PARASITOLOGY 
SYLLABUS 
COMPILED AND 
P U B L I SHED 
A T 
£fl LIS TED TECHfllClflflS SCHOOL 
LETTEBOIfIn GEIIERfIL HOSPIIBI 
FOR, USE OE STUDENTS 
PUBLISHED mi 
REPUBLISHED 1942 
REPUBLISHED 1943 
REPUBLISHED MAY.1944 TABLE OF CONTENTS 
Page 
Chapter 1. Introduction 1-4 
Chapter 2. Sporozoa 5-17 
Malaria: 
P. viVax 6 
P. malaria 8 
P. falciparum 9 
Differential chart and 
doubtful species 10 
Mosquito dissection 11-14 
Coccidia 14-16 
Chapter 5. Rhozppoda(amoeba)17-23 
E, histolytica 17-20 
E. vcoli 20 
E. gingivalis 20 
D. nana 21 
I. buetschlii 21 
D. fragilis 22 
Differential charts 23 
Chapter 4. Mastigophora and 
ciliata. 24-37 
Mastigophora (flagellates) 
mucous surface flagellates 25-29 
C. mesnili 25 
T. hominis 26 
G. lamblia 27 
E. intestinalis 28 
T. intestinalis 29 
Coprozoic 29 
Blood and tissue flagellates 30-35 
L* donovani 31-32 
L. tropica 33 
T. gambiense & rhodesiense 54 
T. cruzi 34-35 
T. lewisi 35 
Differential charts of 
trypanosomes 35 
Ciliata (ciliates) 
B. coli 36—37 
Chapter 5, Laboratory methods 
used in study of Protozoa, 
Protozoa infecting blood & tissue 
1, collecting of specimen 38 
2, unstained wet mounts 38 
3, thin smear 39 
4, thick smear 39 
Stains and staining 39-44 
Wright’s stain 40 
Giemsa stain 40 
Culture and Miscellaneous 44-48 
Protozoa infecting mucous 
surfaces 
1. Collection of specimen 46-51 
2. Fixation and staining 52-59 
3. Culturing 59-53 
4. Methods of study 63-66 
Page 
Chapter 6. Helminthology 67-10?. 
1. Cestodes (tapeworms) 
D. latus 69-70 
S. proliferum 70 
T, saginata 70-7? 
T. solium 72-74 
E. granulosa 71-75 
H. nana 75-76 
Rare species infesting 76-77 
man 
?. H ematode s (roundworms) 
A. lumbricoid.es 78-79 
E* vcrmicularis 79-81 
T, spiralis 31-8? 
T. trichiurus 85 
S. stercoralis 85-85 
A. duodenale 85-86 
N. Americana 86-38 
A* braziliense 88 
A, caninum 88 
W. bancrofti 88-89 
D. perstans 89-90 
F. ozzardi 90 
O. volvulus 90-91 
Loa loa 91 , 
D. medinensis 91-9? 
5. Trematodes (flukes) 
S. mansoni 95 
S. hematobium 95 
S. japonicum . 94 
Blood flukes differential 
table 94-95 
Table differentiating; 
F. hepatica 
F. buski 
C, sinensis 
P. westermanni 96 
4. Laboratory methods used 
in examining for Helminths 
Feces 97-99 
1. Examination for ad tts 97 
?. Examination for larvae 97 
5. Examination for eggs 97-99 
Blood 99 
Urine 99 
Sputum 99 
Skin 99-100 
Soil 101 
Stoll egg counting method 98 
Halls, swab for pinworm 99-100 
Baermann apparatus 101 
Shipment of material for 
diagnosis at a central 
laboratory 101-10? MEDICAL PROTOZOOLOGY 
CAPT. WILLIAM S. STONE, ‘M.C. 
CHAPTER I 
INTRODUCTION 
DEFINITION OF MEDICAL PROTOZOOLOGY 
Medical Protozoology is the science ■which deals with those members of 
the phylum Protozoa that infect man, plus those additional members vdiich may 
accidentally contaminate the body surfaces, secretions or excretions and may 
thereby be confused with the truly infectious protozoa. 
THE PROTOZOA 
The Protozoa are unicellular animals, capable within the limits of that 
cell of carrying out all the functions necessary for their independent ex- 
istence and propagation. 
I. THE PROTOZOAN CELL 
The protozoa are usually microscopic in size and in a given species 
may vary considerably in this respect, depending upon the suitability of 
their nutritional environment and upon the stage of their life cycle. The 
protozoa may be of almost any conceivable shape, and the exact form of the 
body may be regarded as an adaptation to their mode of life and environment. 
Their structure varies greatly, but in general, it is made up of a cytoplas- 
mic body and one or more nuclei. 
1. CYTOPLASM 
The cytoplasm is usually well differentiated into a denser, clear, 
outer layer, the ectoplasm, and a more fluid, granular, inner layer, the 
endoplasm. 
a. Ectoplasm 
The ectoplasm is primarily protective in function and is some- 
times very tough and resistant. It may even develop special organdies of 
defense, i.e., the trichocysts of Paramecia. Certain protozoa have the 
ability, under adverse environmental conditions, of secreting a very resis- 
tant chitinoas-like cyst wall. This cyst wall £orms a covering for the re- 
sistant stage of the individual and is in direct contact with the ectoplasm. It is capable of preventing dessication and preserves the life of the organ- 
ism until conditions are favorable for the resumption of a vegetative exis- 
tence. Movement« likewise, is a function of the ectoplasm, and in this con- 
nection, specialized organelles of locomotion may be formed, e.g., pseudo- 
podia in the amoeba, flagella in trypanosomes, and cilia in Balantidium coll, 
The capture and ingestion of food is also a function of the ectoplasm. This 
is accomplished by use of its organelles of locomotion to bring it in contact 
with its food. It may directly engulf the solid particles at any point on 
its body surface, as do amoebae, or ingest the particles through special 
openings (cytostomes) in its surface, as do Balantidium coli. In addition, 
certain protozoa ingest their food wholly or in part by direct osmosis 
through the ectoplasm, e.g., malarial plasmodia and trypanosomes. Excretion 
is also a function of the ectoplasm. This activity is accomplished by spec- 
ial organelles, i.e,, contractile vacuoles, cytopyges or anal pores, in some 
instances, v/hilc in others waste material is directly extruded from any point 
in the ectodermal surface. 
b. Endoplasm 
The endoplasm carries out the process of digestion and as- 
similation of the food materials after they have been taken into the cell 
body. Digestion of solid particles is carried out by the endoplasm forming 
a digestive vacuole about the ingested particle. The endoplasm then secretes 
a digestive ferment into the vacuole, which breaks the solid material down 
into simple products that can be assimilated and used in the enabolism of the 
cell. In the vegetative organism the endoplasm acts as a circulatory system 
and is constantly flowing in a more or less circuitous manner. In certain 
protozoa special supportive structures are formed by the endoplasm, i.e., 
axostyles in Giardia lamblia. 
2. NUCLEUS 
The nucleus is usually round or oval in shape when viewed from one 
plane, but it is actually spherical, elliptical or disc-like in conformation. 
In size, it is usually l/lO to l/ 6 the diameter of the cell. It is usually 
fixed in its position within the cell, but in the more primitive forms, it 
may move with the endoplasmic currents. The nucleus is the center of life in 
the cell, since when it is injured or destroyed, the cell dies. It is re- 
sponsible for reproduction, controls growth and reactions to environment, 
and contains the hereditary determinative factors that perpetuate a given 
species upon reproduction. The structure and relationship of the component 
parts of the protozoan nucleus afford the most valuable moans of separating 
and distinguishing closely related species. Therefore, it is of greatest 
importance to pay close attention to preserving and observing the nuclear 
structural details. The protozoan nucleus is made up of a clear, achromatic 
nuclear membrane filled with a semitranslucent nuclear sap. Within the 
nuclear sap is distributed an intricate, delicate network of linin 'ilaments. 
Chromatin, a dense, dark staining material, containing the factors of the 
cell regulating heredity, growth and reproduction is distributed in granules 
upon the nodal points of the linin network and upon the inner surface of the 
nuclear membrane. The arrangement and size of these chromatin granules with- 
in the nucleus is typical for each individual species. Situated in or near 
the center of the nucleus is a larger dense staining granule known as the Karyosome. It. differs from the karyosome. of the metazoan cell in that it 
contains, in addition to chromatin, a substance known as plastin. It 
bably contains the centrosome also. 
3. REPRODUCTION. 
V/hen dividing, the majority of Protozoa, in some modified fashion or 
other, undergo mitotic division of the nucleus. However, every possible 
graduation exists between simple direct division or amitosis (fission) -and 
true mitosis, as seen in the metazoa. 
THE METAZOA 
In contrast to the protozoa, the metazoa are multicellular animals, the 
cells of which are differentiated, each performing a special function, i.c., 
muscle cells contract, bone cells support, gland cells secrete. The cells 
of the metazoan are also interdependent upon one another for th.ir existence, 
as is evidenced by the rapid death of any group of cells separated from the 
remainder of the body. 
I. CELL.STRUCTURE 
The Metazoan cell structure is similar to the protozoan cell with the 
following exceptions; 
1. THE CYTOPLASM 
The cytoplasm is not differentiated into ectoplasm and endoplasm. It 
secured its food and gets rid of its waste products by the process of osmosis 
with the surrounding media. It does not develop food vacuoles, but certain 
cells (e.g. Macrophages) may phagocytize solid substances harmful to the 
metazoan existence. These cells then form vacuoles about the ingested, harm- 
ful body and attempt to destroy or neutralize it by secreting destructive 
ferments into the vacuole. The cytoplasm possesses a definite idiosomc or 
sphere. The centrosome is usually extra nuclear. 
2. THE NUCLEUS 
The nucleus possesses one or more nucleoli, which have a structure and 
location similar to that of the karyosome in the protozoan nucleus, but the 
nucleoli are composed of plastin only. The karyosome of the- metazoan nu- 
cleus is composed of a few chromatin granules only, and is located upon a 
nodal point in the linin network. 
3. REPRODUCTION 
In the metazoa, cell division is always preceded by mitotic division 
of the nucleus. 
The general structures of the Metazoan and Protozoan cells are illus- 
trated in Figure 39, CLASSIFICATION OF THE PROTOZOA 
Classification according to Thomson and Robertson. 
Phylum . • • . 
Protozoa 
Subphylum . . 
Plasmodroma 
Ciliophora 
Class . . . . 
Sporozoa 
Ciliata 
Rhizopoda 
Mastigophora 
. . -tt 
I. SUBPHYLUM PLASMODROMA 
This includes the most important organisms which infect man and other 
animals. This group is distinguished by the fact that syngamy is performed by 
complete fusion of the male and female gametes, a process known as copulation. 
In this subphylum there are only 3 classes of medical importance. They are; 
1. SPOROZOA 
In this class are found, the most important protozoan parasites of man, 
e.g., malaria. The formation of sporozoites occurs after the sexual process 
of syngamy, e.g., in malaria it occurs in the stomach of the female anopheline 
mosquito, after which the bite of the mosquito is infective for man. 
2. RHIZOPODA 
This class is also known as the Sarcodins. They are amoeboid, and 
movement is produced by means of pseudopodis. 
3. MASTIGOPHORA 
The organisms included in this class move and feed by the aid of a 
specially developed ectoplasmic structure, a flagellum, the number of which 
varies according to the species of flagellates in question. 
11. SUBPIIYLUM CILIOPHORA 
The characteristic feature of this group is the possession of special 
ectoplasmic structures called cilia. Syngamy is accomplished in this group by 
juxtaposition of the gametes, followed by exchange of nuclear chromatin. This 
process is called "conjugation” to distinguish it from the process of copu- 
lation in the Plasmodroma, 
1. CILIATA 
The members of this class have cilia on the surface of their bodies at 
all stages of development and most of them possess a definite mouth or cyto- 
stome. CHAP1 TER II 
THE SPOROZOA INFECTING MAN. 
CLASSIFICATION OF THE SPOROZOA. 
CLASS; SPOROZOA. 
Subclass 
Order 
Suborder 
Genus 
Species 
Coccidiomorpha 
Coccidiida 
Haemospor- 
idiidea 
Plasmodium 
P, vivax 
P, falciparum 
P. malarias 
Plasmodium in monkeys, 
birds, etc. 
Eimeriidea 
Isospora 
I. hominis (I, belli) 
man 
Eimeria 
E. gubleri (?) °n 
E. perforans) 
E. stiedao ) Habblts 
E. clupearum) „. , 
E. sardinae ) 
All of the known sporozoan parasites of man are included in the above 
abbreviated classification. The organisms of this group are characterized 
by: (1) The asexual cycle takes place by a process known as schizogony, 
which is usually intracellular, i.e., malaria in the red blood cells, coccid- 
iosis in the epithelial colls of the intestinal mucosa. (2) Male or micro- 
game tocytes and female or macrogametocytes are formed in the sexual cycle. 
Prior to actual sexual union, the female gametocyte becomes a female gamete 
by extrusion of polar bodies (chromatin), the male gametocyte produces a 
number of male gametes. There is complete fusion of the male and female 
gametes. 
THE GENUS PLASMODIUM (MALARIA) 
The parasites of this genus are pigmented, amoeboid organisms, living 
on or within the rod blood corpuscles of man or some other vertebrate host. 
The asexual cycle (schizogony) and the formation of gametocytes (gametogony) 
are confined entirely to the red blood cells, whereas sporogony is found in 
the insect vector. In the case of human malaria, man is the intermediate 
host and the anopheline female mosquito is the definitive host. The defini- 
tive host is the one in which the sexual cycle takes place, while the inter- 
mediate host is the one in which only the asexual cycle and the development 
of gametocytes are found. Figure 40 shows the life cycle of the malarial 
parasite in man and in the mosquito. The malarial parasites of man are: 
I. PLASMODIUM VIVAX (GRAS-SI AND FELETTI, 1890). 
This is the organism of tertian or benign tertian malaria, the most 
common malarial fever of man. This parasite is distributed geographically 
over the tropical and subtropical areas, and over certain portions of the 
temperate zone during the warm months. P. vivax is the hardiest of the mal- 
arial parasites and in infected localities in the temperate zone it predom- 
inates in the spring and early summer. 
1. LIFE CYCLE IN MAN 
a. SCHIZOGONY. 
The sporozoites are injected into the blood stream by the 
infected female anopheline mosquito, where they rapidly parasitize reel, blood 
cells, round up, and assume the typical signet ring form. These ring forms 
are about l/ 3 to 1/4 the diameter of the infected red cell and when stained 
by Wright’s or Giensa’s method, the chromatin stains red and the cytoplasm 
blue. In about six hour's, the parasite increases in size by one-third and 
shows marked amoeboid movement within the infected cell. The infected red 
cell has now enlarged until its diameter is from 101.1 to ISM end its color is 
paler than the uninfected red cells. If the infected red cells are properly 
stained, fine pink dots or stippling (Schuffner’s dots') are usually present 
in the cytoplasm, and minute granules of light brown pigment are seen near 
the chromatin of the parasite. The growth of the parasite is fairly rapid 
and by the end of thirty-six hours it fills two-thirds of the enlarged, stip- 
pled, parasitized rod cell. After about forty hours, the parasite fills the 
cell and its chromatin usually divides into 12 to 1G segments. Small yellow- 
ish brown granules of pigment are now scattered throughout the body of the 
parasite. The pigment then clumps toward the center of the parasite whose 
cytoplasm then divides into equal sections, one about each segment of the 
chromatin. The small bodies resulting from the division of the mature schi- 
zont are called "merozoites.” At the end of forty-eight hours the merozoites 
rupture the red cell membrane, (at which time the patient’s rigor begins), 
and then each liberated merozoitc seeks out and parasitises a new red blood 
cell, to repeat the cycle. The above description depicts the asexual life 
cycle of P. vivax which, following the time relationship with the rigor, can 
be demonstrated in the blood stream. At any given time, however, practically 
all of the stages in the life cycle can be demonstrated in the blood stream, 
although one stage always predominates. The probable reason for this is that 
infections in man are due to more than one bite by infected mosquitoes, or 
that in a given single bite, some of the sporozoites instead of being inocu- 
lated intravenously are inoculated subcutaneously, thus delaying the start 
of their life cycle. 
b. GAMETOGONY 
The gametocytes or sexual forms develop from certain undif- 
ferentiated merozoites. The stimulus for their formation is unknown, but it 
is probably a response to developing immunity on the part of the host. Four days are required for the development of the ring forms into mature gameto- 
cytes, However, the gametocytes must be from seven to ten days old before 
they are infectious for the mosquito# When fully grown the gametocytes are 
rounded in form and have a fairly homogeneous cytoplasm. They appear in the 
peripheral blood stream within about seven days after the initial" fever. 
Their life in the blood stream is about ten to twenty days# They are prob- 
ably incapable of reproducing themselves or of initiating the asexual cycle- 
in man without first undergoing sporogony in the anopheline female mosquito. 
(1) The male gametocyte (microgametocyte) is 7 Mto 8 M 
in diameter and occupies an enlarged red cell without completely filling it, 
Schuffner’s dots may be present in the margin of the red cell. The cyto- 
plasm of the parasite stains faint blue-grey and contains numerous granules 
of brown pigment. The chromatin stains red and is scattered over a fairly 
wide area, sometimes in the shape of a band, near the center or edge of the 
parasite. 
(2) The female gametocyte (macrogametocyte) is larger, 
8 M to 10 M, fills the parasitized cell more completely and its cytoplasm 
stains a darker blue than the male gametocyte. The chromatin of the female 
gametocyte is in one compact clump, usually near the sdge of the parasite, 
and the pigment is in coarse brown compact granules. There are usually 
from 3 to 6 female gametocytes for every male gametooyte in the peripheral 
blood. 
2, SPQROGOI& IN TOE FEMALE ANOPHELINE MOSQUITO 
The gametocytes are drawn into the mosquito’s stomach along with 
its blood feed. Within twenty minutes, the male gametocyte develops eight 
to ten cytoplasmic processes, which soon exhibit violently lashing flagel- 
lated ends. These cytoplasmic processes soon separate from the original 
cell body and are then called ’’microgametes.” While this process has been 
going on in the male gametocyte, the female gametocyte extrudes two polar 
bodies containing one-half of its chromatin. It is now ready for fertiliza- 
tion and is called a ”macrogametc,” The microgameto penetrates the cell wall 
of the macrogamete, the chromatin of each fuses and the parasite now becomes 
a motile zygote or ookinete. The ookinete now penetrates the wall of the 
mosquito’s stomach, rounds up, becomes immobile, and is then called an 
’’oocyst,” The chromatin of the oocyst undergoes multiple divisions, until 
there may be as many as 10,000 separate granules, which are grouped together 
in small clumps. The oocyst now contains from ten to twenty group:: of these 
chromatin granules and is now known as a ”sporoblast,” Each chromatin 
granule now develops a fusiform cytoplasmic process and becomes a sporozoite. 
The sac containing the sporozoites is now called a ’’sporocyst,” The sporo- 
cyst ruptures, liberating the motile sporozoites, which soon gain access to 
the mosquito’s salivary glands. Then, if the opportunity presents itself, 
the mosquito is ready to infect a new individual, Sporogony in the female 
anopheline mosquito requires about seven to twelve days for its completion, 
if the temperature is about 20° C, and the air is about 70$ saturated with 
moisture. The life cycle of P, vivax in man showing its relation to the tem- 
perature curve of the host is given in Figure 41. •11. PLASMODIUM MALARIA# (LAVEtRAK, 1881) 
This parasite causes quartan malarial fever and it is the rarest 
of the three species in man. ' Except for infected transients, it is probably 
confined to the tropical or subtropical portions of the world. In sub- 
tropical regions, the greatest number of new cases of this infection occurs 
during the fall months. The symptons of this infection are much more severe 
than would be expected from the number of the parasites infecting the red 
cells in any given case. In any given infection, all stages are usually 
present in the blood stream at one time, although one stage always predom- 
inates. 
1. LIFE CYCLE IN MAN. 
a. SCHIZOGONY 
Schizogony in Plasmodium malariae is similar to that of P. 
vivax with the following exceptions; P. malariae requires 72 hours to com- 
plete its asexual circle, During the first 6 hours of its grovrbh it has the 
largest ring forms of all the types of malaria, and v/hen it is 24 to 48 hours 
old, it develops typical band forms* Its segmenting forms only develop from 
8 to 12 merozoites. The infected red cells are not enlarged and Schuffner•s 
dots are not present. The pigment present is coarse and stains dark brown 
to black. 
b. GAMETOGONY. 
Gametogony in Plasmodium malariae is similar to that in P. 
vivax, except that the organisms are smaller and the red cells arc not en- 
larged, and do not contain Schuffner’s dots. The time required for its 
gamctocytcs to develop is approximately seven days. 
2. SPOROGONY 
The life cycle in the female anopheline mosquito is the same as 
for P, vivax, except that it takes about three times as long to complete 
the cycle. The life cycle of P. malariae in relation to the temperature 
curve of -the host is shown in Figure 42, 111. FLASLIODIULI FALCIPARUM (WELCH, 1897) 
This is the organism of estivo-autumnal, subtertian, malignant 
tertian or pernicious malaria. Its distribution is similar to that of 
Plasmodium malariae, but it is second only to P, vivax in the frequency of 
its occurrence. It is the most severe of all the malarial fevers of man 
and requires prompt early diagnosis and treatment. 
1. LIFE CYCLE IN MAN. 
a. SCHIZOGONY 
Schizogony is similar to that of P. vivax with the follow- 
ing exceptions: (1) The ring forms are smaller when they first parasitize 
a red blood cell. (2) There arc more apt to be multiple infections of the 
red cells and ring forms may show two dots of chromatin. (3) The ring 
stage may persist for about 24 hours in the poripherial circulation during 
which time it doubles or triples its size. (4) Ring forms and m: Lure gam- 
etocytos (crescents) ordinarily are the only forms found in the p-: ipheral 
blood. In severe, overwhelming infections by this parasite bizarre fila- 
mentous and other forms may occasionally be present in the peripheral blood, 
(5) Developing schizonts and gametocytes are usually found only in the cap- 
illaries of the internal organs. These organisms have a marked tendency to 
clump together and stick to the endothelial cells, thus bringing about cap- 
illary blockage. (6) Segmenting forms of P, falciparum develop 16 to 32 
merozoites. (7) The parasitized red blood cells are not enlarged. (8) 
Schuffner’s dots are not present in the parasitized cells, but large granules 
staining purplish red, called "Llaurer1s dots” or malignant stippling are 
present. 
b. GAMSTOGONI 
The gametocytes are crescent-shaped with rounded ends and 
they are greater in length than the diameter of the red cells which they have 
parasitized. The male ganetocyte stains bluish-gray and has a central nuc- 
leus with diffuse chromatin. The pigment present is in coarse grains and 
stains brownish-black. It is scattered between the grains of chromatin. 
The tips of the male crescent are usually more rounded than those of the 
female crescent. The female ganetocyte (crescent) stains sky-blue and its 
nucleus is made up of a compact mass of chromatin and pigment. 
2. SPOROGONI 
The life cycle of P, falciparum in the female anopheline mosquito 
is essentially the same as that of P. vivax, except that l/o to 1/2 again 
as much time is needed to complete the cycle. Figure 43 gives the life 
cycle of P. falciparum in relation to the temperature curve. IV. DOUBTFUL SPECIES ADD SUBSPECIES OF MALARIAL PARASITES OF MAN. 
The doubtful species and subspecies of the malarial parasites of 
nan are: 
1. PLASMODIUM FALCIPARUM QUOTIDIANUM. (Craig, 1909) 
This organism is differentiated from P. falciparum infections 
clinically by more or less constant fever with daily exacerbations. The 
ring forms of the parasite are smaller than those of P, falciparum and only 
12 to 14 nerozoites are produced at segmentation. 
2, PLASMODIUM OVALE (Stephens, 1922) 
This organism is identical with P. malariae, except for the ab- 
sence of band forms. In addition all parasitized red blood cels are oval 
in shape. 
3. PLASMODIUM VIVAX, VARIETY MINUTUM (Emin, 1914) 
This organism is identical with P. vivax except that the para- 
sites never enlarge the parasitized red cells. The schizonts only fill 
two-thirds of the parasitized red cell and only 4 to 10 merozoites are 
produced at segmentation. Table 85 presents the characteristics differen- 
tiating the three main species. V. PLASMODIA OF MONKEYS 
These organisms are identical morphologically, in general terms, 
with those of man, but repeated attempts to transmit them to man and to 
infect monkeys with human malaria have been unsuccessful insofar as main- 
taining the organisms through successive transfer is concerned. 
VI. THE EXAMINATION OF MOSQUITOES FOR MALARIA 
Various surveys in heavily infected areas in different parts of 
the world seldom reveal infection of more than 0.2$ to 2$ of the chief an- 
opheline vector. In tropical countries all of the anopheline mosquitoes 
found infected with malarial parasites are not necessarily human malarial 
vectors, for the parasites present nay be those of monkey malaria which is 
morphologically identical with that of human malaria, and a differentiation 
between the two can only be made by transmission to man or the species of 
monkeys present in the area. Therefore, in mosquito surveys, to determine 
the malarial vectors of any given locality in which malarial fever is present 
either in an epidemic or endemic form, great care should be taken to examine 
a sufficient number of the suspected species of anophelines to draw an 
accurate conclusion. As a routine at least 100 to 400 females of a given 
species should be examined. The procedure for examining them for the pre- 
sence of malarial infection is as follows; 
1. COLLECTION OF MOSQUITOES 
Capture the anopheline mosquitoes near human habitation and pre- 
ferably near the houses of known cases or carriers. Keep the mosquitoes 
alive until the time of examination. 
2. KILLING MOSQUITOES FOR EXAMINATION. 
Place four to six mosquitoes in a glass jar containing a small 
amount of cotton.moistened with chloroform. As soon as they are dead re- 
move them from the jar and place between layers of cotton, moistened with 
water until they are dissected. 
5. TECHNIQUE OF DISSECTION. 
a, Materials required 
(1) Clean slides and cover glasses, 
(2) Two straight dissecting needles in needle holders, 
one to have a two-edged cutting point. 
(3) Pipettes or droppers. 
(4) A pair of fine pointed scissors to clip off the 
wings and legs, 
(5) A pair of fine pointed tissue forceps to handle 
the dead mosquito. 
(6) Alcohol lamp, 
(7) Physiological saline solution. 
(8) Physiological saline solution deeply tinted with 
methylene blue. b. Precautions. 
(1). Mosquitoes should be dissected within one hour after 
they have been killed, as autodigestion of tissue rapidly sets in and it will 
be almost impossible to secure proper specimens if more than two or three 
hours are allowed to elapse before beginning the dissection. 
(2). Never let the preparation become dry while dissec- 
tion is in progress; always keep the specimen in a drop of physiological 
saline * 
(3). "When the alimentary canal is removed be careful not 
to break up the malpighian tubules as granules liberated by so treating them 
will greatly confuse the picture. 
c. Procedure. 
(1). Select one of the dead female anopheline mosquitoes, 
identify its species, record the results and then remove the wings and legs. 
Place the body on a glass slide in a drop of saline solution tinted with 
methylene blue so that its ventral surface is toward you and the tip of the 
abdomen points to your right. 
(2), The salivary glands lie in the forequarter of the 
thorax; to remove them, hold the body in place by gently pressing the tip 
of a dissecting needle on the anterior half of the thorax, then with the 
shaft of the second needle press on the head, gently drawing it away from 
the thorax. By this procedure the pair of very minute, blue-stained, re- 
tractile, trilobate glands are usually extracted attached to the head. If 
the glands fail to come away with the head, they may sometimes be expelled 
by gentle pressure with a dissecting needle on the anterior and middle por- 
tions of the thorax, or as a last resort by teasing the thorax to shreds. 
Remove the glands from the head by means of the cutting-edged dissecting 
needle, then by securing them on the needle tip, transfer them to a small 
drop of unstained saline on another slide and cover with a cover glass. 
(3). To obtain the stomach, place the tip of one needle 
on the 7th and the other on the Bth abdominal segments, break the chitinous 
connection between these segments by gentle teasing, then transfix the tho- 
rax with one of the needles and pierce the last segment of the abdomen with 
the other; now using gentle traction on the last abdominal segment, draw 
out the viscera into the saline solution. Secure the stomach by means of 
the shaft of the needle in the left hand, then with the right hand, using 
the needle with the cutting edge, trim off the malpighian tubules and all 
structures lying posterior to them. Now pick up the stomach on a needle tip 
transfer it to a large drop of untinted saline on a clean slide, straighten 
it out and cover with a cover glass. Withdraw enough of the saline from 
the preparation by means of a slip of filter paper so that the cover glass 
no longer floats, then gently heat the slide over the alcohol lamp until the 
stomach becomes distended due to the heat expansion of its gaseous content. 4. EXAMINATION OF THE SPECIMEN 
a. The Stomach. 
Center the stomach with the long axis perpendicular to the 
observer, in a low-powered microscopic field, then place the point of a dis- 
secting needle against the left edge of the cover glass and hold it sta- 
tionary, Now move the slide to the left by means of the mechanical stage, 
which will cause the stomach to revolve and in its revolution all its ex- 
ternal surface can be examined for projecting cysts. All suspicious spher- 
ical bodies found attached to the 'stomach wall should be examined under 
higher magnification. The species of large cysts can not be recognized, 
but in small and medium sized cysts, the pigment is fairly characteristic. 
P. falciparum cysts have dense clumps of large, black, pigmented granules 
and P, vivax cysts have delicate strands of yellow to brown, fine-pigmented 
granules, while P, malariae cysts have clumps of coarse, dark-brown, pig- 
mented granules. For the exact morphology of malarial parasites in the wall 
of the mosquito stomach see Figure 44, b. The Salivary Glands 
Place the point of a dissecting needle on the cover 
glass directly over the salivary glands, then by gentle repeated pressure 
crush the glands. The crushed glands and adjacent fluid are then examin- 
ed under the high power or oil immersion objective for malarial sporo- 
zoites, The species of sporozoites can not be determined from their size 
or morphology. If in doubt about the presence of sporozoites, the cover 
glass should be removed, the preparation allowed to dry, after which it 
is stained by the Wright or Giemsa method. Examination of the stained 
smear will establish whether or not sporozoites are present. 
VII. LABORATORY DIAGNOSIS 
Chapter V. 
For the laboratory diagnosis of malaria see Laboratory Methods, 
THE COCCIDIA 
The coccidia are sporozoon parasites of the lining cells of the in- 
testine of vertebrates. One species, Eimeria stiedae, parasitizes the 
cells lining the bile ducts of the liver. For a typical life cycle in this 
group see Figure 45. There are only two species of coccidia that parasitize man. They are: 
I• ISOSPORA HOMINIS (I. BELLI). 
This infection in man is found throughout the tropical and sub- 
tropical world, but the majority of cases reported to date have arisen in 
countries around the eastern end of the Mediterranean, There have been 
more than 150 cases of this infection recorded. This infection is char- 
acterized by abdominal discomfort for 3 to 5 days followed by diarrhea 
with considerable tenesmus, which persists for 3 to 6 weeks. The patient, 
although greatly debilitated, usually recovers. After about the third 
week of the diarrhea oocysts are passed in the stools. They are covered 
by a doubly refractive wall, are ovoid in form and measure 15 x 30 M, 
The cyst is- smooth, colorless and very resistant to fixatives. A micropile 
at one end of the cyst can sometimes by made out. The oocyst when passed, 
contains a single ball of cytoplasm. This soon divides into two masses 
which secrete a v/all about themselves. Each cytoplasmic mass then divides 
into four sausage-shaped sporozoites. This cycle requires about 2 days 
for its completion, at which time the sporocysts are infective if again 
ingested. The oocysts continue to be passed during the period of diarrhea, 
although they are never in greater numbers than two or three per slide. 
They are graphically illustrated in Figure 46, 11. EIMERIA GUI3LERI (?) 
This is a coccidial infection of man involving the epithelium 
of the bile ducts in the liver. There have been only a few cases recorded 
in medical literature and this parasite has never been thoroughly studied. 
111. EIMERIA CLUPEARUM AND E. SARDINAE 
These organisms cause coccidiosis in various species of fish. 
When fish infected with these.organisms are ingested by man, the oocysts 
of the parasites are liberated in the gastro-intestinal tract, but are 
unable to infect the epithelium and are eliminated in the feces. Therefore 
finding the oocysts of these species in the feces is of no significance. CHAPTER 111 
RHIZOPODA (AMOEBAE) 
CLASSIFICATION 
CLASS: RHIZOPODA 
ORDER 
GENERA 
SPECIES 
HABITAT 
Amoebida 
Entamoeba 
E. coli 
G, I, tract man 
E. histolytica 
u it n 
E. gingivalis 
Buccal cavity man 
Endolimax 
E, nana 
G, I, tract man 
lodamoeba 
I. butschlii 
n it n 
Dientamoeba 
D. fragilis 
n »i n 
Suppinia 
S. diploidea 
Coprozoic 
Dima s t i gamoeba 
D. gruberi 
ii 
Amoeba 
A. proteus 
Free living 
The amoebae infecting man show fairly marked differentiation of the 
cytoplasm into a clear, hyaline outer zone or ectoplasm and a granular 
inner zone or endoplasm. They move by means of psoudopodia and, with the 
exception of E, gingivalis and D, fragilis, they are transmitted from in- 
fected to new cases in the form of resistant cysts. 
The amoebae of medical importance may be conveniently grouped into 
the pathogenic amoebae, the commensal amoebae, and the confusing contam- 
inating coprozoic and free living amoebae. 
THE PATHOGENIC AMOEBA OF MAN 
I. ENTAMOEBA HISTOLYTICA (SCHAUDINN, 1905) 
E, histolytica is the only known pathogenic amoeba of man. It 
is now recognized that it is universal in its distribution and while in- 
fection is most prevalent in the tropical and subtropical regions, it is 
by no means uncommon in the temperate zone. It is estimated that from two 
to ten percent of the population of the United States harbor this parasite. 
It is the organism causing amoebic dysentery and its sequellae in • ui. 
1. LIFE CYCLE 
The stages of the life cycle of E, histolytica may be divided in- 
to the free vegetative forms or trophozoites, the precystic and the cystic 
forms. a. Trophozoites. 
The trophozoites of E. histolytica vary in size, depend- 
ing upon the suitability of their environment and the rapidity of their 
multiplication. At times, in any given specimen or culture, it is possible 
to demonstrate vegetative forms of E, histolytica in sizes varying from 
8 to 40 M. The average size, when rounded up, is about 25 Min diameter. 
The ectoplasm is very well differentiated from the endoplasm; this is par- 
ticularly true when the amoeba is not moving, but sending out a pseudopod 
and then withdrawing it to send out another. Under these conditions it 
is glassy clear. When the amoeba is rapidly moving, however, there is 
only moderate differentiation of the ecto- and endoplasm. The endoplasm 
is finely granular and has very little color. Although E. histolytica will 
ingest bacteria, starch and other kinds of food in culture, in the body it 
is a true tissue parasite and lives on body cells and fluids to the exclu- 
sion of all other types of food. In the fresh diarrheal stool, E, histoly- 
tica very rarely contains ingested bacteria in its endoplasm. Under these 
conditions, the only ingested solid particles are red blood cells or leu- 
cocyte fragments. Except in individuals undergoing degeneration, the only 
vacuoles present in the endoplasm are food vacuoles. The nucleus is about 
1/6 the diameter of the cell, is disc-like in shape, and moves with the 
endoplasmic currents, occasionally tilting slightly as it floats along. 
In the active living trophozoite it is sometimes very difficult to visual- 
ize, but as it is usually in that portion of the endoplasm that flows into 
the new-formed pseudopod, it can be observed in this position contrasted 
against the ectoplasm. When the nucleus is stained with iodine or hemo- 
toxylin the chromatin is seen to be distributed in fine fairly even gran- 
ules about the inner side of the nuclear membrane. The linin net work is 
free from chromatin and the karyosome is a small centrally located granule. 
Reproduction is by binary fission, which can be easily observed in warm 
stage preparations from rapidly growing cultures. It is usually an equal 
division of nuclear and cytoplasmic substances, but in rapidly,growing 
specimens there may be marked unequal divisions of the cytoplasm, thus giv- 
ing rise to the small organisms which were formerly thought to be a dif- 
ferent strain. When not undergoing division, precystic changes, or in un- 
favorable environment, the vegetative E. histolytica moves rapidly, progres- 
sively. and more or less continuously. The organism moves like a slug, and 
has the ability to cling to a surface and make progress even against or 
across currents in the surrounding media. The movement of amoebae, against 
currents or solid particles in the media, is accomplished by extending a 
pseudopod as far as possible, and then after fixing the tip of this pseudo- 
pod on a solid surface, the amoeba will push out a new pseudopod from a 
point near the tip of the previous pseudopod which is now used as a fixing 
point. Thus, with one stationary point, the amoeba is able to push out the 
new pseudopod and allow the remainder of its body to flow into it until the 
tip of the first pseudopod is the rear most part of the amoeba. The pro- 
cess is then repeated. Moving E, histolytica usually have considerable 
debris trailing along behind each one. 
b. Precystic stage. 
When the environmental conditions gradually become adverse for its continued vegetative existence, E, histolytica rounds up, becomes 
practically immotile and extrudes all undigested solid food particles. 
The chromatin granules in the nucleus become coarser and the nucleus is 
now quite visible in the fresh state when viewed under high power. Rod- 
shaped chromatoid bodies are sometimes formed in the later part of this 
stage when the ectoplasm is about to secrete a cyst wall. This stage is 
known as the precystic stage, but when the adverse environmental changes 
are sudden, the amoeba is uable to undergo this stage and merely rounds 
up and dies. 
c. Cystic Stage. 
Following the pre-cystic stage a definite, clear, hyal- 
ine cyst wall is secreted by the ectoplasm. Cysts are not formed in the 
tissue of the host; they are present only in fecal excrement and in cul- 
tures. The cysts vary in size from 5M to 20 M and their cytoplasm is clear, 
translucent and finely granular. In about ZO% of the new-formed cysts 
there is a large refractile red-shaped body wdth rounded ends, the chroma— 
toid body. This body and its shape are characteristic for E. histolytica. 
and if it can be observed in a cyst, a diagnosis of E. histolytica can be 
made. When present, the chroma toid body can be visualized by reduced 
light in the fresh unstained state. It stains a bluish-black with hema- 
toxylin, but it is not visible in iodine preparations. The iodine stain 
may show, in new-formed cysts, small glycogen vacuoles that are light ma- 
hogany in color. Both the glycogen vacuoles and chromatoid bodies gradual- 
ly disappear as the cysts age. When the cyst is first formed, there is only 
one nucleus present, and it is of the same type as that in the vegetative 
forms. This nucleus soon divides, then each daughter nucleus divides again 
giving a total of four small characteristically shaped nuclei, each lying 
on a different plane in the spherical cyst. The average cyst contains four 
nuclei and it is this four nucleated cyst with or without the red-shaped 
chromatoid body that is characteristic for this species. The cysts, upon 
being passed in the feces, are fairly resistant to mild dessicatlon and 
ordinary disinfectants (ordinary water chlorination will not kill them). 
In the feces as passed, cysts will probably live not longer than from 4 to 
10 days. However, if the fecal matter is diluted with water, as in sewage, 
and the temperature does not exceed 20° C,, they may be viable for periods 
up to 200 days. They are destroyed, by heating to 50° C., for five minutes. 
The cyst is the only state in which amoebae can pass through the gastric 
.juices and still remain viable. For this reason, contamination of food, 
drink or mouth parts by cysts of E, histolytica is the only natural way 
that this infection can be acquired. In addition to the ordinary means of 
contamination, flies and cockroaches may ingest and pass viable cysts in 
their fecal excrement. 
Wien the cysts of E, histolytica are ingested, th.y pass 
unharmed down the intestinal tract until they reach the cecum. Thv_ e ex- 
cystation takes place and the four amoebae resulting from each cyst seek a 
tubular gland where they propagate and soon destroy enough of the cells of 
the host to plug the gland. The small pin-point yellow abscess so formed 
is now freed of most of the bacteria and the amoebae digest away the tissue 
cells and undermine the mucosa. They invade the surrounding lymph spaces, the connective tissue planes, and thrombose the neighboring capillaries. 
The spreading necrotic area thus formed soon sloughs out and leaves a 
small undermined ulcer. The amoebae on the surface of this ulcer are 
washed out into the intestinal contents by the fecal currents, where they 
either invade new glands, become encysted, or are passed as trophozoites, 
thus completing the cycle. If the host resistance is poor, new ulcers 
will be formed in rapidly increasing numbers, and the individual will be- 
come aware of gaseous distension and vague abdominal discomfiture, which 
is soon followed by symptoms of dysentery. If the host’s resistance is 
good, only a limited number of ulcers will be formed and then a chronic 
course ensues (knov/n as the carrier state). However, if the host’s resis- 
tance is lowered, it may at any time become an acute case. Liver abscess 
is stated, to occur in about 15% of all untreated cases infected with E. 
histolytica. 
THE COMMENSAL AMOEBAE OF MAN 
The commensal amoebae of man are of importance in that they may be 
confused with E, histolytica, and because their presence indicates human 
fecal contamination at some time of the mouth parts, fluids, or food in- 
gested by the infected individual. 
I. ENTAMOEBA CPU (GROSSI, 1879) CASACRANDI AND BARBAGALLO, 1895. 
E. coli is a harmless saprophyte living in the lumen of the human 
large intestine. This is the most common amoebic infection of man's gastro- 
intestinal tract and is the one most easily confused with E, histolytica. 
1. THE LIFE CYCLE OF E. COLI 
The life cycle of E, coli is very similar to that of E, 
histolytica with the following exceptions: (1) E, coli is not pathogenic, 
(2) Its cytoplasm is coarsely granular and is not as well differentiated 
into ecto- and endoplasm, (3) It rarely ingests red cells and is usually 
filled with bacteria, starch granules, mold spores, etc. (4) Its nucleus 
is easily visible and contains coarse irregular-sized grains of chromatin 
on the inner surface of the nuclear membrane. (5) The linin network may 
have a few grains of chromatin suspended in it, and the karysome is larger 
and eccentrically located in the nucleus* (6) E, coli moves sluggishly and 
usually does not make a great deal of progress, and it is very easily kept 
in the microscopic field as compared to E, histolytica. (7) The average 
cyst of E. coli usually has 8 small fairly typical nuclei. The chromatoid 
bodies when present have sharp pointed acicular ends (demonstrated by care- 
ful focusing) as compared to the rounded ends of E. histolytica. (8) When 
first formed, the cysts usually contain larger and more marked glycogen 
vacuoles. 
11. ENTAMOEBA GINGIVALIS (GROS, 1894) Brumpt, 1913. 
Entamoeba gingivalis is a harmless saprophytic amoeba found in the 
pus pockets of pyorrhea alveolaris, tartar deposits, cavities of carious 
teeth and in any buccal erosion or abscess. 1. LIFE CYCLE 
The life cycle of. E, ginnivalis is confined to the buccal cavity 
of .man, and is the only amoeba living in this location. The troph- oites 
are almost identical in appearance with those of E, histolytica except 
thab they are usually filled with dark pyknotic bodies and are more slug- 
gish in their movements, C:/'sts have never been demonstrated for this 
amoeba. Animals susceptible to infection with E, histolytica, when inocu- 
lated per rectum with E, gingivalis, have never revealed any pathogenicity 
in this species. 
111. ENDOLIMAX NANA - (WENYON AND O'CONNOR, 1917) BURG, 1918 
E. nana is a small non—pathogenic amoeba which inhabits the 
lumen of the human colon. Next to E, coli this is the most common amoebic 
infection in man's gastrointestinal tract. This amoeba is of no impor- 
tance clinically, except that it may be confused with small strains of E. 
histolytica. 
1. LIFE CYCLE 
This organism is the smallest amoeba infecting man, measuring 
from 6 Mto 12 M. The life cycle is similar to E. histolvtica with the 
following exceptions: (l) It is a slow moving, sluggish organism showing 
more changes in shape than progressive motion. (2) All the chromatin of 
the nucleus is collected into a large irregularly-shaped karyosome which 
is \isually eccentrically located in the nucleus, (o) There is no chro- 
matin on the inner surface of the nuclear membrane. (4) The cysts are oval 
or irregular in shape and usually contain 4 small nuclei similar to those 
present in the trophozoite. (5) There are no chromatoid bodies present in 
the cyst. 
IV. lOPAMOEBA BUTSCHLII - (PROWAZEK, 1912) DOBELL, 1919. 
I, bitschlii is one of the rarer non—pathogenic amoeba inhabit- 
ing man's colon. 
1. LIFE CYCLE. 
This organism usually varies from 9Mt013 M in size. Indivi- 
duals infected with this organism usually pass many irregularly-shaped 
cysts, but rarely trophozoites in their feces. The trophozoites resemble 
E. coli in form and habit, except that the nucleus has a large oval or 
round eccentrically located karyosome containing all the chromatin. The 
cysts are very irregular in shape and contain either one or two typical 
nuclei. Their most characteristic feature is that, when they are stained 
with Cram's iodine (double strength) large, deeply staining, mahogany brown 
glycogen vacuoles are found to be present. These glycogen vacuoles may 
nearly fill the cyst and displace the nuclei to one side. The glycogen 
bodies get smaller with age and may eventually almost disappear. V. DIENTAMOEBA FRAGILIS - JEPPS AND DOBELL, 1918. 
This is the rarest intestinal amoeba of man, and is a harmless 
saprophyte inhabiting the colon. It is extremely sensitive to changes in 
environment and rapidly degenerates after being passed in the feces. 
1. LIFE CYCLE 
This organism ranges from 3Mto 12 Min size. The endoplasm is 
finely granular and the ectoplasm well differentiated. This organism us- 
ually does not move, but merely changes shape by extruding pseudopods which 
when fully extended, spread laterally. The majority of this species have 
two nuclei; in fact, the uninucleated amoeba are out numbered four to one. 
The chromatin is arranged in a ring of from 4 to 6 granules located in the 
nucleus halfway between the central point and the nuclear membrane. There 
is no chromatin on the nuclear membrane. Cysts have never been d.monstrat- 
ed for this species. 
DIFFERENTIAL DIAGNOSIS 
The principle points of differentiation between the various species 
of amoebae infecting man are illustrated in Figure 47 and listed in 
Table 86. THE COPROZOIC AMD FREE LIVING AMOEBAE. 
I. THE COPROZOIC AND P'REE LIVING AMOEBAE, that may contaminate the 
body secretions or excretions and thus may be confused with the amoebae 
that truly inhabit the mouth or gastro-intestinal tract of man, are char- 
acterized by the possession of at least one contractile vacuole, e.g., 
Amoeba protous. This structure is never present in the amoebae infecting 
man. 
THE LABORATORY DIAGNOSIS 
X. THE LABORATORY DIAGNOSIS of these organisms is accomplished by: 
(a) Direct microscopic examination of the fresh excretions or secretions. 
(b) Preparation and examination of stained specimens, (c) Cultures, (d) 
Differentiation from’confusing objects. The exact methods in performing 
these procedures are given in Laboratory Methods, Chapter V. CHAPTER IV 
THE MASTIGOPHORA AND CILIATA INFECTING MAN. 
CIASSIFICATION 
CLASS: MASTIGOPHORA (FLAGELLATES) 
Subclass 
Order 
Genus 
Species 
Habltat 
Phytomas ti gina 
Euglenoidida 
Coproraonas 
C, subtilis 
Coprozoic 
Zoomastigina 
Pr o tomonadida 
. ■ 
Embadomonas 
Tricercomonas 
Chilomastix 
Trichomonas 
E, intestin- 
alis 
T, intestin- 
alis 
C. meanili 
T. hominis 
Infests mucous 
surfaces of man 
Infests mucous 
surfaces of man 
Infests mucous 
surfaces of man 
Infests mucous 
surfaces of man 
Bodo 
Leishmania 
Trypanosoma 
B. caudatus 
L. donovani 
L. tropica 
T, gambiense 
T. rhodesi- 
ense 
T. cruzi 
Coprozoic 
Blood stream and 
tissues of man 
Blood stream and 
tissues of man 
Blood stream and 
tissues of man 
Blood stream and 
tissues of man 
Blood stream and 
tissues of man 
Diplomonadida 
Giardia 
G• lamblia 
Infest small 
intestine of man 
CLASS 
CILIATA (CILIATES) 
Spirigera 
Heterotrichida 
Balantidium 
B, coli 
Infects large 
inte: of 
pig and man. 
THE MASTIGOPHORA (FLAGELLATES) 
The organisms of the class Mastigophora are characterized during the 
major part of their existence by the possession of single or multiple, long 
hair-like processes called flagella, -which they use for locomotion in the 
liquid media of their habitat. The flagellates may be conveniently divided into the following groups: (1) Those saprophytic upon the mucous surfaces 
of man; (2) Those contaminating the excretions of man after they have been 
eliminated from the' body (coprozoic flagellates); (3) Those infecting the 
blood stream and tissues of man. 
I. THE SAPROPHYTIC FLAGELLATES OF THE MUCOUS SURFACES OF MAN. 
1. Chilomastix mesnili- (Wenyon) Alexeieff, 1910. This organism 
is probably universal in its distribution. It is a harmless saprophyte in- 
festing the lumen of the large intestine of approximately four percent of 
the world’s human population. It is a flexible, pear-shaped organism, 10 M 
to 15 M in diameter, with the small, posterior end tapering into a fine 
caudal process or tail. The large, rounded, anterior end is penetrated by 
a spiral groove which extends about one-third the distance to the caudal 
extremity. At the bottom of this groove is found the cytostome. The nuc- 
leus is located in the anterior end and is round or oval in shape. Its chro- 
matin is often dumped on one section of the inner surface of the nuclear 
membrane. The blcpharoplasts are in apposition with the most anterior por- 
tion of the nuclear membrane and give rise to three anterior free flagella 
and one flagellum directed backwards and attached to the edge of the spiral 
groove. The movement of this flagellate is characteristic. It moves in a 
slow deliberate manner with slight oscillation in its direction and slow 
rotation of its body. Its transmission to new hosts is accomplished by con- 
tamination of food or water with resistant cysts passed in the feces of in- 
dividuals harboring this infestation. The cysts are shaped like a water- 
melon seed and they are about 10 Min size. For a graphic representation of 
the morphology of this organism, see figure 48, 2. Trichomonas hominis - Davaine, 1860. Synonyms: T. intes- 
tinal! s , T. vaginalis, T. elongata and T. buccalls. This is the most 
common saprophytic flagellate of man. The concensus of opinion is that 
it is a harmless saprophyte. It may infest the buccal cavity, the lumen 
of the colon or the lower portion of the genito-urinary tract. T. hominis 
varies from 5M to 20 Min size and is round or oval in shape. The nu- 
cleus is located in the anterior end and there are from three to five free 
flagella. One flagellum is directed backwards and is attached to the free 
margin of a linear undulating membrane which runs the full length ~f the 
organism. A special, clear, elastic, rod-like supporting structure (called 
the axostyle) runs longitudinally through the center of the organism and 
may extend beyond the posterior body limits forming a posterior spine. 
This flagellate has a fast nervous type of movement and is constantly dart- 
ing about. The body rotates rather rapidly and under oil immersion with 
reduced light or on darkfield examination, the undulating membrane is clear- 
ly visible. Cysts have never been demonstrated for this organism. See 
figure 49 for a graphic representation of the morphology of this organism. 3. Giardia lamblia - Stiles, 1915, This is the third most 
common saprophytic flagellate.of man. It inhabits the lumen of the upper 
portion of the small intestine and free living forms are never present 
in the stools except in the presence of a diarrhea. There is still con- 
siderable controversy prevalent in regard to the pathogenicity of this 
organism, but it is probably a harmless saprophyte. G. lamblia is pear- 
shaped, tapering to a point at the small posterior end, and it is about 
7 Mbyl4 M in size. The anterior portion of the ventral surface is con- 
cave and acts as a sucking disc for attachment to the intestinal mucosa. 
G. lamblia is bilaterally symmetrical, all the organelles and other struc- 
tures are paired or lie equally on both sides of the middle line. There 
are two oval, vesicular-shaped nuclei with central karyosemes which are 
sometimes rod-shaped or are in separate granu3.es. The nuclei lie in the 
anterior rounded portion of the organism, dorsal to the ventral sucker and 
are symmetrically located so that one nucleus is on each side of the mid- 
line. Two blepharoplasts are situated near the mid-line of the body just 
anterior and mesial to the nuclei. Two axostyles (strengthening 2 <ds) 
arise from the blepharoplasts and pass backward to the caudal end ,;f the 
organism. Four pairs of flagella arise along the mid-line of the ventral 
surface, the anterior pair cross each other before emergence from, the 
cytoplasm, A dark-staining, comma-shaped rod sometimes lies across the 
posterior part of the axostyles. 
The cysts are oval in shape, about 7 M by 10 M in size, 
and contain two to four nuclei and rudimentary axostyles plus comma- 
shaped bodies. There may be considerable variation in the content and 
relationship of the internal structures. See figure 50 for a graphic 
representation of the morphology of this organism. 4, Embadononas intestinalis - 'Kenyon and O’Connor, 1917. This 
is a rare, non—pathogenic intestinal flagellate that has occasionally- 
been found in human feces. It is a small (4 Mby9 M) very actively 
motile flagellate, pyriform in shape and when viewed from the side is 
said by some to be slipper-like in appearance. The cysts are very small 
(2.5 Mby 7 M) and are difficult to find. For morphology see figure 51. 5. Tricercomonas intestinalis - Wenyon and O’Connor, 1917. 
This is a rare, non-pathogenic intestinal flagellate of man found mostly 
in the Federal Malay States. It infests the lumen of the colon and is 
about 4Mbylo M in size. It is an extremely active flagellate with a 
very plastic, almost amoeboid body. For morphology see figure 51, above. 
• COPROZOIC FLAGELLATES 
The coprozoic flagellates are free living organisms that may acci- 
dentally contaminate fecal specimens after they have been passed. They 
usually possess contractile vacuoles and nuclei math a very large, cen- 
trally located karyosome containing practically all of the chromatin. 
3odo caudatus and Copromonas subtilis are the two most frequently encount- 
ered organisms in this group. For their morphology see figure 52. THE FLAGELLATES INFECTING THE BLOOD AND TISSUE OF MAN 
The flagellates infecting the blood and tissue of nan can be divided 
into the Leishmania and the; Trypanosomas. These flagellates undergo mor- 
phological changes during certain stages of their life cycle and in discus- 
sing the life histories of these parasites, it will be necessary to refer 
to a given stage by the name designated for it. For the sake of clarity, 
definitions of each stage are given below, and are illustrated in figure 
55, (1) Leishnania. The body is round or oval and the 
kinetoplast, which is made up of two structures, the parabasal body and 
the blepharoplast, lies near the centrally located nucleus. There is no 
free flagellum or undulating membrane. 
(2) Leptomonas. The body is elongated•oval in shape with 
the nucleus in the center and the kinetoplast in the anterior tip. There 
is a free flagellum, but no undulating membrane. 
(3) Crithidia. The body is identical with that of the 
leptomonas except that the kinetoplast is almost in apposition with the 
anterior surface of the nucleus. There is a short undulating membrane 
between the kinetoplast and the anterior tip of the organism. The flagel- 
lum runs from the blepharoplast along the free edge of the undulating 
membrane and may or may not become free at the anterior end. 
(4) Trypanosome. This is the same as the crithidia form, 
except that the kinetoplast is in the posterior tip of the organism and 
there is an undulating membrane running the full length of the body. The 
flagellum may or may not have a free anterior end. 
(5) Metaeye]ic forms. In insect vectors the trypanosomes 
undergo a cyclic development. The organisms that occur’ at the end of the 
cycle are identical with the blood trypanosomes, except that they are 
smaller. 
Polymorphism or monomorphism in referring to trypanosomes 
does not mean variation in shape or size of the body, but indicate whether 
or not forms in the blood stream that are not undergoing division ■.re uni- 
form in regard to the type of flagellum they possess. Thus, if both free 
and bound forms of flagella are present they are polymorphic, and if only 
one form is present, they are monomerphic. 
Multiplication is by means of longitudinal fission. The 
kinetoplast divides first, then the nucleus, and lastly the cytoplasm. 
I. LEISHI.IANIA 
These organisms produce a fairly common parasite infection of 
man throughout the tropical world. They have a life cycle in various 
vertebrate hosts and one in the Phlebotomas fly. The latter requires about 
ten days for its completion, after which tine, the fly’s salivary secre- 
tions are infected with the Leptononad forms of the parasite and when the 
fly feeds on a susceptible vertebrate, they probably are inoculated into 
the wound produced by the bite. Leishmania cause two types of infection 
in nan, one, the visceral form, is confined to the endothelial cells of 
the capillaries of the internal organs, and the other, the cutaneous form, 
is primarily located in the endothelial cells of the capillaries and lymph 
spaces of the skin. 
1, LEISHMANIA DONOVANI - LAVEEAN AND MESNIL, ISO 3. This organism 
causes visceral Leishmaniasis, known clinically as Kala Azar or Dum Dum 
fever. « a. Morphology 
In the vertebrate host L. donovani are small ovoid or 
round bodies with a vesicular nucleus and a kinetoplast, but never have a 
free flagellum. Their size varies from 1M to 5M in diameter. With 
Wright’s or other polychrome stain of the Romanowsky type, the cytoplasm 
stains a pale blue, the nucleus appears as several bright red granules, 
the parabasal body stains deep purple, and the axonene stains a faint pink. 
These tissue forms known as leishmania are practically always found within 
the cytoplasm of endothelial cells or macrophages. There may be as many 
as twenty of these parasites in one cell. In culture or in the invertebrate 
host, Phlebotomus papataci. the small leishmania forms elongate, develop a 
free flagellum and assume the leptomonad form. For a graphic representa- 
tion of the morphology of this organism and of L. tropica, see figures 54 
and 55. b. Laboratory diagnosis. 
See Laboratory Methods of the United States Array. 
2. LEISHMANIA TROPICA - WRIGHT, 1903. Synonym L. braz.liense. 
This organism causes cutaneous leishmaniasis, commonly known as Espundia, 
Oriental sore, Delhi boil or Bubas. 
Its morphology is identical with that of L, doncvani« In 
the cutaneous lesions it is found in the endothelial cells cr macrophages 
which are present in great numbers in the dilated tissue spaces of the in- 
flammatory lesion. The parasites may be quite scarce, but persistent 
search will usually reveal their presence in lesions typical for this dis- 
ease. 
a. Morphology. 
b. Laboratory diagnosis. 
See Laboratory Methods, Chapter V. 
11. THE TRYPANOSOMES. 
The trypanosomes are generally shaped like a sickle and are 
pointed at one or both ends. The convex margin has an undulating membrane, 
in the margin of which is imbedded the flagellum. The flagellum arises 
from a snail granule, the blepharoplast. near the posterior extremity of 
the organism. It then passes along the free margin of the undulating mem- 
brane and may emerge at the anterior end of the organism as a free flagel- 
lum of variable length. Just posterior to the blepharoplast, there is us- 
ually a dark, shining, round or rod-like body known as the parabasal body. 
The nucleus is usually round or oval and centrally located. The cytoplasm 
is usually clear, but nay contain granules of volutin which stain red with 
polychrome stains. Figure 56 shows diagrammatically a trypanosome and its 
internal structure. The trypanosomes of medical importance are: (1) T, gambiense 
and T. rhodesiense. the causative organisms of African sleeping sickness; 
(2) T, cruzi, the causative organism of Chagas1 disease or South American 
trypanosomiasis; (o) The confusing trypanosome, T, lewisi.- This is a 
universal natural infection in rats, transmitted by the rat flea and is 
non-pathogenic for man. However, in the event rats are used as laboratory 
animals for inoculation with T, cruzi. T, lewisi, if already present in 
the rats1 blood streams, may be confused with it. 
1. TRYPANOSOMA GAMBIENSE ~ DUTTON, 1902. 
This is the most common organism causing African sloeping 
sickness of man. 
a, Morphology. 
This trypanosome is characterized by marked polymorphism 
in the blood of mammals. It varies in length from 13 Mto 40 M; long forms 
•with a free flagellum and short forms with no free flagellum may be present 
in the same blood smear, plus all graduations between the two. The nucleus 
in T. gambiense is always centrally located. 
b. Mode of transmission. 
T, gambiense is transmitted to a new host through the in- 
vertebrate vector, the tsetse fly, the most important species being glos- 
sina palpalis. This transmission may be accomplished by: (l) Mechanically 
If the tsetse fly feeds first on an infected individual and then almost 
immediately upon a non-infected individual, the fly may inoculate fresh in- 
fected blood from one to the other. (2) After cyclic development. After 
G, palpalis has fed upon an infected case of sleeping sickness, the try- 
panosomes taken in the blood feed, multiply and undergo a cyclic develop- 
ment in its mid-gut. In a few days they pass forward along the gut and 
eventually gain entrance to, and infect the salivary glands. They are now 
crithidlal in form. In from twenty to thirty days, they develop into 
metacyclic trypanosomes and are now capable of being transmitted through 
the salivary secretions of the feeding fly to a new host. When once in- 
fected with T. gambiense, the tsetse fly remains so for life. 
2. TRYPANOSOMA RHODESTENSE, - STEPHENS AND FANTHEM, 19'0. This 
organism’s morphology is identical with that of T. gambiense, exc. u that 
in some of the short stumpy forms, without free flagella, the nucleus is 
situated in the posterior third of the body and may even be behind the 
parabasal body. Sleeping sickness due to T. rhodesiense is a much more 
severe infection than that due to T, gambiense. 
3. TRYPANOSOMA CRUZI - CHAGAS, 1909. This is a fairly common 
trypanosome infecting man in tropical -and subtropical South and Central 
America, It is an acute, fairly severe disease in children and is charac- 
terised by fever, thyroid, splenic and lymphatic glandular enlargement, 
followed by marked anemia and emaciation. In the adult, the infection us- 
ually runs a chronic course, with thyroid enlargement followed by symptoms 
of myxodema. The heart or any other organ may give rise to symptoms due to 
local invasion by this parasite. a. Morphology and life cycle in man. 
T. cruzi as seen in the peripheral blood is about 20 M 
in length. Its body tends to be shaped like the letter C. and the post- 
erior end is sharply pointed like a wedge. The parabasal body is very 
conspicuous and oval in shape. There is a free flagellum. The nucleus 
is oval in shape and centrally located. There may be some variation in 
the width of the cytoplasmic body depending upon the age of the trypano- 
some i young forms are narrower than the old ones. After a variable period 
in the blood stream, the mature trypanosome invades muscle fibers or other 
tissue cells, rounds up, becomes leishnania in form and multiplies by 
binary fission. This multiplication of the leishnania forms continues 
until there may be as many as twenty or more parasites in a group. They 
then develop a short flagellum, the body lengthens, the parabasal body 
moves posteriorly and the young trypanosome now invade the blood stream. 
There may be considerable local tissue damage and destruction due to the 
invasion of tissue cells by this parasite. 
b. Life cycle in the Reduvid. 
Probably the most important vector is Conorhinus mogistus. 
About six hours after the reduvid has fed upon an infected individual the 
ingested trypanosomes round up and assume Icishmania forms. These forms 
rapidly multiply as they pass backwards along the intestinal tract. In 
the hind-gut they again develop flagella and in about twenty-five to thirty 
hours become crithidial forms. These forms multiply in the lumen of the 
pighian tubules. The crithidial forms now develop into metacyclic trypano- 
somes and when the reduvid again feeds, he deposits a small drop of fluid 
from his rectum upon the skin of the individual fed upon. This fluid excre- 
ment may contain great numbers of the trypanosomes which are capable of 
passing through mucous membranes or by means of small abrasions into the 
blood stream, thus starting the disease. 
4. TRYPANOSOi.IA. LEWISI - KENT, 1880, 
This is the organism of rat trypanosomiasis. It is cosmo- 
politan in distribution and is not infectious for nan. It is only of sig- 
nificance when it is confused with T. cruzi infections in laboratory ani- 
mals. The distinguishing points morphologically are that the nucleus of 
T. lewisi is in the anterior third of the organism while that of T. cruzi 
is in the middle third, and the parabasal body in T. lewisi is rod-shaped 
while that of T, cruzi is oval. T. lewisi is transferred from rats to 
other laboratory animals with great difficulty, while T. cruzi transfers 
readily. 
5. DIFFERENTIATION OF SPECIES 
The main differential points arc shown in figure 57, 6. LABORATORY DIAGNOSIS 
This is made by demonstrating the trypanosomes in concentrat- 
ed or unconcentrated stained smears, dark field preparations or wet mounts 
of blood. In some types of trypanosome infections smears from lymph gland 
aspirations are most apt to contain the organisms. When the trypanosomes 
are too scarce to be demonstrated by microscopic technique, culture or in- 
oculation into laboratory animals should be resorted to. For technique in 
the above procedures see Laboratory Methods, Chapter V, 
THE CILIATA 
The members, of this class have short, hair-like processes called 
cilia covering their bodies, which they use for locomotion and the captures 
of food. There is only one species in this class that is pathogenic for 
man, namely Balantidium coll. 
I. BALANTIDIUM COLI - (MALMSTEN, 1857) STEIN, 1362 
B. coll is a rare parasite of the human colon, and a common one . 
of the colon of pigs. It is cosmopolitan in distribution. In man it is a 
true tissue parasite. It lives within the lumen of the large intestine' 
primarily, but invades the mucosa and produces large sloughing ulcers, in- 
distinguishable from those of Entamoeba histolytica. It acute cases it 
gives rise to an acute dysentery while, vague abdominal discomfort is a 
common symptom in chronic cases. 
1. MORPHOLOGY 
a. TROPHOZOITES 
This is the largest protozoon parasitizing man. It is oval 
in shape and its size varies from 60 to 220 M in its greatest diameter. 
At the anterior end is a depression called the peristome, which leads into 
the cytestone (mouth) and then the cytopharynx (pharynx). The body surface 
is covered with oblique, parallel row's of cilia. These cilia are greatest 
in number and length around the peristome. The cilia around the peristome- 
beat rythmically toward the opening, thus creating currents which aid in 
the capture and ingestion of food. The endoplasm contains numerous food 
vacuoles and two contractile vacuoles, one near the caudal extremity, the 
other about midway in the bod:/. Periodically in the live specimen those 
contractile vacuoles are seen to contract and expel their contents. At the 
rearmost point of the organism there is a small excretory opening, the 
cytopyge. Within the middle one-third of the endoplasm, there is a large 
oval or kidney-shaped, dark staining body, the macronuqleus, and near its 
concave side is a small typical protozoon nuclear structure with a large 
central karysome, the micronucleus. Reproduction is usually by transverse 
binary fission, although conjugation does occur in this species. Figure 
58 shows the organism and its internal structure, and also its appearance 
in a lymph sinus of the intestinal mucosa. b. CYSTS 
The cysts of this parasite are more or less spherical in 
shape, measure 50 to 60 M in diameter and appear like a rounded tropho- 
zoite with a translucent shell around it. Each cyst contains a single 
organism and when excystation takes place only one daughter B. coll is 
produced. 
c. TRANSMISSION 
Transmission of this parasitic infection from infected to 
non-infected individuals only takes place when food or drink is contamin- 
ated by fecal excrement containing the cysts of B. coli. The cysts are 
only formed in the fecal currents of the colon. IVhen living unencysted 
forms are passed in the feces, they die without undergoing encystation. 
If the living unencysted forms should be ingested, they would be Tilled 
by the acid, gastric juices. Thus ingestion of viable cysts is the only 
natural way that this infection can bo acquired. 
2. LABORATORY DIAGNOSIS 
In acute cases with a mucosanguineous diarrhea living tro- 
phozoites are found in feces. In chronic cases cysts are found in the 
feces, Eor technique see Laboratory Methods, Chapter V. CHAPTER V 
LABORATORY METHODS AND TECHNIQUE 
GENERAL METHODS USED IN THE DIAGNOSIS OF PROTOZOA 
INFECTING THE BLOOD AND TISSUE 
I. COLLECTION OF THE SPECIMEN 
1. PRECAUTIONS 
a. Tine. 
If it is possible, without detriment to the patient, select 
the most favorable time and method to find the suspected parasite in the 
clinical infection under consideration. 
b, Use all available methods and repeated examinations. 
If it is necessary, use all of the methods of examination 
available to establish a diagnosis, and do not be satisfied until repeated 
examinations at favorable times prove or disprove the clinical diagnosis. 
c. Type of slides to use. 
In making blood or tissue smears always use new slides or 
slides that are free from fogging or scratches. 
d. General cleanliness of slides. 
The slides should be clean and free from grease or good 
smears cannot be made. 
e. Chemical cleanliness of equipment 
Slides and other laboratory equipment used in handling 
blood and tissue prior to and during staining should be chemically clean. 
If they have been cleaned in acid cleaning solutions, be sure that they 
are washed in sufficient tap water followed by distilled water so that 
their reaction is neutral. 
2. UNSTAINED WET MOUNTS 
The fresh unstained wet mounts are prepared by mixing one small 
drop of the patient’s blood or aspirated tissue fluid with two drops of a 
0.85% sodium chloride solution in distilled water on a slide, then covering the material with a cover slip. There should not be so much fluid on the 
slide that the cover slip floats or it*will be impossible to keep objects 
in the microscopic field. The cells in the preparation should not be so 
thick as to override one another and red cells should not form rouleaux. 
If they do so, the preparation should be discarded and a new one made using 
a greater dilution of the patient’s blood. 
3. THIN SMEARS 
Thin smears for staining are prepared by securing bleed or 
tissue juice from the patient, and then making thin smears on a clean 
slide as if for a differential white blood cell count. . The smear should 
be so thin that the cells are in a single layer and do not override one 
another. When the smear is too thick, it is usually due to the use of too 
large a drop of the patient’s blood or to the streaking slide being held 
at an angle of less than 35 degrees when the smear was made. The smear 
should not be blotted but allowed to rapidly air dry without the use of 
heat. Do not bloTw the breath upon the smear to hasten drying; it vdll lake 
the red blood cells. If smears arc stained before they are dry, the cells 
will not properly fix, the stain will be precipitated and the result will 
be unsatisfactory. 
4. THICK SMEARS 
Thick smears for staining are made by securing a large drop of 
the patient’s blood on a clean glass slide, then spreading it by the use 
of a corner of another slide so that it covers an area about the size of 
a ten cent piece. The smear is then allowed to thoroughly air dry at 
room temperature or in 37° C, incubator. If the smear is not thoroughly 
dried before it is stained, the parasites will not stay on the slide. 
5. COMBINED THICK AND THIN SMEARS. 
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 one- 
half inch from the thick smear, and then streaking it towards the opposite 
end of the slide. Draw a line with a wax pencil between the two smears 
and they are now ready for staining. 
11. STAINS AND STAINING TECHNIQUE. 
1. PRECAUTIONS 
a. Quality of Stains. 
Only good stains of proven quality should be used. Each 
lot of stain should be carefully tested on known slides before use on un- 
knowns, In general, those that stain leucocytes and red blood cells satis- 
factorily will also stain blood and tissue protozoa. b. Methyl Alcohol. 
Methyl alcohol used in making up stains and in fixing 
smears should bo chemically pure end free from acetone. 
c. Distilled Water. 
Distilled water used in diluting stains and in washing 
stained smears should be neutral in reaction or buffered to a pH of 6.8, 
d. Staining Time, 
The staining time for each step in any given method should 
be varied to give the best final result for any given lot of stain. The 
time quoted for various steps in each stain of this type is the average 
time found most satisfactory at the Army Medical School. 
e. Hoat. 
After blood or tissue smears are stained, do not use heat 
to dry them, it will tend to fade the stain. 
f. Stock Stain. 
Great care.should be exercised in seeing that no water gets 
into the stock stains prior to use in the staining technique, or the stain 
will bo precipitated and rendered unfit for use. 
2. WRIGHTS STAIN AND STAINING TECHNIQUE. 
The stain is the same as that given for blood work in "Labora- 
tory Methods of the United States Army". The technique in staining for 
protozoa is as follows: 
a. The Thin Smear 
(1) Flood the thoroughly dried blood smear with the stain 
for one-half to one and half minutes. 
(2) Dilute the stain on the slide with an equal volume of 
neutral distilled water or enough of the water so that there is produced a 
metallic scum on top of the mixture. Allow this mixture to stand for 
three to five minutes. 
(5) Thoroughly wash the stained blood film with n.utral 
distilled water until it is light pink in color, then blot and allow to air 
dry. 
b, Thick Smears 
Thick smears are best stained by the Giemsa method. How- 
ever, it this stain is not available, they may be stained with 7/right’s 
stain, using the following method: (1) Immerse for 10 minutes the thoroughly dried thick 
smear in a solution made of: 
Formalin 5 cc. 
Acetic:acid 1 cc. 
Distilled water q.s.ad 100 cc. 
This solution fixes the parasites and white blood 
cells, but dissolves the red cells. 
(2) Remove the smear and wash thoroughly in tap water 
following with distilled water. 
(3) Allow slide to thoroughly dry and then stain by 
bright1s method as for a thin smear. 
c. Combined Thick and Thin Smears. 
Proceed as for a thick smear, but, be careful to immerse 
only the thick smear in the acidulated formaldehyde solution. If the 
thin smear comes in contact with this solution, the red cells will be dis- 
solved out and the smear will be useless. 
to stain by Wright*s method is usually to in- 
sufficient 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 
due to improper drying of blood films before starting the stain, introduc- 
tion of water into the stock stain, or too much evaporation of the alco- 
holic stain before dilution. Red cells stained blue, except for the oc- 
casional cells showing polychroraatoph'ilia, are either over-stained, (too 
much time allowed after diluting the stain) or have been insufficiently 
washed during the last stage of the staining process. 
3. DISKSA STAIN. 
This stain is as good as that of. Wright and in addition is 
more permanent. The good commercial stains are usually much more satis- 
factory than those made up in small amount in a laboratory. If it is 
necessary to make up this stain, the formula is as follows: 
Azur II eosin 
Azur II 
Glycerol, C. P., (Sp. gr. 1,26 and 
water content not over 1,5%) 
Methyl alcohol, C. P., acetone free 
0,3 grams 
0.8 grams 
23 cc. 
25 cc. 
Dissolve the Azur II and Azur II eosin in the glycerol which 
has previously been heated to 60° C. Now add the methyl alcohol and allow 
the mixture to stand for 12 hours, then filter and the stain is ready for 
use. The actual staining solution is made by the proportionate mixing of one 
drop of the above stock solution with one cubic centimeter of neutral dis- 
tilled wateri Distilled crater containing 1 cc. of 0,5$ sodium carbonate 
in each 100 cc. is probably the best diluent. Seventy-five cubic centi- 
meters of the diluted stain will stain about twenty-five slides. After 
the stain is diluted, it should be used as soon as possible as it deteri- 
orates and will not stain well after twelve hours. Staining technique by 
the Giemsa method is as follows: 
a. The Thin Smear. 
The thin smear must be fixed by immersion in mctlp l alcohol 
for five minutes. It is then removed, dried and placed in the diluted 
G-iemsa stain for thirty minutes if the stain is for malaria and forty-five 
to sixty minutes if it is for flagellates. Remove from the staining solu- 
tion, rinse with distilled water until the blood film is light pink in 
color, then stand it on end and allow to drain dry. ’When thoroughly dry, 
the smear is ready to be viewed. 
b. The Thick Smear 
The thick smear is stained in exactly the same way as the 
thin smear, except that it is not fixed in methyl alcohol prior to immer- 
sion in the staining solution. 
c. The Combined Thick and Thin Smear 
The combined thick and thin smear is stained exactly in 
the same way as the thin smear except that groat care must be taken to see 
that the methyl alcohol does not get on the thick smear. If it should do 
so, the red cells of the thick smear will not lake out and the thick blood 
smear will be useless for diagnostic purposes. 
4. PROPER STAINING. 
The criteria for a good blood or tissue stain when used to 
detect the presence of protozoan parasites are; 
a. The red cells should be pink to a copper color. 
b. The white blood cells should show clear cut nude a stain- 
ing with good contrast between the nucleus, cytoplasm and cytoplasmic gran- 
ules. 
c. The parasites should show light blue stained cytoplasm with 
bright red or violet chromatin and their morphological structure should bo 
clearly defined. 
SPECIAL LABORATORY METHODS USED IE THE DIAGNOSIS 
of protozoa infecting the blood and tissue. 
I. MALARIA. 
1. COLLECTION OF THE SPECIMEN. a, Time. 
The most favorable.tine to find malarial parasites in the 
blood stream in a clinical case of malaria is the period starting twelve 
hours after one chill up to one hour before the next chill. 
b. Effect of Quinine Thera 
Quinine therapy within four days prior to the taking of a 
sample of blood to be examined for malaria will usually make it very dif- 
ficult or impossible to demonstrate the parasites in the peripheral blood, 
except by the thick smear method. 
2. FRESH UNSTAINED WET MOUNTS. 
The fresh unstained wet mounts aro prepared as outlined under 
general methods. Except for plasraodium vivax. the recognition of malarial 
parasites in these mounts is quite difficult and requires experience. The 
pigment in the clear hyaline body of the amoeboid forms of P, viva:: moves 
quite rapidly and can be easily seen under the oil immersion objective, 
but P. malariae and P. falciparum parasites are less readily seen. For 
the above reason, this method of diagnosing malaria is generally unsatis- 
factory and should not be used except when stains are not available* 
3. THIN STAINED SHEARS. 
The preparation of thin stained smears as outlined under gen- 
eral methods, stained with Wright’s or Giemsa stain, is the method of 
choice in the routine diagnosis of malaria. 
4. THICK STAINED SMEARS AND COMBINED THICK AND THIN STAINED SMEARS. 
The thick stained smear and the combined thick and thin stained 
smears are prepared and stained by Giemsa or Wright*s method as outlined 
under general methods. They are the methods of choice in searching for 
malarial parasites in carriers, clinical cases that have very few para- 
sites in the peripheral blood and in malarial surveys. The appearance of 
the parasites in a stained thick preparation after the red cells have been 
laked out is quite different from that in stained thin preparations. They 
are quite typical, but the body of the parasite may be distorted in shape 
due to the destruction of the red blood cells. Therefore, where possible, 
the novice should not use this method until he or she has become thoroughly 
familiar with the malarial parasites and those confusing objects that are 
found in the thin stained smear, A convenient way of handling and staining 
large numbers of thick smears in a malarial survey is the one outlined by 
Barber and Kemp of the U. S. Public Health Service. "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 
one inch from one end of the slide and the other end is used for 1- baling. 
The slides are assembled in groups, a cardboard 1/16 to 1/8 inch thick 
and ij inches long, is inserted between the slides at the labelled ends 
and the whole fastened together by means of a stout rubber band. The entire 
block may non be stained and dried as a single unit. Figure 59 illustrates 
such a block. CULTURAL METHODS 
The cultivation of the plasmodia of malaria by the methods 
now available is of no practical value in the diagnosis of this infection. 
Its chief use at present is to demonstrate, for teaching purposes, the 
forms of P. falciparum that are usually only found in the capillaries 
of the internal organs. If the technique of malarial culture is desired 
it may be found in the Journal of Experimental Medicine, XVI, 567, 1312. 
6. INOCULATION OF LABORATORY ANIMALS. 
There are no laboratory animals that are susceptible to human 
malaria. 
7. OBJECTS THAT MY BE CONFUSED WITH MALARIA 
Objects that may be confused with malaria in the stained blood 
smears when -superimposed on a red cell are: Blood platelets and precipi- 
tated stain, . Sometimes abnormal red cells showing young nucleatec' forms 
or basophilic degeneration may also lead to confusion. Malarial parasites 
have a definite body shape, internal arrangement and characteristic stain- 
ing properties which, when once•familiar with them, should load to easy 
differentiation from other objects. 
8. LABORATORY DIAGNOSIS. 
The laboratory diagnosis of malaria infection in man consists 
of finding and recognizing the malarial parasites, and their type, ■when 
present in the blood preparations made from suspected malarial fever 
patients. In examining preparations made for malaria the oil immersion 
objective should be used. Malarial parasites are best seen when the light 
coming through the substage is slightly reduced. The proper amount of 
light may be obtained by moving the slide until a blood platelet is center- 
ed in the field, then adjust the substage so that the maximum definition 
of its morphological detail is obtained. In searching for the parasites 
the slide should be covered in an orderly manner moving back and forth 
over the smear so as not to repeat any field previously examined. Never 
make a diagnosis on the first parasite found, cover enough of the slide 
so that if two species of malarial parasites arc present you will find them. 
If in doubt about any single abnormal parasite found, remember that where 
there is one malarial parasite there arc bound to be more .and careful 
search will usually reveal an easily recognizable form. Early diagnosis 
and treatment is of utmost importance if a favorable prognosis is to be 
expected in P. falciparum infections, owing to its tendency to produce 
early and unexpected cerebral complications. Therefore, if P. falciparum 
infections are found in the area in which you are working and you have a 
blood smear in -which you are certain there are malarial parasites, but you 
are unable to determine the type, report the case as positive for ilaria 
so that treatment may be started. Then make- additional smears and careful- 
ly study to determine the type. 11. TRYPANOSOMES 
1. COLLECTION OF THE SPECIMEN. 
a. Time 
The most favorable time to find these organisms in a 
suspected infection is during periods of pyrexia. 
h. Blood. 
In most cases of trypanosomiasis there are usually very 
few organisms present in the blood stream; therefore, it is necessary to 
use concentration methods as well as direct preparations in searching for 
those parasites. Such a concentration method is as follows; 
. A sterile venipuncture is done and 10 cc. of blood are 
withdrawn and immediately mixed with Uo cc. of sterile warm (37° C.) dis- 
tilled water in a5O cc. sterile centrifuge tube. The tube is agitated and 
as soon as hemolysis is complete it is centrifuged at 2500 revolutions per 
minute, or over, for 5 minutes. Then decant the supernatant fluid and oil 
the trypanosomes present in the sample of blood will be found in the resi- 
due which can then be examined microscopically of if it has been kept ster- 
ile, used to inoculate culture media. 
c. Lymph Gland Puncture. 
This method is the one most apt to reveal positive results 
in the diagnosis of trypanosomiasis. Puncture one of the enlarged lymph 
glands under aseptic technique with a Luer syringe equipped with a 19 gauge 
needle and aspirate some of the gland substance. The aspirated gland 
material may then be examined by microscopic, cultural or animal inocula- 
tion methods. 
d. Spinal Fluid Examination, 
Spinal fluid examination is probably only of value in 
African sleeping sickness. Due to the small number of trypanosomes present 
in the spinal fluid it is usually necessary to centrifuge the specimen and 
then examine the residue so obtained to, demonstrate their presence. 
e. Tissue, 
T. cruzi is the only trypanosome that forms a nidus and 
propagates in tissue. The pathological examination of biopsy material 
from localized swellings or lymph glands in suspected cases of this dis- 
ease may reveal its presence. In autopsy material, heart, thyroid tissue, 
brain and bone marrow are most apt to harbor this parasite. 
2, SLIDE PREPARATIONS. 
Fresh unstained wet mounts, thin stained smears and thick stained smears are prepared as outlined under general methods. 
3. CULTURE IN LABORATORY' MEDIA. 
Concentrated laked blood, aspirated contents of lymph glands, 
spinal fluid or biopsy tissue from cases of trypanosomiasis when inocu- 
lated into suitable media may yield cultures positive for trypanosomes. 
The various species are cultured as follows: 
a. T. Cruzi. 
(l) One of the best media upon which to culture T. cruzi 
is that originated by Novy, MacNeal, and Nicolle (N. M. N. medium) and 
is made as follows; 
14 Grams. 
6 grams. 
900 cc. 
Agar 
Sodium chloride 
Distilled water 
Mix and dissolve by means of heat, then tube in 6 cc. amounts and auto- 
clave for 30 minutes under 20 pounds steam pressure. Remove the steriliz- 
ed tubed nedia and allow it to cool to Ug° C. Then under aseptic conditions 
add 2 cc, of sterile defibrinated rabbitfs blood to each tube, mix well 
and slant. Slanted tubes should be placed in the ice box to cool and 
harden so that they will have the maximum amount of water of condensation. 
When cool the cotton plugs in each tube should be covered with a rubber 
cap to prevent evaporation of the water from the media. The tubes should 
be tested for sterility by incubation at 37°0. for 2k hours before they 
are used. Trypanosomes will not grow in the presence of bacterial con- 
tamination. 
(2) Inoculate the suspected material into the water of 
condensation of N. M. N. medium and incubate at 25° C., examining every 
Ug hours for 10 days. Leishmania forms, crithidial and trypanosome forms 
ordinarily found in the reduvid will appear in the culture. Subculture, 
however, is quite difficult. 
b, T. Gamblense and T. Rhodesiense. 
T. gambiense and T, rhodesiense are difficult to culture 
but nay grow on the above N. M. N. medium if ifo glucose is added and the 
culture is incubated at 32° C. The forms that develop in the culture will 
be the crithidial and metacyclic forms ordinarily found in the tsetse fly. 
>4. INOCULATION QP LABORATORY ANIMALS. 
Inoculation of laboratory animals is used to establish a diag- 
nosis when other methods fail. Great care should be used in selecting the 
proper susceptible animal as given in the differential chart on trypanosomes 
and ruling out the possibility of a natural infection existing at the time of inoculation. Material obtained as outlined under the collection of 
specimens is inoculated intraperitoneally into the animal selected. 
5. LABORATORY DIAGNOSIS 
The laboratory diagnosis is arrived at by studying the material 
gathered according to the above outline and correlating it with the dif- 
ferential table plus life histories of the organisms. The presence of 
autoagglutination in apatientfs blood is suggestive of trypanosomiasis. 
111. LLISE/iuJUSIS. 
1. COLLECTION OF THE SPECIMEN 
a. Blood. 
Leishmania are rarely found in the circulating blood and 
when present they are found in the macrophages and polymorphonuclear 
leucocytes. 
b. Liver Puncture. 
When visceral leishmaniasis can not be diagnosed by blood 
examinations, this is the method of choice, and of all methods it ir most 
likely to yield positive results. This procedure is not without dagger, 
however, but can usually be done safely as follows; The excursions of the 
diaphragm are limited by putting on a tight abdominal binder. Then, cau- 
tioning the patient not to move, under a local skin anesthetic, insert a 
2 inch, 16 gauge Luer needle attached to a 10 cc. Luer syringe containing 
2 cc. of saline, through the skin just above the Bth rib in the right mid- 
axillary line. Keep the needle and syringe at right angles to the skin and 
after the needle has penetrated 1 inch inject the 2 cc. of saline and then 
insert the needle to its full length. Then aspirate a small amount of 
material and withdraw the syringe. The aspirated contents may then be used 
to make smears for staining or for inoculation of culture media. 
c. Spleen Puncture. 
Securing material for the diagnosis of visceral leishman- 
iasis in this manner is attended with great danger of hemorrhage. However 
if necessary to make the diagnosis through this procedure, it is performed 
in much the s/me manner as liver puncture. 
d. Skin Lesions. 
Material for diagnosing infection with L. tropica is ob- 
tained by scraping the edge of the cutaneous ulcers or using ft Luer syringe 
with a 19 gauge needle to aspirate some of the contents of a skin nodule. 
This material is then made into thin smears for staining. Material from 
skin lesions is usually too heavily contaminated by bacteria to bo success- 
fully cultured. 2. CULTURE ON LABORATORY MEDIA. 
Culture on laboratory media is carried out by inoculating 
material obtained by the above procedures into the water of condensation 
of a slant- of N. M. N. medium which is then incubated at 25vJ C. The 
material inoculated must be free from bacterial contamination or leish- 
mania will not grew in the culture. Within 3 to 10 days in successful 
culture, the water of condensation will contain numerous rosettes of 
leptomonad forms of the leishmania which may be demonstrated by wet fresh 
mounts of stained preparations. 
3. INOCULATION 0? LABORATORY ANIMALS. 
Localized cutaneous infections can sometimes be produced by 
inoculating material infected with leisbraania into the skin of dogs, cats, 
rats, mice, guinea pigs, or monkeys. The hamster, however, is the animal 
of choice and infection with leishmania can be produced readily in it. 
k. THE ALDEHYDE TEST. 
This test is performed by adding two drops of commercial 
(375) formalin to 1 cc. of serum from the suspected case. If the infec- 
tion is due to L. donovanl, the serum will jell and look like the cooked 
white of an egg. This’test, however, is not specific, for it may give a 
positive result in some cases of tuberculosis, leprosy or malaria. 
5. LABORATORY DIAGNOSIS. 
The laboratory diagnosis of L. donovani or L. tropica i made 
by finding these organisms in the leucocyted and endothelial cells 
stained smears, in cultures or in the lesions of inoculated laboratory 
animals. GENERAL METHODS USED IN THE DIAGNOSIS OF THE PROTOZOAN PARASITES 
AND SAPROPHYTES OF THE MUCOUS SURFACES OF MAN 
I. COLLECTION OF THE SPECIMEN. 
1. PRECAUTIONS. 
a. Collecting the Specimen 
The specimen should he directly collected in clean covered 
receptacles (hed pans, swabs in test tubes containing i cc. of physiological 
saline, syringes, bottles or dropper), preferably sterilized by heat. These 
receptacles should not be sterilized by chemical disinfectants as protozoa 
in the vegetative stage are easily killed and quickly undergo autolysis in 
the presence of only small amounts of such chemicals. If the receptacles 
are not properly cleaned and sterilized there is always the possibility of 
introducing free-living protozoa into the specimen and thus confusing the 
picture 
b. Handling the Specimen Prior to Examination. 
(l) Specimens for Examination Locally 
These specimens should he kept in the original re- 
ceptacle used for collection until examined. The material should he kept 
at a temperature of 37° C. as all vegetative organisms in this group are 
quite sensitive to chilling and are also rapidly killed hy temperatures of 
U5°C. or higher and hy drying. Therefore, when delivered for diagnosis, 
the specimen should have its original moisture content or examination for 
these organisms will he useless. 
(2) Specimens for Shipment. 
shipment without previous fixation. Cysts, found in fecos, are the only 
forms that can be satisfactorily shipped in the fresh state, The cysts 
should be concentrated and washed with tap water until quite free from fo- 
cel material as they rapidly die in the feces as passed, but will live 
from twenty to two hundred and fifty days, depending on the temperature, in 
the washed state. 
(a) Vegetative organisms will not stand routine 
(h) Concentration and Fixation of Mucous Surface 
Protozoal Specimens Containing Trophozoites Preparatory to Shipment. 
-1- Fecal specimens. Thoroughly emulsify 20cc. 
of feces in 200 cc. of w arm (37° C.) physiological saline in a settling flaSc. 
or tall narrow cylinder, allow to stand for five minutes and then decant the 
supernatant fluid into two 50 cc, centrifuge tubes. Centrifuge the material 
at 1850 EPM for five minutes,then decant off the supernatant fluid, save 
the precipitated residue of one tube for fresh examination and to t.o tube 
containing the other precipitate add 25 cc. of fresh Schaudinn’s fixing solution. Thoroughly mix the precipitate and the fixative and allow the 
mixture to stand for at least one hour, preferably hours. 
-2- Cultures and other liquid specimens. 
Pipette the fluid containing the organisms directly into a 50 cc. contrifuge 
tube and then centrifuge and fix the material as indicated above. 
-3- After the one specimen has been fixed by 
Schaudinn’s solution the required length of time, the solution should be 
removed from the specimen by first diluting it with an equal volume of dis- 
tilled water, then centrifuge the mixture so obtained at 1850 PPM for 5 
minutes and decant off the supernatant fluid. Nov/ add 50 cc. of 95f° ethyl 
alcohol to the precipitated residue and it will then be ready for shipment 
in a suitable container. Ship the fixed and unfixed specimens obtained as 
above to the corps area or other central laboratory for diagnosis. 
c. Time of Examining the Specimen After Collection. 
All specimens should be examined at the earliest possible 
opportunity after collection. Those types of protozoa rapidly degenerate 
and the possibility of a positive diagnosis is inversely proportional to 
the elapsed time after collection of the specimen. 
d. Thd Necessity for Multiple Specimens and Examination. 
In patients infected with protozoa of this type, the 
number of organisms present on an infected surface at any given time de- 
pends upon the type of pathology produced, the bacterial flora present and 
the resistance of the host. Because of this multiplicity of governing 
factors, protozoa usually appear in showers; they may be present in great 
numbers one day and relatively scarce the next. Therefore, to arrive' at 
anything like a satisfactory diagnosis in questionable cases, repeated ex- 
aminations, possibly under different conditions, should be made on con- 
secutive days. 
2. SPECIMENS FROM THE BUCCAL CAVITY AND THE GSNITO-URINARY TRACT. 
These are best collected directly from the lesi ns by using 
-swabs moistened with physiological saline, or by aspirating some of the 
mucous secretions by means of a syringe or dropper. This material is then 
immediatly examined microscopically or by culture. 
3. SPECIMENS FROM THE CASTRO- INTE ST INAL TRACT. 
a. Precautions. 
(1) It is practically impossible to find protozoa in a 
stool after an oil cathartic, e.g. castor oil, mineral oil, or following a 
barium meal or enema. Therefore, in the event any of the above has "been used 
examination for protozoa in the feces should be delayed for at least ~f2 hours 
(2) The portions of a stool most nicely to contain para- 
sitic protozoa are those showing blood or mucus. In formed stools, r nail 
flecks of mucus or mucus and blooc> can always be found on the surface of the specimen. In semiformed or liquid stools if the specimen'is exam- 
ined carefully, mucus and bleed can also be found. 
~(3) Specimens collected by means of an enema are us- 
ually unsatisfactory. 
b. How and When to Collect the Specimen 
Feces should be passed directly into a thoroughly clean, 
warmed bed pan and immediately taken to the laboratory for examination. 
The best time to secure a specimen is at about 9 A.M. At this time the 
patient, due to the stimulation of peristalsis by breakfast, will be able 
to cooperate best and the laboratory personnel should be able to give the 
specimen immediate attention. 
£• Stage of Found in Different Types of Stools. 
Formed stools usually contain cysts or precystic protozoa 
only. Semiformed and liquid stools will ordinarily contain only ve eta- 
tive forms and it is thses types of stools that best afford an opportunity 
to diagnose a protozoan infection. If the patient is not already passing 
such a stool, and it is necessary to rule' cut the possibility of a para- 
sitic protozoan infection, give a saline cathartic and collect the second 
or third liquid stool passed for examination. 
d. Concentration Methods, 
This method is only applicable to cysts for which it is 
the procedure of choice. For all practical purposes only formed or semi- 
formed stools will contain cysts and if they can not be demonstrated in 
the unconcentrated specimen the following procedure should be used. 
Select a portion of feces about the size of a walnut and thoroughly emul- 
sify it by means of a tongue depressor in 200 cc. of distilled water con- 
tained in a graduate or settling flask. Allow this material to stand for 
30 minutes to give the heavier particles time to settle out; now decant 
the supernatant fluid into a liter graduate or,flask and add enough dis- 
tilled water to fill the container. The diluted specimen is then placed 
in the refrigerator overnight to allow the cysts to settle to the bottom. 
The following morning decant or siphon off the supernatant fluid and dis- 
card it. The residue is then examined directly for cysts or it may be 
washed one or more times and further concentrated by the following meth d. 
Transfer the residue to a 50 cc. centrifuge tube and dilute with enough 
distilled water to fill the tube, thoroughly mix and then centrifuge the 
speci ipn for two minutes at 2000 RPM. The supernatant fluid is tnen decant- 
ed and’ cysts if present will be found in the precipitated residue. 
e_, Proct oscoo ic Bxaainat i on. 
If the pathologic?.! lesions are in the rectum or oigmoid 
specimens may he obtained by means of the protoscope or sigmoidoscope and 
examined immediately. These are more apt to yield protozoa than the passed 
feces. However, because of the attending discomfort and perhaps pain to the patient, this method should be used only after it has been demonstrated 
that the feces are negative. 
11. PREPARATION OF THE SPECIMEN FOR MICROSCOPIC EXAMINATION. 
1. THE WET FRESH UNSTAINED SPECIMEN. 
a. Feces and Mucus Specimens. 
Warm a clean slide so that it feels comfortable when 
touched to ?the hack of the hand. Then secure a small piece of mucus or 
mucus and blood from the fresh specimen by means of a wire loop or wooden 
applicator and thoroughly emulsify it in one drop of physiological saline 
on the middle of the slide. Now take a clean number one cover slip between 
the thumb and forefinger of the right hand, contact the slide with one edge 
of the cover slip near the drop but not touching it, pusn the slip a Long 
the surface of the slide until its edge contacts the drop, rock it slightly 
from side to side to allow a portion of the fluid to come under the edge of 
the slip, then let the cover slip drop from between the fingers allowing it 
to fall on the side. The fluid portion of the drop on the slide will then 
automatically be drawn by capillary attraction under the slip, while the 
solid particles will be excluded. This method insures a thin even prepara- 
tion of not too great a density (when newsprint is viewed through it the 
words are legible) and insures oven apposition of the cover slip to the 
slide. The preparation is now ready for examination, but if it is to be 
kept on a worm stage for any period of time it should be ringed with vase- 
line. 
b. Culture and Concentrated Cyst Specimens. 
Culture and concentrated cyst specimens are prepared for 
microscopic examination exactly as above except that because of their fluid 
nature they are not mixed with physiological saline prior to applying the 
cover slip. 
staining) 
2. THE STAINED PREPARATION (See technique under fixation and 
IH. FIXATION AND FIXING TECHNIQUE. 
1. PRECAUTIONS. 
a. Type of Fixatives. 
A fixative should he selected that has approximately the 
same surface tension as the material to he fixed, or it will he difficult 
to keep the organisms on the slide, e.g. protozoa should not he fixed in 
solutions that have more than hofo alcoholic content. 
b. Time Needed for Fixation. 
Always allow the specimen to remain in the fixative for at least the minimum time quoted. It is possible to secure some fixation 
in less time, hut the increase in the quality of stains produced after 
adequate fixation more than justifies the additional time allotted to this 
step. 
c. Condition of Fixative. 
The fixative should always hefresh and the number of 
slide preparations fixed in a given amount limited, i. e., seventy-five 
cubic centimeters of fixative will only fix satisfactorily about twenty- 
five slide preparations. 
d. Condition of Specimen to be Fixed. 
The specimen to be fixed should be fresh and the organisms 
should not show any evidence of degeneration. If the specimen is old and 
the organisms are degenerated, good fixation and staining will be impossible. 
2. FIXATIVES. 
a, Schaudinnf s So lut ion! 
Mercuric chloride, HgCl0, C.F., saturated solution 
in physiological saline 65 cc. 
Ethyl alcohol, 95/° 35 cc. 
Acetic acid, glacial, C.P. 5 cc. 
The glacial acetic acid and the ethyl alcohol should 
he added to the hichloride of mercury solution just before use. 
b, Fomol Sublimate Solution: 
Mercuric chloride, saturated solution in physio- 
logical saline 90 cc. 
Formaldehyde (Jlfo) 10 cc. 
3, FIXATION TECHNIQUE. 
a, Flagellates, dilates and cysts. 
If the material to he fixed is not in the liquid state, 
mix enough physiological saline with it to form a thin watery solution. 
Now ruh well - into one half of the surface of a clear, slide one small drop 
of fresh egg white or normal horse serum, than put one large drop of the 
specimen on this surface and spread it-evenly. Allow the preparation to 
dry until the fluid portion of the specimen will no longer run when the 
slide is tilted, then drop the slide, film slide down, into 90 cc. cf cno 
of the above fixing solutions contained in one half of a Petri dish. 
After ten minutes turn the slide over or place it in a Coplin jar three- 
fourths full of the same fixative and allow it to remain for one to two 
hours. ■ ■ ~ b. Amoeba Trouhozoites. 
•.v • , Slide preparations for fixing and staining these organisms 
are made in the same way as under (a) except that as soon as the drop.of 
material is placed on the slide a cover slip is dropped on it. The prepar- 
ation is then allowed to set for three minutes and is then placed in a 
Coplin jar. The fixative is then carefully added and is allowed to act for 
2 hours. After, fixation the cover slips are removed from each specimen and 
the slides are then ready to he carried through the routine of staining. 
This method of putting the cover slip on the preparation makes it possible 
to inspect the slide for amoebae prior to fixation and in addition is a 
great help in keeping the amoebae on the slide during fixation. 
c. Preserving Specimens after Fixation. 
The preparations after fixation should be rinsed in tap 
water and then placed in fOfo ethyl alcohol; they can either be preserved for 
future staining in 951° ethyl alcohol, or stained immediately. 
IV. STAINS AND STAINING TECHNIQUE. 
1. PRECAUTIONS. 
a. Stains. 
A good quality of hematoxylin should be procured and when 
made up it should be thoroughly ripened before use. Test the stains on a 
known specimen before using them on an unknown.. 
b. Age of Material after Fixation 
Specimens should be stained as soon as practicable after 
fixation because fixed specimens gradually lose their ability to take the 
stain. 
c. Drying of the Specimen. 
The specimen should never be allowed to dry at any stage 
in the technique as it causes the organisms to shrink and become distorted 
in shape. 
d. Washing after Destaining. 
If the dostaining agent is not thoroughly washed out of 
the specimen it will fade the stain. 
c. Dehydration of the Specimen for Permanent Stains 
If the specimen is not properly dehydrated before clearing 
in xylol, the xylol will become milky and the slide when viewed microscop- 
ically after mounting in balsam will appe.yr blurred. 2. THE lODINE STAIN. 
This is a temporary stain used for quick diagnosis only and 
it must "be made up fresh every 10 days or it will not stain properly. 
a. Formula: 
lodine 
Potassium lodide 
Distilled water 
Glacial acetic acid 
0,05 gram 
0.10 " 
100 cc, 
1.0 " 
b. Technique for Trophozoites 
This method is principally of value in studying am ebae. 
After findingtrophozoites and studying them in the fresh wet unstained 
mount the nuclear details can be clearly visualized and studied micro- 
scopically if the cover slip is raised at one edge and a drop of the iodine 
stain is thoroughly mixed with the contents under the slip. 
c. Technique for Cysts. 
Mix on a clean slide one drop of the iodine stain with 
one drop of the material containing cysts, apply the cover slip as ex- 
plained under fresh wet unstained mounts and then examine microscopically. 
3. HEMATOXYLIN STAINS. 
a. Heidenhain*s Iron Hematoxylin. 
This is the best stain for protozoa. 
Hematoxylin 1 gram 
Ethyl Alcohol, 95$ 10 cc,. 
Distilled water 90 cc. 
Thymol 1 crystal 
Dissolve the hematoxylin in the alcohol, add the distilled 
water and thymol, then allow to ripen in a clear glass-stoppered "bottle ex- 
posed to the sun for one month. 
b. Har ri s1 Hemat oxylin. 
This is a good stain and is the one rn&st readily prepared. 
Hematoxylin 1 gram 
Ethyl alcohol, 951° 10 cc. 
Dissolve the hematoxylin in the alcohol. 
Alum (ammonium or potassium) 20 grains 
Distilled water ' 200 cc. 
Dissolve the alum in the water by the aid of heat, and add 
the hematoxylin solution. Bring the raisturo to a boil as rapidly as pos- 
sible, and then add one-half gram of yellow oxide of mercury. The solution at once assumes a dark purple color. As soon as this occurs, remove the 
vessel containing the solution from the flame and cool rapidly by plung- 
ing; into a basin of ice water. As soon as the solution is cool it is 
ready for use. The addition to the stain of glacial acetic acid to a con- 
centration of 4 per cent is supposed to increase the precision of the 
nuclear staining. 
c. Mayer * s Hemalum, 
This is a fairly good 
and reliable stain. 
Hematoxylin 
Distilled water 
Sodium iodate, C.P, 
Potassium alum, C.P. 
1 gram 
1000 cc. 
0,2 gra, 
50 Grams 
Dissolve the hematoxylin in the distilled water, add the 
solium iodate and the potassium alum, then allow to ripen in the sun for 
one month in a clear glass-stopnered bottle. Filter the stain before using. 
U. HEMATOXYLIN STAINING TECHNIQUE 
a. Slide Method. 
The fixed and washed slide specimens prepared as outlined 
previously are carried through the successive steps in staining as follows: 
(1) Wash 10 inutes in ~JQijo ethyl alcohol. 
(2) Wash.lo minutes in 70$ ethyl alcohol to which has 
been added sufficient iodine stain to produce a light mahogany color. 
(3) • Wash 10 minutes in ‘JQffo ethyl alcohol, 
(h) Mordant in the following solution(2U hours if us- 
in Heidenhain1s stain, 1 hour if using Harris’ or Mayer's stain), 
Ir on alum (ferric ammonium sulphate), 
kfo aqueous solution 1 part 
Ethyl Alcohol, 10 parts 
.(5) Wash for 5 minutes in ]CP/o ethyl alcohol. 
(6) Stain for 1 hour if using Harris' or Mayer's hemalum 
and for hours if using Heidenhain's iron hematoxylin. 
(7) Remove from stain and wash in two changes of tap water 
(8) Destain in the following: 
Iron alum, 2to kjb aqueous solution 1 part 
Ethyl alcohol, 50$ 10 parts 
Differentiate by agitating each slide separately in the 
above solution until a light grey to blue tinge predominates; control the 
exact point by observing the staining definition of the rganism under the 
microscope every'few minutes. 
(9) Rinse in tap water, then wash for 10 minutes each in 
three separate dishes 'of 70f> ethyl alcohol. 
(10) Begin dehydration by two changes of 95$* ethyl Icohol 
for 5 minutes each. (11) Complete dehydration by two changes of absolute 
ethyl, alcohol for 10 minutes each. 
(12) Replace the absolute alcohol in the specimen by 
two changes of xylol for 10 minutes each. 
(13) Mount in Canada balsam. 
b. Method of Staining Protozoa in Bulk. 
In the staining of mucous surface protozoa of man there 
are almost as many variations in staining technique as there are workers 
in the field. The chief difficulties encountered in fill of the better 
techniqo.es of staining are: (l) keeping the organisms on the slide or 
cover slip during fixation and staining, (2) keeping the organisms free 
from distortion and clear from the debris so that their internal structures 
are not obscured, (3) carrying out proper differentiation of the internal 
structures of the organisms at the time of destaining, (U) having a suf- 
ficient number of -well stained organisms on the slide after staining so 
that a diagnosis will not have to be made on a few more or less typical 
organisms. 
The following staining method not only satisfies the 
above requirements, but also affords a method of concentration, thereby 
making it possible to secure satisfactory stains-, from specimens contain- 
ing very few organisms, that could not be stained by other methods. The 
method of staining differs from other standard hematoxylin staining methods 
only in that the organisms are not fixed and stained on slides or cover 
slips, but are fixed and stained in bulk. The organisms are concert 
trated and then carried through the ■ steps of fixation and staining in 50 
centrifuge tubes. Very few of the organisms present in the origins . speci- 
men are lost during fixation, staining and mounting* The organisms are 
natural and. life like and are not distorted by the reagents or manipulations 
used. 
(l) Precautions, 
(a) There should bo no delay in concentration, ex- 
amination and fixation of trophozoites after they have been secured from 
the patient. Needless to say degenerated organisms will not take a sat- 
isfactory stain. It is not necessary to be so cautious in the immediate 
fixation of cysts, but as a rule the quality of protozoan staining pos- 
sible to secure, is inversely proportionate to the elapsed time after 
securing the organisms from the host or culture, and their subsequent 
fixation. 
(b) Destaining must be checked by frequent micro- 
scopic observations of the organisms being differentiated. Care should 
be exercised to carry the dostaining to the point that there is sharp dif- 
ferential detail between the nuclear structures and the cytoplasm. The 
common tendency with this technique is not to carry the destaining far 
enough. The organisms should bo checked for structural detail after the acid destain has been neutralized and if there has been insufficient de- 
staging they should be carried back into, acid alcohol and further de- 
stained until the desired degree of differentiation has been secured. 
(c) The stained material must be properly dehy- 
drated before clearing in xylol. 
(d) Discard the supernatant fluid decanted off af- 
ter centrifuging, in each step of the procedure, or if the alcohols and 
stains are to be used over again, filter through a Berkefield N filter as 
there is danger of carrying organisms in used alcohols and stains to the 
next case subsequently stained. 
(e) The material being stained may be left for 
2U to Ug hours additional time in any step of the staining procedure with 
the exception of the fixing solution and the acid alcohol destaining re- 
agent. This allows the staining procedure to be carried out without inter- 
fering with the routine work in a laboratory. 
(2) Concentration and Fixation. 
(a) Fecal specimens. Thoroughly emulsify 20 cc, of 
feces in 200 cc. of wain (37° C.) physiological saline in a settlirgflask 
or tall narrow cylinder, allow to stand for five minutes and then decant 
the supernatant fluid into two 30 cc. centrifuge tubes. Centrifuge the 
material at 1850 EPM for five minutes, then decant off the supernatant 
fluid, save the precipitated residue of one tube for fresh examination 
and to the tube containing the other precipitate add 25 cc. of fresh 
Schaudinn’s fixing solution. Thoroughly mix the precipitate and the fix- 
ative and allow the mixture to stand for at least one .hour, preferably 
2U hours. 
(h) Cultures and other liquid specimens. Pipette 
the fluid containing the organisms directly into a 50 cc. centrifuge tuhe 
and then centrifuge and fix the material as indicated above. 
(3) Staining. 
(Between each step in the subsequent procedure the 
material is centrifuged at ISSO RFM for 5 minutes, then the supernatant 
fluid is decanted off and the next solution added to the precipitated 
residue which is then thoroughly emulsified by rotation or stirring.) 
(a) Wash tho fixed material two times with distilled 
water. 
(b) Wash 10 minutes with ethyl alcohol (contain- 
ing enough Gram’s iodine to give it a light brown color.) 
(c) Wash 10 minutes with ethyl alcohol (d) Stain by adding Harris’s or Delafield's hema- 
toxylin for from 1 to 2k hours. 
(e) Wash with tap water. 
(f) Destain by adding about 20 cc. of acid alcohol 
(lfo HCI in 70fo ethyl alcohol) to the stained precipitate in the centri- 
fuge tube. Mix the precipitate and the destaining solution and occasion- 
ally stir the mixture with a wooden applicator stick. From time to time 
check the progress of destaining by taking one drop of the mixture, plac- 
ing it on a slide, applying a cover slip and then observing the progress 
of nuclear differentiation of the organisms under the high dry power of 
the microscope. The organisms will be fairly easy to find in the average 
case and destaining should be allowed to go on until the cytoplas... is 
practically colorless and the nucleus stands cut sharp and clear. As scon 
as the required definition has been obtained, add sufficient ammonia water 
(5 drops in 50 cc. of distilled water) to neutralize the acid alco- 
hol and turn the solution bright blue. 
(g) Wash with tap water. 
(h) Dehydrate with: ~Jojo ethyl alcohol 10 mi utes 
95$ II it it M 
95$ " 11 11 " 
absolute " 11 n 11 
absolute 11 " " n 
(i) Clear in xylol. 
(j) Thoroughly emulsify the stained cleared mixture 
in a minimum of xylol, breaking up all small clumps, then add sufficient 
thick Canada balsam to make a syrupy mixture. Then without further cen- 
trifugation place one drop of the balsam containing a suspension of the 
stained organisms on a clean slide, apply a cover slip and allow to dry. 
Permanent class room sets and multiple perma- 
nent mounts for file and consultation can be easily prepared by making 
more than one preparation in the final step. Stained material suspended 
in balsam can be kept in bottles and mounted on slides whenever desired. 
V. CULTURAL METHODS 
1. MEDIA 
a. The Boeck-Drbohlav Medium (R.S.S. Medium) 
The Boeck-Drbohlav medium is the one most generally used 
for the protozoa found on the mucous surfaces of man. In this medium all 
of the amoebae end mucous surface flagellates found in man, with the excep- 
tion of Giardia lambiia, will survive and multiply and the individual char- acteristics are "better preserved than'in any other medium. This medium is 
prepared from: 
(1) Eggs 
(2) Sterile Ringer's solution (Autoclaved at 15 lbs. 
pressure for 20 minutes and then allowed to cool) 
Sodium chloride 8.0 grams 
Potassium chloride 0.2 " 
Calcium chloride 0.2 " 
Distilled water 1000 cc. 
(3) Sterile horse serum (Must not contain tricresol or 
other preservatives). 
8.0 grams 
Four whole eggs are emulsified in 50 cc. of the sterile 
Ringer's solution. Place k cc. of this mixture into each test tube and 
sterilize in on autoclave as follows: Turn the steam into the outer cham- 
ber of the autoclave until the jacket is hot, then plane the tubes in a 
slanting position in the sterilizing chamber, close the door and be sure 
that the vacuum exhaust valve is also closed. Turn the steam into both 
chambers of the autoclave and open the outside exhaust valve. At the first 
appearance of steam from this valve close it and allow the pressure to 
climb to 15 lbs., at which time shut off the steam and (caution) allow the 
pressure to decline to 0 of its own accord bef ore removing the media. Re- 
peat this procedure on three successive days, storing the media at room 
temperature between sterilizations. Prior to use, U cc. of the liquid por- 
tion of the medium, consisting of sterile horse serum one part and Ringer's 
solution eight parts, are added. The addition of sterile whole wheat pow- 
der at the time of inoculation as outlined under St. John's medium will 
sometimes increase the rate of growth of E. histolytica. 
b. St. John's Wheat Broth Medium, 
This medium is of differential value in the cultivation 
of amoebae in that only E. histolytica can maintain growth in it. However, 
many strains of E. histolytica can not be cultivated in it. The medium is 
made as follows; 
(1) Powdered heart muscle 1 gram 
(2) Locket's solution 1000 cc. 
Sodium chloride 8.00 grams 
Calcium chloride 0.20 " 
Potassium chloride 0.20 11 
Magnesium chloride 0.10 " 
Sodium di-hydrogen phosphate 
(NaHgPOlj.) ' 0.10 " 
Distilled water 1000 cc. 
Sodium bicarbonate O.UO grams 
Mix the above ingredients and boil in a double boil r for 
one hour, filter through medium filter paper and tube in 10 cc. amounts. 
Then sterilize in an autoclave at 15 lbs. pressure for 15 minutes. (3) Whole wheat flour is ground in a mortar until it 
will pass through a 50 mesh sieve (50 wires to the inch), then tube four 
gram amounts and sterilize in an autoclave at 15 pounds pressure for 15 
minutes, followed by drying in a hot air oven at SS°C. for twenty-four 
hours, 
(*4) Just prior to inoculating this medium, take up 
0.05 cc. of the sterile whole wheat powder in a clean, sterile, dry, wide 
bore one cc. pipette and discharge it into the liquid medium by tapping 
the pipette against the inside wrall of the tube. 
(5) When amoebae cultured in this medium show signs 
of degeneration or cyst formation, it is necessary to add 0.5 cc. of 
sterile horse serum to each culture to revive them. 
2. CULTURE TECHNIQUE. 
Precaution, 
Protozoa of this type are quite sensitive to changes in 
the bacterial flora in vitro; they quickly die in the presence of certain 
bacteria. Therefore, sterile precautions in sofar as is possible, should 
always be used in this technique. 
b. Routine Cultures. 
Using an applicator or wire loop inoculate a portion of 
the specimen about the size of a pea, consisting of fresh mucus, mucus 
and blood, or concentrated cysts, into the liquid portion of the me ium 
and thoroughly emulsify it there. Incubate at 3?J*C. 2nd examine at the 
end of 2*4 and *4g hours. Flagellates growing in the medium will be found 
throughout the liquid portion while amoebae will be found in the very 
bottom portion of the liquid fraction. The specimen from the culture to 
bo examined for amoebae is obtained by introducing a clean sterile 1 cc. 
pipette, with the index finger held tightly over the upper end, into the 
culture so that the tip is at the extreme bottom portion of the liquid 
medium, then gently release the finger pressure allowing only 1/10 cc. 
of the material in contact with the bottom to run into the pipette. 
Resume the. finger pressure on the upper end of the pipette and then with- 
draw it from the culture. The material in the pipette can then be used 
to transfer the culture, make fresh wet unstained or stained preparations 
If it is desired to carry on the positive cultures they should be routine- 
ly transferred every *4-8 hours. However, the survival of these organisms 
in culture depends a great deal upon the bacterial flora present. One 
in which E. coli predominates is usually favorable and one in which spore 
bearers, Pseudomonas scruginosa, or Proteus vulgaris predominate is us- 
ually unfavorable. In the latter case transfers should be made at 2*4 
hour periods. 
Cultures for amoebae that are not positive at the end 
of Ug hours should be transferred and further examined as follows; Allow the culture to remain in the incubator. 2 hours, then without unduly dis- 
turbing it, remove all but o*s cc. of the supernatant fluid by means of 
a sterile pipette equipped with a rubber, nipple. Wash the slant with 
the remaining fluid and then transfer itoto new medium. The resulting 
culture should then be re-examined at 24 and 48 hour intervals before 
calling it negative. 
c. Method of Ridding Amoebic Cultures of Blastocyst is hominis. 
(l) Lactic Acid Medium. 
Prepare the following modified Locke’s solution and 
sterilize it in an autoclave at 15 pound pressure for 15 minutes. 
Sodium chloride 3,0 grams 
Calcium chloride 0.24 grams 
Potassium chloride 0.42 grams 
Sodium "bicarbonate 0.20 grams 
Lactic acid, 1,0 N. 0.23 cc. 
Distilled water q.s.ad. 1000.00 cc. 
With sterile precautions, mix 1 part of sterile horse 
serum with 7 parts of the cool, sterile Locke’s solution. Then add 5 cc. 
of the resulting solution to previously prepared sterile egg slants, i.e., 
the solid medium prepared for Boe ck-Drbohlav medium. 
(2) Amoebic cultures containing Blastocyst is hominis are 
treated as follows: Add sufficient sterile 1 to 10,000 neutral acrifla- 
vine to Ringer’s solution to the supernatant fluid of an amoebic culture 
on Boeck-Drbohlav medium to make a 1 to 50.000 solution. Incubate at 
37° C. for 24 hours, transfer the culture, repeat the process and then 
transfer the culture to lactic acid medium. Transfers of the lactic acid 
medium cultures are made at 24 hour -intervals for three successive times 
and then the culture is transferred to straight Boe ck-Drbohlav medium. 
There will still be some Blastocystis hominis present in the culture, but 
the organisms will usually ail be in a degenerated condition and will 
completely disappear upon successive transfers of the culture. In case 
they do not, the entire procedure is repeated. 
VI.LABORATORY ANIMALS. 
Laboratory animals are of no value as a diagnostic procedure is 
this group However, in the case of E, histolytica, the virulence of this 
organism can be tested by injecting cultures or freshly isolated tropho- 
zoites into the rectum of a young kitten by means of small catheter nd 
syringe. An acute fatal dysentery is usually produced. SPECIAL METHODS-USED IN THE DIAGNOSIS OF THE PROTOZOAN PARASITES 
AND SAPROPHYTES FOUND ON THE MUCOUS SURFACES OF MAN. 
X. THE AMOEBAE’. . 
1. FRESH UNSTAINED TCST MOUNT. 
The wet fresh unstained mount prepared as outlined under gen- 
eral methods is examined systematically covering the slide without repeat- 
ing the microscopic fields, Amoebae in the live state have a particular 
frefractive, translucent, granular character which easily differentiates 
them from other objects in the specimen. This property can be exaggerated 
and will greatly aid in picking out the amoebae when the slide is being 
rapidly examined if the high power objective of the microscope is focused 
slightly above the objects in the preparation. If the observer is well 
trained, amoebae may be recognized under the low power objective and then a 
switch made to the high power objective to determine the detailed character- 
istics. If the outline below is followed in studying amoebae it will be 
found to be of great aid in species determination. 
DIFFERENTIAL POINTS TO OBSERVE IN AMOEBAE IN FRESH SPECIMENS 
TO DETERMINE THEIR SPECIES 
1. Size and color. 
2, Differentiation of ectoplasm and endoplasm. 
3. Granularity of endoplasm and presence of cell inclusions. 
Nucleus - visibility, location when in motion, size, 
(active 
5. Motility, type (sluggish 
(progressive or non-progressive 
(single (clear 
6.’ Pseudopodia ( or ( or 
(multiple (granular 
(slow 
7. Flowing of endoplasm into pseudopod ( or 
(explosive 
(red blood cells in endoplasm 
g, Presence or absence of (bacteria in endoplasm 
9, Presence of (food vacuoles 
(contractile vacuoles 
10. Presence and characteristics of chromatoid bodies. (growth on R, E. S. 
11. Cultural characteristics (and St. John’s Medium 
(on successive transfer 
AFTER ADDING LUGOL’S SOLUTION TO MOUNT 
1. Nucleus - size, shape, distribution of chromatin, and size and unifom- 
ity of granules, location of karyoscme. 
2. Presence of glycogen bodies. 
3. Cysts, if present - character of, size, number of nuclei and type, 
visibility, glycogen bodies. 
2. STAINED SPECIMENS. 
Stained specimens should be made of all amoebae where species 
can not be recognized readily in the fresh unstained state. 
3. CULTURES, 
Cultures properly made will increase the number of positive 
findings and allow further study in determining the true species of an 
amoeba. 
U. CONCENTRATION FOR CYSTS. 
Concentration methods for cysts should be done routinely on 
all fecal specimens that are examined. 
5. THE COMPI iB/T@T -FIXATI ON IE ST. 
At the present time this test is still in the experimental 
stage in its development. It probably will become of practical diagnostic 
significance when a true E. histolytica antigen can be developed. The 
preparation of such an antigen requires that the organisms be grown in 
pure culture in the absence of bacteria, and to date this has not been 
accomplished, or at least has not been reported in the literature. 
6. CHARCOT-LEYDSN CRYSTALS. 
Charcot-Leyden crystals are indicative of chronic inflammation 
and are often found in the feces. 
7. DIFFERENTIATION OF AMOEBIC AMD BACILLARY DYSENTERY 
Differentiation of amoebic and bacillary dysentery can pre- 
sumptively be done by the general character of the fecal exudate. However, 
one may complicate the other and amoebic dysentery in the presence of 
severe bacterial secondary invasion of the ulcers may have many of the 
characters of a bacillary dysentery. Therefore, every effort should be ex- 
erted to demonstrate the causative organism in each case. The main differ- 
ential points are shown in Table Sf• TABLE 87 
DIFFERENTIAL DIAGNOSIS TABLE* ON THE FECAL EXUDATES 
IN BACILLARY AND AMOEBIC DYSENTERY 
Exudate 
Bacillary Dysentery 
Amoebic Dysentery 
Blood 
Varying amounts 
Small amounts to actual 
hemorrhage 
Pblymorphoneut ro- 
philes 
About 90$ of exudate. 
Many show nuclear de- 
generation (ringing). 
Cytoplasm frequently 
contains fat, 
Few, Cytoplasm of some of 
those present shows degen- 
erative changes and in such 
the nuclei may appear 
pyknotic. 
Endothelial 
macrophages 
Present in varying 
numbe r s. Actively 
phagocytic, frequently 
contain erythrocytes and 
leucocytes. Undergo 
toxic degeneration; 
"ghost cells". 
Not seen except in cases 
also having bacterial 
dysentery. 
Plasma cells 
Present, relatively more 
abundant early. 
Present in small numbers. 
Pyknotic Bodies 
Proportionately insignif- 
icant, but are found. 
Constitute about 80fo 
of cellular elements. 
E. histolytica 
trophozoites 
Absent unless the two 
diseases are both present 
Present and must be 
found to make diagnosis. 
Amount of exudate 
actual hemorrhage 
excluded 
Massive, a large part 
of the stool. 
Small. 
Bacterial content 
Low 
Very high, usually. 
* The Cytological Diagnosis of Dysenteric Conditions and its Application 
in the Military Service. Major George R. Callender, M.0., Military 
Surgeon, June, 1925» g. CONFUSING OBJECTS. 
a. Animal Tissue Cells. 
Animal tissue cells derived from the host or ingested as 
food may at times appear like amoebae, but carefulexamination of teem will 
easily establish their true nature. Macrophages may be found containing 
phagocytized red blood cells, but examination reveals their typical metazoan 
nucleus and as a rule amoeboid movement is never observed, epithelial cells 
are pale in color and have metazoan characteristics that easily differen- 
tiate them. 
b. Vegetable cells. 
Vegetable cells such as starch granules, pollen granules, 
yeast cells or other cells of this type have a certain definiteness of 
outline and structure which should lead to no confusion. However, yeast 
cells, such as Blastocystis horninis, may be temporarily confused with 
cysts of amoebae. The presence of budding forms and the particular struc- 
ture as illustrated in Figure 59 should cause no difficulty in differen- 
tiating them. 11. P3oA.GELLA.TES AND BALANTIDIUM COLI. 
1. WET FRESH UNSTAINED MOUNT. 
The wet fresh unstained mount prepared as outlined under 
general m thods is the usual method of diagnosing these organisms. The 
movement, shape, size and structure of these organisms usually makes it 
easy to readily determine their species. 
2. STAINED PREPARAT lONS. 
Preparations stained by Harris' or Heidenhain's method are 
used for detailed study of the morphological characteristics. 
3. CULTURES. 
Cultures in R.E.S. medium will sometimes be positive for 
these organisms when they can not bo found in the fresh specimen. {Balan- 
tidium coli is cultured with difficulty, but these organisms are so largo 
that they can hardly be overlooked;for this reason culture is usually 
unn cuc:ty. 
U. CONTUSING OBJECTS. 
Confusing objects are mostly accidental contaminating free- 
living protozoa which are readily differentiated by the typo of nucleus 
and. presence of contractile vacuoles. For details see discussion of these 
organisms. REFERENCE BOOKS 
1. Blacklock, D. B. find Southwell, T,, A Guide to Human Parasitology, 
Baltimore, the Williams and Wilkins Co., 1932. 
2. Boyd, Mark F., An Introduction to Malariology, Cambridge, Massachu- 
setts, Harvard University Press, 1930* 
3. Calkins, Gary N., Biology of the Protozoa, Philadelphia, Lea and 
Fehiger Co., 1926. 
U. Craig, Charles F., The Malarial Fevers, New York, William Wood and 
Co.. 1909. 
5. Craig, Charles F., Laboratory Methods of the United States Army, 
Third Edition, Philadelphia, Lea and Fehiger Co., 1929. 
6. Craig, Charles F,, Parasitic Protozoa of Man, Philadelphia, J. B. 
Lippincott Co,, 1926, 
7. Dobell, Clifford and O’Connor, F. W,, The Intestinal Protozoa of Man, 
New York, William Wood and Co., 1921. 
2. Hegner, Robert and Taliaferro, W. H., Human Protozoology, New rk, 
The MacMillan Co., 192U, 
9. Manson-Bahr, Philip H., Manson’s Tropical Disease, Ninth Edition, 
New York, William Wood and Co., 1931. 
10, Rogers, Sir Leonard and Mdgaw, John W. D., Tropical Medicine, Phila- 
delphia, P, Blakiston’s Son and Co., Inc., 1930* 
11. Stitt, E. R,, The Diagnostics and Treatment of Tropical Diseases, 
Fifth Edition, Philadelphia, P. Blakiston’s Son and Co., 1929. 
12. Thompson, John Gordon and Robertson, Andrew, Protozoology, New York, 
William Wood and Co., 1929* 
13. Wenyon, C. M, Protozoology, New York, William Wood and Co,, 1926, HELMINTHOLOGY 
Phylum Platyhelminthes, Claus, Igyi* 
Class I Cestoda 
The Cestodes or tapeworms are endoparasitic and the adult worms are 
found exclusively in the intestinal tract of the host. The adults of this 
class are flat ribbon-like, segmented'worms with relatively small heads 
as compared to the size of their mature tody segments. The heads hear 
sucking organs for attachment to the mucosa of the host's intestine. In 
addition, the head may have a rostellum which in some species is armed 
with characteristic booklets. The posterior part of the head of the adult 
worm is a proliferating area, continuously supplying young segments. A 
digestive system is entirely absent, nutrition being carried on by direct 
absorption. All of the worms of this class are hermaphroditic (they 
contain both male and female generative organs,). The ova usually contain 
a mass of cells among which arc six minute hooks. This indefinitely 
defined embryo is called an oncosphere or hexacanth embryo. When the 
mature ova are ingested by a suitable intermediate host, the embryos are 
liberated in the gastro-intestinal tract and in most species invade the 
tissue of the host undergoing a period of development and in certain of 
the species propagation as well. When the tissue of the intermediate 
host containing the mature embryo is ingested by the definitive host the 
embryo develops into the adult worm, A single species of animal may be 
the definitive and intermediate host for some of the worms of this class. 
Of the tapeworms found in man, D. latus requires three hosts, H, nana re- 
quires only one, and all the rest require two. It is of greatest impor- 
tance to remember that the eggs of H, nana, T. solium, and E. granulosa 
are infective to man. When he swallows H. nana eggs, both the larva and 
subsequently the adult worm develop in him; when he swallows T. solium 
or E. granulosa eggs, only the larval forms, Cysticereus cellulosae or 
Cysticercus granulosa develops, but not the adult. The larval stages of 
E. granulosa give rise to hydatid disease while that of T. solium gives 
rise to Cysticercus cellulosa cysts in the brain, eye, muscle, liver, etc. 
of man. 
Definition of terms. 
1. The head of any tapeworm, whether adult or'larva, is called the 
scolex. 
2. If present, the terminal disc-like or finger-like anterior 
extremity of the head is called the Tostellun. 
3. The entire body of a tapeworm is called a strobila. 
U. A proglottid is a segment. 
5. Proglottides in which the reproductive organs aro not developed 
or only partly, are said to he immature. Those in which they 
are fully developed and functioning are called mature, while 
those with a uterus containing eggs are referred to as gravid. 
The proglottides nearest to the head are always immature, those 
midway in the body aro usually mature while those making up the 
posterior extremity aro gravid. b. The genital pore is the external opening of the genital system 
of a proglottid. It may be single or double and is situated on the later- 
al sides or center of the ventral surfa.ee. If its location is lateral, 
they may be all on the same side of succeeding proglottides or roughly 
alternate sides. 
7'.' Oncosphere is the term used in designating the embryo within 
the egg which has 'within its cellular -mass six miniature booklets. 
S. The ventral surface of a proglottid is that nearest to which the 
ovary lies. In a majority of the species, this can be determined only by 
making sections, however, if the genital pore is centrally located, it is 
on the ventral side. 
9. Calcareous corpuscles are highly refractile bodies often spheri- 
cal, composed of carbonate of lime, and measuring from 5 to 25 u. They 
are found on or near the surfaoe of the proglottides and scolex. 
10, Vitelline glands are glands which supply the nutritive substance 
or yolk in the formation of an egg. 
11, Shell gland. Originally this gland was thought to be the organ 
supplying the shell for,the eggs. It is now known that this is nt the 
function of this gland. Its real function is not known. 
12. Sparganum. This is a terra applied to all Dibothrio-cephalid 
larvae (plerocercoids), the,adults of. which arc not known. 
Key to Genera of adult ccstodes usually found infecting man. 
1. Large worms with segments each having one genital pore situated 
on one of the lateral margins Taenia. 
2, Large worms with segments each having one genital pore situated 
centrally on flat surface Dibothriocephalus 
3, Small worms with segments up to 5 nan# in length and 3 mm. in 
width, each having two genital pores, one on each lateral 
margin Dipylidiura. 
k. Small worms, minute, thread-like, segmentation only distinct 
under low power magnification, segments each having one genital pore 
situated on one of the lateral margins Hymenolepsis 
Key to species of usual adult cestodes infecting man. 
1. Tapeworms belonging to Genus Taenia, uterus with from sight to 
ten main lateral branches on one side Taenia solium. 
Uterus with lU to }0 main lateral branches cn one 
side Taenia saginata. 
2. Tapeworms "belonging to Genus Dibothriocephalus. Gravid segments 
as broad as long, uterus appears as a brownish patch, rosette-like in 
arrangement in the center of each mature segment.. Dibothriocephalus latus, •3‘ Tapeworms belonging to Genus Dipylidium Dipylidium caninum. 
U, Tapeworms 'belonging to Go mis Hymenopelis - Segments microscopic, 
one genital pore on each segment and all pores on the same side of the 
worm, rostellum of scclsx armed with single row of booklets, length of 
entire worm 25 to Uo ram Hymenolepsis nana. 
5. Tapeworms belonging to Genus Hymenolopesis - rostellum of scolex 
unarmed, length of entire worm 20 to 60 cm. mature and gravid segments 
0.8 to 0.9 , wide Hymenolepsis diminuta. 
Dibothriocephalus latus (Linnaeus, 1758) 
This worm is commonly known as the fish tapeworm due to the fact that 
infestation is contracted by eating raw, salted, pickled or insufficiently 
cooked fresh water fish which have been previously infected by the para- 
site, Man,..dog, cat, and f*x may act as definite hosts. This parasite is 
quite prevalent in the area about the Baltic Sea in Europe, also in Russia 
and Japan. A local focus has been established in the fresh water fishes 
of the lakes of Minnesota and the adjacent region in Canada by immigrants 
principally from Finland, Korway, and Sweden. This tapeworm is very long 
lived (up to. lU years) and sometimes produces a severe anemia not unlike 
that found in pernicious anemia. The anemia is thought to be produced by 
hemolysins liberated from the decomposing gravid segments. It not only 
affects the circulating red blood cells but the blood forming bone marrow 
as well. There is usually a multiple infection in man. The eggs of this 
species are operculated and are more commonly found in the feces than 
those of taenia due to the fact that they are more readily liberated from 
the uterus. 
Morphology. 
The worm is usually 2 to 4 meters in length and its maximum width 
is 1 to 2 cm. The terminal segments are broader than they are long, or 
are nearly square. There may be as many as UOOO segments in the mature 
worm. The gravid segments tend to break off, not singly but in short 
chains. The head or scolex is olive-shaped and has two grooves (b ;thria) 
located one on each side. There are no acetabulae or suckers, nor is a 
rostellum present as is found in the Taenia. The genital pore is located 
centrally on the flat ventral surface and when viewed microscopically is 
divided into 3 sections, the anterior opening being that of the vas de- 
ferens, the middle one the vagina, and the posterior one the uterus. The 
testes are very numerous and are scattered throughout the superficial 
layers of the segment. They cannot be distinguished from the vitalline 
glands without differential staining. The uterus arises as a straight 
tube, thin coils to a rosette form, finally opening in the genital pore. 
In the >ravid segments it appears as a brown, centrally located mass. 
The eggs are operculated, yellow to golden brown in color, and measure 
about 70 to U5 M. They can thus be differentiated from the operculated 
fluke eggs of Fasciolopsis buski and Fasciola hepatica which are about 
150 M. in greatest diameter. The eggs, when passed in the feces, contain 
an unsegraented ovum surrounded by yolk. When the feces containing the 
eggs is diluted with water a hexacanth embryo develops in from 3 to 5 weeks depending on the temperature. Under these conditions, cilia appear 
and entirely cover the embryo or embryophore as it is designated. 
The operculum opens and the ciliated embryophore enclosing the onco- 
sphere, escapes and is then called a coracidiura. The coracidium swims 
about freely by means of its cilia and unless it is ingested by its first 
host, a crustacean Cyclops streniun or Dioptomus gracilis, it dies. 
Within the crustacean host, the cilia are lost and the larvae migrate into 
the coelomic cavity where it undergoes further development and then is 
called a procercoid. After a period of 9 to 10 weeks, it' is 600 to fOO M, 
in length and is now infective for certain species of fresh water fish 
(pike, perch, grayling, etc.) if they should ingest the infected crusta- 
cean. The procercoid larva is liberated from the crustacean by the 
digestive juices of the fish and then migrates through the intestinal 
wall of the fish, coining to rest in the fat of the mesentery or in the 
muscle tissue. It is now called a plerocercoid, and after a period of 
development it reaches a size of l.§ cm. in length‘and 2 mm, in width. 
It is grayish white in color and ribbon-like in shape. It is not 
destroyed by ordinary salting, pickling, or smoking. 
It is infective for man, dot:, or wolf, and if poorly smoked, raw or 
ins-officiently cooked fish are eaten, the plerocerc id larva is liberated 
in man’s gastro-intestinal tract and attaches itself to the mucosa and is 
now a true scolex, It gives rise to a chain of segments forming the 
mature worm. During the early developmental stages it sometimes increases 
in length by as many as 30 segments daily. Within a period of 3 t S , 
weeks from the time of ingestion, mature segments and ova will be passed 
in the feces. 
Laboratory diagnosis finding of characteristic operculated ova 
or proglottids in the feces. There is usually a relative eosinophilia 
(8 to 15$). Anemia with an increased color index may or may not be found. 
Sparganum proliferum (Ijima, 1905)* Stile, 1908 
This larval parasite has been found in certain districts of Japan 
and in Florida. The adult form is unknown, but the plerocercoid larval 
forms occur in large number in cysts in the subcutaneous tissues of man. 
These cysts measure about 3 to 12 mm, in length by 300 M. in width. The 
larva are narrower anteriorly but do not contain grooves as they are not 
fully developed. These plerocercoid larvae proliferate by budding so 
that in the older cysts there may be as many as seven larva present. 
The cysts feel like rice grains or shot in the subcutaneous tissue and 
give rise to a severe acne-like condition, with oedema of the surrounding 
tissue and regional lymph gland enlargement. Occasionally deeper 
structures are involved by the cysts. 
Laboratory Diagnosis. Biopsy with isolation of the plerocercoid 
larva 'from the cysts which, if observed, are usually motile. 
Taenia saginata (G-oeze, 1758.) 
This is the beef tapeworm and is the most common large tapeworm of 
man. It is world wide in its distribution and is contracted by th 
ingestion of insufficiently cooked beef which contains the live Cysticercus bovis (larva). Any person harboring this parasite in his 
intestinal tract is soon aware of its presence due to the fact that the 
gravid proglottids become detached and are motile. They sometimes migrate 
out of the rectum singly or rarely in short chains and are then discovered 
in the under clothing. If passed in the feces they usually show motility 
and will be seen making undulating, progressive serpentine movements about 
the fecal mass. They apparently are seeking to leave the fecal mass as 
far as possible before disintegrating and liberating the mature ova. 
This characteristic would enhance the possibilities of their being 
ingested by cattle due to their distribution over a wider area. The 
motile gravid segments are sometimes mistaken for flukes but careful 
examination readily establishes their true identity. 
Morphology. 
The worm usually contains more than 1000 segments and measures 3 to 
U meters in length. The strobilla tends to be pleomorphic. There is 
considerable variation in size and shape of comparable proglottids and 
at times of the scolices between different individual parasites of this 
species. The scolex is about 1.5 to 2 mm. in diameter and does not con- 
tain a rostellum or booklets. Four round to oval acetabulae or suckers 
arc symmetrically located about the head and serve as the sole organs of 
attachment. The neck is about one half the width of the head and twice 
as long. The gravid proglottids are about 5 to 7 mm, in breadth by 20 
ram. in length. The genital pores are lateral in position and roughly 
alternate sides. The ovary has only two lobes and the uterus in the 
gravid proglottids is an elongated central longitudinal tube with iU to 
30 compound dendritic lateral branches on each side. In such segments 
the other reproductive organs have atrophied and the remaining structure 
is nothing more than an egg sack. The eggs are ordinarily indistinguish- 
able from those of T, solium but if the embryonic membrane is still 
present it has two delicate polar processes which are not found on similar 
ova of T. solium. The eggs are 30 to 38 M. in diameter and are usually 
pale buff in color. The outer membrane or shell is a thick walled struc- 
ture perforated with minute pores which on examination under the 
microscope give the impression of radial striae. Within the shell is a 
fully developed embryo (onchosphere) with throe pair of clearly distin- 
guishable booklets. 
Life history. 
Man is thought to be the only definitive host of this parasite. 
When the eggs or gravid proglottids are passed in the feces of man and 
subsequently contaminate forage, water or pasture land, the ova may be 
ingested by cattle or in rare instances by closely related herbivorous 
animals, (buffalo, giraffe, and llama). Within the gastro-intestinal 
tract of its intermediate host the larva emerges from the shell of the 
egg and penetrates the intestinal wall, gains entrance to the lymphatics 
or blood stream and then finds its way to the connective tissue areas in 
striated muscle. The most common side of localisation is the pterygoid, 
tenderloin, tongue and heart muscles'. When localised in the connective 
tissue of muscle the larva develops into a cysticercus (bovis) which is 
commonly called a bladder worm. The larva is an egg shaped milky white 
object, frequently translucent and measures 7 to 10 mm, in breadth by 
4 to 8 ram, in length. Within the bladder is an invaginated miniature scolex characteristic of this species. In infected beef the Cysticercus 
bovis larva can easily be seen; between the fibers of the muscle proper, 
near the tendinous insertion or near the fascial planes. Cattle that 
become infected with this tapeworm larva, if not reinfected, will destroy 
the larva within a period of one year. The only observable remnant of 
the larvae after that time is a calcified or caseous mass. 
If the live Cysticercus bovis larvae are ingested by man, (this is 
frequently the case when rare beef that has not been carefully inspected 
is eaten),* the scolex evaginates itself when the cystecercal wall is 
digested away and attaches itself to the gut wall where it begins to 
grow. Eight to 12 weeks from the time of attachment gravid proglottids 
and ova are being passed in the feces. 
*U. S. inspected beef is a safe product to use in that the inspections 
are carefully made and in addition storage under refrigerated conditions 
with the temperature low enough and the time long enough to kill any of 
the larvae that may have been missed by the inspection. Storage, fresh 
or pickled,under refrigeration, 30 E,, for 3 weeks, or Is' E, or lower 
for 6 days. 
Laboratory Diagnosis. The findings of large gravid proglottids with 
a single lateral genital pore and with ik to 30 lateral branches to the 
uterus. To visualize the uterine branches fix the proglottid in hot 
(70° c.) 356 formalin for 5 minutes and then place it between.two slides. 
If the mounted specimen is held up to the light, the uterine branches will 
stand out as white lines extending out from the central stem. The no is 
usually no differential increase in eosinophiles. 
Taenia solium (Linnaeus, 1758). 
This is the pork tapeworm, and is sometimes designated as the armed 
tapeworm due to the fact that the r stellum is armed with a double row of 
booklets. It is world wide in its distribution and may be found wherever 
raw or insufficiently cooked pork is consumed. It is most prevalent in 
Europe and certain sections of the tropics but is a relatively rare 
infection in the United States. This infestation iscontracted by in- 
gestion of live cysticercal larvae in park that has been insufficiently 
cooked. These larvae are not killed by cold storage and are resistant 
to even freezing temperatures. Smoking or pickling does not kill them 
but they are readily killed by ordinary cooking temperatures. The eggs 
of this parasite are also infective to man, and if ingested, the larva 
upon being freed from the egg shell invade man’s tissue and form cysts 
(Cysticercus cellulosa) in the muscles and organs of the body. 
Morphology 
The adult worm is usually made up of from 600 to SOO segments and 
measures from two to three meters in length. It is usually smaller than 
T. saginata. Malformed segments or scolices are not uncommonly found in 
specimens of T. solium. The scolex is about 1 mm. in diameter and is 
roughly quadrate in shape. It may rarely show some pigmentation. A rostollum is present and is aimed with a double row of alternating large 
and small booklets numbering as a rule from 22 to 32. Four protuberant 
cup-shaped acetabulae or. suckers are symmetrically arranged in the same 
plane about the head. They, with the rostellum, serve as the organs of 
attachment. The neck is about one-half the, diameter of: the head. The 
immature and mature segments are usually longer than broad and are 
strikingly like those of T. saginata, differing only in minute, detail. 
The ovary which lies in the posterior portion of each mature segment has 
3 lobes, a symmetrical pair and an accessary lobe on the side containing 
the genital pore. (T. saginata has only a two-lobed ovary). The uterus 
in the mature segments is a central longitudinal tube with 8 to 10 
lateral dendritic branches on each side. T. saginata has lU to 30 branches 
to a side. 
The eggs are ordinarily indistinguishable from those of T, saginata 
unless the embryonic membrane is still present. If present it does not 
have delicate polar processes as does T. saginata. 
Life history. The adult stage of this parasite is found only in 
man and there is usually only a single parasite present. Several gravid 
segments are usually passed in the feces together as a short chain. They 
usually show sluggish motility but as a rule do not have enough power to 
migrate from the rectum of their own accord. When the egos of gravid 
proglottids are passed in the feces of.man they remain in the area of 
the fecal deposit and if ingested by other animals (principally pigs 
rarely man, sheep or dog), the larval stage is liberated from its shell 
by the digestive juices and then penetrates through the mucosa and invp.des 
the blood or lymph stream where it ultimately lodges in muscle, or some 
organ of the body, and develops into a cysticercus. This cysticercus 
larva (called Cysticercus p llulosa) develops until it is oval in shape 
and about 5x 10 mm. in sizeL It has an opalescent, grey, milky, trans- 
lucent color. It is readily visible to the naked eye and when observed 
in muscle, imports an appearance known as measly (pork). 
Cysticercus cellulosa infections in man are ordinarily the result 
of the ingestion of eggs of T. solium through either contaminated food 
or water. It is possible for a person infected with the adult T. solium 
to ingest the eggs through contamination of his hands and mouth parts 
from his own fecal excrement. It is the opinion of some helminthologists 
that auto-infection sometimes takes plaice due to gastro-intestinal upsets 
with reverse peristalsis. Eggs are carried from the intestines up into 
the stomach where action of the digestive juices on the egg shells cause 
the larvae to be liberated, whereupon they invade the tissue and eventually 
lodge in muscle or organs of the body, giving rise to Cysticercus 
cellulose, disease. 
When insufficiently cocked pork contaminated with live Cysticercus 
cellulose, larvae is ingested by man, the cyst walls are digested away 
and. the miniature armed scolices evaginates and then attaches itself to 
the intestinal wall. Development of the .adult worm then begins and 
within a period of from 5 to 12 weeks adult segments and eggs are being 
passed in the feces. The adult worms according to Faust may live as 
long as 25 years in the intestinal tract of man. Any person infected 
with T. solium is of great danger to any other person in contact with him 
or his surroundings due to the danger of contracting Cysticercus cellulosa infections. As soon as diagnosed he should he isolated until rid of the 
parasite and his fecal excrement should-he disposed of by burning.or 
thoroughly mixing with a 10$ cresol solution.- 
Laboratory Diagnosis. 
1. The finding of large tapeworm proglottids with a single lateral 
genital pore and 7 to 10 lateral branches of the uterus in the feces of man. 
2. If the proglottids are stained and cleared, a three-lobed ovary 
can be demonstrated. 
, 3* Following treatment the recovery of a large tapeworm with an 
armed scolex. 
Echinococcus granulosa. (Taenia echinococcus) (Batsch-1786, Kudolphi,lBOs) 
This is the tapeworm, the larval stage of which causes hydatid 
disease of man. Hydatid disease is world wide in its distribution, 
principally among people closely associated with livestock and dogs. Dog, 
wolf, jackal, and cat are the definitive hosts of this parasite, while 
sheep, cattle, horses, goats, pigs, and rabbits are the intermediate or 
larval hosts. This parasite is quite common in Australia, Russia, Ice- 
land and temperate South America but is rare in the United States. 
McGath of the Mayo Clinic, in reviewing all of the reported ca.ses in the 
United States (U32) doubts if more than s*j>5*j> of the cases of human infection 
with the larvae of this parasite were contracted in the United States. 
All other cases were either immigrants or people who had spent some time 
in an endemic area elsewhere. 
Morphology. 
Echinococcus granulosa is a minute tapeworm and is usually net over 
one (1) centimeter in length. Unlike other tapeworms the strobila con*- 
sists of a head and only three to four proglottids of which one is 
immature, one or two are mature and only one (the terminal segment) is 
gravid. The head is piriform in shape and is 300 M. in diameter, A 
rcstellum is present and is armed with a double row of 28 to 50 booklets. 
Four symmetrically placed oral ace tabulae or suckers are present. The 
neck tapers backward from the head and is narrowest just anterior to the 
first segment. The second segment is mature and shows fully developed 
sexual organs. All segments' are broador than long and the terminal gravid 
segment contains a uterus which because of lateral svaginations gives the 
appearance of a loosely twisted coil. When the uterus is filled with eggs 
it bursts open and the ova are liberated. This may occur before or after 
the gravid segment is detached from the remainder of the worm. The eggs 
when liberated are surrounded by an.embryonic membrane which soon 
disintegrates. The eggs as found in the feces of dog are indistinguishable 
from that of T. solium or T, saginata. 
Life History. 
Dogs infected by this parasite usually harbor a great number f the 
mature worms in their intestinal tract. When passed in the feces, the eggs are scattered with the fecal excrement and contaminate pasture 
land, forage, or water. There they are usually ingested by their most 
favorable intermediate host (sheep). (Man or any of the herbiverous 
animals may act as intermediate hosts). When the eggs reach the intesti- 
nal tract they break from their shell and penetrate the mucosa and gain 
entrance into the blood stream where they are carried to various parts 
of the body. The greatest number land in the liver. A lesser number in 
the lungs and scattered individuals in the remainder of the organs and 
tissue. By the end of three weeks a larval cyst 250 M. in diameter is 
formed by each parasite surviving, and the body of the host is beginning 
to form a defensive wall of scar tissue about the parasite. By the end 
of 5 months the cysts are 1 centimeter in diameter and now show 
differentiation into an outer cuticular elastic fibrious layer and an 
inner germinal layer. Cysts like evaginations of the germinal layer are 
now protruding inward into the fluid contents of the cyst. These soon 
show invaginations with the formation of scclices. These daughter cysts 
at times become detached and eventually may give rise to granddaughter 
cysts. Thus a great number of scolices are produced from one original 
larva. These granular scclices are sometimes designated as hydatid 
sand. They usually are all alive and infective. If the cysts are rup- 
tured, the scolices will scatter through the tissue and give rise to new 
cysts. In addition, the fluid contents of the cysts are quite toxic and 
may cause shock if taken up by the circulation. The primary cyst formed 
by the larvae may develop into a simple unilocular cyst or at times 
into a multilocular cyst. The multilocular cysts are thought by some 
helminthologists to be due to a separate and distinct species of 
E. granulosa. 
If raw meat containing hydatid cysts is ingested by dogs, the scclices 
are liberated by the digestive juices and develop into mature worms within 
a. oeriod of throe weeks. 
Laboratory Diagnosis. - (Of hydatid disease) 
1. Intradennal test. 0.2 cc. of a sterile hydatid fluid is injected 
intracutaneously, a wheal with an out erythematous zone appears within 
15 minutes and both gradually fade, followed by a delayed secondary rise 
several hours later. 
2. Precipitin test. 
3. Complement fixation test. 
. X-ro.y. 
5. Surgical removal of hydatid cyst followed hy histological 
examination. 
Hymenolepis nana. (v. Seehold, 1852 - Blanchard, 1891) 
This is the dwarf tapeworm and is the most frequent of all tapeworms 
encountered in man* I-t is world wide in its distribution and is 
frequently found in the United States and its possessions. It is closely 
related or identical with the dwarf tapeworm of rat and the mouse. 
(Hymenolepis nana vir. fraterna). Morphology. 
The strobila has a length of only 25 to Uo mm. and its maximum 
diameter does not exceed 1 mm. The head is rhomboid in shape with a 
maximum diameter of ,32 ram. There are four acetabulae or suckers present 
and a rostellum armed with a single row of booklets numbering 20 to 30» 
The neck is long and slender and the proximal segments are very narrow 
and short. The mature and gravid segments are three to four times as 
wide as long. The strobila usually contain about 200 segments and the 
largest gravid ones are about 0.03 ram, in length by 0.9 mm. in width. 
The gravid segments drop off from the worm one by one and then rapidly 
disintegrate, liberating the ova. Gravid segments are seldom found in 
feces. . The eggs are 30 to U5 M, in diameter and are spherical or slightly 
oval in shape. The eggs as seen under the microscope show an outer shell 
and two inner membranes surrounding the onchcsphere or embryo. The outer 
membrane has two diametrically opposite polar thickenings, from each of 
which arise Uto 8 thread-like filaments. (These polar thickenings and 
filaments are not present on the only other helminth egg (Hymenolepis 
diminuta), that resembles that of H. nana.) The onchosphere contains 
three pairs of lancet shaped booklets. 
Life cycle. 
The gravid proglcttids disintegrate in the fecal contents of the 
host1s gut and liberate the egos which, when passed in the feeds, may 
eventually contaminate food or water. Sometimes through carelessness on 
the part of the individual infected, the hands may become contaminated 
with the eggs and by this means carried directly to the mouth. All eggs as 
passed in the feces are infective for man, and when ingested and subjected 
Jdo the action of the festive Juices the.larvae emerge fr .1 the shell and 
penetrate the mucosa of the terminal portion of the ileum where they round 
up and form cysts within the villi. The cysticercus thus formed is called 
a cercocyst. After a period of development the cyst ruptures into the 
gut contents liberating the now formed scolex which attaches itself to the 
mucosa. Within the period of two or three weeks the worm has reached 
maturity and is liberating eggs in the feces. 
Laboratory diagnosis. 
The finding of typical hexacanth eggs in the feces, with outer shell 
and two inner membranes., the outer membrane having polar thickenings and 
filaments, in the feces. 
Bare species of Costodes found infecting Man. 
The adults of Dyoilidium caninum, (common dog tapeworm), Hymenolepis 
diminuta (the common tapeworm of the rat and mouse), Taenia confusa (lile 
history unknown) and various species of Davainea (the common tapeworms of 
birds and fowls), are occasionally found infecting man. The larval forms 
of Multiceps multiceps (European dog tapeworm) and I)iphylobolhrium 
mansoni (Sino Japanese dog tapeworm) rarely infect man. The incidence 
of .these infestations is so small in the United States that for practical 
purposes they need not be extensively studied. They will be briefly discussed or demonstrated in class if further information is desired, 
text hooks such as Faust's "Human Helminthology" or Baylis "Manual of 
Helminthology" should he consulted. 
Phylum Nemathelmintbss Vogt (fide Cams, I 863), 
The round worm parasites of man are found under the Class Nematoda 
Rudolph!, 1808. They are in general unsegraented worms bilaterally symme- 
trical, with three body layers and elongated rounded or filiform bodies. 
An alimentary tract is present with a mouth and usually an anal opening. 
The coeloraic cavity is large and no cilia are present during any stage of 
the life cycle. The sexes are usually separate. 
Definitions. 
Buccal vestibule - The cavity of the mouth. 
Cervical and caudal ala* - Ridge or wing-like expansions of the cuticle. 
Cervical papillae - Minute fingsrlike protuberances of the cuticle in the 
vicinity-of the esophagus. 
Chitin - A hard transparent substance forming teeth rr cutting plates. 
Cuticle - The outer dense translucent covering of the worm; corresponds to 
epidermis in mammals. 
Cloaca - The cavity in male nematodes into which the intestines and ves 
deferens discharge. It is absent in females. 
Copulatory bursa - A strong wing-like clasping organ used in copulation. 
Copulatory spicule - The copulatory organ. 
Dentiguous ridges - Chitinous prominences with serrated or toothed margins 
Gubernaculum - A chitinous elevation in the dorsal wall of the cloaca 
which acts as a guide for the copulatory spicule during copulation. 
Filariform - Applied to the esophagus of a larval nematode when it is long 
compared with the length of the larvae and does not have a posterior 
dilated bulb. Larvae having such an esophagus are designated as 
filariform. 
Rhabditiform - a name applied to the esophagus of a nematode larvae when 
the posterior end is dilated into a bulb'. Larvae having such an 
esophagus are designabed as rhabditiform. 
Oviparous - A female that lays eggs. 
Parthenogenic - A female that produces young without fertilization by the 
male. 
Viviparous - A female that gives birth to living larvae. (Hatchwig of the 
eggs having taken place in the uterus.) Ascaris Lumbriboldes .Linnaeus, 1758. 
This is one of the most common helminths infesting man. It is world- 
wide in its distribution and is particularly prevalent in certain sections 
of the tropics. It is a very common human infestation in the Philippines, 
Porto Rico, Panama and to a lesser degree the southern United States. 
In general it is prevalent in any country within the 60 degrees Fahren- 
heit isotherm where there is adequate moisture and improper disposal of 
human excrement. It is thought by many to be identical with ascaris 
found in hogs, however, human experiments conducted by Japanese investi- 
gators (Koino 1922) seem to disprove this assumption. The sexes are 
separate and the females are about twice as long and one and a half times 
as great in diameter as the males. The eggs of this parasite are 
extremely resistant to chemicals and to a lesser extent to desiccation. 
They are readily destroyed by ordinary cooking temperatures, and are 
inhibited in their development by temperatures below Uo degrees F. 
Freezing, however, does not destroy them, they merely remain dormant 
until favorable temperatures are encountered, Eosinophilia, urticaria, 
intestinal discomfort and obstruction in some form are quite commonly 
encountered in individuals infested with this parasite. Infestations 
are usually multiple although occasionally only a single parasite may be 
present. 
Morphology. 
The females of this species are usually 20 to U5 cm, in length and 
about 3 to 6 mm. in transverse diameter while the males measure from 15 
to 25 cm. in length and 2 to U ram. in transverse diameter. They are 
pinkish to yellow grey in color and give the impression of trans lucent 
outer wall with an inner cavity filled with thread-like organs. On 
each side a greyish white cuticular line extends the whole length of the 
worm. The heads of the male and female are identical and are mado up 
of a mediarn dorsal broad elliptical lip and a symmetrical pair of sub- 
median ventral lips all of which show fine teeth upon their exposed sur- 
faces. Each lip has on its lateral margins a pair of minute papillae. 
There is a small buccal vestibule and a cylindrical muscular esophagus 
10 to 15 mm. long leading directly to the mid intestine. The intestines 
terminate in a rectum which opens directly into the anal pore of the 
female and cloaca of the male. The posterior extremity of the female is 
straight and tapers to a point while that of the male is curved ventrad. 
Two retractile club shaped copulatory spicules are usually visible in the 
curved portion of the posterior extremity of the male and there are 
several preanal and postanal papillae. Further details of the anatomical 
structures of these worms will not be discussed because they are of no 
practical importance in routine identification. 
Life History. 
The mature female ascaris discharge their fertile eggs into the 
feces. If males are also present in the gut the female will have been 
fertilized prior to liberation of the eggs. However, in light infesta- 
tions with this parasite, males or females alone may be the only worms 
present. In such an event the eggs, if present, will be unfertile. It 
is estimated that the genital tubules of a mature female ascaris have a capacity of 27 million ,eggs, The average daily output of eggs from each 
female is about 200,000 (Brorn and Cort, 1927). 
The fertile eggs as passed in the feces are yellowish brown in color, 
oval in shape, with a thick transparent shell and an outer coarsely 
mammillated albuminous covering which is sometimes absent. They measure 
to 75 M, in length by 35 to 50 M. in their lesser diameter. Unferti- 
lized eggs are usually much longer and normally have a thin irrego Lar 
mammilated outer shell, filled with an unorganized mass of highly 
refractive granules. 
When..the eggs are passed in the feces the embryos are undeveloped but 
if exposed to a temperature of 91 degrees 3?. under humid conditions they 
will devei op'into rhabditiforrn sheathed larvae within a period of 9 to 13 
days. After this stage of development has been reached they are infective 
if ingested by man and will readily hatch after passing into the intes- 
tinal tract. Under conditions that exist in tropical and subtropical 
countries the period of development into rhabditiforrn larvae is usually 
about one month. After the rnabditiform larvae have once developed in 
the eggs they are extremely long-lived and may bo still alive and 
infective if kept in moist soil for a period of 5 to 6 years. They will 
still be alive and untile within the eg? after a period of two years in 
2'jo formalin. 
Eggs containing live rhabditiforrn larvae, if ingested by man, pass 
into the intestinal tract where they hatch and shed their sheath. The 
rhabditiforrn larvae are thread-like in shape, tapering at both ends and 
measure 0.2 to 0,3 ram, in length by 13 to 15 M. in transverse diameter. 
The larvae penetrate the mucosa and gain entrance into the blood or lymph 
streams. They are carried by the circulation to the lungs where the 
majority of them ledge and undergo a further period of development until 
they ©re lo to 21 mm. in length. Their stay in the lung is marked by a 
pneumonic reaction on the part of the host and in heavy infections the 
lungs may be almost completely consolidated as in lobar pneumonia. There 
is usually a marked (12 to 3Of>) eosinophilia at this stage. After this 
period of development the larvae migrate via. the bronchi, trachea, epi- 
glottis, esophagus and stomach to the intestines where after 6 to 8 weeks 
they develop into mature worms and the females begin discharging eggs in 
the feces. The period of exposure to development of mature worms with 
liberation of eggs in the feces is 2 to 2-| months. 
Laboratory diagnosis. 
1. Finding of typical mammilated eggs in the feces. 
2. Recovery of the large adult round worms following treatment. 
EnteroMus (Oxyuris) vermicular is (Linnaeus, 1755) Leach, 1853* 
This is the pinworm or seat worn of man and is world wide in its 
distribution. It is encountered particularly in children and individuals 
with careless uncleanly habits. Due to the fact that the eggs are 
infective when liberated by the female pinworm and do not require an extrinsic incubation period, occasional infestations are found in 
families of the upper Because of the infectiousness of 
these eggs and the fact that they will live for about 10 days under 
conditions of desiccation that exist in the average American home, an 
infestation once contracted is very difficult to get rid of. If one 
member of .a family is infested all including other intimate contacts will 
b§. Contaminated bath tubs, wash basins, towels, wash cloths and food or 
dishes are the most frequent sources of transmission from one individual 
to another. Examination of vacuum cleaner sweepings from a house in 
which the individuals are infested with E. vermicularis will reveal, as a 
nfj-e, many viable eggs of this parasite. Pinworm infestations are quite 
common among: married enlisted men and their families but are relatively 
uncommon among soldiers, with 1 year of service or over, living in 
barracks. This is due to the fact that married soldiers usually live at 
home where there is no supervision of sanitation while soldiers in 
barracks are under strict sanitary control. 
Surveys of Washington orphanages, schools and lower working class 
families by Hall and his associates (1936) have shown that from 12 to 
1 &/o are infested with pinworm. Clinical symptoms frequently observed 
are vaginitis, rectal pain, gastro-intestinal upsets, depraved appetities, 
constipation, insomnia, nervous irritabilities and less of weight. 
Heavy infestation with pinworm can be as damaging to physical and mental' 
development of children as light hookworm infestations. 
Morphology. 
Males and females of this species have the same type of head. It 
is slightly bulbous in shape and is made up of three labis which serve as 
the organs of attachment to the mucosa. The labis are capable of being 
retracted into the body. The posterior portion of the esophagus is 
dilated into a bulb and the digestive tract is a straight tube. The females 
are fusiform in shape and about 8 to 13 mm, long by 0,3 to 0.5 mm. in 
transverse diameter. The caudal extremity of the female tapers to a fine 
point and makes up about 1/3 the total length. That of the male is 
sharply curved ventrad, is somewhat bulbous in shape, and near its temin- 
cus is located a retractile, conspicuous copulatory spicule 70 M. in 
length with an acutely curved tip. The anus of the female opens at the 
junction of the middle and posterior thirds of the body, while the vagina 
opens at the juncture of the anterior and middle thirds. 
Life cycle. 
The adult worms live in the coecum, appendix and adjacent parts of 
the colon and ileum. After fertilizing the female, the male dies and 
is passed in the feces. The female in a short time becomes distended 
with eva, releases her hold on the mucosa and passes with the fecal 
currents to the rectum. She does not liberate her eggs into the feces but 
migrates by active crawling through the rectum onto the perianal region or 
even into the vagina if the host is a female. This migration usually 
takes place at night but may occur at any time. It is usually accompanied 
by an intense itching that gives rise to scratching. When the females 
are upon the perineum they rapidly become desiccated and are either 
mechanically crushed by the hosts scratching or literally explode due to 
drying, thus liberating the embry .cnated eggs, all of which are mature and infective* The eggs usually are picked up by the fingers or fingernails 
cf the host while scratching the perineum and are carried eventually tc 
the mouth where they are ingested. Eggs not picked up by the fingers cf 
the host continually drop off into the clothing contaminating the area 
frequented by the individual. This especially is true in bathrooms, 
bedrooms and-bed clothing. Those eggs will remain alive and infective 
for at least 7 days but are usually not viable after 10 days. 
The eggs as freed by the*, female pinworm are oval in shape with one 
side slightly flattened. They are 25 by M. in size and made up of a 
transparent outer albuminous shell and an inner embryonic membrane sur- 
rounding a well developed embryo. 
When the egg is ingested it passes to the small intestine and hatches 
into an embryo 140 to 150 M. in length. After a short period of develop- 
ment it passes to the coecum where it attaches itself to the mucosa and, 
within a period of three weeks from the time of ingestion, is mature and 
ready for egress from the bowel. Reproduction does not take place within 
the human host and if reinfection could be stopped a person would be 
free from the parasite after the one generation (3 weeks). 
Lab o rat ory D iagnosis. 
1. Demonstration of typical eggs from perianal swabbings 
2. Finding of adult pinworms on the perineum or in the feces 
Trichinella spiralis (Owen, 1835) Bailieit, 1855* 
This is the worm, the larval stage of which gives rise to the disease 
known as trichinosis in man, hog, rodents, and carnivorous animals. 
Infection with the larvae of this parasite is cosmopolitan in its extent 
both for man and animals. In most instances man contracts this infection 
by the ingestion of insufficiently cooked pork containing the live larvae 
of 5/ spiralis * Autopsy study of human cadavers in Boston, Buffalo, 
Rochester, Minnesota, Washington, D. C., &ew Orleans, La,, and San 
Francisco show an incidence of infection varying from 3 to As 
embryos and not free eggs are liberated by this parasite, it is impossible 
for man or animals to contract this infection without eating meat contain- 
ing the live larvae. The majority of hogs probably contract the infection 
by the ingestion of uncooked pork scraps in garbage. Rarely the ingestion 
of infested rats may be the source. It has been estimated that from one- 
half to 15$ of hogs are infected with the larvae of T. spiralis, the 
greater percentage being among hogs fed on garbage from large cities. 
Garbage fed hogs are indiscriminately slaughtered with grain fed hogs and 
as their meat is inferior to grain fed hogs they are more often used for 
sausage making. If pork is cheaper than beef it is often mixed with 
beef in preparing hamburger meat for use at roadside cafes, restaurants and 
quick lunch stands, A rare hamburger in such a place stands a good chance 
of containing live trichina larva. Meat inspection cannot elirnin; to 
trichina infested pork from being sold. However, if no raw pork is con- 
sumed and if a cooking temperature of at least 137° is reached in all 
parts of the meat being cooked all the larvae will be killed, Pork used 
in the preparation of sausages which are meant to be consumed without further cooking is considered safe if it has "been stored at a temperature 
not exceeding y D. for 20 days. 
Morphology. 
The female worm measures 3 to 4 mm. in length and about 60 to 75 M. 
in its greatest transverse diameter. It is an elongated round tapering 
worn having its smallest diameter at the oral end and greatest diameter 
at the caudal extremeity. The anus opens at the posterior tip and the 
vulva near the anterior 1/5 of the body. The ova are formed in the ovary 
near the caudal extremity and by the time they have reached the vulva have 
become embryonated and hatched. The larvae as liberated from the vulva, 
are 90 to 100 M. in length and 6 M. in diameter and are capable of 
passing through the capillary beds of the liver and lungs once they gain 
entrance to the circulation of their host. 
The males measure from I.l+ to 1,6 mm, in length by Ho to 50 M, in 
diameter. They are. attenuated anteriorly and slightly bulbous poster- 
iorly. The cloaca opens at the posterior extremity and is guarded by two 
conical papillae. It is avertible during coitus. 
The encysted larvae are found tightly coiled in fibrous or calcified 
cysts measuring O.S to 1 mm, in diameter in or near the tendinous 
insertion of striated muscle. 
Life Cycle. 
When live encysted trichina larva, are ingested by a suitable host 
the digestive juices dissolve the cyst capsule and liberate the larvae. 
The larvae then pass into the small intestines and attach themselves to 
the mucosa where further growth takes place. By the end of the 3 to 5 
days development is nearly complete. The males fertilize the females 
and then die. The viviparous females then migrate further into the mucosa 
and eventually come to lie in one of the lymph channels or partially in 
a small venule. By the end of seven to twenty days embryos are being dis- 
charged into the blood stream, from 1000 to 1500 embryos are liberated by 
each female. 
The small motile larvae pass readily through the liver and lungs to 
the arterial circulation where they are carried to the capiallary bods of 
the luscles. These larvae triple their size and then encyst near the 
tendinous insertion of the muscle fibers. They are present in greatest 
numbers in the muscles of mastication, respiration andjhonation, although 
they may be found in any striated muscle. They may be found in this 
position as early as 9 days following the initial infection and will 
continue to increase in numbers present up to the Uoth day. During the 
period of invasion, fever, leucocytosis, ecsinophilia (Uo to 60 present) 
muscle pain, nervous irritability and sometimes meningitis symptoms may 
be present. 
Laboratory Diagnosis, 
1. Demonstration of encysted trichina larvae in: 
-(a) Biopsied muscle or muscle from cadavers. 
(b) X-ray demonstration of encysted calcified larvae. Trichoca;jljr-iun t.richiuruc (Linnaeus, 1771) Blancnafd, 1895 
This is the humanvhip or thread worm and is quite common in warm 
rapist climates although it may he found elsewhere. It is quite common 
in the United States and possesseions. This worm although very persistent 
when once established in the gut of man does not seem to give rise 
to nearly so much damage as the other helminths. The whip ■ worms of ani- 
mals (dog, sheep, etc.) are not known to he infective for man. 
Morphology 
In shape and general appearance this worm resembles a whip. Its 
anterior 3/5 hair-like and corresponds to the lash while the posterior 
2/5 is thick and fleshy like the stock of a whip. The males and females 
are nearly the same size, 130 to 150 mm. in length, hut sometimes the 
males are slightly smaller. The anterior portion of the worms is nothing 
hut a capillary tube which it buries beneath the mucosa parallel to the 
long axis of the host's gut. The posterior free part of the worm contains 
the digestive and generative organs, the anus opens at tne caudal extremity 
while the vulva opens at the Junction of the bulbous and thread-like 
portion of the body. The caudal tip of the male is curved ventrad and 
emds in a copulatory spicule with a retractile thickly seined sheath. 
The eggs are brown in color, barrel or lemon-shaped with mucous plugs in 
either end. They measure 50 x 25 M. and contain an unsegmented ovum 
when passed in the feces. 
Life Cycle. 
The fertile unsegmented eggs are passed in the feces and ere rot 
infective until the embryo has developed. A period of incubation varying 
from 12 to 60 days depending upon the temperature, oxygen supply and 
humidity is usually required. The eggs are extremely resistant to drying 
and low temperature changes. They are said to retain their vitality for 
5 years in contaminated soil. 
Man is infected by ingesting food or water contaminated with the 
eggs containing mature developed larvae. The larvae are liberated from 
the shells by action of the digestive Juices and after a short period of 
development in the small intestine migrate to the caecum and attach them- 
selves to the mucosa., developing into adults within 6 to 8 weeks. 
Laboratory Diagnosis. 
The finding of characteristic eggs in the feces. 
Strongyloides stercora.lis (Bavay, I 876) Stiles and Hassall, 1902. 
The geographical distribution of this worm is cosmopolitan, being 
most commonly found infesting man in moist warm climates. Strongs- 
loides infestations of animals are not identical in specie with that of 
man. Dogs may be experimentally infested with man's parasite but the 
infestation does not last, Man is probably the only host reservoir for £. Stercoralis. It is not an uncommon infestation in the United States 
and its possessions. It is unusual in that a free living generation may 
form part of the life cycle and that only parthenogcnotic females and 
rhabditiform larvae are found in the small intestines of man or dog. If 
the infestation is heavy and frequent rsinfestation taking place, a 
persistent bloody diarrhea with urticarial skin reactions- will frequently 
be present, but in light infestations vague intestinal discomfiture is 
the only thing complained about. 
Morphology. 
The parthenogenic females measure about 2.2 mm. in length and about 
50 M. in transverse diameter. They are so small that they can only be 
discerned by the use of low power magnification. They live attached to 
or in the mucosa of the jejunum and upper part of the ileum and can only 
be demonstrated by the post-mortem scraping of the mucosa, using a 
dissecting scope to separate them from the tissue. They have a cylindri- 
cal esophagus which extends one-third of the body length and the vulva 0- 
pens at the junction of the middle and anterior thirds of the body. In 
gravid worms the eggs, about 20 to 30 in number end resembling those of 
hookworm, can be seen lined up in a single row in the uterus extending 
from near the anus to the vulva. Fnen the eggs are liberated from the 
vulva they contain a rhabditiform larvae which almost immediately hatches 
and passes into the feces. 
The rhabditiform larvae as found in freshly passed feces are actively 
motile and about 225 M. in length by 10. M. in breadth. They have a 
shallow buccal vestibule as compared with hookworm larvae and are less 
attenuated posteriorly. The esophagus is typically rhabditiform in type 
with a short club-shaped anterior portion and a globular bulbous dicta- 
tion posteriorly. The esophageal bulb is longer than that found in the 
rhabditiform larva of hookworm. The free living adult resemble the 
rhabditiform larva in type, the male measuring 0.7 mm. in length by 
U5 M. in width. The female 1 rnm. by 50 M. The caudal extremity of the 
male is curved ventrally and bears two minute brown curved copulat ry 
spicules about Uo M, in length, The position of the -uterus, vulva and 
eggs in the free living female is similar to that of ..the parthenogenic 
female. The filariform larvae are about 500 M, in length and differ from 
the filariform larvae of hookworm in that the esophagus extends to about 
one-half the body length and the tip of the tail is slightly notched. 
Life Cycle. 
The parthnogenic female liberates her embr/onated eggs on the mu- 
cosa of the small intestine. They almost immediately hatch, liberating 
the rhabditiform larvae which are actively motile and are passed in the 
feces, (l) If the conditions are favorable in the area where the feces 
are deposited the larvae develop into free living males and females. If 
feces containing the rhabditiform larvae is, allowed to stand for as long 
as 30 hours free living adult forms may be present. The females after 
reaching maturity end being fretilized by the male’s oviposit about 50 to 
75 embryonated eggs which hatch in a very short time liberating 
rhabditiform larvae. The larvae then undergo a period of development 
and after moulting become filariform in type. (2) If conditions are unfavorable the rhabditiform larvae develop directly into filariform 
larvae. The filariform larvae are infective and if they come in contact 
with the skin of man or dog they will penetrate it and gain entrance to 
the blood or lymph stream. They are carried by the blood to the lungs ■ 
and after a period of development'break into the areolae, migrate to 
the bronchi, -trachea, epiglottis, esophagus, and stomach to the jejunum 
where they develop into parthenogenic females. 
Laboratory Diagnosis. 
For practical purposes the finding of rhabditiform nematode larvae 
in the freshly passed feces of man is diagnostic. 
Ancylosto-ia duodenale (Dubin, Creplin IBUS. 
This is the "old world hookworm". It is tropical and subtropical in 
its distribution and at times may be found in colder climates under 
special conditions, such as fecal contaminated mines, where moisture and 
temperature are favorable. The adults live in the jejunum and du-denum 
of man, occasionally in the pig and rarely in certain carnivorous animals, 
They are larger than the adults of IT. Americana and have a less m rked 
dorsal flexion of the head. This parasite is relatively uncommon in the 
United States and is only found in areas where immigrants from southern 
Europe live and take inadequate precautions in the disposal of human 
excrement, 
Morphology. 
The mature adults are small, pimp, rigid round worms, yellowish 
white in color and the anterior extremity is slightly flexed dorsally. The 
buccal capsule or mouth is large, gaping and armed with six ventrally 
placed pointed dog-like teeth. The middle pair are small and difficult 
to demonstrate while the remaining four teeth are large, close together 
and conspicuous. The sub-ventral surface of the mouth cavity has two 
teeth-like lancets with rounded tips. The esophagus is club-shaped and 
measures about one-sixth of the length of the worn. It terminates 
posteriorly into the intestine which is a straight tube running caudally 
to the rectum. A muscular valve is present at the esophageal intestinal 
junction. A single, minute cervical papilla is present on each lateral 
margin of the cuticle, opposite the middle of the esophagus. The female 
worm measures 1.2 cm. in length and 600 M. in its greatest transverse 
diameter. The vulva is at the junction of the middle and posterior thirds 
of the body. The rectum is near the caudal tip. The generative organs 
consist of a pair of long curved tubes, one situated for the most part 
posterior to, and the other mostly anterior to the vulvas The ovary is 
the distal portion of each tube, the oviduct the middle portion and the 
proximal or terminal portion is the uterus.’ The uteri unite to form a 
common vagina which in turn opens into the vulva,. 
The male worn measures about 9 mrn. in length and is about M. in 
its greatest transverse diameter. The male genital organs consist of a 
very long coiled tubular testis which occupies the middle third of the 
body, a vas deferens, a spindle-shaped seminal vesicle, and a long 
ejaculatory duct opens into the rectum which is continuous with the cloaca and copulatory bursa. Two protrusile long bristle-like copulatory spicules 
are situated in a sac-like structure on the terminal portion of the 
intestine. These copulatory spicules can be protruded into the copulatory 
bursa and are guided by a chitinous-like thickening called the gubernacu- 
lum. The copulatory spicule's are not fused in A. duodenale, while in 
N. Americana they fuse and the terminal portion of the fused spicule is 
armed' with a fishhook-like barb. The copulatory bursa is made up of two 
wing-like projections at the caudal extremity of the worm and is-supported 
by thick fleshy rays. The dorsal rays are tripartite and the clefts 
shallow, that of Necator americanabipartite and the clefts deep,- 
The eggs are oval in shape and have a colorless transparent shell. 
They measure about 60 by 35 M. and when observed in freshly passed feces 
contain a four to eight celled embryo. A clear space is present between 
the embryo and the shell. The eggs of A. duodenale, N. Americana and 
A. braziliense are for practical purposes indistinguishable. 
Life History, 
The eggs as passed in the feces when deposited under favorable condi- 
tions on the ground develop into rhabditiform larvae, 250 M. in length 
within 12 to 2k hours. The larvae are actively motile and feed on organic 
matter at the edge of the fecal mass. At the end of ~f2 hours they become 
sluggish, the skin or cuticle is moulted and the larvae are 500 M, in 
elngth. Growth continues and the esophagus becomes filariform in shape 
by the end of U to 7 days. The larvae then moults again but does not 
cast its skin. The larvae enclosed in its old skin does not feed, is 
actively motile and may migrate as much as k inches of its own volition. 
The larvae are bluntly rounded anteriorly and their tails taper rapidly to 
a single point. Filariform larvae of S. stercoralis have a tail with a 
bifid tip. The larvae developed to this stage are infective and live a 
month if temperature and humidity conditions are favorable. In putrify- 
ing fecal matter in which conditions of anaerobiasis exist they quickly 
die. If the filariform larvae come in contact with the skin of man they 
penetrate it moulting the third time in the process, and gain access to 
the circulation, Inhere they are carried to the lungs. They then break 
through into the alveolae and migrate via the bronchi, trachea, epiglottis, 
esophagus and stomach to the intestines where they moult the third time 
and develop a small buccal cavity aimed with k teeth. They attach them- 
selves to the gut wall, grow to 4 ram, in length, moult the fourth time 
and then continue growing without further moults to the adult sexually 
mature worm. Fifteen to twenty days are required for the development time 
in the gut and five weeks from the time the filariform larvae enter the 
skin the adults are mature in the gut and passing eggs in the feces. 
A.duodenale females lay about 2 to 2& times as many eggs per day as 
N. americana. It has been estimated that 28,000 eggs per day is the 
average output of one female. 
Laboratory Diagnosis. 
1, The finding of typical eggs in the freshly passed feces, followed 
by the recovery of typical adults in the feces after treatment. Nocator americana* (Stiles, 1902) Stiles, 1906. 
This is the so-called new world hookworm although it in all probabil- 
ity was imported to the Americas by slaves from Africa during the coloniza- 
tion period. It is the most common helminth infestation in the Ur.itod 
States where it is practically all limited to the area comprising tne 
Southern States. Various surveys of this area have shown that 20 to 
kofo of the rural population are infested by this parasite. It is frequent- 
ly found in tropical and subtropical regions in other parts of the world. 
Morphology. 
only differs from it in the following characteristics: 
All hookworms are much like Ancylostoma duodonale. N. Americana 
1. The average adults are slightly smaller. 
. Length 
Breadth 
Male's 
Female s 
Males Females 
A. duodenale 
9 mm, 
12 
500 M. 600 M. 
N, amerleans 
g " 
10 " 
300 " U50 " 
2. The head is only slightly dorsally flexed in A. duodonale and is 
markedly sc in N. americana.. 
3, A. duodenale has two cervical papillae present on the cuticle at 
the level of the middle of the esophagus. They are absent in N, americana. 
h. The buccal capsule is conspicuously large in A. duodenale and is 
aimed with 6 teeth and 2 subventral lancets. The buccal capsule of 
needle-like subventral and two subdorsal lancets. 
5. The vulva in the female, A. duodenale, is located at the juncture 
of the posterior and middle thirds. It is near the middle of the '.mdy in 
N. americana. 
6. The dorsal rays of the male copulatory bursa are tripartite with 
shallow clefts in A. duodena.lo and bipartite with deep clefts in IT. 
americana. 
7. The mole copulatcry spicules are fused in their terminal portion 
and end with a fishhook-like barb in N. americana.. They are unfused in 
A. duodenale and no barbs are present. 
8, The eggs of N. Americana are larger and narrower than A. duodenale, 
but for practical purposes are indistinguishable. 
Life Cycle. 
The same as for A. duodenale. Laboratory Diagnosis, 
1. Demonstration of typical eggs in the feces and recovery of adults 
in feces after treatment. 
2. All cases of hookworm disease in the U. S. should he considered 
due to N. amcricana until proven otherwise. 
Ancylostoma braziliense♦ Gomez de Faria, 1910. 
This is the common tropical hookworm of days and cats. It is tropical 
and subtropical in its distribution. It is quite frequently found 
infesting those animals in the southern United States, but is a rare 
intestinal infestation in man. If the filariform larvae come in contact 
with human skin they will penetrate it and give rise to a local dermatitis 
known as creeping eruption. This skin condition is due to the migration 
of the larvae in the deeper layers of the skin, and the secondary bacte- 
rial infection that accompanies it. The larvae do not gain access to the 
circulation and probably never reach the intestines. 
Ancylo stoma can inurn (Ercolani, 1859) Hall, 1913* 
This is the most common hookworm of dogs and cats. It is cosmopol- 
itan in its distribution and gives rise to a condition in dogs known as 
kennel anemia. The larvae may give rise to mild forms of creeping 
eruption in the skin of man but probably never infest his intestinal 
tract. 
Wuche re ria banc red t i (Cobbold, 187?) 
This is the parasite responsible for most cases of elephantiasis and 
other types of localized parasitic lymph blockage. (However, only a 
small percentage of individuals infested with this parasite develop 
clinical elephantiasis.) W. bancrofti is tropical or subtropical in its 
distribution being; particularly prevalent in certain sections of Africa 
and some of the Islands of the Pacific. It is found in Porto Rico, 
Panama, Philippines, Guam and in a limited area around Charleston, South 
Carolina and the coastal region between that city and Savannah, Georgia. 
The adult worms lie ceiled in the lymph vessels just distal to lymph 
glands. When once developed they seem to be unable to pass or circumvent 
the lymph gland proper. Their mass and shape acts as an effectual plug 
in blocking the lymph vessels leading into the glands. This blockage is 
responsible for the lymph stasis and most of the pathological findings 
in this disea.se. The blockage may be acute'with rapid swelling followed 
by sudden recession of the swelling due to movements of the worm opening 
the lymph channels. Ultimately the accompanying chronic inflammatory, 
reaction with scar tissue formation leads to chronic lymph blockage. 
Morphology. 
The adults are creamy white filariform worms with smooth cuticle, 
cylindrical shape and gradually tapering ends. The head end is knob-like while the tail is bluntly rounded. The females measure 8 to 10 cm. in 
length by 0.2*4 to 0,3 mm, in breadth. The males *4 cm. in length bp 
0,1 mm. in breadth. The adults are extremely difficult to-recover from 
-the tissue. Attempts at extraction usually cause-then to break into 
two or mere pieces. They are best studied by using histological stained 
sections of tissue containing the parasites. 
Life Cycle. 
The females liberate the sheathed embryos into the lymph vessels. 
(The sheath of the embryo is not a retained moult but the stretched shell 
from the ova.) All females present, probably mature, and liberate their 
larvae simultaneously. The larvae measure 130 to 320 M. in length by 
7.5 to 10 M. in diameter. The column of nuclei representing the primitive 
gut does not extend to the tip of the tail. The internal structures 
are undifferentiated. The larvae are motile and gain access to the peri- 
pheral circulation with a nightly periodicity starting at about *4 p.m. 
and increasing in numbers in the peripheral circulation until about 2 a.m. 
When periodicity is present few if any can be found during the day. In 
certain areas there is no periodicity or if present corresponds to the 
biting habits of the insect intermediate host. Nocturnal can be changed 
to diurnal periodicity by changing the sleeping habits from night to day 
time for two or three weeks. 
If the larvae in the peripheral blood are ingested by suitable species 
of mosquito (Culex fatigans, Aedes varlegatus, and Anopheles costalis 
incriminated to date) the larvae moult, migrate through the intestinal 
wall of the mosquito end eventually lodge in the thoracic muscles where 
they become quiescent, thick and short. After a period of a few days 
they develop their intestinal tract and generative organs. Growth 
continues, the cuticle is moulted and the larvae now about 1 mm, in 
length by 20 M. in breadth migrates from the thoracic muscles to t.x. 
labium in which it lies. One to three weeks time are required for the 
mosquito cycle. When the mosquito again feeds the larva is dropped on 
the skin, and after penetrating it, migrates to the lymphatics and de- 
velops into an adult. 
Laboratory Diagnosis, 
1. The finding of sheathed nematode larvae with the column of 
nuclei not extending to the tip of the tail, in the blood, urine or 
chylous fluids of man. 
2. Biopsy of lymphoid' tissue with demonstration by histological 
means of the adults and larvae. 
Dipetalunema perstans (Manson, 1891) 
This is the filarial parasite the larvae of which have no periodicity 
in their appearance in the peripheral blood. It is found principally in 
the tropical sections of .Africa and .South Anerica. The adults are found 
mainly in the retroperitoneal;connective tissue and lymph channels. Morphology 
The adult females measure 7 cm. in length by 130 M. in breadth. ’The 
cuticle of both sexes is smooth when viewed microscopically. ' The heads 
of these species is crowned by two epalet-like appendages on the cuticle. 
The larvae as seen in the peripheral blood are unsheathed and the 
column of nuclei extend to the tip of the tail. 
Life Cycle. 
The life cycle is the same as for W, bancrofti except that certain 
midges, Culecoides austeni and C. grahami are the insect intermediate 
hosts and vectors. 
Laboratory Diagnosis. 
The finding of unsheathed nematode larvae in the peripheral blood 
with a column of nuclei extending to the tip of the tail. 
Filaria Ozzardi (Manson, 1297) 
This filarial parasite is found in the West Indies and parts of 
South America. The life history and pathogenicity are unknown. The 
adults live in the retro-peritoneal tissue, abdominal wall and lymphatics 
The whole adult male specimen''has never been studied. The females are 
about 7 cm. long by 230 M. in breadth. The tail is terminated by a pair 
of large papillae. Unsheather larvae 200 x 5 M. without a column of 
nuclei extending to tip of tail are found in the blood stream. There 
is no periodicity in their occurrences. 
Onchocerca volvulus (Louckart, 1893) 
This is the filarial parasite which is responsible for the develop- 
ment of nodular indurated swellings (calabar swellings) on the exposed 
surfaces of the body. These localized areas of swelling are most 
frequently found about the scalp and arms. They are due to the adult 
filaria. nesting in the ares, and discharging their larva into the cuta- 
neous lymphatics. The larvae are found in the cutaneous nodules bat 
seldom if ever in the blood stream. This infestation is found 
principally in West Africa, South America and Central America. It is 
quite prevalent in the highland coffee plantation area of Guatemala and 
is a frequent cause of disfiguring face lesions and blindness. This is 
a chronic disease slow in progress but of long duration. 
Morphology. 
The females measure up to cm. in length by UOO M. in breadth. 
The males 3 cm, in length by 150 M. in breadth. The cuticle of both 
sexes is raised in minute annular and oblique thickenings with serrated 
edges, which are more prominent towards the tail. The larvae are 
unsheathed, t 10 M. in size and do not have a column of nuclei to the tip of the tail. 
Life History, 
The adult females liberate their larvae into the lymph spaces of 
the subcutaneous nodules. These nodules tend to ulcerate and are a ; 
favorite place for flies to alight and feed. The simuliuiir flies are 
the intermediate hosts of this parasite. S. damnosum in Africa and S>. 
mooseri and others in Central and South America. These flies do not 
feed on blood but bite and chew the superficial tissue ingesting the 
tissue, lymph, blood and serum exudate. In feeding on a nodule due to 
0. volvulus they also ingest the larvae. After a period of development 
in the simulium flie’s gut and thoracic muscles the larvae migrate to the 
labium and are then infective if deposited on the skin by subsequent 
feedings of the fly. .a 
Laboratory Diagnosis. 
1. Smears from aspirated contents of skin nodules or skin scrapings 
of ulcers showing unsheathed nematode larvae 350 x 10 M.. nuclei of tail 
not extending to tip. 
2. Biopsy of nodules showing typical parasites. 
Loa loa (Guigot, 1778) 
This is the diurnal fiiaria parasite of West Africa and is rarely 
found in other countries except in people who have resided in West Africa 
and later migrated. The adults are peculiar in that they .wander about 
the body and may bo found in various tissues. They are most often seen 
making progressive motion under the conjunctival tissue of the eye. 
They are capable of moving in tissue at the rate of l/ 2 inch a minute. 
The cuticle in both sexes is covered with small wart-like projections. 
The larvae are sheathed 200 x 10 M. in size and the nuclei of the tail 
extend, into the tin. 
The life cycle is the same as that described for W, bancrofti except 
that the chrysops flies are the intermediate hosts and insect vectors. 
Dracunculus medinensis (Linnaeus, 1?58) 
This is the guinea worm and is found in the tropical and subtropical 
regions of India, Africa and South America. It is principally found in 
carnivorous animals in South America -and to a lesser extent in man. The 
adults live in the subcutaneous connective tissue and fascial pianos ‘of 
muscle, chiefly of the arms, legs, ankle and foot. 
Morphology. 
The adult males are about 2 cm. in length but have only been found 
on two occasions. The adult females measure about 120 cm. long by 1,5 ram. in breadth. The head has a chitinous shield and the vulva is situated 
very close to it. The tail is sharply bent into a hook shape. 
The larvae are unsheathed, ~fDO x 25 M. and have a long tapering tail 
Life History. 
When the female is gravid and the larvae are ready to be liberated 
she approaches the skin surface on an extremity and causes an ulcer to 
fora. When the ulcer is open and brought in contact with water the female 
protrudes her head and discharges a milky fluid containing myriads of 
embryos into the water. The embryos seek out the crustacean cyclops and 
develop in its body cavity to the infective stage. If the infected 
cyclops is ingested in drinking water the larvae escape in the intestines, 
invade the tissue and develop into adults. After fertilizing the female 
the male dies. The female then wanders in the tissue to one of the 
extremities where she comes to the surface and causes a surface skin ulcer. 
Class Trematoda, Rudolph!, 1808. 
The trematodes or flukes (except schistosomes in which the sexes are 
separate) are mostly parasitic hermaphroditic unsegmented, flak worms 
varying from 1 mm, in length to several centimeters. Two ventral 
prominent suckers are present near the head of all species and in the 
genus Heterophyes a third sucker is present around the genital pore. The 
digestive system consists of a mouth, esophagus and a bifurcated 
intestine which ends blindly. Anal openings are not present. The 
excretory system consists of flame cells, collecting tubules and excretory 
bladder. The male reproductive system consists of usually two various 
shaped testes (Genus Schistosoma U to 8), a pair of va.s cfefferens and a 
vas defferens ending in a cirrus. The female genital organs consist of 
an ovary (usually fan shaped) a branched tubular oviduct, one branch of 
which ends blindly, the other in its subterminal portion is designated 
as the uterus and the terminal portion the vagina. The genital pores 
are inconspicuous and located just in front of or just behind the central 
sucker. The life cycle of these parasites involves two or more he *s 
with a definite period of development in each. For the trematodes of 
medical importance man is the definitive host while certain molluscs 
emd sometimes crustaceans and fish act as intermediate hosts. There is 
always a period of parthenogenic multiplication in the mullusc while 
crustaceans or fish act only as carriers for encysted forms infective 
for man. 
With the exception of Schistosoma japonicum in the Philippines end 
Schistosoma mansoni in Porto Rico and rarely Fasciola hepatica, the 
trematodes infections of man are not contracted and are seldom encountered 
in the U. S. or possessions. They will only be briefly discussed and 
demonstrated. 
Definition of Terms 
Cirrus - the male copulatory organ or penis. Digenetic - Name applied to trematodes with a parthenogenic multipliua>;, 
tion in the larval stages as well as the usual sexual multiplicat ion 
in the adult stages. 
Distomata - A term applied to trematodes with two suckers close together. 
Gynaecophoric canal - The channel, formed by infolding of the lateral 
margins of the body in male schistosomes, in which the female lies. 
Miracidium - The ciliated larva which develops in all trematode egos and 
which is infective to raulluscs only. This is the first larval stage. 
Sporocyst - The second larval stage occurring in mulluscs. It is sac or 
ribbon-like in shape and contains masses of germ cells. In schistosomes 
■ only, a sexual multiplication occurs in this stage. 
Redia - this is the third larval stage and occurs only in molluscs. A 
sexual multiplication occurs in this larval stage of all species except 
schistosomes. 
Cercaria - The final stage of larval development. It alone is infective 
for the definitive host (man, dog, cat, sheep, hogs, etc.). 
Schistosoma mansoni, Sambon, 1907. 
This is the fluke of Africa, the West Indies and South America. 
Porto Rico is the only United States territory in which it is found as a 
local parasite. The adults inhabit the hemorrhoidal plexus and the portal 
circulation of man. They are seldom found in other venous areas. They 
deposit their .ova in the small venules of the,rectum and rarely those of 
the bladder. The majority of pathological lesions due to this parasite 
are in the rectum or liver. These lesions are typified by chronic 
inflammation, granulation, fibrosis, ulceration and scar tissue contrac- 
tions. Numerous large spindle shaped eggs with lateral spines are found 
in or on the inflammatory tissue, some are liberated into the rectum, 
rarely into the bladder and others undergo degeneration and calcificat ion 
in the tissue. Diarrhea, ulcerative proctitis and hepatitis with ultimate 
cirrhosis are frequent complications of this disease. 
Schistosoma haematobium (Bilharz, 1852) We inland, IBSS 
This is the blood fluke causing Bilharz disease, which is character-.; 
ized by haemature, cystitis, urethritis and hepatitis with cirrhosis. It 
is found in parts of Africa, Arabia, Palestine, and Mesopotamia, The 
adults inhabit the portal vein and its radicles and especially the 
vesical plexuses of man. The large broadly spindle shaped eggs with 
posterior terminal spine are found usually in the urine, rarely in the 
feces. This disease has never been known to have been contracted in the 
United States or its possessions. Schistosoma japonicum, Katsurada, I^OU 
This is the blood fluke causing schistosomiasis in Japan, China 
and certain areas in the Philippine Islands. The adult worms are found 
in the small veins of the large intestine particularly in the hemorrhoid- 
al plexuses, hut ra,ay occur in the gastric, mesenteric and other veins. 
Man, horses, cattle, pigs, dogs, cats, and experimentally mice may he 
infested hy this parasite. The eggs are large, oval to .spherical in 
shape with a small know-like projection on one side which is sometimes 
hard to demonstrate. The eggs are found in the feces, ulcerative lesions 
of the colon and rectum and in the liver. The disease resulting from 
infestation with this parastie is characterized hy diarrhea, colitis, 
and practitis followed hy cirrhosis of the liver, ascites and edenr . 
A detailed description of the morphology and life cycles of the 
schistosomes will not he given hut will he summarized in the following 
table: 
Males 
Length Breadth 
Type of cuticle Number of testes 
s. 
s. 
s. 
haematobium 
mansoni 
Japonicum 
1 cm. 1 mm. 
1 cm, 1,3 mm. 
1 cm. 500 M. 
Fine tuherculated U 
Coarse tuherculated 8 
Smooth 8 
Females 
Length Breadth 
Position of No. of ova Ova depos- 
ovary in body in uterus ited in 
s. 
Haematohium 
2 cm. 250 M. 
Posterior •§• 20:30 Singly 
s. 
Mansoni 
12:16 ran. 170 M. 
Anterior 1:3 Singly 
s. 
Japonicum 
2 cm. UOO K. Middle 30:50 In masses 
(10:30) 
Ova 
Shape Whore found Length Breadth 
S. 
haematohium 
Spindle shaped 
Terminal spine 
Urine 120;l60 M. U0;60 M, 
Rarely feces 
s. 
mansoni 
Spindle shaped 
Lateral spine 
Feces lUO:l65 M. 60;J0 M. 
s. 
Japonicum 
Broadly oval 
with small sub- 
terminal knoh 
on surface 
Feces , JO;100 M, 30j6p M, Fresh water molluscs acting'as mb e mediate hosts. 
_S. haematobium - Bulinus Physopsis africana, P. globosa and 
Planorbid dufouri. 
S_. mans on i -■ Planorbis boissyi,. P, pfeiffori, P. olivaceus, P. guadaloupen- 
sxs. 
S. .japonicum - Oncomelania noxophora, 0. femes ana, 0. hupensis. 
A representative life cycle for- the schistosomes may be summarized 
as follows; 
The eggs as passed in the urine or feces contain a ciliated larvae 
called a miracidium. When the eggs come in contact with water they hatch 
liberating the ciliated miracidium which swims rapidly about searching 
a suitable mollusc host. If it does not find one within a few hours it 
dies. When the miracidium finds its mollusc host it penetrates the 
tentacles and losing its cilia, and other special organs for which it has 
no further use, it migrates to the visceral mass and becomes a sporocyst. 
The sporocyst multiplies by formation of daughter sporocysts until by the 
end of a few weeks the visceral mass of the snail is permeated with these 
minute, elongated, delicate, tubo-like larvae, Five to seven weeks after 
infesting the snail the sporocysts stop producing further progeny and 
develop into the final larval form, the cercaria, which are infective to 
man. The cercaria are forked tailed, glider-like larvae and are cm cable 
of swimming rapidly. They will swim toward anything agitating the water 
such as a man wading or swimming. Upon gaining contact with the skin of 
nan they penetrate it, shed their tails, gain access to the circulation 
and are carried to portal circulation by the blood stream where they 
develop into adults within a period of three weeks. When mature the 
females enter the male gynaecophoric canal and remain there in copulation 
until ready to oviposit. When this time arrive the females leave the 
males and work their way against the blood currents until they reach a 
small venule in the hemorrhoidal or vesicle plexus. They force their 
way into a vessel that is slightly smaller than their transverse diameter 
by stretching it, deposit their eggs and withdraw. The eggs with spines 
are deposited so that the spines face the direction of the blood flow. 
The blood pushing against them force the spine into the vessel wall 
which soon causes a pressure necrosis and rupture into the feces or 
urine. S, japonicum eggs are deposited in>a mass blocking the venule* 
this leads to thrombosis, necrosis and ulceration with discharge of the 
ova into the feces. 
A summary of the life history of other flukes that may be encountered 
in man in the United States or its possessions is given below: Summary of Life History of 
Flukes Infesting Man 
F.hepatica 
F. buski 
C.sinensis 
P.westermani 
Egg;** 
) 
/A f 
WyS 
Egg contains miraci- 
diura when passed in;- 
Feces 
Feces 
Feces 
Sputum 
Egg reaches fresh 
water and the miraci- 
Snail (Lim- 
naea trun- 
Snails 
(Planorbis 
Snails 
(Bithynia 
Snail (Mel- 
ania liber- 
dium escapes, swims 
about and invades a 
mollusc;- 
catuia). 
schmackeri, 
Segmentina 
Nitidellus) 
Spp.). 
tina}. 
Miracidiura loses its 
cilia and specials or- 
gans & becomes spore- 
cyst.Migrates to vis- 
ceral mass of mollusc 
Yes 
Yes 
Yes 
Yes 
i 
\ 
i 
Sperm cells develop 
within the sporocyst 
& produce rediae:- 
Yes 
Yes 
Yes 
Yes 
Rediae product daugh- 
ter rediae inside them 
selves; these escape 
via birthpore & per- 
meate visceral mass 
of the mollusc:- 
Yes 
Yes 
Yes 
Yes 
Mature rediae develop 
within themselves cer- 
cariae, these likewise 
escape via birthpore 
to visceral mass of 
mollusc;- 
Yes 
Yes 
Yes 
Yes 
Cercariae leave, snail 
& swim about in water, 
enter second host of 
larva which is;- 
No second 
larval host 
cercariae 
encyst on 
grass. 
No second 
larval host 
cercariae 
encyst on 
water plants 
Fresh 
Vat or 
fish, 
various 
species. 
Fresh water 
crabs and 
crayfish. 
Definitive host;- 
Sheep, rare- 
ly man. 
Mein and 
Pig- 
Man, 
cats & 
dogs. 
Man, dog, 
cat, tiger 
wolf, pig. 
Definitive hosts 
eats and chews;- 
Grass con- 
taining 
encysted 
larva. 
Water 
plants 
contain- 
ing en- 
cysted 
larva. 
i 
Raw fish 
Raw crabs 
or crayfish. 
The live cercariae 
thus swallowed, each 
become adults in the;-’ 
Bile ducts 
Small in- 
testine , 
1 
Bile 
ducts 
Lungs Laboratory Methods Used in Examining for Helminths. 
Feces, 
1, Examination for adult helminths: 
A careful inspection of the fecal specimen will disclose any 
large helminth present as Ascaris lumbricoides or the large tapeworms. 
It will be difficult, unless great numbers are present, to demonstrate by 
such a procedure, small helminths, such as hookworms, pinworms, or the 
dwarf tapeworm. For this type of worm a method of concentration should 
be used. The procedure is as follows; secure a large specimen of feces 
and dilute it with tap water until a thin solution is obtained. The 
fecal solution should then bo strained through wire screen or gauze to 
get rid of the fecal matter, any small helminth present will be retained 
on the strainer. Wash the material retained on the strainer with 
additional amounts of tap water and transfer it to a shallow flat 
bottomed white enameled pan. The transfer is best done by inverting the 
strainer over the pan and then washing the material into the pan with 
about 100 cc. of tap water. A cotton applicator or long handled c ael’s 
hair brush is then used to go over the material bit by bit to detect any 
helminth present. 
2. Examination for larvae; 
A small bit of feces is selected and emulsified in a. drop of 
water upon a glass slide. A cover slip is applied to the emulsion and 
the preparation examined under low power magnification. The amount of 
feces used in making the preparation should be such that when the cover 
slip is applied ordinary news print can still be vusualized through it. 
Examine the specimen carefully, if Strongyloides larvae are present they 
can be easily found due to their active movement and the commotion 
created by them. 
3. Examination for eggs; 
a. Hon-operculated eggs. 
(l) Direct smear method. Examine hy the fresh wet pre- 
paration the same as for larvae. If Schistosome eggs are suspected 
select a hit of bloody mucus if present. 
specimens) 
(2) Concentration method. (Should be routine on all 
(a) Reagent 
25 per cent sucrose (cane sugar) solution in 
tap water, to which is added phenol as a preservative. Make vu 
1000 cc. at a time and keep on hand for routine use. (b) Equipment 
(l) Test tubes l 6 x 150 mm. (standard 
bacteriological culture tubes). 
(2) Applicator sticks. 
(3) Steel wool. 
(U) Wire loop, loop to be l/U inch in dia- 
rnater and bent at right angles to the shaft of the wire. ■ 
(s_) Glass slides and coverslips, 
(,c) Procedure. 
, Select a piece of feces the size of a hazel 
nut and place it in the’bottom of a test tube. Add 2 cc. of the sugar 
solution and thoroughly emulsify the feces by means of an applicator 
stick. When the feces is liquid in consistency, fill the tube to within 
one-eighth inch of the top with additional sugar solution. If there is 
much floating debris drop a small piece steel wool into the tube t sink 
it. Let the specimen stand in a vertical position for 30 minutes. Using 
the wire loop remove the uppermost layer of the solution and place it on 
a glass slide, coverslip it and examine. 
b, Operculated eggs. 
Use the fresh 7/01 preparation method and the concentration 
method given for Protozoan cysts. 
c. The Stoll egg counting method, (Used in estimating the 
number of helminths present by the egg count of the feces). 
(1) Method. 
Weigh 5 grams of feces into a large test tube 
graduated at 75 cc. Fill the test tube to the 75 cc. mark with N/lO NaOH 
(U grams of NaOH to 1000 cc. of water is sufficiently accurate). Add 
several glass beads, close the tube with a rubber stopper and shake until 
a homogeneous solution is obtained. Transfer 0,15 cc. of the suspension 
to a 2 x 1 inch slass slide and coverslip it with a number 2, coverslip 
(22 xkO ram.). Count the number of eggs present, the average of two such 
counts should be the count selected. 
(2) Interpretation, 
(a) Counts on liquid and mushy stools must be 
converted to correspond to formed stool amounts. 
(l) To convert mushy stool counts to for .ed 
stool averages multiply by two. (2) To convert liquid stool,counts to formed 
stool averages multiply by four. 
(b) When formed stool's are used iri making up the 
suspension the count of a 0,15 cc. sample of the mixture' multiplied by 
100 gives the number of eggs per gram of feces. 
(c) UU eggs per gram of feces indicates one female 
he cat or americana. present. 
Blood. 
Ova are not found in the blood. Larvae present arc best demonstrated 
by (l) thick blood films made «.t the optimum time, stained with Giemsa 
stain; (2) Drawing of 10 or more cc. of blood at the optimum time, laking 
it with distilled water, centrifuging the laked blood and examination of 
the sedimented residue; (3) if great numbers of the larvae are present 
they may be demonstrated by making wet mounts of the blood and examining 
the preparation direct without staining it. Most larvae are actively 
motile and can be easily seen under low power magnification. 
Urine, 
Filaria larvae or Shistosome ova are the only helminth material 
likely to be encountered. They are best demonstrated by examining the 
centrifuged sediment of a specimen of urine. 
Chylous fluid. 
Filaria larvae may be present and are best demonstrated by examining 
the sediment of a centrifuged specimen. , 
Sputum. 
Examine by: (l) The wet mount method selecting a blood tinged 
portion if available, (2) Add four volumes of N/10 sodium hydrcocic 
thoroughly mix and allow to‘digest for 2 to U hours, centrifuge ana 
exanin the sedimented residue. 
Skin. 
1. Examination for eggs. 
The only eggs likely to be found are those of pinworm.' They 
are most likely to be detected in swabbings from the perianal region. 
Hall’s method is the best available and is as follows: a. Material 
(l) Light weight cellophane (similar to that used on 
cigarette packages) cut into one inch squares. 
(2) One-eighth inch glass rod cut in U inch lengths and 
fire polished on each end. 
(3) Glass vials 10 x 60 mm. with a one hole rubier stopper 
to fit. 
(U) Rubber bands made by cutting l/ 8 inch pieces from 
l/g inch rubber tubing. 
b. Assembly, 
Insert a glass rod through the rubber stopper of a vial 
so that it comes to within l/U inch of the bottom. Remove the stepper 
containing the glass rod from the vial and fold a square of cellophane 
over the end which gees in the vial. Secure the cellophane in place with 
one of the small rubber bands. Replace the prepared swab into the vial 
and it is ready for use. 
c. Securing the specimen. 
The specimen should he secured from the patient the first 
thing in the morning before he has load the opportunity to tako a hath or 
defecate. To secure the specimen remove the swah from the vial and jswah 
the perianal region with the cellophane end. Replace the swah hack in 
the vial, label and send to the laboratory for examination. 
d. Examining the specimen. 
Place a drop of N/lO sodium hydroxide on a clean 
glass slide. Remove the swah from the vial, holding it vertical place the 
cellophane end into the drop NaOH. using a fcreep slip the rubber 
hand up the glass rod and then straighten out the cellophane so that it 
is like a coverslip on the drop of NaOH. Remove the rod, place an 
additional drop of RaOE on top of the’ cellophane and apply a coverslip. 
Examine the preparation under lev/ power magnification. 
2, Exanination for larvae. 
Onchocerca larvae may he present in skin nodules and can ho 
demonstrated hy studying aspirated . lymph, tissue scrapings, tissue juice 
or hy making stained histological sections. 
Other tissue. 
The demonstration of helminth parasites in tissue is done by; (l) 
histological study of biopsy material. (2) Digestion of the tissue with 
10 to 20 volumes of a solution containing pepsin and o,jj> hydrochloric 
acit. Followed by concentration of the larvae present by using the Baermann apparatus. (3) Direct examination of the tissue by pressing 
it into a thin layer between two heavy glass slides. All of the above 
methods are of value in demonstrating trichina larvae. 
Soil. 
1. Eggs. 
Soil contaminated with helminth eggs such as Ascaris is host 
examined hy the floatation concentration method given unuer feces, 50 to 
100 gram samples of solid should be used. 
2. Larvae. 
The recovery of helminth larvae from soil is best done by 
using the Baermann apparatus. Large samples of the soil being studied 
should be used. 
Serological Diagnosis. 
This means of diagnosing helminth infections is as a rule unsatis- 
factory. It should only be used when other methods are not applicable. 
It should only be done in large laboratories where special precautions 
can be taken to insure satisfactory antigens and controls. 
The Baermann apparatus. 
It consists of; (l) A glass funnel 6to 10 inches in diameter 
fitted with a short piece of l/U inch rubber tubing and a Hoffman clamp. 
(2) A round basket U to S inches in diameter and 3 inches deep made of 
1 ram. wire screen. (3) Cheese cloth cr gauze. 
The sample of soil or digested tissue to be tested is placed in 
the cheese cloth lined basket and the basket suspended in the funnel. 
Water heated to 100 degrees F. is run into the funnel until its level 
just touches the bottom of the basket. The material is allowed to stand 
and after a period of 1 to U hours, live larvae if present will be found 
in the water in the lower portion of theffunnelslls stem. A piece of ice 
placed on top of the specimen will hasten the migration of the larvae. 
The larvae are observed by either drawing of a small portion of the water 
and examining it direct under the microscope or by drawing of a 10 cc. 
sample, centrifuging it and then examining the sediment. 
Shipment of helminth material for diagnosis at a central laboratory. 
1. Specimens of large helminths. 
Kill and fix the specimens in hot (85) degree Centigrade) 
3 per cent formalin in tap water. Select a suitable container and ship 
the specimen in a solution of 5 per cent formalin and 2 per cent glycerin 
tap water. 
101 2. Fecal specimens, larvae or eggs. 
(a) Fixed specimen. 
Use at least two volumes of hot (S5 degree Centigrade) 
5 per cent formalin in tap water for fixation and then ship in a suitable 
container. 
(b) Unfixed specimen. 
When the distance which the material will have to he 
shipped is not too great an unfixed specimen should always he shipped 
If possible pack the specimen hottle in a container with a small amount 
of dry ice. 
(c) Specimens on glass slides such as filaria smears take 
adequate precautions to prevent breakage. File: 222 revised 
HEMATOXYLIN STAINING TECHNIQUE TOR FECES 
For fixed preparations- a smear is made with a paste brush on a clean 
flamed slide. If the fecal material is too dry, moisten slightly with 
normal saline. If it is too liquid, ■ smear a drop of egg albumen on the 
slide first, allow to dry slightly, and then brush over with specimen* 
Without allowing the slide to dry immerse it immediately in fixing fluid. 
SOLUTIONS 
l 
SCHAUDINN'S FLUID: 
a. . Stock solution: 
Mercury bichloride, saturated solution in normal 
saline (saturation is 5-73 grams ner 100 cc.) 
Alcohol 95 per cent 
2 parts 
1 part 
This solution will keep indefinitely. 
b. Add 2 cc, glacial acetic acid to Ug cc. of 
stock solution. 
* This solution is unstable and should be made up 
fresh before use and discarded immediately 
afterwards. 
2 
HEMATOXYLIN STAIN; 
a. Stock solution 1: 
Hematoxylin 
5 grams 
1000 c.c. 
Distilled water 
Place in clear glass bottle., cork and ripen in 
a light place for 1 month or longer 
.3. 
CAKBOL XYLOL: 
Xylol 
Liquefied Phenol (Merk’s Blue label grade) 
3 parts 
1 part 
■ u 
MORDANT: 
Ferric ammonium sulfate 
Distilled water 
2 grams 
100.0 c.c. 
5. 
DSCOLORIZEE; 
Ferric ammonium sulfate 
Distilled water 
0.5 gram 
100.0 c.c. METHOD 
Fix wet smears in Schaudinnfs fluid, heated to 
60° C. in water bath 30 min 
(SchaudinnTs fluid may be used cold - S hours) 
JCfj> Alcohol tinged to wine color with iodine 5 min. 
70jo Alcohol 5 min. 
50$ Alcohol 5 min. 
Wash in running tap water 2 min. (or more) 
Mordant (2*/> Ferric Ammonium Sulfate, heated to Uo° C.) 10 min. 
Wash in running tap water 10 min. 
Hematoxylin stain, heated to Uo° C. 10 min. 
Wash in running tap water 5 min.(or more) 
Decolorizer (0.5$ Ferric Ammonium Sulfate) 5 min. 
Wash in running tap water for a minimum of - 10 min. 
Graded alcohols - 50- 95i° 5 min. each 
Absolute alcohol .... 5 min. 
Carbol-Xylol !;• 5 min. 
Xylol 5 min. 
Mount with a thin layer of balsam,- thinned with 
Xylol, and cover slip. 
NOTE: 
The decolorization process is quite variable. So make 
more than 1 (one) slide and after decolorising for 
5 minutes, take 1 of the slides and rapidly run through 
the alcohols and xylene solutions leaving it in each for 
about 10 seconds. Mount the slide with halsam and a 
coverslip and examine under the microscope. Rarely, if 
ever, will the slides he too light; if so, place in the 
stain again and decolorize less than 5 minutes. Usually 
the slides will he Just right or a little dark. If too 
dark, decolorize for another minute and test the slides 
again.