ten to a certain power, and that 
is correct. The definition of a log follows directly from 
this fact, for a log is the power to which ten is raised to give 
that number. 
We have now considered the meaning of all the words in 
the definition of pH, but no doubt it still doesn’t make a great 
deal of sense, sc let us see how it works. To do so, however, 
we will have to digress somewhat again. 
It is well known that water instead of being merely 
JijO is really a reversible reaction, i.e., H+ t 0H~. 
Further, it is known by experiment in this case that (H+) x 
(0H“) » l/lO1 . (The brackets () around the ion means 
”concentration of,”) From the equation above it can be readily 
seen that for each molecule of water that ionizes, one ion of 
hydrogen is formed. Therefore, the concentrations are equal. 
Thus we can say (H+) x (OiT) = (H+) ; (H+)2 = l/lO or (H+l = 
By recalling a little more arithmetic we write 1/10 , 
Reducing all the above paragraph into a nutshell, we can say that 
in neutral water (H+) = 10~'. 
Now let us recall our definition of pH, pH = the log 
of the reciprocal of the (H+). (hydrogen ion concentration). 
Let's see,inwater (H+) =* 10""' “ l/lCr . the reciprocal of 
l/lO' = = 10 , and the log of 10' = 7. 
23. 
ph 
1 
2 
3 
U 
5 
6 - 
7: 
e' 
9 
10 
11 
12 
13 
U - 
ACID 
SOLUTIONS 
NEUTRAL SOLUTIONS 
ALKALINE 
SOLUTIONS 24. Titration - 
a. It is merely a convenient method of measuring the 
amount of one solution that will react with a fixed amount of 
another solution. The fixed amount of the one solution is 
measured with a pipette into a beaker, and if necessary 
diluted with water. The standard solution, or titrating reagent 
is placed in a burette, and added carefully drop (gtt) by drop, 
until the reaction is complete. This is found by previously 
adding an indicator to the solution in the beaker which changes 
color when the reaction is complete, or commonly alluded to as 
when the "end point" has been reached. 
b. The following table shows some of the most 
commonly used indicators, their color changes and the pH at 
which this change takes place; 
Indicator 
Color Change (Acid to Base) 
pH Range 
Methyl Orange 
Pink to yellow 
~~ 
Methyl Red 
Pink to yellow 
4.8 - 5.4 
Phenol Red 
Yellow to red 
7.0 - 7,4- 
Phenolphthalein 
Colorless to red 
7.8 - 8.0 
Congo Red 
Blue to pink 
3.3 - 4.6 
Litmus 
Red to blue 
5.0 - 6.0 
Resorcin Yellow 
Yellow to Orange 
11.1 -12.7 
25. Buffer Action - this is a term developed along with 
knowledge of hydrogen ion control. Briefly, buffers are sub- 
stances which, when they are added to solutions, decrease the 
rapidity with which pH changes on dilution or on addition of 
hydrogen or hydroxyl ion. Buffer action is a sort of masking, 
by means of whick the total acidity or alkalinity of a solution 
may be altered without marked change of pH, 
The mechanism of buffer action is difficult to explain 
without going into mass action phenomena; and it will suffice 
here to note that buffers act by selectively repressing or 
decreasing the extent of ionization. Remington (page 543) gives 
a more complete explanation of buffer action. Buffers are 
seldom of direct application in laboratory,except in the pre- 
paration of some solutions, Each of these is a problem in itself. 
26. Types of Solutions: 
a. Simple solution of reagents - is one in which a 
designated weight or volume of reagent is placed into a 
specified solvent. When no solvent is designated, distilled water is always intended. Here caution is definitely to be observed 
in that the proper reagent is selected, also accurate weighing 
and measuring of both the reagents of the solute and solvent is to 
be observed, 
b. Percentage solutions - in this type of solution 
simple arithmetic in percentage is the vital knowledge necessary 
in that all volumes are calculated on the basis of 100, And 
if this can be kept in mind, little cause for confusion will 
arise. Percentage solutions are rough calculations, hence Gms. 
and cc's are interchangeable. For example, a 1% can be made of 
any reagent by placing 1 Gm. or 1 cc and adding sufficient water 
to make 100 cc. Or in other words, 1 Gm. or 1 cc qs. to 100 cc. 
In fractions of % for instance, ,25% can be made by weighing er 
measuring, 1/4 Gm., 250 mg., .25 cc qs. to 100 cc with water. 
In like manner, where the % is greater than one, such as a 20$, 
20 Gms, or 20 cc is qsed up to 100 cc, as one would state in 
rather a common laboratory vernacular. 
c, Normal Solutions - this type of solution is one 
containing one equivalent weight (chemical equivalent) of a 
reagent qs (i.e,, as much as necessary of distilled water) to 
1000 cc. (1 liter). 
(1) An equivalent weight or chemical equivalent 
is the amount of a reagent that either contains or reacts with 
1 atomic weight of hydrogen, 1.008 Gm,, this to be expressed in 
Gms. 
(2) The cautions to be observed in the making of 
this solution is the accuracy of calculation, namely, the accuracy 
of atomic weights, the addition of proper molecular weights, the 
accurate number equivalents, the conversion of Gms. to cc’s, if 
the reagents are liquid, and the proper calculation of various 
assays of the reagents. As an example: 
4N-H2S0, - Sulfuric acid- 
H2 = 2.016 
S *32.060 
40 *64.000 
98.076 t 2, since there are 2 H's * 
49.038 Gms. » 1 normal qs. to 1000 cc 
4N *49.038 x 4 * 196.152 Gms, qs to 1000 cc 
cc x Sp G(specific gravity) * Gm. Using the above equation, one can convert the calculated Gras, 
in 4N to the number of cc to be used, provided the Sp.G, is 
known. In the case of the particular the Sp.G, is 1,84. 
The solution then follows: 196.152 Gm. 184 - 106.604 cc qs 
to 1000cc.This would be correct if the assay of the would 
be 100%. In this case it is 96% to 98%. Hence 97% should 
utilize in its calculation thus: 106.604 *.97 * 109.901 cc qs 
to 1000 cc, will give an accurate 4N H^SO^. 
d. Molar Solutions - is a solution containing the 
molecular weight in Gms. of the reagent qs to 1000 cc, with 
distilled water. Here no equivalent weight or chemical equiva- 
lent are necessary. However, conversion from Gms. to ccfs and 
a calculation for percentage of assay should be effected. 
e. Miscellaneous Solutions: 
(1) Alcoholic Solution - is one in which the solvent 
is alcohol, ethyl, 95%. 
(2) Aqueous Solution - is one in which the solvent 
is distilled water. And whenever no reference is made to the 
type of solvent, distilled water is intended. 
(3) Isotonic Solution - is one having the same 
osmotic pressure as some other solution with which it is 
compared. Isotonic Salt Solution (.9%) has the same osmotic 
pressure as the salt solution in body cells or body tissue, 
(4) Hypotonic Solution - is one having an osmotic 
pressure less than the comparing solution. 
(5) Hypertonic Solution - is one having an osmotic 
pressure greater than the comparing solution. BLOOD CHEMISTRY 
I. Protein Free Filtrate 
A, Solutions; 
1. Sodium Tungstate, 10$ (Na0 WO,. HO) 
* 4 2 
2. Sulfuric Acid, .66N ) 
3k Sulfuric Acid, 10$ 
4i Potassium Oxalate, 2% 
5* Benzoic Acid, ,25$ 
B, Procedure; 
1, 1 vol, of oxalated blood 
and 
' 2. 7 vol, of distilled water (to lake blood),shake well 
add 
3. 1 vol. of 10$ Ka WO . H O, mix 
add 2 4 2 
4. 1 vol. of ;,66N H SO , shake 
(changes pinx to dark brown), and if not add 
5. drop by drop 10$ until right color, filter. 
C, Action; 
The 2/3 N acid is intended to be equivalent to the 
sodium content of the tungstate so that when equal volumes are 
mixed substantially the whole of the tungstic acid is set free 
without the presence of an excess of H?S0 , The tungstic acid 
set free is nearly quantitatively taken up by the proteins and 
hence, the blood filtrates obtained are free of protein and only 
slightly acid. 
II. Blood Sugar Determination 
A. Solutions; 
1, Stock Sugar Solution, 1$ Anhydrous Dextrose 
Solution in ,25$ Benzoic Acid, 
2, Working Standards; 
Stock Sugar Sol.’ . 
a. Weaker; 5 cc ♦ .25$ Benzoic Acid qs. to 500 cc. 
Stock Sugar Sol. 
b. Stronger; 5 cc + .25$ Benzoic Acid qs. to 250 cc. 3. Alkaline Copper Solutions 
4.0 gm, Anhydrous Sodium Carbonate 
4.00 cc. distilled in 1 liter flask 
'; 7,5 gm. Tartaric Acid, when dissolved add 
4..$ gm. Crystalized Copper Sulfate 
qs, to 1000 cc with Distilled 
4.. Molybdate - Phosphate Solutions 
35 gm. Molybdic Acid 
5 gm. Sodium Tungstate 
200 cc 10% Sodium Hydroxide Solution in 1000 beakei 
200 cc distilled 
Boil vigorously 4-0*minutes, then cool 
Add distilled H O qs to 350 cc. 
125 cc Phosphoric Acid (85% Solution) 
Add distilled qs to 500 cc. 
B. Procedures 
1. 2 cc Protein Free Filtrate in blood sugar tube 
2. 2 different standards in 2 blood sugar tubes 
3. To all add 2 cc of Alkaline Copper solution 
4v Boil for 6 minutes in boiling water bath 
5. Cool for 3 minutes without shaking 
6; cc of Molybdate-Phosphate Solution 
7. Add distilled up to 25 cc., mark 
8. Invert several times, mix well, • 
9. Compare in colorimeter, 
C. Calculations 
Weaker Solution: 
(20) 
Reading of Standard x 100 « mg. per 100 cc 
Reading of Unknown 
Stronger Solutions 
(20) 
Reading of Standard x 200 « rag, per 100 cc 
Reading of Unknown Ill, Blood Sugar Determination by Lewis and Benedict 
A. Solution: 
1. Picric acid, chemically pure. For each determination 
0.5 Gm. will be required, 
2. Sodium carbonate, saturated solution {22%), Each 
determination requires 2 or 3 cc. 
3. Standard dextrose solution, a 0,02$ solution of 
pure dextrose in saturated aqueous solution of picric acid. 
B. Procedure: 
1. Place 2 cc of oxalated blood in a centrifuge tube, 
preferably with round bottom to facilitate stirring; and add B 
cc of distilled water, 
2. Mix well, and let stand until the blood is 
completely laked, 
3. Add 0,5 Gm, of dry picric acid and stir well with 
a slender glass rod. Let stand for five minutes, with occasional 
stirring. 
A. Centrifugalize, and filter the supernatant fluid 
through a small filter paper into a dry test tube, 
5. Transfer 3 cc of the filtrate to a 10 cc volumetric 
flask (a tall test tube with a 10 cc mark or an accurately 
graduated centrifuge tube will answer). Add 1 cc saturated 
solution of sodium carbonate, 
6. Place 3 cc of the standard dextrose-picric-acid 
solution in a similar flask or tube, and add 1 cc of saturated 
sodium carbonate solution. 
7. Heat both tubes in a beaker of boiling water for 
fifteen to twenty minutes. Cool to room temperature. 
8. Make up the unknown and the standard solution to 
10 cc with distilled water. Mix well. 
9. Compare the unknown with the standard in a colorimeter. 
The calculation is based upon the fact that the unknown 
represents 0,6 cc of blood and the color standard 0,6 mg, of 
dextrose. With the plunger type of colorimeter or the Denison 
Laboratory Colorimeter, the following formula may be used; Heading of Standard x 100 « mg. of dextrose in 100 
Reading of Unknown cc of blood. 
When the blood sugar is high, a standard containing 
double the amount of dextrose-picric-acid solution must be used, 
and this requires that the final result be multiplied by 2, 
\ 
IV. Marshall Test; Determination of sulfanilamide, sulfapyridine, 
sulfathiazole, sulfaguanidine, and sulfadiazine in blood. 
A. Solutions; 
\ 
1. A solution of trichloracetic acid containing 15 gm. 
dissolved in water and diluted to 100 cc. 
2. A 0.1% solution of sodium nitrite. Should be 
prepared fresh each day. 
3. a,An aqueous solution of N-(1-naphthyl) ethylene- 
diamine dihydrochloride containing 100 mg, per 100 cc. This 
solution should be kept in a dark colored bottle. If kept in 
ice box when not in use, it will keep for one week.(See footnote) 
4. A solution of saponin containing 0.5 gm. per liter, 
5. 4N hydrochloric acid, 
6. A solution of ammonium sulfamate, containing 0.5 gm. 
per 100 cc. 
7. A stock solution of sulfanilamide, sulfapyridine, 
sulfathiazole, sulfaguanidine, or sulfadiazine in water contain- 
ing 200 rag. per liter. The chemically pure, dry, finely 
powdered drug should be used, not tablets. 
8. Working Standards - these are made according to the 
following table; 
,5 cc stock, add 18 cc Trichloracetic Acid, qs to 
100 cc * .01 .* 2 equation factor. 
1, cc stock, add IB cc Trichloracetic Acid, qs to 
100 cc « ,02 s U equation factor. 
2,5 cc stock, add 18 cc Trichloracetic Acid, qs 
to 100 cc = .20 • 10 equation factor. 
5.0 cc stock, add 18 cc Trichloracetic Acid, qs to 
100 cc * .10 * 20 equation factor. 
(Footnote) 
3.b,The alternate solution, and the one most fre- 
quently used is 1 cc of Dimethy-a-naphthylamine qs to 250 cc 
with Ethyl Alcohol 95%. Caution to be observed in the use of 
this solution is, that in the procedure 5 cc of this solution 
is used instead of 1 cc of solution 3.a, above. B. Procedure: 
1. 1 cc or 2 cc oxalated blood. 
2. 15 cc or 30 cc Saponin Solution, ,05$. Wait 2 minutes 
or until laked. - • . 
• f , ; *'*’* * 
' ■ ‘ ' • I ■ , 
3. 4 cc or Bcc Trichloracetic Acid,15$; filter, 
4. 10 cc Filtrate; also 10 cc of Standard 
5. 1 cc Sodium Nitrite Solution, .1$ , same here. 
(This to be prepared fresh each day); wait 3 minutes. 
6. 1 cc Ammonium Sulfamate Solution ,5$ same here; 
wait 2 minutes. 
7. 5 cc Dimethyl~a~naphthylarain Solution same here. 
(1 cc of N~(1-Naphthyl} ethylenedtamine dihydrochloride is used 
at times if available.as an alternative solution). . 
8. The unknown is then compared with the appropriate 
standard, as treated above, in the colorimeter, 
C, Calculation: 
1. Reading of Standard x Equation factor = mg.$ of free 
Reading of Unknown (see 8,page 47) sulfonamide 
2. Total sulfonamide determination is obtained by using 
5 cc of the filtrate in a calibrated test tube, and adding 1. 
cc of 4N-hydrochloric acid solution. The mixture is heated on a 
boiling water bath for 2 hours. Taking care not to allow the 
volume go below 3 cc, cool and adjust the volume to 10 cc. This 
gives the final dilution of blood 1:40,* The subsequent dia- 
zotization procedure is as described above. 
3. The use of conversion factors can be considered in 
the calculation, if standards are not of the same sulfonamide as 
the sulfonamide in the blood specimen. Some -of the most 
frequently used factors are-in the'following table. These are 
utilized by multiplying the already calculated mg.$ by the 
respective conversion factor. Factors Used in the Marshall Determination 
UNKNOWN 
Sulfanil- 
amide 
Sulfa- Sulfa- 
pyridine nilyl 
Sulfa- Sulfa- 
thiazole methyl 
Sulfa- 
diazine 
Sulfanilamide 
1.4,7 
2.07 
1,48 . 
1.54 , 
1.50 
Sulfapyridine 
0.68 
. 1*45 . 
1.04 
1.11 
Sulfanilyl- 
Sulfanilaraide 
O.'iS 
0.69 
• 75 
0.80 
Sulfathiazole 
0.68 
0.96 
1.33 . 
. 1.08, 
Sulfamethyl- 
Thiazole 
0.65 
0.90 
.92 
V, Modified Method of Hall, and United States Army Laboratory 
for the Determination of Alcohol in Blood, Urine and 
Spinal Fluid, . . 
A, .. Solutions: , 
1. Anstie1s Reagent. modified, stronger. 
Potassium dichromate (reagent quality) 3*70 grams 
, . Distilled water ....... 150 cc 
Concentrated Sulfuric Acid (C.P. 
quality). • * . • 280 cc 
Distilled water . .to make . , . . , . $00 cc 
Dissolve the potassium dichromate in the 1$0 cc of 
distilled water and add slowly, with constant.stirring, the 
sulfuric acid. .Finally, dilute to $00 cc with distilled water, 
(Note: this solution is about 11% stronger than the ordinary 
modified Anstie1s reagent, and should not be confused with that 
solution. It should be plainly.labeled stronger Anstie’s reagent. 
It may, however, be readily converted into modified Anstie1s 
reagent by diluting 9 parts of the stronger Anstie’s reagent with 
1 part of distilled water, and may thus be used in carrying out 
the determination as given heretofore, if desired, instead of 
making up the two separate solutions). ./i 2. Standard Alcohol Solution; 
Absolute Ethyl Alcohol 2.53 cc 
Distilled Water .... to make 100 cc 
Place about 50 cc of distilled water in a 100 cc 
volumetric flask and add the alcohol directly to the water to 
prevent its evaporation. The alcohol can be conveniently measured 
by means of a 1 cc serological pipette, transferring two full 
1 cc proportions and finally the 0.53 cc portion. 
3. Scott-Wilson Reagent 
Mercuric cyanide 5 gm. 
water , . ,300 cc 
Sodium Hydroxide 90 gm. 
water . 300 cc 
Silver Nitrate .,1,4-5 gm. 
water .200 cc 
Add Sodium Hydroxide thoroughly cooled to Mercuric 
cyanide; mix thoroughly; then add Silver nitrate to the mixture 
with constant stirring. This solution will keep for six months. 
If it becomes cloudy or a precipitate forms, filter. Do not 
pipette this solution - it is very poisonous. 
4. Preparation of Standards: 
Arrange 9 test tubes of uniform diameter and color 
(color comparison tubes are better) in a test tube rack and place 
9 cc of stronger Anstie’s reagent (Solution ) and distilled 
water in the amounts shown in the following table; 
Tube 
No. Alcohol 
Distilled 
Corresponds to 
Alcohol in 
the 
Solution( ) 
Water 
Specimen 
1 
None 
1.0 cc 
Negative 
2 
0,1 cc 
0,9 cc 
0.5 milligrams 
per 
cubic 
centimeter 
3 
0.2 cc 
0.8 cc 
1.0 " 
t! 
ii 
it 
4 
0.3 cc 
0,7 cc 
1.5 « 
ti 
it 
ii 
5 
0.4 cc 
0.6 cc 
2.0 « 
n 
it 
it 
6 
0-5 cc 
0.5 cc 
2.5 » 
n 
n 
it 
7 
0,6 cc 
0.4 cc 
3.0 « 
it 
ii 
it 
8 
0.7 cc 
0.3 cc 
3.5 " 
ti 
ti 
ti 
9 
. 0.8 cc 
0.2 cc 
4.0 " 
it 
n 
it 
The contents of each tube must then be thoroughly 
mixed. This may be accomplished by drawing the contents of each 
tube up into a 10 cc pipette .and allowing it to run back into 
the tube several times. If desired these standards may be kept for several 
weeks if tightly stoppered and kept in a vertical position in a 
test tube rack. The solutions in the standards must not come 
into contact with the stoppers, as both cork and rubber stoppers 
contain reducing substances which may cause a change in color 
of the standards. 
Each standard should be labeled with the number of 
milligrams of alcohol to which it corresponds (e.g*, tube No. 1 
should be labeled "Negative"; tube No, 5 should be labeled 
"2.0 mg."; etc.). 
It is useless to try to make standards for readings 
greater than 4.0 milligrams-of alcohol per cubic centimeter, as 
the Anstie’s reagent is apparently completely changed by that 
amount of alcohol, and no difference can be detected between the 
4.0, the A.5 and the 5,0 mg.,per cc standards, no matter by which 
method they are prepared. Should specimens be encountered which 
have 4.0 mg, of alcohol per cc, or more, a second determination 
should be made using half the quantity of the specimen (2 cc 
instead of 4 cc) and the result multiplied by two to give the 
final reading. 
B. Procedure: Urine, blood or spinal fluid: 
Arrange two 27 x 210 mm. tubes with two-holed rubber 
stoppers and inlet and outlet tubes. The inlet tubes should 
extend nearly to the bottom of the tube and the outlet tube just 
below the stopper. Using well-washed rubber tubing, connect the 
inlets and outlets' in such a manner that a current of air may be 
aspirated through the specimen tube over into the tube containing 
Anstie's reagent. 
In the specimen tube place 4 cc of specimen, 2 to 4 cc of 
Scott-Wilson reagent, and sufficient water to make 10 cc. Half 
way between the upper level of the fluid contents and the bottom 
of the stopper, place a wad of glass wool. 
In the second tube, place 10 cc of the Anstie's reagent. 
Stopper the tubes, and adjust the suction so that a 
reasonable current of air .is aspirated through the tubes. Immerse 
the tubes in a water bath previously brought to boiling. 
Continue the boiling and aspiration for 12 to 15 minutes. 
Cool the dichromate solution and compare with the 
standards which read directly in milligrams per cc. TABLES AND EQUIVALENTS 
1 myriagram « 
10,000 grams 
1 kilogram - 
1,000 grams 
1 hectogram = 
100 grams 
1 decagram « 
10 grams 
1 gram » 
wt. of 1 cc of water 
1 decigram 55 
1/10 part of 1 gram « ,1 gram 
1 centigram * 
1/100 part of 1 gram = ,01 
gram 
1 milligram = 
1/1000 part of 1 
gram 52 .001 gram 1 
mgm. 
Oj/ 
s fluid 
ounce 
J = fluid 
dram 
0 * pint 
m = minim 
cc - cubic 
centimeter 
Min. 
cc 
Min. 
iY 
1 
= ,06 
60 m = 
5 
= .30 
8 
rfy' 
10 
= ,60 
16 
1 © 
15 
= 1. 
(.92) 
32 
1 qt. 
20 
* 1.25 
2 © 
1 qt. 
30 / 
* 1.90 
4 qts. = 
1 gal. 
1 fluid % 
* 4. 
(3.75) 
1 fluid "Y 
= 30.00 
(29.57) 
16 fluidly 
= 473.17 
» 1 pt. 
32 fluid 
= 946.33 
= 1 qt. 
Grams 
Grains 
.0010 
s 
1/64 
.0162 
ss 
1/4- 
.0324- 
* 
1/2 
.065 
s 
1 
.100 
= 
1 1/2 
.500 
s 
7 1/2 
1. 
s 
15 
2. 
s 
30 
Grams 
i Y 
s 
28.350 
16 7 
= 
453.60 
* 1 lb. SUPPLEMENT TO BLOOD CHEMISTRY 
VI, Non-Protein Nitrogen - all tabes in which NessLer1 s have been 
in must be rinsed with concentrated HNOg. 
5 cc, Blood filtrate (in pyrex tube); graduated at 35 
and 50 cc, 
1 cc. Diluted acid mixture (50 cc. of 5$ Copper Sulfate 
300 cc, of 85$ Phosphorous Acid 
100 cc. of Con, H2SO4) 
(Dilute 1/2 of this solution with l/2 of Distilled HgO) 
Boil vigorously with glass bead until tube fills with 
vapors, cover end of tube with funnel; continue heating 3 min., 
at least until solution is clear. 
Cool 90 seconds 
15 cci to 20 cc. Distilled HgO with tube at 45° angle 
Cool to room temperature qs, to 35 cc, 
15. cc; Nessler*s Solution (stopper) 
(Add simultaneous with standard) 
Standard must contain 0,3 mg, of N per cc, 
(,4?16 Cm, Ammonium Sulfate qs, to liter) 
3 OCi Ammonium Sulfate standard in 100 cc, Vol, flask 
2 cc. Dil, phosphoric sulfuric acid mixture 
60 cc, Dist. HgO. 
30 cc, Nessler*s Solution 
to 100 cc# with Diet* HgO. 
Calculation: 
- R§ x 30 = mg. $ 
RU 
or 
Place unknown at 30 in colorimeter,standard then ® 
NPN per 100 cc, • • 
1 ’ •* * 
Significance: ■-« r . 
Normal NPN,;of blood, is 25 to 35 Mg, $. NPN is a mixture 
of urea, uric acid, creatinine, ammonia, amine acids and a mixtur 
of other undetermined compounds.. 
Raised in terminal nephritis; 
'Lowered in uremia; urea determination more important. VII. Blood Urea 
Unknown - 5 cc, Blood Filtrate in 25 cc. Vol, flask 
15 cc. Bist. HgO 
2.5 cc, Nessler1s 
qs. to 25 cc. with Bist, H^O. 
Standard- 3 cc. Standard Ammonium Sulfate in 100 cc, Vol. 
flask (10 cc. a 1 mg. N) 
60 cc. Bist, HoO 
10 cc, Nessler*s Sol. 
qs, to 100 cc. with Bist, HgO 
Calculation: 
RS x 15 = mg, # 
RU 
Signlgicance: 
Normal urea of blood is 10 to 15 mg, #, Early 
nephritis may rise to 30 to 40 mg, #. In uremia the urea is 
retained. High readings may be found in: 
Bichloride noisoning 
Bouble polycystic kidneys 
Intestinal obstruction 
Prostatic obstruction 
Lead uolsonings 
Certain infections 
Cardiac failure 
The amount of urea gives an indication of surgical 
risk, 
VIII, Creatinine 
Unknown - 10 cc. free filtrate 
5 cc. alkaline picrate solution 
(15 cc, saturated picric acid solution) 
(3 cc. 10# NaOH Sol.) 
Standard - 5 cc. standard (5 cc. s.03 mg.) 
(In 500 cc.vVol. flask 3 cc. of the standard 
Creatinine Solution add 50 cc. of .IN to 
HOI, dilute to 500 cc. with Bis*. HgO) 
(Standard Creatinine Solution -,1 Cm, of 
Creatinine in 100 cc. of .1N-HC1) 
15 cc. Bist. HgO 
10 cc. Alkaline Picrate Solution (see above) 
Stand 15 minutes 
b2 Calculation; 
RS x 1,5 - mg, io 
'• RTJ 
Significance; , 
Creatinine is the least variable nitrogenous 
constituent of the blood. Normal 1 to 2 mg. $, 
. 1 Early nephritis* 2 to 4 mg. Chronic nephritis and 
uremia 4 to 35 mg, $, marked impaired kidney function, 
4 to 5 mg, Anything over 5 mg. $ is an unfavorable 
• prognosis. 
IX, Chlorides 
10 cc. Blood Filtrate 
10 cc, Dist, H20 
10 cc. Silver Nitrate Solution (l cc, = 1 mg, NaCl) 
(2,905 Gm. Silver Nitrate in Dist, HpO, then qs* to 
1000 cc.) The Silver Nitrate and HNO3 are not to be 
added simultaneously. 
' ' 5 cc. Con, HNO3 
Stand in dark 5 minutes 
,3 Gm. Ferric Ammonium Sulfate 
Titrate with Standard Thiocyanate Solution (until 
definite salmoh-red * v hot yellow 
(1.7 Gm. KCNS qs. to 1000 cc*) 
or 
(1*4 Gms, NH4CNS qs. to 1000 cc.) 
; • t Calculation:' 
cc. of AgN03 minus cc. of Thiocyanate Solution x 
by 100 * mg, 
. I 
Example: 10 cc, NaCl - 5,5 cc. Thiocyanate ~ 4,5 x 
100 « 450>mg.:$. 
Significance.; 
Normal-450 to 5Q0 mg, $ in whole blood 
570 to 62Q mg, $ in plasma 
Chiefly obtained from the condiment salt in food. 
About,5 Gm, is needed daily; excreted chiefly by kidneys, 
some by skin and intestines. When chlorides are retained, 
water is also retained in the body; decreased when body 
fluids are lost. C02 on exposure to air; chlorides go from 
cells to plasma. Acid into blood, as in acidosis, 
chlorides go from plasma to cells. X. Plasma Proteins 
A. Functions of "blood. 
1. To carry food from intestine and O2 from lungs to 
tissue cells. 
2. To carry waste products from tissue cells to 
excretory organs: kidneys, lungs, intestine, skin. 
3. To carry hormones, 
4. To aid in the defense of the "body against disease, 
5. To aid in the maintenance of Kf, HgO, temperature 
and all other equilibriums in the tissues, 
B. Plasma! contains 9$ solid material. Of this 9$, 7.5$ 
is -protein - most important constituents of blood -olasma. 
Constituents are: 
1. Fibrinogen 
2. Euglobulin 
3. Pseudoglobulin 
4. Albumin 
5. Nucleoprotein 
6. Seromucoid 
Fibrinogen is concerned with clotting; responsible 
for Sp. Gravity viscosity;colloid osmotic pressure, 
which has the important part to do with regulating the 
water exchanges between the blood stream and the body 
tissues. 
Suglobulin and Pseudoglobulin have to do with the 
reaction in immunology only. 
XI. Calcium in Blood 
Normal-9 to 11.5 mg. 
Slightly higher in children than in adults; decrease in 
late months of -pregnancy, After -oarathyroidectomy, 
calcium falls to a low level. At 3.5 to 7 mg, $ will 
cause tetany. In severe nephritis, calcium will fall 
to 7, mg, ‘fo. Red blood cells have no calcium. 
XII. Icterus Index 
Centrifuge blood, pipette off serum 
1 cc. of serum in large test tube 
Pour few cc, of Standard Bichromate Solution in another 
tube (1:10,000) 
Dilute serum with .9$ Saline to about same depth of color 
Note amount of saline used. 
Dilution will be 1 cc, of serum plus number of cc, of ,9$ 
saline. 
b4 Calculation: 
HS x Dll. of Unknown = Icterus Index 
RU 
Significance: 
Normal - 4 to 6 
Above normal occurs in disease of the liver or 
biliary tract; also Hemolytic diseases such as 
Hemolyticanemia, Pernicious anemia; low values are 
found in secondary anemia. 
b5 LABORATORY TECHNICIANS MANUAL 
PART IV 
Parasitology . . . , , ......... .Sec. I & II 
Parasitological Methods) 
) Sec. Ill 
Examination of Feces ) 
Some Arthropod Vectors of Disease .Sec. IV CONTENTS 
Section I -------------- General Introduction 
The Protozoa 
Section II -------------- The Helminths 
Section III -------------- Examination of Feces 
Laboratory Diagnostic 
Procedures 
Section IV ------------- - Some Arthropod Vectors 
of Disease SECTION I 
GENERAL INTRODUCTION 
THE PROTOZOA PARASITOLOGY 
General 
A parasite may be defined as an animal or plant that lives within or 
upon another organism (the host) at whose expense it obtains some ad- 
vantage without compensation. Broadly speaking, parasitology includes 
the bacteria, spirochetes, filterable viruses and fungi, as well as the 
protozoa, helminths and arthropods. However, it is customary to consider 
in this field only animal parasites which infect or infest the host, or 
serve as transmitters of pathogenic organisms. 
Animal parasites are common in all countries, but are most numerous 
in the tropics. Some parasites cause serious disturbances in the host 
•while others may give rise to no symptoms or apparent damage even though 
present in large numbers. Thus we see that the presence of a parasite 
does not necessarily imply pathogenicity. We are interested in non- 
pathogenic parasites only insofar as it is necessary to distinguish them 
from pathogenic species. 
Parasitoses that are duo to protozoa or helminths are known as 
infections, while those due to arthropods are termed infestations. In 
certain infections man is the only host. In others there may be two or 
more hosts in which the host either matures or passes part of its life 
cycle. The host harboring the parasite during its sexual stages of 
development is called the definitive host. The intermediate host harbors 
the parasite during its larval or intermediate stages. 
The signs and symptoms produced in man by pathogenic animal parasites 
are many and variable as to degree, and although the presence of a parasite 
may be suspected, a definite diagnosis can be made only through identi- 
fication of the causative agent or its products from the body excreta, 
fluids or tissues. 
Classification of Animal Parasites 
Animals that arc a.liko in all respects arc classed together as a 
species. The male and female of a given species may be very unlike, but 
through mating they produce young that have characteristics similar to the 
parents. The term genus is of wider application and may include one or 
more species, being made up of animals that are similar in general structure. 
Genera thaw have certain characteristics in common make up a family. 
Families are grouped into orders, orders into classes and, finally, several 
classes may make up a phylum, the largest classification unit, of which 
there are several in the animal kingdom. In some cases there may be a 
further breaking down of these groupings into sub-classcs, super-families, 
sub-families, tribes, etc. 
In naming a species wc always write the genus name first, commencing 
with a capital letter, followed by the species name which begins with a 
small letter. In some cases it will bo noted that an author’s name and 
date will follow, thus: Ascaris lumbricoides Linnaeus, 1758, The phyla 
of modical importance are: Protozoa, Platyhelminthes, Nomathelminthos 
and Arthropoda. PHYLUM - PROTOZOA 
cuss 
ORDER 
(OMITTED) 
GENUS 
SPECIES 
Sarcodina (Rhizopoda) 
Move by means of 
pseudopodia 
Endamoeba 
Endolimax 
lodaraoeba 
Dientamoeba 
Trypanosoma 
E« histolytica 
E« coli 
E. gingivalis 
E. nana 
I. butschlii 
D. fragilis 
( T. gambiense 
( T. rhodesiense 
( T. cruzi 
Flagellata (Mastigophora) 
Move by means of 
undulating membrane or 
flagella 
Leishmania 
Trichomonas 
Chilomastix 
Embadomonas 
Enteromonas 
Giardia 
( L. donovani 
( L. infantum 
( L. braziliensis 
( L. tropica 
T. hominis 
T. vaginalis 
C. mesnili 
E. intestinalis 
E# hominis 
G. lamblia 
Infusoria (Ciliata) 
Move by means of 
numerous fine cilia 
which are shorter than 
flagella. Have con- 
tractile vacuoles 
Balantidium 
Nyctotherus 
B. coli 
N. faba 
Sporozoa 
No motor organs. 
Parasitic in cells or 
tissues. Reproduce 
by spores 
Eimeria 
Isospora 
Plasmodium 
Sarcocystis 
E. stiedae 
I. hominis 
( P, vivax 
( P. malariae 
( P. falciparum 
( P. ovale 
S. tenella The Protozoa are the simplest forms of animals, being composed of a 
single cell. Protozoal cells are made up of protoplasm which is divided 
into nucleus and cytoplasm. In some instances the cytoplasm may be 
separated into an outer hyaline portion, the ectoplasm and an inner 
granular portion, the endoplasm. The ectoplasm is concerned with pro- 
tecting the organism and with the procurement of food, excretion and 
sensation. It also gives origin to the structures responsible for 
locomotion. The type of motor organ (organelle) serves as the main 
basis for classifying protozoa. Types of organelles are; pseudopodia, 
flagella, undulating membranes, cilia. 
The endoplasm is concerned with growth and reproduction. Contained 
in the endoplasm is the nucleus ?*hich is necessary to reproduction and 
the maintenance of life. In many protozoa it consists simply of a 
chromatin mass without definite structure. In others, some or all of the 
following parts may be observed; nuclear membrane; chromatin granules; 
karyosome; linin network; ccntrosomc. Additional structures seen in the 
endoplasm of the protozoa include; contractile vacuoles which expand and 
contract at regular intervals; food vacuoles; chromatoidal bodies; 
ingested materials such as food particles, bacteria, etc. 
In the flagellates, besides the nucleus, there may be a secondary 
nucleus, the kinotoplast which is composed of two parts; the parabasal 
body and the blepharoplast. The flagellum may arise from the latter. 
The portion of the flagellum immediately arising from the blepharoplast 
is called the axonemo. 
Food is obtained with the aid of the organs of locomotion and may be 
ingested through the ectoplasm or through a rudimentary mouth (cytostome). 
Respiration is accomplished by absorption of oxygen and elimination of 
carbon dioxide through the ectoplasm, (aerobic), or by breaking down 
complex substances in the endoplasm (anaerobic). Excretion takes place 
in one of the following ways; by diffusion through the ectoplasm; by 
expulsion from vacuoles; during reproductive activities. 
Substances secreted by protozoa include ferments, enzymes, toxins 
and pigments. The ferments are active in digestion, and the other sub- 
stances may in some cases be responsible for damage done to the host. 
Some organisms also secrete a substance which hardens to form a protective 
coating or cyst, Encystment serves to protect the organism from adverse 
conditions. In some instances reproduction take place during the 
encysted state. 
Reproduction may bo sexual or asexual, A simple division of the 
organism into two parts is called fission. When it divides into many 
parts, each with separate cytoplasm and nucleus, we have schizogony. In 
sexual reproduction there is usually an alternation of generations with 
one phase of the life cycle being completed in each of two different 
hosts; sexual in one, asexual in the other. Class - Sarcodina 
(Tho Amoeba) 
Endamooba histolytica 
This parasite is world wide in distribution but is mono common in 
the tropics and in regions whoro sanitation is poor. On the basis of 
surveys it is estimated that 5$ to 10$ of the people in the United States 
harbor this organism. Infection with Endamoeba histolytica is known 
as Amebiasis or Amebic Dysentery, This is the most important protozoan 
parasite found in the intestine of man. The portal of entry and primary 
site of infection arc the mucous membrane of the lower small intestine 
and the mucosa of the entire largo intestine. Extension to the liver 
and other organs may occur, resulting in amebic abscess. 
Infection takes place by ingestion of food or drink containing 
cysts. The cysts pass through the stomach unchanged, but through the 
action of intestinal secretions the cyst wall becomes permeable and 
four motile amoeba emerge. With the aid of a cytolytic substance which 
they secrete, they penetrate the intestinal mucous membrane whore they 
multiply and give rise to characteristic '‘bottleneck1’ ulcers of variable 
size and extent. 
Symptoms produced by this invasion are extremely variable but arc, 
in general, proportionate to the degree of ulceration. There may be 
alternating periods of diarrhea and constipation. In a severe case there 
will be an excessive number of bowel movements attended by abdominal 
pain with a progressive weakening of the patient. Bowel discharges may 
consist almost entirely of blood and mucus with shreds of raucous membrane. 
When an active diarrhea exists, organisms that are moved out do not 
have time to become encysted. One should, therefore, look for motile 
trophozoites in diarrheal stools. Since the typo of motility is import- 
ant in identifying the trophozoite, stool examination must be done within 
20 to 30 minutes after passage, keeping the specimen warm, otherwise 
motility may be lost. 
When the diarrhea ceases and stools become formed, the organism then 
has time to become encysted. Cysts are very resistant to adverse 
conditions, and there is, therefore, no immediate hurry in examining 
formed stools. 
The laboratory diagnosis of Endamoeba histolytica is justified if 
amoeba are found in the feces which show the following characteristics: 
Trophozoites 
1. Active, progressive motility, directional in character, 
2. Hyaline, finger-shaped pseudopodia, 
3. Ingested red blood corpuscles. 
4 Life Cycle of Eimeria perforans 
In the intestinal epilhelium of 
a Rabbit 
1-7 Schizogong 
6-13# Macrogametogony 
14. Oocyst 
15-19. Microgametogony 
Metazoan Ceil 
Protozoan Cell D.fragilis 
E.gingivalis 
projgg&a',; 
E.nana 
GRAPHIC DIFFERENTIATION OF AMOEBA 
Lbrtschlii 
E,coli 
E.histolytica 
Pre-cysticForms 
Cystic Forms 
Trophozoites  
TABLE 86—CHARACTERISTICS OF THE AMOEBAE OF 
MAN 
STAINED TROPHOZOITES 
E. HISTOLYTICA 
E. COL 1 
E. NANA 
1. 3UTSCHL11 
0, FRAG !LI A 
Average Size 
20 to 35 u 
15 to 30 u 
6 to 10 u 
9 to 13 u 
3 to 12 u 
Nuclear membrane 
(stains faintly 
or not at all) 
L ined wi th minute, 
fairly even sized 
grains of chroma- 
tin which stain : 
deep 1y. 
Lined with coarse irre- 
gularly sized grains 
or tars of chromatin 
which stain deeply. 
Chromatin on nu- 
clear membrane in 
thin line and 
stains poorly. 
A few poorly staining, 
widely separated, 
chromatin grains on 
nuclear membrane. 
Chromatin on nuclear mem- 
brane in thin line and 
stain) poorly. Nucleus 
frequently double. 
Karyosome 
Short rod or globule 
of small diameter, 
centrally suspended 
within the nucleus. 
Regular outlinc. 
Stains deeply and 
uniformly. 
Short rod or ball or 
irregular out 1inc, 
usually eccentric. 
Diameter greater than 
that of E, Hi stoly- 
tica. Stains deeply 
and uniformly. 
Very large, central 
or eccentric, com- 
posed of 1, 2 or 
more deeply stain- 
ing masses in a 
1ighter staining 
matrix. Outline 
often irregular and 
oblong. 
Similar to that of 
E, Nana but larger 
and more apt to con- 
tain a poorly stain- 
ing central portion. 
Causes the nucleus 
to appear 1 ike an 
eye with a widely 
di1ated pupi1, 
Coopoead of severe! 
minute deeply stain- 
ing, 4iscrete grains. 
Llnin network 
(stains faintly 
or not at all) 
Contains no chroma** 
tin grains between 
the karyosome and 
nuclear membrane. 
Sometimes contains 
grains of chromatin. 
Region just without 
karyosome halo often 
appears cloudy after 
staining. 
It is not often dis- 
cernible, Consists 
of a few short 
lines from the kar- 
yosome halo to the 
nuclear membrane, 
(Karyosome usually 
the only structure 
visible in the nu- 
cleus.) 
Like a web when de- 
fined by an excel- 
lent stain. 
Not demonstrable 
THE LIVING TROPHOZOITES 
Motion 
Active progression 
in a definite di- 
rection. Form is 
Most stains are not 
actively progressive 
but merely change in 
Some organisms like 
that of IE, Histoly- 
tica (except the 
Many stains in cul- 
ture 1 ike that of 
E, Histolytica, 
Slow indefinite progres- 
sion or merely change 
In conformation. 
elongated in motion. 
conformation. 
amoeba is very 
small) but the ma- 
jority merely change 
in form and do not 
move progressively. 
The majority, how- 
ever, are 1 ike E. 
coU,  
E. HISTOLYTICA 
E, CPU 
£. NANA 
1. BUTSCHL11 
O.FjUGILIA 
Pseudopod it 
Finger-like with smooth 
outline when not in 
progressive motion., Ec- 
toplasm is clear, glass- 
like and easily discern- 
ible? Chen in progres- 
sive motion the ecto- 
plasm may not be clear- 
ly differentiated. One- 
third to one-quarter of 
the parasite is ecto- 
plasm. 
Usually blunt, but it 
may be 1 ike E, His- 
tolytica, The ecto- 
plasm is usually not 
clearly differen- 
tiated, One-quarter 
to one-fifth of the 
parasite Is ectoj» 
plasm but it is of** 
ten poorly differen- 
tiated from the endo* 
plasm even when the 
amoeba is in motion. 
Like E. Histolytica. 
One half to one- 
third of the para- 
site is ectoplasm 
and is easily dif- 
ferentiated. 
Like jE» Histolytica or 
very broad with coarse- 
ly indented outline. 
One-half to one-third 
of the parasite is ec- 
toplasm and Is easily 
differentiated. 
Often comprises one- 
half of the organism. 
Outline is often ?n- 
dentad. 
Color 
Faint Green 
Gray 
Gray 
Faint Green 
Gray 
Vtsibility of 
ncucteus 
(oi|-immers?on 
lens) 
Usually difficult to 
visualize except v/hen 
the nucleus passes 
into the pseudopodia 
and is contrasted a- 
gainst the clear ecto- 
plasm. 
Quite clear, it is 
much more readily 
seen than that of 
E,- Histolytica, 
The karyosome may be 
defined with ease. 
The karyosome may be 
defined with ease. 
Difficult to distin- 
guish from ingested 
bacteria. 
Endoplasmic in- 
clusions of di- 
agnostic signif- 
icance. 
Red blood cells are 
typical and diagnos- 
tic, Degenerated and 
culture forms contain 
bacteria. 
A voracious feeder 
but usually does not 
ingest red blood 
cells. Bacteria 
and starch grains 
are the principal 
inclusions. 
Bacteria 
Bacteria 
Bacteria 
1 
CYSTIC SJAGE 
Average Size 
3 to 20 u 
10 to 30 u 
5 to 8 u 
7 to 15 u 
Nuclei number 
4 to 4, rarely more. 
Mature cysts contain 
4 nuclei. 
1 to 8, rarely more 
Mature cysts con- 
tain 8 nuclei. 
I to 4, rarely more 
Mature cysts con- 
tain 4 nuclei. 
1 or 2 
Visibility of 
nude] in the 
unstained liv- 
ing state. 
Poor but discernible 
with the oll-lmmer- 
sion lens. 
Good 
Good 
Good Average Size 
5 to 20 u 
10 to 30 u 
5 to 8 v 
7 to 15 u 
Shape 
Generally spherical 
or nearly so. 
Generally longer than 
broad and one side 
may be less curved 
than the other. 
Irregularity of shape 
is common. Generally 
oval, 
Great irregularity of 
shape and outline is 
common. 
Reserve food in-’ 
elusions (these 
disappear in oid 
specimens and 
are not constant 
in young cysts.) 
Bar-shaped chromatoid 
bodies in 0-90 per 
cent of cysts. Some- 
times a small amount 
of glycogen Is pre- 
sent in young cysts. 
It is diffuse and 
stains a light brown 
with iodine. 
Acicular chromatoid 
bodies present In 
about 10 per cent 
of cysts, A large 
amount of glycogen 
may be present and 
push the nucleus 
against the cyst 
wail. 
Small granules or 
masses of volutin 
and glycogen may be 
present. Neither 
is characteristic. 
f/.asses, grains of rods 
of volutin may be pre- 
sent but these are not 
characteristic. The 
glycogen, almost in- 
variably present, is 
large in amount, 
smoothly outlined and 
stains a deep brown 
with iodine. This 
iodine body is char- 
acteristic and diag- 
nostic. 
* Cystic stage of 
0» f 1 ijt is unknown. Cysts 
1. Nuclei 1 to U in number, 
2. Minute, centre.lly located karyosomes, 
3. Large chromatoidal masses with rounded ends, 
Blastocystis hominis, a vegetable organism should not be mistaken 
for the cyst of E. histolytica. It is spheroidal and from 5 to 30 
microns in diameter. It consists of a central vacuole surrounded by a 
rim of cytoplasm containing many nuclei which stain black with 
hematoxylin. 
The symptoms and signs produced in acute Amebic Dysentery are very 
similar to those in Eacillary Dysentery, Although laboratory 
identification of the causative organism is necessary for a definite 
diagnosis, presumptive evidence may bo gained by an examination of the 
fecal exudate. In the case of Amebic Dysentery it will be found that 
the exudate shows few pus cells with a relatively high bacteria content, 
while in Bacillary Dysentery about 90% of the exudate consists of pus 
cells, and the bacteria content is usually low, 
Methods to bo used for laboratory examination are: 
1. Microscopic examination of 
a. Fresh and stained foccs, 
b. Material from amebic abscesses, 
c. Stained tissue sections, 
2. Cultivation. 
3. Complement fixation. 
Non-Pathogenic Intestinal Amoeba 
Two other common, but non-pathogcnic amoeba, of worldwide 
distribution, arc E. coli and E, nana. Life cycles of both arc 
similar to that of E. histolytica and they arc transmitted in the same 
manner. 
I, butschlii and D, fragilis arc apparently of wide distribution, 
but are least commonly found of the non-pathogonic amoeba, (For chief 
differential features of intestinal amoeba, sec adjoining table). Class - Ciliata 
Balantidium coli 
This ciliato is the cause of Balantidic Dysentery, a chronic 
affection of the largo intestine, resembling Amebic Dysentery, but 
usually loss severe. It is commonly found in the intestine of pigs and 
apes, but is of relatively rare occurrence in man. Infection takes 
place through ingesting food or drink contaminated with feces containing 
cysts of B. coli, 
B. coli is of widespread distribution, human infection having been 
observed in many countries of Europe, the Philippines, China, Africa, 
Central and South America, and in many of the United States, 
Laboratory diagnosis depends upon finding the organism in the feces. 
Methods used for detection are the same as for intestinal amoeba. 
The trophozoite can usually be found and is too largo to be overlooked. 
It is an actively moving oval organism, 50 to 100 microns in length 
and 4-0 to 70 microns in width, It is covered with cilia which arc in 
constant motion, and has a funnel-shaped mouth (cytostome) at the 
anterior end. Included in the endoplasm are: a largo bean-shaped 
macronucleus and nearby a smaller round micronuclous; food Vacuoles; 
two contractile vacuoles, which pulsate at regular intervals - a largo 
one anteriorly and a smaller one posteriorly* 
Class - Flagellata 
Intestinal and Vaginal Flagellates 
The common intestinal flagellates arc Chilomastix mosnili, Giardia 
lamblia and Trichomonas hominis. With the exception of T. hominis they 
exist in both vegetative and encysted state. No intermediate host is 
necessary and it is doubtful whether the human species are found in 
animals. These parasites are commonly regarded as non-pathogonic, 
although they may at times bo associated with diarrhea. They arc classi- 
fied according to the number of flagella and whether or not they have an 
undulating membrane and blepharoplast, 
T. vaginalis is a common parasite of the female vagina where it may 
give rise to a catarrhal type of inflammation with loucorrhca. 
Methods for laboratory examination are essentially the same as for 
the amoeba. The flagella may bo demonstrated by staining a slide 
preparation with a drop of iodine and examining under darkfiold 
illumination. The chief diagnostic features of each will be found in 
table adjoining. 1. Paristtme, 
2. Cytostome. 
3, Cytophar^mx. 
4, Fo»d Vacu#l3. 
5* Contractile Vacutle* 
6. Micr#nuoleus. 
7, Macr•nucleus, 
Cytopyge, 
9. Ciiia. 
Balantidium coll •* Trichomas hominis (Davaine i860) 
1. Flagella. 
2. Nucleus. 
3. Undulating Membrane. 
4-. Basal fibre, 
5. Axostyle . 
6. Blepharoplast, 
7. Food Vacuole. 
Trophozoit 
Cyst 
Chilomastix mesnili Giardia lamblia 
1. Crossing anterior flagella 
2. BlephoroDlast. 
3. Nucleus. 
4. Sucker 
5. 4xostyle. 
6. Parabasal body. 
GysC 
J Latent View' 
/TrophozDite 
Cysts 
Trophozoite 
Copromonas sabtilis 
Cyst 
Trophozoite 
ibjnro ohoz o 11 e 
w 
V 
Cyst 
Dodo candatus 
Trophozoite DIFFERENTIAL FEATURES OF SOME OF THE COMMON 
INTESTINAL AND VAGINAL FLAGELLATES OF MAN 
CHILOMASTIX 
MESNILI 
GIARDIA IAMBLIA 
TRICHOMONAS 
VAGINALIS 
TRICHOMONAS 
HOMINIS 
Shape 
..Elongated Pear 
Pear-shaped • 
Pear-shaped 
Pear-shaped 
Size 
10 to 15 Micra 
12 to 15 Micra 
15 to 18 
Micra 
10 to 15 Micra 
Flagella 
A-3 anterior 
1 in cytostome 
B-A anterior, 2 
caudal, 2 ventral 
5-X anterior, 
1 posterior 
5-3 to 5 
anterior, 1 
posterior 
Undulating 
Membrane 
None 
None 
Present 
Present 
Spiral 
Groove 
Present 
None 
None 
None 
Sucking 
Disk 
None 
Present 
None 
None 
Mouth 
Cavity 
Present 
None 
Present 
Present 
Nucleus 
1 
2 
1 
1 
Cyst 
Yes 
Yes 
No 
No 
Cyst- 
shane 
Lemon-shaped 
Oval 
... 
... 
Size of 
Gy at 
7 to 9 Micra. 
9 to 12 Micra 
* 
Cyst - No, 
of Nuclei 
1 
L 
Motion 
Jerky.progressive 
Jerky.progressive 
Progressive 
Progressive 
Specimen 
Feces 
Feces, bile 
Vaginal swab 
' Feces 
Patho- 
genicity 
None 
Questionable 
Questionable 
None Class - Flagcllata (Cont'd) 
Blood and Tissue Flagellates 
Family - Trypanosomidae. This family is divided into six genera, 
two of which will bo considered! Trypanosoma and Leishmania. 
Genus - Leishmania 
Members of this genus have only leptomonas and leishmania 
forms in their life cycle, (See adjoining figure). They have both 
vertebrate and invertebrate hosts. There are throe recognized species 
parasitic in man, morphologically identical. In man a typical leishraan 
donovan body appears ovoidal, 2 to 3 microns in longest diameter and 
contains a nucleus and kinctoplast. Those organisms arc usually 
enclosed in monocytes, polymorphonuclear leukocytes or endothelial cells. 
With Wright’s Stain the cytoplasm is pale blue and encloses a rather 
largo red nucleus. The coll membrane may be indefinite. The 
kinctoplast stains rod and appears a.s a rod lying at right angles to 
the nucleus. In the invertebrate host and in cultures, the ovoidal 
body becomes spindle-shaped, and a single flagellum arises from the 
kinctoplast at the anterior end. Reproduction is by longitudinal 
binary fission for all members of the family Trypanosomidae, 
1, Leishmania donovani 
This species is the cause of Kala Azar or Dumdum Fever, 
a disease characterized by enlargement of the spleen and liver; long, 
irregular fever; anemia, with progressive loss of weight and strength. 
Approximately 90% of untreated cases end fatally in two to three years. 
With early treatment a majority of cases can bo cured. Transmission 
is thought to take place through the sand fly. Possibly some cases are 
transmitted by the oral route. 
Distribution! Eastern India, North China, countries 
bodering the Mediterranean, the Sudan, ??cst Africa, Iraq, Southern 
Russia, 
Laboratory Diagnosis! the presence of the parasite must 
bo demonstrated, 
a. Smears may be made from the blood, liver, spleen, 
lymph glands, bone marrow and stained with Wright’s 
Stain for microscopic examination, 
b. Blood culture 
c. Presumptive Tests (Globulin Precipitation 
(Aldehyde Test 
(Antimony Test LeJshinanla donovanl In the Kupfer Cells of the liver 
teiahuanla tropica In Sr.ear from Cutaneous lesion 2, Leishrnania tropica 
This organism is the cause of cutaneous leishmaniasis 
(Oriental Sore), Ulcers may appear on any exposed part of the body. 
They are usually about one inch in diameter and have rounded edges. 
Healing may take place in from several months to a year, leaving a 
disfiguring scar. One attack results in immunity. L, tropica 
apparently does not invade the viscera. Uncomplicated cases never end 
fatally. 
Distribution: India, Persia, Palestine, Africa, 
countries around the Mediterranean, Costa Rica, Brazil, Peru, 
t 
Transmission: sand fly, direct contact. 
Laboratory Diagnosis: find the parasite in material from 
edge of ulcer. Culture may also be used, 
3, Leishrnania brazilionsis 
Infection with this species results in muco-cutaneous 
leishmaniasis, an ulcerating infection similar to Oriental Sore, except 
that the lesion tends to spread to tho mucous membranes of the mouth, 
nose and throat, and may result in deformities, loss of voice, etc. 
Distribution: Central and South America, 
Transmission: Sand fly. 
Laboratory diagnosis: As for Leishrnania tropica. 
Genus - Trypanosoma 
Members of this genus have leishrnania, loptomonas, crithida 
and trypanosoma stages in their life cycle. (See adjoining diagram) 
An insect as well as a vertebrate host is probably necessary 
for each species. Transmission may bo in one of tho following ways; 
Through tho mouth parts of the intermediate host while 
feeding on blood of the definitive host; 
Through ingestion of infected feces of intermediate 
host by vertebrate; 
By infected fo'c'os of insect being rubbed into wound 
made by bite. A typical trypanosome stained with Wright’s appears as a 
spindle-shaped body, 15 to 30 microns or more in length with a delicate 
reddish membrane arranged into folds running along one side. The 
cytoplasm stains pale blue and may contain dark blue volutin granules. 
The nucleus lies near the middle and stains reddish purple. The 
kinetoplast is dark red and lies at the posterior end. The single 
flagellum arises at the posterior end (blcpharoplast), runs along the 
edge of the undulating membrane and terminates as a free flagellum 
at the anterior end. 
In fresh blood preparations, trypanosomes arc colorless and 
through their rapid movement, may give a spinning motion to red blood 
corpuscles. 
Apparently all trypanosomes are harmless to their invertebrate 
hosts. Throe species arc of medical interest: T, gambiense, T. 
rhodcsicnsc and T. cruzi, 
1, Trypanosoma gambiense 
Causes African sleeping sickness, a disease characterized 
in the early stages by irregular fever, lymph gland enlargement, anemia 
and weakness. In the later or sleeping stage, nervous and mental 
symptoms appear, the victim becomes emaciated, is apathetic and may 
sleep most of the time. Treatment is effective in a large percentage, 
if given early. After the appearance of the sleeping stage, most cases 
progress to coma and death within a few years. 
2, Trypanosoma rhodcsicnsc 
Is the cause of a more virulent form of African sleeping 
sickness. The disturbances caused are similar to those listed for T, 
gambiense, but the disease usually ends fatally within one year unless 
early treatment is given, 
T. gambiense and T. rhodcsicnsc arc similar in appearance. 
Laboratory differentiation is based upon finding posterior nucleated 
forms in laboratory animals that have been inoculated with the 
trypanosomes. Apparently a larger percentage of T. rhodcsicnsc will 
develop the posterior nucleated forms, 
3, Trypanosoma cruzi 
This parasite is the cause of Chaga’s Disease or South 
American Trypanosomiasis, It lives in the blood as a typical 
trypanosome, in the tissue colls of man as a leishmania and in the 
intestinal tract of certain insects as a crithidia. In man the parasites 
arc located in the rcticulo-endothelial cells of the spleen, liver and 
lymphatic tissues, and in tho cells of the heart, muscles. Diagram of a Trypanosome 
Forms Found in Life of 
1* Leischmania. 3. Crithidia 
2. Leptomonaa* 4., Trypanosoma. DIFFERENTIAL CHART TRYPANOSOMES OF MEDICAL IMPORTANCE 
0 i sease 
P reduced 
African Sleeping Sickness 
of Man. 
African sleeping 
sickness of roan 
Virulent form. 
Chagas disease - South 
American, Trypanisomiasis 
of Man. 
Trypanosomia- 
sis of Rats 
Non-pathogen ic 
of Man. 
CuIture 
Culture Media - NNN plus 
glucose 32°Cs Ponselles* 
medium at 25°C. 
Cultivation difficult 
Same as for T. 
gambiense 
Culture Media 
NNN 25°C, 
CuIture 
Media 
NNN. 25°C. 
Suscepti- 
ble Lab- 
Animals 
All except monkeys. 
A 11 except monkeys. 
Guinea pigs, 
rabbits, dogs 
monkeys. 
bats, mice, 
cats. 
White Rats, 
Mice with 
difficulty 
I nverte- 
brate Host 
(Vectors) 
tsetse FIy,G.marsitans,G. 
palpalis,G.brevipalis,G. 
oal 1 idipes,G.tachfnoids 
(Bite infective after 20-30 
day cyclic development) 
Same as for T. 
gambiense 
Kissing Bug, T. 
infestans,T.sordioa,Rhod- 
nius prolixus, possibly 
Cimex lectularius. 
Fleas. (C. 
canis, Pulcx 
irrltans,etc) 
Vertebrate 
Host 
Man and probably Antelope 
Man and probably 
Antelope 
Man. Armadillo, probably 
Cat, O'possum 
Rats 
Le i shm§n ia 
Forms in 
Moo^6 °r 
No 
No 
In Tissue 
In Blood 
Dividing 
Forms in 
Per i.Blood 
Yes 
Yes 
Never 
Yes 
Forms in Blood 
fj > 
% If 4 M 
D % * 
10 days 
K) 
Drawings 
from Blooc 
Smears 
f) 
J% 
]>WSft 
and 
T i ssue 
i 
Parasites same as 
T. gambiense except 
in laboratory 
animals in which 
posterior nuclear 
forms develop. 
d 
Forms in 
)t 
tTt> AT 
p—" 
Bl 
Forms in Brpoc 
in Jf^irst 1 oT\ 
(f.>days^ 
c'-s<.r 
ParabasaI 
Body.Size 
4 Shape 
SmalI.Inconspicuous 
Small. Inconspicuous 
Small. Inconspicuous 
Conspicuous,Large and Oval 
Large and 
Rod-shaped 
Position 
of Nucleus 
Middle 1/3 
Middle 1/3 
Anterior 1/3 
Type 
blood stream) 
Polymorphic 
Monomorph ic 
Monomorphic 
Length 
13 to 40 
18 to 35 
|6 to 24 
20 to 30 
Geographic 
D i str}bu- 
tton 
Tropical Africa 
East Africa (Rho- 
desia, Nyasaland, 
Tanganyika,Mozam- 
bique) 
South America and probably 
Central America 
Cosmopolitan 
Spec i es 
T. gambiense 
T.rhod iense(probably 
a virulent strain of 
T. gambiense 
T. cruzi 
T. lewisi Chaga’s Disease is found in acute and chronic forms. 
The acute stage usually lasts 20 to 30 days and is characterized by 
fever, swelling of the face, enlargement of the thyroid and lymphatic 
glands, and of the liver and spleen. If the victim survives, the 
disease may become chronic and last for many years, giving rise to a 
variety of symptoms, ( 
During the acute stage the trypanosomes may be found 
in the peripheral blood. In the chronic form the trypanosomes disappear 
from the blood, but the loishmania forms are present in the tissues. 
For additional information on those parasites, soo 
Differential Chart of Trypanosomes, Methods used for 3.aboratory 
diagnosis are outlined in Section III, 
Class - Sporozoa 
Genus - Plasmodium (Malaria Parasites) 
P, vivax (tertian) 
P, malarias (quartan) 
P. falciparum (malignant tertian, Estive-Autumnal) 
P, ovale (mild, similar to tertian) 
Malaria is a group of closely related infections due to protozoan 
parasites that live chiefly in the blood, and are transmitted by 
Anopheline mosquitoes. Three common species of the genus plasmodium 
are associated with malarial fever in man: Plasmodium vivax, P. malariae 
and P, falciparum. A fourth species, similar to P, vivax, has been 
identified. Since this parasite (P. ovale) is comparatively rare, it 
will not be described here. 
This infection typically is characterized by severe paroxysms of 
chills, fever and sweating, -which may occur at regular intervals corres- 
ponding to the period of time necessary for the asexual cycle of 
development. The paroxysms may occur daily (quotidian), on alternate 
days (tertian) or at 3~day intervals (quartan), However, in many 
instances, these symptoms are neither regular in occurrence nor clearly 
defined. Frequently the only symptom noted is an irregular fever. 
Anemia of varying degree occurs in all typos of malaria, but is usually 
most pronounced with P. falciparum infections. Uncomplicated infections 
with P, vivex, P. malariae and P. ovale rarely end fatally - most 
malaria deaths being due to P. falciparum. Cerebral malaria and 
Blackwatcr Fever may also occur with infections caused by P. falciparum. 
In nature, malaria is transmitted only by the bite of the female 
Anopheles mosquito. Experimentally, the infection may be transferred by 
injecting blood of an infected person into another. More than 120 species of Anophelino mosquitoes have been identified. About 20 
species of this group carry malaria in different parts of the world. 
The chief carrier in southern United States is Anopheles 
quadrimaculatus. On the Pacific coast, in Canada and Europe, the 
main carrier is A. maculipennis. 
Malaria is one of the most prevalent of all preventable diseases; 
it is the scourge of the tropics. It has a wide geographic 
distribution, occurring from the Arctic Circle to the Equator, but 
is more prevalent and more virulent in warm, moist climates. The 
Tertian form has the widest distribution, being common in temperate 
regions, as well as in the tropics. Quartan malaria is least 
commonly found of the three main types, except in Central and West 
Africa, Estivo-Autumnal malaria is the prevailing type in most 
tropical regions and is generally confined to hot, moist climates. 
Repeated infections with malaria leave a pronounced resistance. 
It is well known that mosquitoes do not bother some individuals - 
possibly on account of body odor. Negroes are much more resistant to 
Tertian malaria than whites, indicating an immunity to this particular 
plasmodium. 
The Tertian parasites may remain latent in the spleen or other 
organs for several years. Exposure, overwork, fatigue, etc., may then 
cause an attack. In badly infected regions, as high as 60-70% of the 
population may be carriers. 
There are two cycles in the life history of the malaria plasmodia: 
(1) the asexual cycle in Man (Schizogony); (2) the sexual cycle in 
certain species of female Anopheles mosquitoes (Sporogony), All of 
the known species infecting man go through the same.developmental 
stages, but with the exception of P. vivax and P, ovale, which are 
similar, different time periods are required, 
l 
The Asexual Cycle 
Only P. vivax will bo described. The differences in microscopic 
appearances of the other types will be found in Differential Table of 
Malaria Parasites. 
When the infected Anopheles female secures a blood meal, she 
injects saliva containing sporozoites into the wound. Apparently the 
sporozoites are carried by macrophages to the reticulo-endothelial 
system, where they reproduce by segmentation for the first U or 5 days 
after entering the body (exo-erythrocytic stage). About the 5th day PLASMODIUM FALCIPARUM 
LIFE CYCLE IN MAN AND IN FEMALE ANOPHELINE MOSQUITO 
A, LIFE CYCLE IN MAN 
lo SCHIZOGONY (ASEXUAL CYCLE) 
a, 1-3 FORMS IN THE PERIPHERAL BLOOD 
b„ 6-12 FORMS IN THE CAPILLARIES OF THE INTERNAL ORGANS 
II GAMETOGONY (DEVELOPMENT OF GAMETES.) 
Va, 1-3 UNDIFFERENTIATED FORMS IN PERIPHERAL BLOOD 
b, 4-10 DEVELOPMENT OF GAMETOCYTES FROM UNDIFFERENTIATED 
c, H-1,2 MALE AND FEMALE GAMETOCYTES IN PERIPHERAL BLOOD 
g. 10-11 RUPTURING'HR? 
LIBERATOROZ01 TfS YmJc 
h. 12 SPOROZOITES IN GLANDS 
BL LIFE CYCLE IN FEMALE 
! ANOPHELINE MOSQUITO 
/ I, AFOROGONY 
I a. I-FEMALE GAMETOCYTE 
j WITH EXTRUDED POLAR 
/ BODIES , 
b, 2-EXFLAGELLATION OF 
MALE GAMETOCYTE AT 
c, FERTILIZATION OF jfr 
FEMALE GAMTOCYTE 
GY MALE GAMTOCYTE 
d, ZYGOTE OR MO- • 
TILE OOK1= 
NETES % 
eD 7-ZYGOTE PENETRATES STOMACH 
WALL, ROUNDS UP AND BECOMES OOCYST 
f,8-9 THE NUCLEUSDIV IDES INTO MULTIPLE 
GRANULES, EACH GRANULE DEVELOPES 
A CYTOPLASMIC PROCESS AND BECOMES 
A SPOROZOITE, Mosquito Stomach snowing Malarial Oocysts In 
War lous Stages of Development 
(low power magnification) 
Portion of Mosquito Salivary Gland Showing 
Infection with Malarial Sporozoites 
{Oil Immersion magnification) they invade red blood corpuscles, and proceed to grow and segment, 
destroying the infected erythrocytes in the process. Forty-eight 
hours are required from the entrance into the erythrocyte until the 
bursting forth of daughter parasites (merozoites) in the case of P, 
vivax. Liberated merozoites re-enter red cells to repeat this cycle, 
and after about two weeks from the time of infection (incubation 
period) enough parasites have been produced to cause symptoms. When 
regular bouts of chills and fever occur, the time of chill corresponds 
to the rupture of parasitized red cells. All stages of development 
may be seen in the blood at one time, but usually one form 
predominates. The various recognizable stages of development are 
listed below. 
Trophozoites 
In its earliest form, the parasite appears as a small hyaline 
ring in the infected erythrocyte. In about 6 hours ameboid activity 
may be seen in fresh preparations. With Wright’s or Giemsa’s stain 
the ring form shows a blue ring of cytoplasm with a small red nucleus. 
Growth of the parasite continues and the infected rod cell 
becomes pale and swollen. With good staining, fine pink dots 
(S.chuffner ’ s) will usually bo seen in the infected erythrocyte, and 
small yellowish brown pigment granules arc present in the cytoplasm 
of the parasite. At the end of about 36 hours tiro-thirds of the rod 
cell is filled by the parasite, ameboid motion is lost and the pigment 
granules collect near the center, 
Schizont 
After about 4-0 hours the parasite fills the cell and its 
chromatin usually divides into 16 to 24 irregular masses distributed 
throughout the cytoplasm, 
Merozoites 
Just before segmentation, the cytoplasm of the parasite 
divides into equal -sections, one about each segment of chromatin. At 
the end of 46 hours the mature merozoites rupture out of the red 
coll and each one then seeks to enter a now erythrocyte to repeat the 
cycle. The pigment is liberated into the blood stream to bo destroyed 
by phagocytes. Stained mature merozoites, before liberation appear as 
a collection of oval or round blue bodies, each having a bright red 
or purple chromatin dot near the periphery. Gametocytos 
Some of tho liboratod merozoites which enter red cells do 
not go through the asexual cycle of segmentation as outlined, but 
gradually enlarge to become sexual forms (gametocytes). The reason 
for this difference in development is unknown, but it probably 
represents an effort of tho parasite to overcome increasing 
resistance of the host. These arc the forms responsible for 
initiating the sexual cycle in the mosquito. It takes U days for the 
development of the gametocytos from ring form to maturity, and they 
must bo 7 to 10 days old before they arc infectious to tho mosquito. 
Stained male forms (microgamctocytcs) are 7 to B microns in diameter 
and do not completely fill the enlarged red cell. The cytoplasm 
appears pale blue and contains scattered brown pigment granules. The 
nucleus stains rod, is diffuse and located near the center of the 
parasite. The female form (macrogametocytc) is larger, filling the 
red cell; tho cytoplasm is darker blue and the nucleus is compact and 
not centrally located. There are usually more female than male forms. 
The Sexual Cycle 
When the female Anopheles takes a blood meal from an infected 
human host, all forms of the parasite may be ingested. Upon reaching 
the mosquitoes’ stomach, tho male forms send out 8 to 10 flagellar- 
like processes, each containing nuclear material. These break off 
(microgamotos) and each seeks out a matured female (now macrogamctc), 
which is fertilized. The resulting organism is called tho zygote. 
This becomes motile (ookinete), and moves into tho stomach wall where 
encystmcnt takes place (oocyst). The chromatin in the cyst undergoes 
division with tho formation of as many as 10,000 granules in small 
clumps. Each chromatin granule becomes a cigar-shaped sporozoite. 
The cyst ruptures and motile sporozoites liberated into the body 
cavity make their way to the salivary glands. It requires 7 to 12 
days for this cycle with favorable temperature and humidity, Tho 
mosquito apparently is not harmed by these parasites. 
Laboratory Diagnosis 
A definite diagnosis of malaria can only be made by finding the 
causative parasite in thick or thin blood films. Blood for examination 
should be taken during the period from 12 hours after a chill until 
1 or 2 hours before tho next one is expected. When only ring forms arc 
found, additional blood films should be taken 8 or more hours later, 
in order to determine the species. 
Laboratory diagnostic procedures arc outlined in Section III,  
Total deaths 
(absolute numbers) 
U.S.Army 1920-1 93* 
Inclusive 
2 
(8.32?) 
(4. 
ON 
21(87.62?) 
Total admissions 
(absolute numbers) 
U.S.Army 1920-193< 
Inclusive 
10,714 
(75.1?) 
128 
(0.9?) 
3,432 
(24?) 
Sea 
len 
pic 
•he 
of 
ofA 
ce in 
a I re 
re al 
malar 
sxi;.- 
Vftn" 
ta are 
Spring and early 
Summer 
Late 
Fall 
Late Summer and Early Fall 
=™F” 
z 
o , 
o 
o 
g 
to 
Time required 
to complete,, 
cycle St 20°C 
Relative Humi- 
dity ?0 
7 to 
2 days 
20 to 
24 days 
|6 to 20 days 
>- 
z 
Length of 
Life Cycle 
Four days. Infective for 
mosquitoes in 7 days. 
Seven days. Infective for 
mosquitoes in 10 days. 
Four days. Infective for 
mosquitoes in 7 days 
g 
o 
o 
r> 
f— 
g 
< 
o 
Shape 
o*o*Round} t^d^Oval 
o*o*Round} c^cfOVal 
Crescent shaped with male 
having ends more rounded 
- 
Size 
0* 
%u to 10^ 
6/oto 75/-^ 
I2/4.X 
2.5y~ 
En 
o 
0* 
7/*-to 
55/* to 65 /“* 
12yt*x 
Ut 
CO 
5-t 
co 
Differential 
characters of 
schizonts 
See Illustrations 
Band Forms, See 
11 lustrations 
See illustrations 
ll 
'4 
Color 
Light Brown 
Dark brown or 
bl ack 
Dark brown or 
bi ack 
z 
i 
o 
Type 
Fine Grains 
Large amt. coarse grains 
Coarse grains 
a. 
P resence 
and Amt. 
Yes, Large 
Amts, 
Yes. Large amts. 
Yes. Sma11 
amt. 
O 
>• 
z 
o 
o 
o 
Stages present 
in peripheral 
bjood at any 
o i ven t ime 
Al1 stages, but one 
predominates 
Al1 stages, but one 
predominates 
Ring forms &.gametgcyte? only. 
Other forms in capillaries of 
internal organs except in 
severe cases 
4 
, Q 
X 
8 
Nurpber 
zoites 
of Mero 
formed 
12 to 
|6 
8 to 
12 
12 to 32 
tH 
Length of 
Life Cycle 
48 hours 
72 
hours 
48 hours 
H 
iH 
Mot 11i ty 
Active 
ameboid 
Sluggish 
ameboid 
Little or none 
-d 
3 
U1 
Mature, 
form?,in 
, Fills 
ce 1 1 
Fills 
ce 11 
Fills only 
2/3 cell 
-» 
5 
to 
Ring forms 
first hour 
1»5/4* to 1, 
75/~ 
to 2, 
5>~ 
O.Wo 
.5^ 
-1 
UJ 
o 
o 
Presence of 
Multiple In- 
fection 
Uncommon 
Uncommon 
Fairly coi 
nmon 
LU 
cc 
o 
UJ 
StlpplIng 
Fine granules 
Pink granules 
of Schuffner 
Not present 
A few baso- 
philic gran. 
Few coarse 
grains 
illo|1!ng of 
Maurfer stains 
M 
H* 
CO 
Co 
or 
Pale.yellow- 
ish Red 
Pale,brown- 
ish red 
Dull green 
Greenish red 
6r normal 
Furpli 
Brassy Copper 
FurpI{sh Red 
or normal 
<* 
Lw 
<C 
a. 
Size 
Enlarged to 
0/4-ur 12^/* 
Not enl 
arged 
Not en 
1 arged 
to 
Unstained 
Stained 
Unstained 
Stained 
Unstained 
Stained 
UJ 
to 
UJ 
E 
to 
P. vivax (tertian or 
benign tertian.) 
P, Malaria 
(quartan) 
P. falciparum {aestivo- 
autumnal malignant tertian, 
subtertian or pernicious 
tertian) LIFE-CYCLE OF P. FALCIPARUM IN MAN SHOWING RELATION TO TEMPERATURE CURVE. Life Cycle of P* Malaria Jn !‘an Showing Relation to Tenperature Curve* Life Cycle uf P. vivax in Man Showing Relation to Temperature Curve, SUMMARY TABLE 
SHOWING 
SOURCE OF MATERIAL FOR LABORATORY EXAMINATION AND 
CHIEF DIAGNOSTIC CHARACTERISTICS OF PROTOZOAN PARASITES 
SOURCE 
PARASITE 
CHIEF DIAGNOSTIC CHARACTERISTICS 
Leishmania 
donovani 
Oval, red-nucleated bodies in Reticulo- 
endothelial cells 
Plasmodium 
malariae 
RBC not enlarged; rings, bands, ovals 
Schizonts 6-10 merozoites 
Plasmodium 
falciparum 
Small rings, crescents, does not enlarge 
red cells 
BLOOD 
Plasmodium vivax 
RBC enlarged; pale, Schuffner’s dots 
Schizonts 15-20 merozoites 
Trypanosoma 
gambiense 
Active, slender trypanosome 
Trypanosoma 
rhodesiense 
Active, slender trypanosome 
Trypanosoma 
cruzi 
Short, stumpy trypanosome 
Balantidium 
coli 
Large moving ciliate; bean-shaped macro- 
nucleus, globular micronucleus, contractile 
vacuoles 
INTESTINE 
Chilomastix 
Flagellate; cytostome, spiral groove, 
lemon-shaped cyst 
Endamoeba coli 
Trophozoite sluggish, no RBC, contains 
bacteria, mature cyst 8 nuclei 
Endamoeba 
histolytica 
Trophozoite active, ingested RBC, no 
bacteria, mature cyst 4- nuclei SOURCE 
PARASITE 
CHIEF DIAGNOSTIC CHARACTERISTICS 
INTESTINE 
Giardia lamblia 
Pear-shaped flagellate, sucking disk, 2 
nuclei, oval cyst 
Trichomonas 
hominis 
Poar-shaped flagellate with undulating 
membrane 
SKIN AND 
MUCOUS 
Lgishmania 
donovani 
Oval, red-nucleated bodies in Reticulo- 
endothelial cells 
MEMBRANES 
Leishmania 
tropica 
Oval, red-nucleated, intracellular bodies 
Endamoeba 
histolytica 
Trophozoite active, ingested RBC, no 
bacteria, mature cyst A nuclei 
Plasmodium 
falciparum 
Small rings, crescents, does not enlarge 
red cell 
BRAIN 
Trypanosoma cruzi 
Short, stumpy trypanosome 
Trypanosoma 
gambionso 
Active, slender trypanosome 
Trypanosoma 
rhodosionsc 
Active, slender trypanosome 
MUSCLES AND 
TISSUES 
Trypanosoma cruzi 
Short, stumpy trypanosome 
LYMPH NODES 
Lgishmania 
donovani 
Oval, red-nucleated bodies in Reticulo- 
endothelial cells SOURCE 
PARASITE 
CHIEF DIAGNOSTIC CHARACTERISTICS 
LYMPH NODES 
Trypanosoma 
gambicnse 
Active, slender trypanosome 
Trypanosoma 
rhodesiense 
Active, slender trypanosome 
SPLEEN 
Leishmania 
doncvani 
Oval, red-nucleated bodies in Reticulo- 
endothelial cells 
LIVER 
Endamoeba 
histolytica 
Trophozoite active, ingested RBC 
no bacteria, mature cyst 4- nuclei 
Leishmania 
donovani 
Oval, red-nucleated bodies in Reticulo- 
endothelial cells SECTION II 
THE HELMINTHS PHYLUM - PLATYHELMINTHES 
(The Flatworms) 
Class - Trematoda 
Members of this class are commonly known as flukes. They are 
flat, unsegmented, leaf-shaped and have an alimentary tract but no 
anus. Most species have reproductive organs of both sexes in one 
parasite (hermaphroditic). The developmental cycle is a complicated 
one of alternation of generations, the intermediate host usually being 
a snail, mussel or crab. The ova are opcrculatcd in most, an 
important exception being the Schistosoma, 
Human infection with the flukes is largely confined to the tropics 
and the Orient, Laboratory diagnosis depends upon identification of 
the ova in the appropriate body fluid or discharge, 
f 
Additional information regarding the common flukes of man will 
be found in Table on adjoining page. 
Class - Cestoda 
The ccstodos or tapeworms arc segmented, ribbon-shaped and have 
no alimentary tract. The adults consist of a scries of flat, rectangular 
segments (proglottides) attached to a smaller segment, the head or 
scolex which is equipped to anchor onto the intestine of its host. 
The segments arise from the scolcx by budding. Each segment is 
hermaphroditic and in mature ones the uterus is filled with ova. The 
testes usually consist of a great number of small glands scattered 
throughout the segment. Ducts from those unite to form a single vas 
deferens leading to the genital pore which is found on one lateral border 
of the segment, or upon its flat surface. Sperm from the sa.me or 
another segment reach the genital pore and are carried to the oviduct 
where the ova arc fertilized. They then pass into the uterus where they 
may remain until the segment disintegrates or until passed out through 
a birth pore. 
There arc no circulatory, respiratory or digestive systems. 
Nourishment is obtained by absorption. Excretion takes place through 
four excretory canals which run the full length of the chain of segments 
and empty posteriorly. Near the lateral borders arc two or more nerve 
cords derived from the scolcx which also run the entire length. 
As a rule the tapeworms pass a larval or encysted stage in the 
tissues of an intermediate host, only the adult stage occurring in man. 
Exceptions to this are Taenia solium and Hymenolepis nana. In the 
case of T. solium the cystic stage may take place in man, favored  
FASCIOLA 
HEPATICA 
FASCIOLOPSIS 
EUSKI 
CLONORCHIS 
SINENSIS 
PARAGONIMUS 
TJESTERLIANTT 
SCHISTOSOMA 
HAEMATOBIUM 
SCHISTOSOMA 
MANSONI 
SCHISTOSOMA 
JAPONICUM 
Common Name 
Liver 
Fluke 
Large Intes- 
tinal Fluke 
Chinese 
Fluke 
Lung 
Fluke 
Blood 
Fluke 
Blood 
Fluke 
Blood Fl^ike 
Adult lives; 
Liver 
Intestine 
Bile ducts 
Lung 
Pelvic veins 
Mesenteric 
Veins 
Mesenteric 
Veins 
Spread by: 
Water 
plants 
Water 
chestnut 
Raw fish 
Raw crab 
Wading in 
infected 
water 
Wading in 
infected 
water 
Wading in 
infected water 
Specimen 
Feces 
Feces 
Feces,bile 
Sputum 
Urine 
Feces 
Feces 
Ovum 
HO by 
80 Miera 
Operculated 
130 by 80 
Micra Oper- 
culated 
30 by 15 
Micra, Vate 
shaped 
Operculate d 
90 by 55 
■ Micra 
Opercu- 
lated 
150 by 60 
Micra. No 
Operculum 
Terminal 
. spine 
150 by 60 
Micra, No 
Operculum 
Lateral 
spine 
80 by 60 Micra. 
No operculum. Rudi- 
mentary spine 
(small. liQQk) 
DIFFERENTIATION OF THE COMM FLUKES OF MAN Helminths# Diagram of diagnostic material. A, egg of the whipworm (trich- 
ocephalus trichiurus); B, egg of the pinworm (Enterobius vermicularis); C, egg 
of the broad fish tapev/orm (Diphyllobotbrium latum); D, egg of the dwarf 
tapeworm (Rymenolepis nana); E, egg of the Chinese Liver Fluke (Clonorchis 
sinensis); F, G, H, fertilized, unfertilized, and decorticated eggs respectively 
of the large intestinal roundworm (Ascaris lumbricoides); 1, egg of hookworm 
(Necator americanus or Ancylostoma duodenale); J, fore-part of rhabditoid larva 
of hookworm showing long buccal cavity. Compare with short buccal cavity of 
rhabditoid larva of Strongyloides stercoralis, K; L, egg of either the pork 
tapeworm (Taenia solium) or the beef tapeworm (T. saginata); M, N, gravid seg» 
ments of the pork and beef tapeworms respectively, shov;ing difference in the 
number of uterine branches - fewer than fifteen in the pork tapeworm; more 
than fifteen in the beef tapeworm; 0, egg of hookworm showing fully developed 
embryo. Such eggs are commonly found in constipated stools of hookworm patients 
P, egg of the Oriental lung fluke (Paragonimus westermani); Q, egg of the Ori* 
ental blood fluke (Schistosoma .japonicum); R, egg of Manson!s blood fluke (S. 
mansoni); S, egg of the vesical blood fluke (S. hematobium); T, egg of the 
sheep liverfluke (Fasciola hepatica) or the large intestinal fluke (Fasciolop- 
sis buskl). Approximate magnifications, E X600, M and N X3; all others 
X300, Compiled and modified from various sources. tissues being the eye and brain (cysticcrcus ccllulosac), Apparently 
H, nana requires no intermediate host. There is, therefore, danger 
of direct infection (oral) from handling feces containing the ova of 
either of these parasites* 
The portal of entry for all known tapeworm infections, except 
ocular sparganosis, is through the mouth. Eating raw or incompletely 
cooked meat of the intermediate host, which is infected with encysted 
larvae, is the source of infection with T. saginata, T. solium and 
D. latum. 
The important tapeworms of man belong to three genera: Taenia, 
Hymcnolcpis and Diphyllobothrium* 
1, Taenia saginata 
This, the beef tapeworm, is the common tapeworm of the United 
States, and is widely distributed over the world. The scolex is about 
the size of a large pin head and is surrounded by four sucking discs. 
The terminal segments which become detached and appear in the feces, 
are about 18 to 20 mm, long and U to 7 mm, wide. The length of the worm 
varies from several to 30 or more feet. Cattle provide the intermediate 
host, the eggs or segments on reaching the soil being swallowed by 
cattle when grazing or when drinking contaminated water. The larva is 
sot free in the intestine of the cow and makes its way to the muscle 
or other tissue where it becomes encysted, 
Man is infested by eating raw or improperly cooked meat 
containing the cysts. On entering the stomach the cyst wall is digested, 
the scolex is sot free, becomes attached to the intestinal wall and 
grows to maturity in 2 to 3 months, and the cycle is repeated. Since 
the head or scolex is the ancestor of the worm, it is important to make 
certain that the head ha.s boon passed. 
Detection of tapeworm scolex: following the vermifuge ad- 
ministered by physician, the stool is saved in a container together with 
the stool passed before administration of the drug, and taken to the 
laboratory. The entire quantity of feces is passed through a $20 sieve. 
The scolex is identified by examining a segment between two glass 
slides and holding it to the light. 
Laboratory diagnosis will depend upon finding the segments or 
ova in the feces. The ova are spheric or ovoid, yellow to brown in 
color and have a thick radially striated shell. Vegetable cells 
(generally present) arc often mistaken for these ova. DIFFERENTIAL CHARACTERISTICS OF THE IMPORTANT TAPEWORMS 
OF MAN 
DIPHYLLOBOTHRIUM 
LATUM 
HYMENOLEPIS 
NANA 
TAENIA 
SOLIUM 
TAENIA 
SAGINATA, 
TAENIA 
ECHINOCOCCUS 
Frequency 
in U. S. 
4 
1 
3 
2 
_5 
Length of 
Adult Worm 
10-30 ft-. 
1/2 to 4 1/2 
. . Cm. 
6-12 ft. 
Suckers 
2 (grooves) 
4 . . 
4 ... 
4 
L 
Hooks 
None 
Present 
Present. 
None 
Present , 
Number of 
Proglottids 
3000-4000 
Up to 200 
800-1000 
1000-2000 
-3 
Mature 
Broader than 
Broader than 
Longer 
than 
Longer 
than 
Longer than 
Proglottid 
long 
long 
broad 
broad 
..broad 
Uterus 
Central rosette 
Irregular 
sac-like 
7-12 . 
branches 
15-30 
branches 
Loose coil 
in end 
proglottid 
Genital Pore 
Middle 
Lateral on 
one side 
Lateral, 
Alter- 
ant e 
Lateral, 
Alter- 
nate 
Lateral 
alternate 
Ova 
70 by 45 Miera, 
Operculated 
45 by 35 
Miera,Globu- 
lar, 2 mem- 
branes, 
booklets 
35 by 35 
Miera, 
Radially 
striated 
cortex, 
booklets 
35 by 35 
Micra, 
Radially 
striated 
cortex 
booklets 
Intermediate 
Host 
Cyclops and fish 
None 
Hog, man 
Cattle 
Man, sheep 
Stage in Man 
...Mult 
..-Mult 
Adult 
.■Larval 
Adult 
Larval 2, Taenia solium 
The pork tapeworm is comparatively rare in this country. 
Its life cycle is similar to that of the beef tapeworm - the pig 
being the intermediate host. The ova of Taenia saginata and Taenia 
solium are so closely similar that they are practically 
indistinguishable. Best method of differentiating the two is to 
recover mature segments from the feces. These should be cleaned 
and relaxed by shaking in the physiological salt solution. Segments 
may then be pressed between two glass slides and held up for 
examination in front of a strong light. Count the number of uterine 
branches. (Sec Table of Differential Characteristics), 
3. Diphyllobothrium latum 
Infection with the fish tapeworm occurs only in those 
regions where the species of fresh water fish that provide the 
intermediate host for this parasite, are found. This infection is 
common in Japan and many countries of Europe. It has been reported, 
not infrequently, in the United States, 
The ova are usually easily found in the feces where the worm 
is present. They are brown in color and filled with small spherules. 
The shell is thin and has a small hinged lid at one end. The lid 
may be demonstrated by making sufficient pressure on cover glass 
to force it open, 
4. Hymenolopis nana 
The dwarf tapeworm is 1 to 4.5 cen. in length. It is mono 
common in tropical countries, but is found in northern latitudes 
and is probably the most common of all the tapeworms in the United 
States. Transmission is direct, no intermediate host being required. 
Laboratory diagnosis must usually depend upon discovery of 
ova in the feces, since the worms themselves are partly disintegrated 
as a rule when they leave the body. The ova are colorless, semi- 
transparent, nearly spheric and contain an embryo surrounded by two 
distinct membranous walls, between which is a broad zone of 
gelatinous substance. Three pairs of parallel booklets can usually 
bo seen inside the inner membrane, 
5, Taenia echinococcus (Echinococcus granulosus) 
The dog is the normal definitive host for this parasite. 
Usual intermediate hosts are cattle, sheep and hogs, but man and 
other mammals may be infected. The infection in man is known as 
Hydatid Disease and occurs wherever man lives in such close association 
with dogs that the hands or food become contaminated with their feces. It is common in Iceland, Central Europe, Africa, Arabia, South 
America and Australia, The infection is rarely found in man in 
the United States, but is common in swine in some parts of the 
country. 
Larvae develop from ingested ova and become encysted in 
various organs of the body, particularly the liver and the lungs. 
The cysts eventually cause serious disturbances as a result of 
pressure and tissue destruction, and unless completely removed 
surgically, death usually occurs in a few years. Diagnosis 
depends upon; (1) finding free scolices in fluid obtained from 
a cyst, or (2) an intradermal reaction (Casoni’s Test), which 
consists of injecting a small drop of cyst fluid intradermally, 
and noting the reaction obtained. PHYLUM - NEMATHELMINTHES 
(Roundworms) 
Class - Nematoda 
The nematodes are. worms varying in length from 1 mm, 
to SO mm, in different species. They are unsegmented and possess 
a tubelike alimentary canal which has a distinct esophagus near 
the mouth. All are covered with a cuticle of varying thickness, 
formed from the ectoderm, which is frequently ringed. Inside the 
ectoderm is the body cavity containing clear fluid within which the 
reproductive organs lie. The sexes are as a rule separate. The 
male can usually bo recognized by its smaller size-and by the curved 
posterior end which may have an umbrella-like expansion - the 
copulatory bursa. The genital opening of the female lies ventrally 
and may be located close to the mouth or near the tail. 
Vascular and respiratory systems are lacking. The nervous 
system consists of a nerve ring around the esophagus, dorsal and 
ventral nerve cords, and circular bridges connecting the cords. The 
excretory system usually consists of two tubes which discharge near 
the head, A few nematodes are viviparous (wuchorcria, trichinolla), 
but in most cases the female deposits ova of characteristic appearance. 
The life cycles vary with the different species. As a rule, 
the larvae are different from the adult. An intermediate host is 
necessary for only a few species, but usually a larval stage takes 
place before a new host is infected, 
1, Ancylostoma duodonalo - Old World type ) Hookworm 
Nocator amcricanus - Now World type ) 
Hookworm infection (ancylostomiasis) is a chronic affection 
characterized by gastro-intestinal disturbances, anemia, mental 
and physical lethargy. It is common in tropical countries, but is 
also found in subtropical and even northern latitudes where unhygienic 
conditions exist* The hookworm inhabits the duodenum and upper part 
of the sma.ll intestine. Multiplication docs not occur in the body. 
The severity of an infection is proportionate to the number of worms. 
Ancylostoma duodenale is common in southern Europe and in 
Egypt, and is not infrequently found in America, Nocator amcricanus 
is common in central and southern Africa and in subtropical America 
and southern United States, The principal point of differentiation 
between the two species is that the Nocator is provided with two 
pairs of plates in the buccal cavity (mouth), while the Ancylostoma 
has two pairs of hooklike teeth. The Nocator is smaller than_ 
Ancylostoma, the male being 7 to' 9 min, long, the female 9 to 11 mm.  
ASCARIS 
ANCYCLOSTOMA DUODENALE 
, AMERICANUS. 
ENTEROBIUS 
VERMIGULARIS, 
STR01GYLOIDES 
, STEF, GOB ALLS., 
TRICHINE LLA 
, SPIRALIS... 
TRICHURIS 
TRICHIURA 
FILARIA 
..BANCROFT L...... 
Disease 
Ascariasis 
Hookworm disease 
Binworm 
infection 
Strongyloidi- 
asis 
Trichinosis 
Whip worm 
infection . 
Filariasis 
Elephantiasis 
Geographical 
Distribution 
— 
Dosmopolitan 
Sub-tropics and 
tropics 
Cosmopolitan 
Sub-tropics 
and tropics 
Cosmopolitan 
Cosmo- 
politan 
JrcplcLS 
Location 
in Man 
Intestine 
Intestine 
Intestine 
Intestine 
Inte.st.ine 
Intestine 
Adult- 
Lynphatics, 
Larva,Blood 
Size (mm) 
Male 
150-300 
6-10 
2-5 
,7 
1.5 
30-45 
4Q 
Female 
200-350 
10-13 . . 
6-13 
2-2 
i35-5D. 
B3 
Definitive 
Host 
Man, hog 
Mon 
Man 
Man 
Man, Carni- 
vorous 
mammals . _ 
Man,monkey, 
hog 
Man 
Ovum 
Broadly ovoid 
60 by 45 
Liera, Mam- 
millate 
covering. 
Brownish 
Oval, blunted ends, 
60 by 40 Micro. Thin 
shell, clear zone. 
Segmented cytoplasm, 
Transluscent 
Oval,flattened 
on one side, 
55 by 25 
Micro, Thin 
shell. Con- 
tains embryo 
Transparent 
Ova not 
found in 
stool. 
Living larva 
None 
Ovil vdth 
plugs in 
both ends 
50 by 25 
Miera, 
Bruwnish 
None 
Infective 
Stage 
Embryonatcd 
ovum 
Filariform larva 
Embryonatcd 
ovum 
Filariform 
larva 
Encysted 
larva 
Emaryon- 
atad ovum 
Microfilaria 
carried by 
-iaosauit-Q- 
Site of entr 
r Intestine 
Skin 
Intestine 
Skin 
Intestine 
Intestine 
Skin 
-DSY.ftlPPP&flt-. 
Indirect to 
intestine via 
intestine, 
lymph,blood, 
lungs,throat 
Indirect to intestine 
via lymph, blood, 
lung, throat 
Direct 
Indirect to 
intestine via 
lymph,blood, 
lung,throat 
Direct larva 
encyst in 
muscle 
Direct 
To lymph 
nodes via 
lymph stream 
DIFFERENTIATION OF IMPORTANT ROUND WORMS OF MAN Tho life histories of the two arc probably the same. Tho 
ova pass out with the feces and under favorable conditions of 
warmth and moisture, develop an embryo which hatches in a few days# 
The resulting larvae, after several developmental changes, are 
ready to infect a now host, Tho chief point of entry is through 
the skin of the feet. From the skin the larva migrates through the 
lymphatics, or blood, to the heart and lungs. It then pierces 
tho alveoli of the lungs and passes into the bronchioles, bronchus, 
trachea and pharynx, and eventually roaches tho intestine in 
which it develops into tho adult. 
The severe anemia produced when worms arc present in largo 
numbers is probably due to;abstraction of blood by the worms and 
hemorrhage from bleeding points left when they change position in 
the intestine; continued bleeding - presumably due to a secretion 
in tho buccal glands that prevents coagulationj toxic secretion of 
parasites; secondary microbic infection. 
Tho blood picture may show a red blood cell count of 2 to 3 
million, 35-50% hemoglobin, a low color index, and 10-25% 
eosinophils. The laboratory diagnosis usually depends on finding - 
ova in the feces, since adult worms arc seldom seen except after a 
vermifuge. The-ova arc nearly always typical, showing a thin, 
but very distinct shell, a clear zone and a finely, granular, 
segmented protoplasm, 
2, Ancylostoma braziliense 
This is a parasite of dogs and ca.ts, and occurs in North 
and South America, Ceylon, India, Siam and the Philippines, Human 
infection usually takes the form of "creeping eruption" - a painful 
and itching dermatitis. Infective larvae arc able to penetrate tho 
skin and burrow extensively through tho subcutaneous tissue, but 
apparently do not progress farther into the body, Tho adult worms 
(in dogs) a.ro slightly smaller than A, duodonalo and can be 
distinguished by their characteristic teeth - a small median pair 
and an outer pair that are larger. The ova arc not distinguishable 
from those of A. duodenale, 
3. Strongyloidos stcrcora,lis 
This parasite may be found in warm, moist regions throughout 
tho world. It is considered to be harmless, or at worst, as causing 
a mild catarrhal enteritis. The infection is acquired by filaria- 
form larvae penetrating tho skin, or through their being ingested. 
Upon reaching the alimentary tract, the females inhabit the walls 
of tho duodenum and jejunum. Their eggs hatch in the tissues and 
larvae are passed out in the feces. When an active diarrhea exists, 
the ova may be passed in the feces. They are similar to the hookworm 
ova in appearance. Laboratory diagnosis depends upon finding the larvae. It 
should be borne in mind that hookworm ova may hatch in 24- to 4-8 
hours after the feces has been passed. The following points will 
aid in differentiating the two larvae: 
Hookworm Strongyloides 
Ova found in fresh feces - Larvae usually 
Larvae have deep mouth cavity - Mouth cavity shallow 
Genital anlage small, - Large, conspicuous 
inconspicuous 
4-. Ascaris lumbricoides (Roundworm) 
This parasite inhabits the small intestine and its presence 
is characterized by gastro-intestinal disturbances, malnutrition, 
anemia, cachexia and nervous symptoms. The male measures 15-20 cen. 
in length, and 3 rnm. in diameter. The female is about twice as 
large as the male. It is the most common parasite of the intestine, 
especially in tropical countries. Found more frequently in children 
under 10 years of age, but occurs also in adults. 
Eggs of the parasite which are in the feces are unsegmented, 
oval in shape and provided with a rough, irregular protective shell. 
Under suitable conditions, the eggs develop into an embryo and larva 
in the soil or \mter in about 2 weeks. The egg does not hatch until 
it enters the intestine of man or some susceptible animal. The 
larva is set free in the host and attains adult stage in U to 6 weeks, 
after which the eggs arc discharged and the cycle repeated. Man 
is infected by drinking polluted water, eating contaminated fruits 
and vegetables, or by soiled fingers. Playing on the ground is a 
common source of infection for children. 
Laboratory diagnosis: discharge of the adult Yjorm with the 
feces is common. In nearly all cases, diagnosis can bo made by 
detection of the eggs. 
5. Entorobius vermicularis 
This, the pinworm, is worldwide in distribution. It matures 
in the small intestine and cecum and in the adult stage inhabits the 
colon and rectum, especially of young children. Infection results 
from ingestion of embryonated eggs. Gravid females usually migrate 
through the anus and deposit eggs in perianal folds, causing pruritis 
ani and vulvi. Adult worms arc not infrequently found in feces, 
particularly after copious enema. The ova arc rarely found in the 
feces. They are best found by scraping the skin at margin of anus 
with a dull knife, or with NIH swab. The N I H Anal Swa* 6. Trichuris trichiura (whipworm) 
This parasite is 3.5 to 5 ccn. long. Its anterior portion 
is slender and threadlike, the posterior portion thicker. It is one 
of the most common intestinal parasites in this country, and is of 
world wide distribution. It lives in the large intestine with its 
slender extremity embedded in the mucous membrane. As a rule, these 
worms do not cause any symptoms, however, the damage they do to the 
intestinal mucous membrane may favor bacterial invasion. Infection 
results from ingestion of embryonatod ova. 
The worms arc rarely found in the feces. The ova are com- 
paratively small, brown, ovoid in shape, and have a button-like 
projection at each end, 
7. Filaria 
Wuchercria bancrofti 
This is the most important member of the filaria group. 
It is distributed throughout the world in.temperate and tropical 
regions. Many species of Acdcs, Anopheles and Culex mosquitoes serve 
as intermediate hosts, Man acquires the infection during the bit of 
an infected mosquito. The adults normally live in the lymphatics 
in the region of lymph nodes and the female deposits microfilariae 
which are carried to the blood stream. Lymphangitis may be produced 
with an associated high fever, enlargement of lymphatic glands and 
inflammatory swelling of the parts affected. Repeated attacks may- 
result in permanent swelling (elephantiasis). 
Diagnosis is based upon demonstration of the micro- 
filariae in blood films. Blood for examination should bo taken at 
night since in most cases the parasites are found in the peripheral 
blood only at this time, 
Acanthochoiloncma perstans 
Infection with this parasite occurs during the bite of 
an infected midge (Culicoidos), The adults inhabit chiefly the 
mesentery and retroperitoneal connective tissue. Apparently the 
infection is symptomlcss. Occurs in tropical Africa and South America, 
Diagnosis depends upon recovery of microfilariae in the blood stream, 
Loa loa 
Human infection is acquired through the bite of infected 
Chrysops flics. The adult worms normally live in the subcutaneous 
tissues through which they migrate back and forth, causing a temporary 
inflammation (fugitive swelling). This parasite is found mainly in 
Central Africa. Diagnosis can bo made by recovery of microfilariae from 
peripheral blood during the day; by removal of adult worms from their 
tunnels; or by the filaria-group serological test. Onchocerca volvulus (the blinding filaria) 
The parasite is transmitted during the bloodmoal of 
infected black flics (Simulium). The adults inhabit the subcutaneous 
lymphatics and are usually enclosed in fibrocystic nodules. Several 
months are required for development of nodules after infection. 
Larvae deposited in the nodules may spread in the skin for a 
considerable distance, and in cases over $ years duration, may 
penetrate tissues of the eye, eventually causing blindness, 0, 
volvulus is common in Central Africa, Western Guatemala and Southern 
Mexico, Diagnosis may be made by demonstrating the presence of 
adults or microfilariae in excised nodules, 
Dracunculus medinensis (Guinea worm) 
Distributed in parts of Africa, India, Arabia, West 
Indies, Brazil and Guiana. Infection occurs through swallowing water 
containing infected crustaceans (cyclops). Larvae liberated in the 
stomach make their way to connective tissue a.nd v/hen mature migrate 
to a position just under the skin, frequently near the ankle joint, 
A blister forms on the skin just over the head of the worm. When 
the blister breaks and upon contact with fresh water, larvae are 
passed through this opening into the water. 
The adult males vary in length from 12 to 30 mm,, 
while the females are much larger and may reach a length of 1 meter. 
Diagnosis is based upon finding the worm under the skin, 
8. Trichine11a spiralis 
Infection with this parasite is known as Trichinosis, It 
occurs mainly in the United States and Europe, Infection is acquired 
by eating raw or improperly cooked pork containing encysted larvae. 
Ingested females deposit larvae in the lymphatics of the small 
intestine, whence they are carried to all parts of the body to become 
encysted in striated muscle. The cysts arc oval and about 0,/+5 mm, 
by 0.2$ mm, in size. The encysted larvae may live 10 to 20 years, 
but eventually die and the cyst becomes calcified. 
This parasite affects chiefly rats and hogs, although other 
animals arc susceptible. Hogs become infected by eating rats, 
and in turn, rats acquire the infection through eating scraps of 
pork around abattoirs. 
The symptoms produced depend upon the number of parasites 
ingested and may be very mild or very severe, and end fatally. During the second and third ucok after infection in severe eases, 
there is high fever and prostation. Larvae nay then be found in 
the circulating blood. From the 10th day on larvae begin to migrate 
into the nusclo, giving rise to severe muscle pain, stiffness and 
disability. 
Diagnosis must usually depend upon finding the encysted 
larvae in bits of excised muscle (deltoid or pectoral), or upon 
intradernal test. 
Laboratory procedures for the diagnosis of Helminth 
infection arc outlined in Section III. SECTION III 
EXAMINATION OF FECES AND 
LABORATORY DIAGNOSTIC PROCEDURES EXAMINATION OF FECES 
The normal stool consists chiefly of water, undigested and 
indestible remnants of food and enormous numbers of harmless bacteria. 
The quantity of feces varies with the amount and types of foods 
eaten, but normally averages about 200 Gms, a day. One or two 
stools per day may be considered normal, but one stool every three 
or four days may not be incompatible with health. 
Macroscopic Examination 
1. Odor - may be reported as normal, slight, sour, putrid, 
very foul, etc. The odor is influenced by diet and disease. It is 
stronger with meat than with a vegetable diet, and may be very 
slight when milk is the only food, 
2'. Color 
a. Light or dark brown is the normal color and is due to 
bilirubin. 
b. Yellow (may be due to milk diet, rhubarb, senna, santonin, 
unchanged bilirubin), 
c. Green (leafy vegetables, calomel, biliverdin), 
d. Clay (deficiency of bile), 
e. Acholic (undigested fat, jaundice), 
f. Dark reddish brown (excess of cocoa or chocolate). 
g. Black (iron,bismuth, charcoal, blood), 
h. Red (undigested blood, beets). 
3. Consistency - fluid, serai-solid, formed, hard .With diarrhea 
or after cathartics, the stools are mushy or liquid. Constipation 
results in hard stools. Ribbon-like stools may result from some 
obstruction in the rectum. 
4-. Mucus - report as strings, balls, casts or ribbons, and 
approximate amount. Excessive quantities of mucus signify irritation 
or inflammation. 
$. Blood - localized, diffuse, color, amount* 6, Concretions - should be searched for while washing fccos 
through a sieve with water. Gallstones are usually faceted but may 
be definitely identified by testing for cholesterin and bile pigments. 
7. Parasites - Some parasites may be detected by washing feces 
through a sieve and examining suspicious objects in a flat-bottomed 
dish over a dark background. (See also Section on Parasit.Lab, Methods). 
Chemical Examination 
1. Reaction - acid, alkaline, neutral.Test v/ith litmus paper. 
2. Occult blood - chemical tests for microscopic amounts of 
blood depend upon the reaction of iron in the liberated hemoglobin 
with the reagent employed. To avoid false positives, the patient 
should have been on a meat free diet for 72 hours prior to testing, 
a. Benzidine Test 
This test is most reliable when run on other extract 
of feces. (See D'Riva's Concentration Method, Section III). 
(1) Dissolve knife-tip of benzidine in 2 cc. of glacial 
acetic acid and add 1 cc. of hydrogen peroxide. 
(2) Make a smear of feces on a slide and pour above 
reagent over it. The appearance of a blue color in a few seconds 
indicates a positive test. 
b. Orthotoluidinc Test 
Reagent - IS orthotoluidine in glacial acetic acid. 
(1) To 1 cc.of watery feces, add 1 cc. of reagent, 
(2) Add 1 cc. of hydrogen peroxide {?>%)• A positive 
tost is indicated by the appearance of a bluish-green color, 
3. Bile - since bilirubin is produced from hemoglobin, an 
estimation of urobilin in the feces gives some information as to the 
rate of blood destruction. In obstructive jaundice, urobilin is 
reduced in amount or absent from the feces. 
a. Schmidt's Qualitative Test - emulsify a little feces in 
a saturated aqueous solution of mercuric chloride. Observe after 6 
to 2U hours. Urobilin, the normal pigment, gives a salmon pink color; 
bilirubin, green, (This test takes place at once if the emulsion is 
boiled). b. Gmelin’s Test - make a thin smear of feces on filter 
paper and touch with a drop of yellow nitric acid. The appearance 
of a rainbow of colors with green on the outside indicates a positive 
test. 
Microscopic Examination 
To got a uniform mixture and representative sample for 
examination, it is best to rub up a portion of feces about the size 
of a walnut, in water. This may then bo used for the propagations 
listed below, 
1. Place a drop of feces suspension on a slide and apply a 
cover glass. This preparation should bo just thick enough that 
newsprint can still bo road through it. Examine with both low and 
high power, covering the slide systematically. The beginning student 
may first use this preparation for studying the elements found in 
normal feces, such as vegetable cells, plant hairs, etc, (See 
diagram in text). Other structures that may be observed in this 
preparation arc listed below, 
a. Degree of digestion of muscle fibers. If striations 
are visible, digestion is imperfect, 
b. Red blood corpuscles arc rarely scon unless their 
source is the rectum, colon or anus. Bleeding from the small intestine 
or higher, must usually bo detected by a chemical test. Red cells 
may be present in clumps in amebic dysentery, 
c. Pus cells are present in ulcerative conditions of 
intestine. They may bo seen more clearly by a.dding a drop of 30% 
acetic acid to the slide, 
d. Epithelial cells in small numbers are practically always 
present, but may bo so degenerated as to bo unrecognizable. 
Excessive number indicates inflammation or irritation, 
o. Crystals of various types may bo found, but arc not of 
much significance. Triple phosphates, calcium oxalate, fatty acids 
and Charcot-Lcydon crystals may be scon, 
f. Ova and larvae of parasites may be detected in this 
preparation. (See also parasitological methods). 2, Mix a drop of foe os emulsion on a slide v;ith a drop of 
Lugol’s iodine solution, apply cover glass and examine for 
protozoan cysts and starch grains. Undigested starch granules turn 
blue, partly digested ones appear reddish, 
3. Mix a drop of the emulsion with 1 or 2 drops of Sudan III 
and examine for fats. Flakes or droplets of neutral fats stain 
orange rod; normally no appreciable amount is present. Fatty acids 
appear as flakes or noodles and stain very faintly. Soaps appear as 
yellowish flakes or coarse crystals, 
4* Examination for parasites and ova should consist first of 
direct smear with and without Lugol’s solution, and if unproductive, 
this should bo followed in order by flotation preparation and a 
concentration method, (See parasitological methods) 
Flotation Solutions 
a. 
Glucoso 
500 Gms. 
Water 
320 cc. 
b. 
Sodium Chloride (sat, sol,) 
c. 
Zinc Sulfate- (ZnSO^) 
Specific Gravity 
1,180 
d. 
Glycerin and saline, equal parts PARASITOLOGICAL LABORATORY METHODS 
I. LABORATORY METHODS FOR THE INTESTINAL PROTOZOA 
A, Collection of Specimens 
Successful demonstration of the intestinal protozoa 
depends to a great extent upon the care with which the sample of feces 
is collected. If the trophozoite forms are to be identified, it is 
essential that the examination be made as soon as possible after a 
fluid specimen is passed, preferably within thirty minutes. It is 
well to have the specimen collected in the laboratory where the diagnosis 
is to be made. A clean, dry, covered receptacle should bo used. 
Admixture with urine or antiseptics should be avoided. If a fluid 
specimen must be transported from ward to laboratory some moans of 
keeping it at body temperature should bo supplied, A container has been 
devised which consists of a fluted cylindrical base in which warm water 
is placed, and an enamelled bowl of slightly smaller diameter which 
fits in double-boiler fashion within the cylinder, A projecting edge 
holds the bowl at the top of the cylinder* 
If the patient is not having an active diarrhea, a liquid 
stool specimen is obtained by administering a saline laxative* A 
specimen obtained by an oil cathartic is to bo avoided. It is well to 
wait at least 72 hours after such a cathartic has taken effect before 
collecting a sample for examination. Microscopic droplets of oil are 
often confused with cysts. Specimens collected by an enema are also 
undesirable. 
Formed stools for examination for cysts may be shipped to 
the laboratory if no facilities are available locally. Examination 
should be made within two days after the specimen is passed, 
B, Preparation of the Specimen for Microscopic Examination 
1. Direct Examination 
Direct examinations for intestinal protozoa arc made by 
emulsifying the feces with a drop of physiological saline on a glass 
slide. The mixture is smeared across the slide to the width of two 
cover glasses, A cover glass is placed immediately over one half of 
the smear. To the other half a drop of iodine stain is added, and a 
cover slip applied. In this manner the material may be examined, both 
stained and unstained, on the same slide. 
With Lugol’s and D’Antoni’s iodine stains* the cytoplasm 
of the protozoan cysts appears lemon or greenish-yellow, while the cyst 
wall, nuclear membrane, and karyosomes are unstained and have a grecnish- 
refractilc appearance. Chroraatoidal bodies, if present, appear as 
unstained refractilo bars within the cytoplasm while glycogen vacuoles 
stain a yellowish-brown. 
*Sce section on formulae for composition of all stains and reagents 
mentioned in this chapter. With Kofoid's oosin-iodinc stain, best results are 
obtained by placing a small drop of stain adjacent to the focal sus- 
pension and placing a cover glass over both drops, A stained area 
and an unstained area result. In the clear area living organisms 
and unstained cysts appear. In the stained area the bacteria, fecal 
particles, and yeasts (except the larger forms) stain at once. 
Protozoan cysts stand out clearly as bright spherules which soon 
become tinged with the iodine to varying shades of yellow, while their 
glycogen inclusions, when present, turn light to dark brown according 
to their mass. As the stain penetrates, the nuclei become more 
clearly defined. This method provides a clear picture of the smear 
because of its differential staining characteristics. 
If the first slide examined is negative, at least three 
other samples should be selected from different parts of the stool. 
Those portions containing blood or mucus are most likely to reveal 
parasitic protozoa. 
In a fresh warm stool tho parasites appear actively 
motile. Such motility may be maintained during the examination by tho 
use of a warm stage apparatus. Examinations, especially for the 
vegetative forms of the amebas, may bo aided by the use of a drop of 
vital stain which the trophozoites readily absorb without interfering 
with their motility. 
Most infections of any importance arc sufficiently heavy 
to bo demonstrated in direct preparation. To discover the cysts in 
light infections, it is desirable to use a concentration method, 
2, Concentration Methods 
a. Simple Sedimentation 
Whore laboratory facilities are limited, the simple 
sedimentation method may be used, A portion of the stool about the 
size of a pecan is emulsified in a test tube in about ten parts of 
lukewarm water. If no centrifuge is available, the tube is allowed to 
stand until the sediment settles out. The supernatant is decanted and 
2 or 3 cc, of water are added to tho sediment which is then broken up, 
Tho tube is nearly filled with water, tho contents mixed wall and the 
process of centrifuging or settling is repeated. Those stops should be 
repeated until the supernatant is clear, 
b. Zinc Sulfate Flotation Method 
A more efficient method which may be used with limited 
equipment is the flotation method, A solution of high specific gravity 
is used, and the cysts of the protozoa are floated to the surface of 
tho liquid. Most commonly used is a aqueous solution of zinc 
sulfate. The specific gravity of this solution is about 1,18, Tho 
technic is as fellows; (1) Suspend a portion of the stool, about the sizo 
of a pecan, in 10 quarts of lukewarm water, 
(2) Strain through wet cheesecloth in a small 
funnel into a Wasserman tube. 
(3) Centrifuge at 2500 rpm for 45 to 60 seconds. 
Pour off the supernatant, add 2 to 3 cc, water, break up the sediment, 
and add more water, 
(4) Repeat stop number 3 until the supernatant is 
clear, 
(5) Pour off the last supernatant, add 3 to 4 cc, of 
the zinc sulfate solution, break up the sediment, fill to within 
one-half inch of the top of the tube, and centrifuge at 2500 rpm for 
45 to 60 seconds. 
(6) Remove the diagnostic material at the surface of 
the tube with a wire loop. Add a drop of iodine stain and make a 
cover-slip preparation. 
In the absence of a centrifuge the fecal material may 
be emulsified in plain water and allowed to settle out repeatedly until 
the supernatant is clear. Then the remaining sediment may bo mixed 
with a zinc sulfate solution in a shallow dish. After an hour or more, 
during which time the material has been mixed frequently, the 
diagnostic surface film may be removed by the application of a cover 
glass to the liquid. 
Other solutions may be substituted for the zinc sulfate 
as long as the specific gravity remains at about 1,18 to 1,2, A 24$ 
solution of sodium chloride or a 40% solution of ordinary cane sugar 
may be used, 
c. DcRiva’s Method 
Then the materials and equipment are available, the 
most desirable concentration method is that described by DoRiva: 
(1) Emulsify approximately 1 gram of feces in 5 cc, 
of 5$ acetic acid. 
(2) Strain through wet cheesecloth into a graduated 
centrifuge tube, 
(3) Add an equal volume of other and shako vigor- 
ously for 30 seconds tc 1 minute. (4) Centrifuge at 2500 rpm for 5 minutes. 
(5) Remove the sediment from the bottom of the tube 
with a capillary pipette and make a cover-slip preparation. 
Four distinct layers result; First,the ether layer, 
which may be used for an occult blood test; second, the detritus plug, 
composed of layers of bilo, soaps, and protein material; third, the 
acetic acid solution; and fourth, the sediment which will contain the 
cysts or ova. 
In the course of a routine stool examination, one should 
make at least four direct preparations to be examined with and 
without iodine stain, and in addition, one of the concentration methods 
should be performed and the sediment viewed direct and with iodine 
stain, 
C, Special Staining Methods 
For routine purposes the temporary iodine stains are usually 
sufficient. For some diagnostic work and. for permanent mounts, it is 
desirable to use a stain which clearly defines the cell structures. 
The standard stains for such work contain the dye hematoxylin. 
The fcccs to be stained may be smeared, or stained in bulk. 
To prepare a smear for demonstration of trophozoites, a bit of tho 
fcccs is spread on a cover glass. The specimen is usually sufficiently 
fluid so that the preparation of a saline suspension is unnecessary. 
The cover-slip is then immediately dropped, smear down, in o. watch glass 
of Carnoyfs or Schaudinn’s fixative. The fixative is allowed to act 
for 20 to 30 minutes. If tho smear has been made properly on a 
perfectly clean cover glass, there will be little loss of the tropho- 
zoites during fixation. 
Solid or semi-solid specimens may bo emulsified in saline, 
smeared on cover glasses and treated as above. If difficulty is 
experienced in making tho trophozoite material adhere to tho cover 
glass, it may be smeared on a glass slide which has been rubbed with 
egg albumin or horse serum, A cover glass is then applied and tho smear 
placed in an empty Coplin jar. The fixative is slowly poured into tho 
jar. The smear is allowed to remain in the fixative from 20 to 30 
minutes. The slide is then removed and the cover glass is taken off. 
After fixation the smears arc stained according to ono of the 
methods given below; 
1, Technic for Hcidcnhain’s Iron-Hematoxylin Stain a. Remove smears from fixative and rinse. 
b. Mordant in 5% aqueous iron alum for at least 4-5 minutes, 
c. Rinse in water, 
d. Stain in Heidonhain’s Iron Hematoxylin for at least 
4.5 minutes, 
e. Rinse in water, 
f. Differentiate in 2 to 5% iron alum, controlling the 
degree of definition with the microscope, 
g. Wash in running water for 2 to 5 minutes, 
h. Dehydrate in ascending grades of alcohol, (50%>, 
70%, 95%, absolute), 
i. Clear in xylol, 
j. Mount in Canada balsam. 
Since this stain must be used rcgrossivcly, i,c,, the 
material is grossly overstained and then differentiated, its success 
depends entirely on accurate differentiation, A common error is to 
fail to carry the process of differentiation far enough. A 2% solution 
of iron alum extracts the stain more slowly, and is most convenient 
for the beginner. 
If the smears are accidentally over-differentiated, they 
may bo replaced in the hematoxylin bath until they are jet black, then 
rodifforentiated, 
Iron hematoxylin is a permanent stain. It never fades, 
provided the iron alum is properly rinsed out of the smear after 
differentiation. A large number of counterstains may be used with the 
hematoxylin stain, 
*Usc 90% alcohol in several changes after Carnoy's, 
If Schaudinn’s or any other fixative containing mercuric chloride is 
used, it is necessary to remove the deposit of mercuric chloride before 
staining. This is done in the following manner: 
(1) Immerse in 70$ alcohol for 10 minutes, 
(2) Immerse in 70% alcohol to vdiich enough iodine 
stain has been added to impart a mahogany color for 10 minutes, 
(3) Immerse in 70$ alcohol for 10 minutes. 2. Tochnic for Harris* Hematoxylin Stain 
a. Remove fixative and rinso in tho propor medium. 
b. Stain in Harris’ Hematoxylin for at least 45 minutes. 
c. Wash in tap water* 
d. Destain in acid alcohol (1% HC1 in 70% alcohol) 
Check the degree of differentiation with tho microscope. 
e. When the desired definition has boon reached, transfer 
to dilute ammonia water (5 drops of ammonium hydroxide in 50 cc, of 
water). Allow to neutralize until the smear is completely blue, 
f. Dehydrate in ascending grades of alcohol, 
g. Clear in xylol and mount in balsam, 
3. A Method for a Rapid Hematoxylin Stain 
This method is especially useful in diagnostic work, 
whore permanence is not required, 
a. Make a smear of the feces. 
b. Fix while wet, and rinse according to the fixative, 
c. Mordant for 2 to 3 minutes in 5% iron alum at 56 
degrees Centigrade, 
d. Rinse in water, 
e. Stain in Hematoxylin for 1 to 2 minutes at 56°C, 
f. Rinse in viator and allow to stand until blue-black. 
g. Dehydrate in 95% alcohol 30 seconds to 1 minute, 
followed by absolute alcohol or acetone for 1 minute, 
h. Clear in xylol and mount in balsam. 
Tho Hematoxylin used in this staining method Is made by 
adding 0,4 cc. of 10% alcoholic solution of Hematoxylin and 0,8 cc, of 
glacial acetic acid to 40 cc. of distilled water. Many workers prefer to stain protozoan material in bulk. 
This method overcomes certain difficulties usually encountered when 
smears are used: First, keeping the organisms on the .slide or cover 
glass during fixation; second, keeping the organisms free from 
distortion and clear from debris so that the internal structures are 
not obscured; third, carrying out proper differentiation of internal 
structures of the organisms on the slide at the time of differentiation; 
fourth, having a sufficient number of well stained organisms on the 
slide after staining so that tho diagnosis will not have to be made 
on a few more or loss atypical organisms. The bulk staining method 
also affords a method of concentration while staining. Very few 
organisms are lost, and distortion is hold to a minimum, 
4. Bulk Staining with Heidcnhain’s Iron-Hematoxylin 
a. Fix with Carnoy’s in 50 cc. centrifuge tubes for at 
least 30 minutes, 
b. Decant tho supernatant, (Centrifuging between stops 
is necessary only when Washing, or after differentiation), 
c. Wash ¥dth 9C$ alcohol, several changes, 
d. Wash with 70% alcohol, 
e. Wash with 50% alcohol, 
f. Stain with Hoidenhain's Iron-Hematoxylins 
(1) Mordant in 5%> iron alum for 45 minutes or more, 
(2) Rinse in water, 
(3) Stain in Hematoxylin for 45 minutes or more, 
(4) Rinse in water, 
(5) Differentiate in 5% iron alum, 
(6) Wash with several changes of water, 
g. Dehydrate in ascending grades of alcohol, 
h. Clear in xylol, 
i. Add a balsam-xylol mixture. 
The degree of differentiation is controlled by making a 
cover glass preparation of the material at intervals while it is do- 
staining, and examining with tho microscope. Harris’ Hematoxylin may bo used, following the same 
general principles used with Keidonhain'st 
a. Fix and rinse., 
!• 
b. Stain in Harris’ Hematoxylin for /+ 5 minutes or more, 
c. VJash in tap viator, 
d. Destain in 1% HC1 in 70% alcohol, checking continually 
viith the microscope to control the degree of differentiation. 
e. Neutralize with dilute ammonium hydroxide, 
f. Dehydrate in ascending grades of alcohol, 
g. Clear in xylol, 
h. Add a. balsam-xylol mixture. 
D, Culture Methods 
1, Eoock-Drbohlav Medium. (Dobell and Laidlaw’s Modification) 
Four eggs arc carefully washed, brushed with alcohol, and 
broken into a sterile flask containing glass beads. 50 cc, of sterile 
Ringer’s solution are added and the mixture thoroughly shaken until 
a homogenous suspension is secured. The mixture is tubed in U cc. lots, 
or enough to produce a slant of about 1 to 1 l/2 inches when coagulated 
by heat. The tubes arc then slanted and placed in an inspissator and 
kept at 70°C,, until the slants are solidified. The tubes are then 
autoclaved at 15 pounds pressure for 20 minutes. After autoclaving, 
the slants arc covered to a depth of about 1 cm, with a mixture of 8 
parts of sterile Ringer's Solution and 1 part of sterile inactivated 
human blood serum. To insure sterility, the mixture of Ringer's 
Solution and blood serum should be passed through a Berkfcld filter 
and incubated at 37°C, for at least 24- hours before it is used. A 
bit of sterile rice powder should be added to each tube when culturing 
the amoobas. 
The tubes arc inoculated with an applicator or wire loop, 
selecting a pea-sized portion of the specimen containing fresh mucus 
or mucus and bloodj a bit of concentrate containing cysts may also bo 
used. The culture is incubated at 37°C. raid examined at 2% hours and 
at 4.8 hours. Flagellates will bo found throughout the fluid portion 
of the media. Amoebas will bo found at the base of the fluid portion, 
A sterile 1 cc. pipette is used to withdraw 0.1 cc. of the 
fluid for examination. Routine cultures are transferred every 4-B 
hours. If a new culture is not positive after 4-S hours, all but 
0.5 cc. of.the fluid portion is removed; the slant is washed with 
the remaining fluid, and transferred to a new tube of media. The 
resulting culture should be examined at 2k and 4-8 hours before being 
pronounced negative. 
The amoebas are quite sensitive to the presence of 
certain species of bacteria, especially Pseudomonas aeruginosa and 
Proteus vulgaris, and die readily in their presence. Other organisms, 
particularly Sscherechia coli are beneficial in the culture. It is 
essential that aseptic technic be followed in preparing the culture 
media, 
II, LABORATORY METHODS FOR BLOOD AND TISSUE PROTOZOA 
A, Methods for Examination for Malaria 
1. Fresh, Unstained Blood 
With practice, the malaria parasites may be detected and 
diagnosed from the study of a fresh specimen. A cover glass is touched 
to a drop of blood, and placed on a slide. The preparation is then 
examined with the 1,8 mm. objective. Movement of the pigment granules 
within the parasite may be detected without the use of a warm stage 
in the tropics. Such examinations require the use of subdued light. 
If the preparation is to be studied for any length of time, the cover 
glass should bo ringed with vaseline. 
2. Thin Smear Method 
Blood smears are prepared as for leukocyte counts, except 
that they are spread more thinly to provide an undistorted picture of 
the erythrocytes. The smears are stained as for'differential leucocyte 
counts. The nuclei of the leukocytes must be heavily stained if the 
parasites are to bo well defined, 
3. Thick Smear Method 
a. Slides for this purpose must absolutely bo clean and 
free from grease, 
b. Four small drops of blood are placed on a slide so 
that each drop marks the corner of a square which could be covered by 
a dime. The drops are coalesced with an applicator or a toothpick. 
Some workers use a single large drop, which is then spread over the same 
area as that obtained in the four-drop method. If too much blood is 
used and the resulting smear is too thick, the blood film will crack 
and pool from the slide. If too little blood is employed, the parasites 
will be, too sparse. The exact amount of blood to bo used is easily 
determined after short experience. c. The smear is allowed to dry long enough to make it 
adhere, but not long enough to prevent clear staining of the para- 
sites. 1 l/2 hours in an incubator at 37°C. is usually a sufficient 
time. The smears should be protected from dust and flies while 
they are drying, 
d. Tifhcn film is quite dry it is ready for staining; 
Proceed as follows: 
(1) Dohemoglobinize by flooding slide with a ( 
solution of 0,1% magnesium sulfate in distilled water. Allow to stand 
5 to 10 minutes. 
Note: an alternate solution that can be used 
for dehcmoglobinizing and fixing where more permanent mounts are 
desired, is the following: 
Formalin 5 cc 
Acetic acid 1 cc 
Distilled water qs ad 100 cc 
Allow slide to remain in this solution 10 
minutes (thick film only). 
(2) Flush by pouring tapwater carefully over the 
slide from one end, out of a measuring glass, A white streak con- 
sisting of leukocytes, platelets and the stroma of erythrocytes should 
be left, 
(3) Drain tapwater from slide and allow to dry while 
resting obliquely against some object, 
(A) When slide is quite dry, it may be laid hori- 
zontally and flooded with a mixture of 2 drops Giemsa’s Stain in 2 cc. 
of water. (Water used here should have a pH of about 7. If this is 
not available, use a l/lOOO solution of magnesium sulfate in distilled 
?jater). 
(5) Allow stain to remain on slide for 20 to A0 
minutes or more, as indicated by previous staining experience, 
(6) Flush by pouring water carefully over the slide 
from one end, taking care not to allow metallic looking film floating 
on the stain to come in contact with bloodfilm, 
(7) Dry and examine. (Slide should never be dried 
with artificial heat or by placing in sun), 
The slide should not be loft in the stain for 
less than 20 minutes. If after this period, the slide is too strongly 
colored, this may be taken as an indication for further dilution of 
the stain on the next occasion. If the thick drop is properly stained, the 
microscope reveals a colorless or slightly yellowish background, not 
blue. Platelets will appear red-violet; leukocytes will be strongly 
colored; stroma of erythrocytes should be visible as a blue network. 
Malaria parasites will have a red nucleus and 
dark blue cytoplasm. Where present, Schuffner’s or Maurer’s dots 
should bo visible. Films that are stained too lightly do not show 
these dots and the cytoplasm will be a pale blue. When staining is 
too strong the parasites are shriveled and may be covered with red 
streaks (fibrin) and blue coloring matter. Films that are old or have 
been subjected to the action of light ¥/ill be more or less fixed and 
will be unusuable, 
e. The thick smear may be combined with the thin smear 
on the same slide. In this case, the thin smear is immersed in methyl 
alcohol for fixation, but is not dehemoglobinized, and both smears 
arc stained with Giemsa’s, The thick smear may be used to determine 
the presence of the parasites, and their morphology may be studied 
in the thin smear, 
U* Barber and Komp Method for Handling Largo Numbers of 
Thick Films in a Malaria Survey 
“In handling large numbers of thick smears, it is convenient 
to carry out the technique in groups of 25 slides. With this in mind, 
the thick film is placed about 1 inch from one end of the slide and the 
other is used for labeling. The slides are assembled in groups, a 
cardboard one sixteenth to one eighth of an inch thick and 1 1/2 inches 
long, is inserted between the slides at the labeled ends and the whole 
fastened together by moans of a stout rubber band. The entire block 
may now bo stained and dried as a single unit. 
The combined thick and thin smears for staining are prepared 
by making a thick smear on one end of the slide and a thin smear 
starting l/2 inch from the thick smear,and then streaking it toward the 
opposite end of the slide. Draw a line with a wax pencil between the 
two smears and they are now ready for staining. Proceed as for a thick 
smear but bo careful to immerse only the thick smear in the acidulated 
formaldehyde solution. If the thin smear comes in contact with this 
solution, the rod cells will bo dissolved out and the smear will be 
useless. 
Failure to stain by Wright’s method is usually duo to 
insufficient lapse of time after diluting the stain with distilled water, 
or to contamination of the stain, or other reagents, or material, with 
acid. The precipitation of granules of stain on the blood film is 
either duo to improper drying of blood films before starting the stain, introduction of water into the stock stain, or too much evaporation 
of the alcoholic stain before dilution. Rod cells stained bluo, 
except for the occasional cells showing polychromatophilia, are 
cither overstained (too much time allowed after diluting the stain), 
or have been insufficiently washed during the last stage of the 
staining process,'* 
5. Concentration by the Method of Bass and Johns 
The equipment required, length, and difficulty of this 
procedure do not make it desirable for routine work. It is valuable 
as a check on therapy, and will uncover cases not easily demonstrated 
by the preceding methods. It utilizes the principle that the 
parasitized erythrocytes arc lighter than the normal colls. Best 
results arc obtained with estivo-autumnal crescents and with the adult 
stages of the other species, 
a. Draw venous blood and place it in a tube containing 
the proper amount of dry potassium oxalate. (2 mg. per cc, of blood) 
b. Centrifuge at 2500 rpm for 5 minutes, 
c. Withdraw most of the plasma with a capillary pipette, 
and place it in a small tube, 
d. Carefully skim off the leukocyte layer and the upper 
layer of erythrocytes and place them in a tube 12 cm, by 1 cm., inside 
measurements. Add an equal volume of plasma, 
0. Mix and centrifuge as before, 
f. Draw off the leukocyte layer and the upper erythrocyte 
layer into a long capillary pipette of about 3 to 4 mm, bore. Mix 
by forcing in and out on a slide. Finally draw the well-mixed upper 
cell layer into the pipette, and seal the tip in a flame, Nick with a 
file and break above the blood column, 
g. Place the tube thus formed in a centrifuge tube, pack 
with cotton, and centrifuge as before, 
h. Nick with a file and break 1 to 2 mm. below the bottom 
of the leukocyte layer, 
1. From the upper section of the tube, with a capillary 
pipette small enough to enter the bore of the capillary tube, remove 
the small amount of red cells together with a little plasma and tho 
leukocyte layer, 
j. Mix well, smear and stain with Wright's or Giomsa's. B, Methods of Examination for Trypanosomes 
1. Fresh Preparations: the organisms are best demonstrated 
by the use of darkfield illumination. The living parasites actively 
displace the surrounding red cells, 
2. Thin Films: the films are prepared and stained in the 
standard manner. 
3. Thick Films: it is possible to employ thick films 
prepared as for malaria, but considerable distortion of the organisms 
occurs, 
A. Concentration Method: blood may be concentrated by 
centrifuging 10 cc, of oxalated blood and making smears of the leukocyte 
layer. The smears are stained with Wright's or Giemsa's. 
5. Lymph Node Aspiration: Smears may be made from lymph 
node fluid obtained by aspiration and stained with Wright's or Giemsa's. 
This method is used when examination of the peripheral blood is 
negative and often demonstrates the organisms in the early stages of 
the disease, 
6. Spinal Fluid: spinal fluid is obtained by lumbar puncture 
and centrifuged for 15 minutes. Smears are made of the sediment and 
stained with Wright's or Giemsa's. This method is useful in the 
later phases of sleeping sickness when other methods arc negative, but 
should not bo expected to demonstrate organisms before the encephalitic 
stage is well developed, 
7. Animal Inoculation: white rats are injected intra- 
pcritoneally with 1 cc, of blood or tissue juice. Daily blood 
examinations are made. In positive cases, the trypanosomes will appear 
in the blood of the animal between the third and fourteenth days and 
remain quite constantly, 
C. Diagnostic Methods for the. Loishmanias 
1, Spleen and Liver Punctures: material should be obtained 
only by an experienced medical officer. It is spread on a slide in a 
thin layer and stained with Wright's or Giemsa's, and examined v/ith 
the 1,8 mm. objective. In positive cases, Lcishman-Donovan bodies 
will be found within the rcticulo-ondothclial colls, 
2, Peripheral Blood: Lcishman-Donovan bodies arc found in 
the blood in only about 20% of cases. They will be found, if present, 
within the monocytes or occasionally within the polymorphonuclear 
leukocytes. 3. From Ulcerations: in cases of Oriental Sore, the 
Loishman-Donovan bodies may be demonstrated in endothelial cells 
obtained from scrapings from the ulcer margins. 
A. Culture: 
NNN Medium (Novy - MacNcal - Nicollc Medium) 
Agar 1A gm. 
Sodium chloride 6 gm. 
Distilled Water 900 cc. 
Mix and dissolve by means of heat. Tube in 6 cc, amounts. 
Autoclave 30 minutes at 20 lb, pressure. Remove and cool to A8°C, 
Add asoptically 2 cc. of sterile defibrinated rabbit’s blood to each 
tube, mix well, and slant. The tubes should be cooled in the ice box 
to produce a maximum amount of water of condensation. They should be 
capped Y/ith rubber stoppers to prevent excess evaporation of this 
water of condensation. Incubate for 2A hours to test sterility. 
Inoculate into the wator of condensation and incubate at 22 to 25°C. 
for 3 to 1A days. In cultures, the Lcishmania will be found in the 
Loptomonas or flagellated stage, 
III. EXAMINATIONS FOR HELMINTHS 
The ova of the intestinal helminths may be recovered by direct 
examination of a saline emulsion of the stool, or by one of the methods 
of concentration discussed under the topic “Methods for Intestinal 
Protozoa”, The life cycle of the parasitic helminths must be con- 
sidered Y;hon selecting the proper specimen for examination. Thus, 
rust colored flecks in the sputum would be examined for the ova of 
Paragonimus westermanii, and urine for the ova of Schistosoma hematobium, 
A, Examination for Filaria 
Diagnosis of Filaria bancrofti, Loa loa, and Filaria perstans 
is established by examining the peripheral blood as follows: 
1, Direct Method: a drop of blood obtained at the appropriate 
hour is covered with a cover glass and examined under low power for 
microfilaria. They may be easily detected by following the disturbance 
of the surrounding rod cells, 
2, Concentration Methods 
a, 1 cc. of blood is added to 2 cc. of a 2% solution of 
acetic acid. It is mixed well, centrifuged, and the sediment spread 
on a slide, A cover slip is placed over the wet smear, and the 
preparation is examined under low power. b. 20 drops of blood aro added to 10 cc. of physiological 
saline plus a few drops of a 10% solution of saponin. After hemolysis, 
the mixture is centrifuged and the sediment examined for living 
microfilaria. 
c, A solution of % formalin, 5 parts, and saturated 
alcoholic gentian violet, 2 parts is added to the blood. The mixture 
is then centrifuged, the supernatant discarded, and the residue covered 
with water. It is then rccentrifugod, and the sediment examined for 
microfilaria, 
B. Examination for Strongyloidcs stcrccralis' 
* L 
In the identification of the rhabditiform larvae of Strongy- 
loides it is a good policy to base the diagnosis on living forms, as 
certain vegetable spines and hairs are sometimes confused with the 
larvae. Usually the larvae may bo found in a direct fecal film, but, 1 
concentration methods give a much richer yield. In a solid stool 
specimen the larvae may bo found by making a small depression in the 
focal mass, filling it with water, and keeping it in a warm place for' 
12 to 2A hours. The larvae collect in the water. Some mca.ns of 
keeping the stool moist must be provided, 
C, Examination for Entorcbius vermicularis 
The ova of Entorcbius vorndcularis aro not normally recovered 
in abundance from the feces. Swabbing or mildly scraping the perianal 
regions yields a larger number of eggs. Adults may be captured 
follovdng enemas, or females may bo recovered during their normal 
nocturnal migration from the bowel. 
The NIH swab is most used in the recovery of the 
ova of Entorcbius vorndcularis. It is prepared as follows: 
1. Fix a glass rod (A rnm.) in a rubber stopper in such a 
manner that it will reach almost to the bottom of a 15 x 85 mm, test 
tube when the stopper (No, 00, onc-holo) is fitted in place. A short 
length of the rod should protrude from the top of the stepper, 
2. Fasten a piece of cellophane 1 inch square over the tip 
cf the glass red with a rubber band made from rubber tubing' of the 
appropriate diameter, 
3. Use of the NIH swab:. 
Swabbing should bo done in the morning before the patient 
has bathed or defecated. The dry cellophane-covered tip is stroked 
firmly with an outward motion ever the perianal folds and across the 
anal opening. The cellophane is released by sliding the rubber band 
towards the stopper. The square is mounted in water on a glass slide 
and examination is made for the characteristic ova.. Defects in the 
cellophane resembling the ova in outline should be recognized, as they 
arc confusing and may lead to error. D. Examination for Tapeworm Proglottids 
When the segments of tapeworms arc found in the stool, it 
is necessary to determine whether they arc from the beef or perk 
tapeworms. This is done as follows: 
1, Clean and relax the segments by shaking them in 
physiological saline, 
2, Press the specimen between two glass slides, 
3, Count the lateral branches of the uterus by holding the 
segment up tc a strong light. The branches should be counted at their 
bases since there is some subdivision at the distal point, 
IV. FORMULAE FOR REAGENTS USED IN PARASITOLOGICAL LABORATORY METHODS 
A. Lugol’s Iodine Stain 
Iodine 1 gm. 
Potassium Iodide 2 gm. 
Distilled Water 100 cc, 
B. Kofcid’s Ecsin-Icdinc Stain 
Ecsin saturated in saline 2 parts 
Iodine solution* 1 part 
Physiological saline 2 parts 
C. D!Antoni’s Standardized Iodine Solution 
A standardized solution of potassium iodide is prepared by 
the specific gravity method: 
1. Place 100 gm, of Merck’s or Baker’s potassium iodide in 
a chemically clean 1000 cc. volumetric flask. Add distilled water 
tc the mark. 
2. Yveigh a clean, dry 25 cc. volumetric flask to the 4-th 
decimal place, 
3. Fill the 25 cc, volumetric flask tc the mark with the 
potassium iodide solution and weigh tc the 4-th decima.1 place, 
« 
*Physiclogical saline 100 cc. 
Potassium iodide 5 gm. 
Iodine crystals tc saturation A. Subtract the weights in H2“ and 113U tc obtain the weight 
of the 25 cc. of potassium iodide solution. Theoretically this should 
be 26.925 gm. The actual weight will bo slightly less than this, due 
to the deliquescence of the potassium iodide. 
5. The difference in weight is divided by the theoretical 
weight, and the quotient expressed in terms of percentage. This 
quotient is subtracted from 10 (the percentage desired) to give the 
actual percentage of the solution. 
6. The following proportion may then be set up: 
100 « x . where x z grams of potassium iodide. 
Actual % of soln. 10$ 
7. 100 subtracted from the number of grams obtained from x 
gives the number of grams of potassium iodide to be added to the above 
solution to give a standardized 10$ potassium iodide solution. 
Tho staining solution is prepared by adding 1.5 grams of 
powdered iodine crystals tc 100 cc. of a 1$ potassium iodide solution 
which is obtained from the standardized 10$ solution. The resulting 
solution is allowed to stand for L, days and is then ready for use. It 
must be filtered before using, and should not be allowed to remain 
unsteppered, as volatilization of the iodine will occur. The stock 
solution keeps for long periods of time without deterioration# 
D. Vital Stain 
This stain is based on the 1$ aqueous neutral rod solution 
recommended by Stitt, 
It has been found that when the neutral red is prepared in 
physiological saline in a concentration of 1$, and 6 drops of a saturated 
solution of Janus green in physiological saline are added to each 5 cc, 
of the neutral red solution, an efficient vital stain for protozoa is 
produced, 
E. Heidenhain*s Iron Hematoxylin Stain 
1, The Alum Bath. (Used as mordant and differentiator). 
Iron and ammonia alum (NH/)?.Feo(S0.), ,2A Ho0) 5 gm. 
Distilled water * 100 cc. 
2, The Hematoxylin Bath 
Hematoxylin .5 gm, 
96$ or absolute alcohol 10 cc. 
Distilled water 90 cc. A good brand of hematoxylin is essential* Dissolve it 
first in the alcohol; then add the water. The solution takes a few 
weeks to ripen. Best results are obtained by placing in a stoppered 
flask and exposing it daily to the sunlight. Frequent agitation 
hastens the ageing, 
F, Harris’ Hematoxylin 
Hematoxylin 1 gm, 
ethyl alcohol 10 cc. 
Dissolve 
Alum (ammonium or potassium) 20 gm. 
Distilled Water 200 cc. 
Dissolve the alum in the water with the aid of heat and add 
the hematoxylin solution. Bring the mixture to a boil as rapidly as 
possible and add 0,5 gm, of yellow oxide of mercury. The solution at 
once becomes a dark purple. As soon as this occurs, remove the vessel 
from the flame and cool rapidly by plunging into a basin of ice water. 
As soon as the solution is cool it is ready for use. The addition to 
the solution of glacial acetic acid in a concentration of U% brings out 
the nuclear components more clearly, 
G, Schaudinn’s Fixative 
Mercuric chloride, cone, aq, soln, 2 parts 
Absolute alcohol 1 part 
H, Carnoy’s Fixative 
Absolute alcohol 6 parts 
Chloroform 3 parts 
Glacial acetic acid 1 part 
I, Ringer’s Solution 
Sodium chloride 9.0 gm. 
Calcium chloride 0,2 gm. 
Potassium chloride 0.2 gm. 
Distilled water 1000 cc. SECTION IV 
SOME ARTHROPOD VECTORS OF DISEASE SOLE ARTHROPOD VECTORS OF DISEASES OF MAN 
1 
VECTOR 
ETIOLOGICAL AGENT 
DISEASE 
METHOD .OF INFECTION 
Cyclops 
(Guinea worm) Dracun- 
culus medinensis 
Oral (water 
D iphyllobothrium 
latum 
Oral (Thru fish as 2d 
intermediate host) ...... 
Crabs 
Paragonimus westermanii 
Oral . 
Body Louse 
(Pediculus 
humanus) 
Eorrelia recurrentis 
Relapsing fever 
Contamination of bite 
Rickettsia quintana 
Trench fever 
Contamination of Mta... 
Rickettsia prowazeki 
Typhus fever 
Contamination of_hite_ 
Eat Flea 
(Zenopsylla 
cheopsis) 
Pasteurella pestis 
Bubonic plague 
Contamination of bite 
Rickettsia prowazeki 
Typhus fever . 
Contamination of bite 
Kissing Bug 
(Triatoma) 
Trypanosoma cruzi 
Chagas1 
disease 
Contamination of bite— 
Mosquitoes 
Anopheles 
(70 species) 
Anopheles 
(22 species) 
Aedes (18 
species) 
Aedes aegypti 
Aedes (8 
species) 
Culex (5 
species) 
P,falciparum, P. 
malarias. P. vivax 
Malaria 
Bite 
Filaria bancrofti 
Filariasis 
Invasion of bite 
Virus of yellow fever 
Yellow fever 
Bite 
Virus of Dengue 
Dengue fever 
Bite 
Filaria bancrofti 
Filariasis 
Invasion of Bite 
Filaria bancrofti 
Filariasis 
Invasion 
Sand Fly 
(Phlebotomus) 
Virus of Pappataci 
fever 
Pappataci 
fever 
Bite 
Lcishmania tropica 
Oriental Sore 
? 
Leishmania donovani 
Kala Azar 
? 
Leishmania brazilionsis 
Esoundia 
? 
Tsetse Fly 
(Glossina) 
Trypanosoma gambiense 
African Sleep- 
sing Sickness. 
Lite 
Trypanosoma rhodosi oner 
Bite 
Ticks 
(Dermacentor) 
Pasteurella tularonsis 
Tularemia 
Bite 
Rickettsia rickcttsi 
Rocky Mountain 
Spotted Fever 
...Bite — 
M’ites 
(TrombigulaJ 
Rickettsia japonica 
Tsutsugamushi 
Fever 
Bite _ . Fig, 1. Crayfish ( a crustacean) 
Fig, 2. Myriapods. A, Centipede; B, Millipede. Fig. 3. Comparison of scorpion and 
whip-scorpion. 
A, Scorpion; B, Whip-Scorpion. 
Fig, 4-. Black widow spider 
( an arachnid). F19 5 !»cb*nrtite of mon 
Pig,6. Ticks A, Soft-bodied tick; 
B, Hord-bodied tick Fig,7. Insects. A, Cockroach (Orthoptera): 
B, Beetle (Colenptera); C, Moth 
( Lepidoptera ); D« Ant (Kvmenoptera). Fig,8. Lice (Anoplura), A, Body louse; 
B, Crab louse. 
Fig,9* Flies, A, Larva of house-fly; 
B, Pupa of house-fly. GEOGRAPHICAL DISTRIBUTION OF SOME ANOPHELINE 
VECTORS OF MALARIA 
NORTH 
AMERICA 
CENTRAL AND SOUTH 
AMERICA AND"WEST 
INDIES 
EUROPE 
AFRICA 
ASIA AND 
EAST 
INDIES,. 
OCEANIA AND 
AUSTRALIA 
A. cruciens 
A. crucians 
A. 
hyrcanus 
A. 
umbrosus 
A. 
hyrcanus 
A. annulipes 
A. maculi- 
pennis 
A, pseudopuncti- 
pennis 
A.maculi- 
pennis 
A, rnaculi- 
pennis 
A.maculi- 
pennis 
A. punctulatus 
A.puncti- 
pcnnis 
A, apicimacula 
A.His- 
panic la 
A. 
funestus 
A. 
umbrosus 
A. bancrofti 
A.pseudo- 
puncti- 
pcnnis 
A. intermodius 
A.super- 
pictus 
A.gambiae 
A. barbi- 
rostris 
A, subpictus 
A. quadri- 
raaculatus 
A. psoudornaculipes 
A. His- 
paniola 
A. culici- 
. facies 
A. punctimacula 
A, phar- 
aensis 
A. flu- 
viatilis 
A* albimanus 
A, super- 
pictus 
A. macu- 
latus 
A. albitarsis 
A. minimus 
A, argyritarsis 
A, phil- 
ippinensis 
A. darlingi 
A.stephensi 
A, tarsimaculatus 
1A. super- 
|pictus 
A. gambiae (imported 
♦ 
i 
, ! DIFFERENTIATION OF MOSQUITOES 
ANOPHELES 
CULEX 
AEDES 
Adult 
Antennae 
Adult 
of males are busy, those 
less branched 
Adult 
of females 
Palpi of female as long 
as proboscis 
Palpi of female short 
Palpi of female short 
Palpi of male long and 
spatulate 
Palpi of male are long 
Palpi of male longer 
than proboscis 
Wings spotted 
Plain wings 
Plain wings 
Scutellum arculate 
Scutellum trilobate 
Scutellum trilobate 
Rests at 50 to 80 degree 
angle with the surface 
Rests in a plane 
horizontal to the 
surface 
Rests in a plane 
horizontal to the 
surface 
Body parts in a straight 
line 
Head and proboscis form 
an angle with the line 
of the abdomen and 
thorax 
Head a.nd proboscis 
form an angle with the 
line of the abdomen 
and thorax 
Eggs 
Individual, boat-shaped, 
with side floats 
Eggs 
Aggregated into a 
floating raft 
Eggs 
Individual,surrounded 
by an air chamber 
Larvae 
No distinct respiratory 
siphon 
Larvae 
Distinct respiratory 
siphon 
Larvae 
Short respiratory 
siphon 
Horizontal position in 
water 
Rests at an angle 
with the surface of 
the water 
Rests almost vertically 
in the water Palpi 
Palpi Palpi 
Palpi 
Male Female 
Male Female 
Fig. 10. Mosquitoes. Comparison of various stages of anopheline 
and colicine mosquitoes. 1, Anopheles. A, eggs; B, larva; 
C, pupa; D, adult; E, wing of adult; F, mouthparts of adult 
male and female, 
2. Aedes. A, eggs; B, larva; C, pupa; D, adult; E, wing of 
adult; F, mouthparts of adult male and female. 
3. Gules, A, typical raft of eggs. F:g* 11, wings #f Anopheles mosquitoes. A, A.crucIans; 
B, A.punctipennls; C, A.maculipennis; 
D, A, qiiadrlmaculatus: E, A. pseudepunctipennis; 
F; A, albimanus. Stiga or 
breathing pore 
A, quadrimaculatus 
Palmate hairs 
Dorsal view of an Anopheline Larva 
A. albimanus 
Ventral and Dorsal views of an Anopheline Larva 
A. punctipennis 
A. pseudopunclipenni* 
Hypopygium of male Anopheles albinaus and 
A* punctipennis 
Fig. 12. Anopheles mosquitoes* Structural characteristics of 
larva. Hypopygia of males. Fig. 13. Flies. A, Crane-fly; B, Horse-fly, 
Fig. U. Fly mouthparts. A, Head of stable-fly showing biting 
mouthparts; B, Head of house-fly showing non-biting 
mouthparts. Fig,15. Stigmal plates of fly larva. A, Blow-fly (Calljphora); 
•B, Green-bottle fly (Lucilia); C, Blue-bottle fly (Cynomyia); 
D, Screw-worm fly (Cochliomyia); E, Bot fly (Gastercphilus): 
F, Warble fly (Dermatobia); G, Flesh fly (Sarcophaga); 
H, BJack blow fly frfoormia); 1, Biting stable fly (Stomoxys); 
J, Non-biting Stable fly (Musclna): K, Flesh fly(Wohlfahrtia)? 
L, House fly (Ivlusca): M, Cattle bot fly (Hypoderma);^ 
W, Sheep bot fly (bestris). Fig. 16. Bedbug (Heterpptera). 
Fig. 17. Kissing-bug (Heteropteral. Fig. IS. Fleas (Slphonaptera). A, Human flea (Pulex irritans); 
B, Dog flea (Ctenocephalus cunis); C, Chicken flea(Echidnophaga. 
galling.cea): D, Temperate zone rat flea (Geratophyllus fcsciatus): 
E, Tropical rat flea (Xenopsylla cheopig); F, Heads of human 
flea (left ) and tropical rat flea (right) showing arrangement 
of stout bristles in relation to eyes, (Note that in humrn flea j 
a stout bristle is directly below the* eye, whereas in the tropi- 
cal rat flea it is. in front of the eye.) INSTRUCTIONS FOR PREPARATION AND SHIPMENT 
OF ENTOMOLOGICAL SPECIMENS 
AS DIRECTED BY BTH SERVICE COMMAND LABORATORY 
FORT SAI.I HOUSTON, TEXAS 
1. General - One function of the Service Command Medical 
Laboratory is to accomplish procedures for which a local laboratory 
is not equipped. One of the phases of laboratory work in which it can 
be of especial value to the local installations is in the identification 
of insects and other arthropods which nay be of medical importance. 
The responsibility of the surgeon of each station and command, with 
regard to control of mosquitoes (AR 4-0-205, paragraph 21), includes the 
investigation of the character of the mosquito population. In this, 
and also in connection with problems pertaining to other arthropod 
pests (AR 4-0-205, paragraphs 22-27 inclusive), the Service Command 
Laboratory is prepared to assist by making identifications of the 
organisms involved. 
In order that satisfactory reports may be made on samples submitted, 
co-operation is essential, particularly when specimens must be sent 
considerable distances. Every effort will be made by this laboratory 
to make identification of any material sent in, but it is believed that 
attention to the following instructions will eliminate the bulk of 
unsatisfactory, unidentifiable specimens. 
Improper packing, careless initial handling of specimens, and lack 
of labeling are the most usual causes for complaint. By observing only 
a few precautions, these difficulties can be largely eliminated. 
2. Preparation and Shipment of Mosquito Specimens, 
a. Adult Mosquitoes. 
(l) Hand Collections - Hand-caught adult mosquitoes 
captured by devices such as chloroform tubes, aspirators, 
(Figures 3 and 4-), or insect nets may be killed by 
exposure to chloroform vapour for five minutes. The 
period of exposure to strong chloroform vapour should 
be a full five minutes, inasmuch as those exposed too 
briefly may be merely stupefied. The insects may then 
bo placed in pill boxes or other suitable containers 
between layers of cellucotton, crumpled cleansing 
tissue, or other soft packing materials (Figures 1 and 2), 
Cotton should never be used, for the loose, fluffy 
fibers become badly entangled with insect appendages and 
scales. Too much packing material should not be used - 
a small amount crumpled into place, and enough to pre- 
vent movement of specimens will suffice. Mosquitoes 
must not be packed in too tightly; each specimen should be well separated from the next. However, the insects 
may be packed in layers to increase the capacity of 
the container. Usually 25 to 50 specimens may be 
considered as the maximum to be packed in a box. The 
characters necessary for the proper identification of 
mosquitoes consists of minute scales, hairs and 
appendages which are readily broken or scraped off by 
rough handling; it is therefore desirable to use light 
forceps in transferring specimens. If no forceps are 
available, do not use fingers, but slide specimens into 
the box from the original container used in capture 
or from a piece of paper on which they have been 
deposited. Once a batch of specimens has been packed, 
the box should not be reopened, for the mosquitoes 
become dry and brittle in a few hours and break with 
the slightest movement. (At the receiving laboratory, 
the boxes are placed in a humidifier before opening). 
The locality of capture, method of taking, name of 
collector, and date should be written on each box. 
In addition, an Sth SvG Lab. Entomology form should be 
filled out and sent in with the sample. Hand-caught 
mosquitoes are usually taken from diurnal resting 
places (caves, cattle underpasses, culverts, old 
buildings, etc.) while in the act of biting, or by 
insect net in the field. The manner in which the 
Entomology form is filled will differ slightly for each 
situation, but is demonstrated by the sample given 
below. 
ENTOMOLOGY 
Station ;>>l_:„.±*Jjdtz..... Date — 
Collection Data: Map Locality: ' .. .> '*• 
Charactor of Site:(Woods,Stables,Fond,Marsh,etc.) 
Date:_/ o Time: // ' A ui Weathers 
Hand Collections Type: -■? v.:^i. -»*- - ks &Xs; 
If Trap Used: Location of Traps 
Time Operated P.M. To A.M. Type of traps 
Larval Collection: No. Dips: No. of Collectors: 
Remarks: C / o / c o S\ *4. 
‘<XlXt'SjL. J £,' 4.'; ljcL*ck*j ' 
tc _ _ . 7 
Collected By: ~F Qj7^-)y‘zr7C. / &t s 
(Name, Rank, Designation) 
(Report on Reverse Side) 
8SC Lab 856-SAASFD-2-29-U-20,000 (2) Trap Collections - To impart the best information, 
traps should be operated regularly throughout the 
season. Unless unusually great numbers of mosquitoes 
are entering the traps, the receiving jar (cup) may 
be emptied once a day -- during the early morning. 
The entire collection should be spread out on large 
sheets of unite paper into a thin layer (use forceps). 
The mosquitoes may then be recognized and separated 
from the other insects. The mosquitoes thus set 
apart should oe packed into pill boxes as described 
in 2a >' 1) above. Each shipment of mosquitoes should 
be carefully labeled and accompanied by an 8th SvC 
Lab. Entomology form. A sample of this form as it 
should be filled out for trap collections is given 
bclou. 
SliTOLOLCGY { 
Station ~ TQ£. 4 Dato .. i£. t3.Hl' i 
i o ! 
Collection Data: Map Locality: '**? "y '? c <&■1 -X :- 1 y ■•" | 
Character of Site: (Woods,Stables.Lend,Liarsh,etc.)X*,*o <£••;*. j 
Date: T ~ 7 to 7 ~ * ,- f ? 4 Time: Weather: 4-* 
Hand Collection! Type: 
If Trap Used! Location of Trap; ■ ee r r ».-> fr; '-X ~X?' - i/ 
Time Operated £ P.M, To 8 A.M. Type of Trap; "/lew 
Larval Collection: No. of Dips: No. of Collectors: J, 
Remarks: LX v m'. »•-. . /*!-•■ .■* _ •*. ~v,:l. a*■*■■■ --■ \,4U<>■-■ I 
V. •- wy: s j cf. *. -y .. -y fif! 
i ii _3 „ _ 
Collected By: j-~i fl y\V , • <-5 , «gj-W (Zy. 
(Name, Rank, Designation) 
(Report on Reverse Side) 
8SC Lab 856-SAASFD-2-29-44-20,000 j 
(3) Living Adults - Then in special instances it may be 
desirable to send in a feu matured females to be 
examined for natural infection uith malarial 
parasites, they may be shipped short distances by using 
the bottle shorn in Fituro 6, if the glass tube is 
covered at its leuer end uith gauze or gauze is sub- 
stituted for the cork. When such special examinations 
are requested, complete data concerning the point of 
collection, attendant circumstances and the reason 
for making the request should accompany the shipment. 
A feu blades of grass or strips of crumpled paper are 
included in the container to serve as supports or 
porches for the insects. In the transport of adults 
there is no need of including the uater in the shipping 
container as shorn in Figure 6, b. Larval Mosquitoes, 
(1) Larvae Sent in After Killing - Larvae are chiefly- 
identified by the characteristics of hairs on body and 
head, and other similar minute differences. Hence, 
these, as well as the adult specimens, must be handled 
very carefully. Since characters of young larvae are 
not adaptable to ready identification, large larvae 
should always be included, e.g,, the stage immediately 
preceding the pupa and emergence of the adult. If 
possible the larvae should be killed by dropping into 
hot water 150°F. (not boiling Yjatcr) or into 70% 
alcohol. Following this treatment they may be forwarded 
in small shell vials containing 70 - 95% alcohol or 10% 
formalin (a 1 to 10 dilution of the common commercial 
formalin)• The vial should be almost completely filled 
with liquid, allowing a moderate sized bubble for 
expansion; this prevents undue splashing and violent 
agitation of specimens, or better yet, the vial may bo 
prepared as shown in Figure 5. Each vial should contain 
within (not glued to the outside) an adequate label 
identifying the catch, A pencil should be used to fill 
in the information to insure permanency. Larvae from 
one source only should be included in a single container# 
The form accompanying the shipment and identified by 
the same number as that within the vial should be filled 
out as shown in the sample below# 
ENTOMOLOGY 
Station Date . * Q (/ y' j 
Collection Data; Map Locality; V> ■ ■y/' U-Ct' 
Character of Site; (Woods ,Stables,Fond Jfcrsh,etc. );*!> w -X*-... S'<<Jrf\T.. 
Date; W W / 7 <■=•' V Time; /ol3c *', /v? Weather: 
Hand Collection; Typo; v 
If Trap Used; Location of Trap; *• 
Time Operated P.M. To A.M. Typo of Trap; 
Larval Collection; No. of Dip; Ho. of Collectors; ~£j 
Remarks: <n ? ■ ■•>c•* >>J*So 
p • y ■&?£■-■<. L - O.U 4Quo... 
(ToXLectWBy f <3 ~ ~' _ 7££~^?rr Z^~ITZZr 
(Name5 Rank, Designation) 
(Report on Reverse Side) 
8SC Lab 856-SAASFD-2-29-44.-20,000 Caution: Postal authorities require that vials of this kind, 
containing liquids, be inclosed in a screw-capped tin 
cylinder, which in turn is placed inside a paper mailing 
carton. 
(2) Live Larvae - It is often desirable to forward live 
specimens of the larvae for identification. Live 
specimens of this kind may be reared to the adult 
stage in the laboratory and identification is 
facilitated. Large larvae are preferable, as less 
time will be required for emergence and mortality is 
reduced. A 120 milliliter wide mouth histological 
bottle, which has been modified as shown in Figure 6 
by inserting a glass tube in the cork, is used as a 
shipping container. Water from the natural habitat 
is used to ship the larvae and the container is filled 
to approximately l/A its capacity. Not more than 25 
larvae should be placed in each container, Every 
effort should be made to insure promptness in transit 
and that the container is kept cool. At the height 
of the summer season it is probably inadvisable to 
ship live larvae more than 200 miles. The bottle is 
shipped in the mailing case described in the footnote 
of 2b(l) above. Proper labeling and identification 
of the container is essential and larvae from a single 
source only should bo included in each container. The 
information to be included with the shipment should be 
the same as that listed in Section 2b(l). 
3. Preparation and Shipment of Arthropods Other Than Mosquitoes* 
a. Flies and gnats, such as horse flics, deer flies, black 
flics (Buffalo gnats), biting muscids, etc,, may bo killed 
and packed for shipment in the manner described for 
mosquitoes in Section 2a(l) above. 
b. Ticks, mites, bedbugs, lice, fleas and Triatoma bugs may 
bo killed directly upon collection by plunging into 
alcohol, and preserved for shipment in the same fluid. 
Before placing in shipping container, a plug of absorbent 
cotton should be forced down the vial until it almost 
contacts the specimens. This is done to prevent movement 
among specimens which may damage structures needed for 
identification. The vial is then filled with 7C$ alcohol 
to a point l/A inch below the stopper, and a label is 
inserted. This label should be made out in pencil and 
should state the locality, host (if any), collector, and 
date of collection. The vial should be mailed in a 
standard mailing tube for liquids as indicated in footnote 
caution, Section 2b(l). Figure 2, 
Figure 1. 
Figure A. 
Figure 3. 
ASF, Eighth Service Command Laboratory 
Fort Sam Houston, Texas 
March, 194-4- Figure 5. 
ASF, Eighth Service Command 
Laboratory, Fort Sam Houston, 
Texas,.March, 19UU 
Figure 6. REFERENCES 
War Department Training Manual 8-227 
Medical Protozoology and Helminthology - Army Medical School 
Laboratory Methods of the United States Army - Simmons 
Clinical Parasitology, 3d Edition - Craig and Faust 
Clinical Diagnosis by Laboratory Methods, 8th Edition - Todd & Sanford 
Practical Bacteriology, Hematology and Parasitology, 9th Edition - 
Stitt, Clough & Clough 
Approved Laboratory Technic, 3d Edition - Kolmer & Boerner 
Textbook of Clinical Pathology - Kracke 
Synopsis of Clinical Laboratory Methods - Bray 
All illustrations supplied by courtesy of Army Medical Museum, 
Washington, D. 0.