Ifl/lanuat oj HUMAN PROTOZOA ^anuai of HUMAN PROTOZOA With Special Reference to Their Detection and Identification By RICHARD R. KUDO, D.Sc. Associate Professor of Zoology The University of Illinois CHARLES C THOMAS PUBLISHER 1944 Springfield • Illinois Baltinutre • Maryland All rights reserved, including the right of reproduction in whole or in part, in any form. Published by Charles C Thomas Copi/right, 1944, by Charles C Thomas 220 East Monroe Street, Springfield, Illinois First Edition 'RINTED in the UNITED STATES OF AMERICA Preface THIS MANUAL is bascd upon the author's laboratory not^s that have been in use in an emergency course offered at the University, in which the detection and identification of protozoan parasites of man and simple microscopical technique employed for this purpose, are studied and practised. There are now available several excellent trea- tises on parasitic protozoa of man which deal comprehen- sively with the morphological and developmental details, incidence of infections, geographical distribution, patho- logical changes brought about on human body, diagnosis, treatment, etc. As no one of these meets the need of the class, the present manual has been prepared. It contains onh' the essential information in order to serve as a practi- cal guide or companion book in detecting and identifying the human protozoa. The text has been compiled from the materials which have been accumulated bv the author in the last twenty- five years. In addition, the author has consulted the works of Mr. C. Dobell, Dr. R. P. Strong, Dr. C. M. Wenyon, and manv others, to whom he expresses his indebtedness. To supplement the descriptions in the text, a number of origi- nal drawings which have been especially prepared for this work, are inserted. Except the living specimens, all draw- ings were made with the aid of a drawing apparatus under an oil immersion objective, and depict precisely as the specimens appeared under the microscope. The author is under obligation to the authors of papers that contain the illustrations from whicli all or parts of figures 7, 8, 14, 15, VI MANUAL OF HUMAN PROTOZOA 16, and 28 have been redrawn. He further wishes to ex- press his appreciation to Mr. Charles C Thomas for his whole-hearted cooperation and thorough care in the mak- ing of the booklet. Richard R. Kudo Urbana, Illinois January, 1944 Contents Preface v Chapter 1 Introduction 3 2 Protozoa parasitic in the digestive tract 8 Sarcodina 8 1 Entamoeba histolytica 9 2 Entamoeba coh 15 3 Entamoeba gingivahs 19 4 lodamoeba biitschhi 21 5 EndoHmax nana 24 6 Dientamoeba fragihs 26 Keys to the genera and species of human amoebae 28 Differential diagnosis of the intestinal amoebae 30 3 Protozoa parasitic in the digestive tract (con- tinued) 31 Flagellata 31 1 Retortamonas intestinalis 31 2 Enteromonas hominis 34 3 Tricercomonas intestinalis 35 4 Chilomastix mesnili 36 5 Trichomonas hominis 38 6 Trichomonas elongata 41 7 Giardia intestinalis 41 Keys to the genera and species of flagellates living in the digestive tract 45 Differential diagnosis of intestinal flagellates . . 46 vn 5838:i viii MANUAL OF HUMAN PROTOZOA 4 Protozoa parasitic in the digestive tract (con- tinued) 47 Sporozoa • . 47 1 Isospora hominis 47 Ciliata 49 1 Balantidium coli 49 5 Technique for detection and identification of protozoa parasitic in the digestive tract 53 Collection of material 53 Microscopical examination 54 Fresh preparation 55 Permanent preparation 58 6 Coprozoic protozoa and objects present in the faeces 62 Coprozoic protozoa 62 Objects present in the faeces 67 7 Protozoa parasitic in the circulatory system .... 72 Flagellata \ 72 Trypanosoma 72 1 Trypanosoma gambiense 73 2 Trvpanosoma rhodesiense 75 3 Trypanosoma cruzi 76 Leishmania 78 1 Leishmania donovani 79 2 Leishmania tropica 81 3 Leishmania brasiliensis 82 8 Protozoa parasitic in the circulatory system (con- tinued) [ 83 Sporozoa 83 Plasmodium 83 1 Plasmodium vivax 86 CONTENTS ix 2 Plasmodium malariae 89 3 Plasmodium falciparum 92 4 Plasmodium ovale 94 Differential diagnosis of die diree common species of Plasmodium, as seen in Giemsa- stained thin blood films 96 9 Technique for detection and identification of protozoa parasitic in the circulatory system .... 97 10 Objects which may be confused with blood-in- habiting protozoa in stained films 102 11 Protozoa parasitic in the muscle and the repro- ductive organ 106 In the muscle 106 1 Sarcocystis lindemanni 106 In the reproductive organ 108 1 Trichomonas vaginalis 108 Reference books 110 Index Ill il/lanuat ot HUMAN PROTOZOA Chapter 1 Introduction UNFORTUNATELY mail is liost to soiiie twenty-seven spe- cies of parasitic organisms that are known as protozoa or unicellular animals. These protozoa nourish themselves b\' absorbing the body fluids or by feeding directly on tissue cells or on solid particles present in the digestive tract. The\' grow and multiph' and the daughter indi\iduals in turn grow and multiph-. This phase of a protozoan is called the trophozoite or vegetative stage (Fig. 1, i^). The bodv of a trophozoite is made up of the nucleus and cvtoplasm. The nucleus is vesicular in stiaicture, except in Balantidium coli, a ciliate, in which the macronucleus is a large compact one (Fig. 13, ^). The xesicular nucleus (Fig. 1, 2-4) is composed of the nuclear membrane which en- closes the nucleoplasm. The chromatin granules are either attached to the inner surface of the membrane or sus- pended in the nucleoplasm. In addition there is usually an endosome which is a conspicuous bod\^ and stains difi^er- ently in various protozoa. The endosome is either sus- pended in the nucleoplasm or attached to the nuclear membrane. The cytoplasm is frequenth differentiated into an outer layer, the ectoplasm, and an inner mass, the endoplasm (Fig. 3, i> 2). The nucleus, food vacuoles, etc., are ordinarily located in various parts of the endoplasm. In Sarcodina, Flagellata, and Ciliata, the trophozoite mo\es about ac- 3 4 MANUAL OF HUMAN PROTOZOA tively by means of pseudopodia, flagella, and cilia respec- tively. The fluid food substances are taken in throusb the entire body surface, while the solid food particles may be engulfed by pseudopodia or taken in through a permanent opening, the cytostome. The osmotic regulation is also carried on through the body surface in the majority, and there occurs no contractile vacuole in the human protozoa except in Balantidkim coli (Fig. 13, l). The trophozoite multiplies as a rule by binary fission which produces two daughter individuals. The species of Plasmodium however multiply by multiple division or schizogony (Figs. 21, 22). In this, the nucleus divides re- peatedly without cytoplasmic division so that several to many nuclei are produced within a trophozoite, and finally each nucleus becomes the center of a daughter individual. When the parent body breaks up, there are formed many daughter trophozoites. On this account, this type of tropho- zoite is called a schizont. After multiplying a certain number of times, the tropho- zoites of the protozoa which depend on man as the only host and not transmitted by invertebrates, encysts. It be- comes gradually less active, and secretes a resistant wall. Thus the cyst (Fig. 1, 5-9) is formed. The cyst wall thus produced protects the inner protoplasm against low tem- perature, desiccation, and other unfavorable external con- ditions which the cyst encounters after being voided in the faeces from the intestine. When a cyst finds its way into the digestive tract of man in food or water, excystment takes place, and young organism emerges and develops into the trophozoite. The encystment however does not take place in forms such as Trypanosoma, Leishmania, INTRODUCTION 5 and Plasmodium, which hve in man and also in blood- sucking invertebrates. In the human protozoa which belong to Sarcodina and Flagellata, sexual reproduction is not known, asexual re- production by the trophozoite and in some in the cyst, being the sole mode of multiplication. In the ciliate, Balan- tidium coli, conjugation has been reported to occur by some observers. In Plasmodium in addition to the schizogony mentioned above, certain schizonts develop into macro- gametes and microgametes which unite in pairs and pro- duce numerous zveotes. The zvo"ote formation and subse- quent development into an oocyst take place in the female anopheline mosquito. As to the effects of the protozoa upon the human body, information is at present not complete for all of them. In the broad sense of the term, all protozoa living in man are parasites. However, a ti'ue parasite is one which actually lives at the expense of human body and brings about cer- tain pathological conditions in it. The true protozoan para- sites of man and the diseases for which they are responsible are as follows: Entamoeba histolytica Amoebic dysentery, amoe- biasis Balantidium coli Balantidial dysentery, bal- antidiosis Trypanosoma gamhiense Central African sleeping sickness Trypanosoma rhodesiense East African sleeping sick- ness Trypanosoma criizi Chagas' disease MANUAL OF HUMAN PROTOZOA Leishmania donovani Leishmania tropica Leishmania brasiliensis Plasmoditini vivax Plasmodium malariae Plasmodium falciparum Plasmodiufu ovale Isospora hominis Sarcocystis lindemanni Kala-azar, visceral leish- maniasis Oriental sore, cutaneous leishmaniasis Espundia, naso-oral leish- maniasis Benign tertian malaria Quartan malaria Subtertian or malignant ter- tian malaria Ovale or mild tertian ma- laria Coccidiosis Sarcosporidiosis The remaining thirteen species of human protozoa ap- pear not to invade any living tissues of host. While un- doubtedly absorbing a certain amount of fluid substances in human systems, they seem to subsist also on micro- organisms which abound in the lumen of the intestine. As far as we can find, they do not bring about any noticeable damage upon the host, and are therefore to be called the commensals. Giardia intestinalis inhabits the duodenum and other parts of the small intestine by being attached, when not swimming about, to the gut epithelium, and thus may cause abnormal conditions of the gut epithelium over a wide area. A number of observers are inclined to think that this flagellate is a pathogenic protozoan. The known protozoa which inhabit the human body represent all four major groups of protozoa. Here they will INTRODUCTION 7 be considered for convenience under the following three headings: 1. Protozoa parasitic in the digestive tract. 2. Protozoa parasitic in the circulatory system. 3. Protozoa parasitic in the muscle and the reproductive organ. Chapter 2 Protozoa parasitic in the digestive tract THERE are fifteen species of protozoa which have been reported to inhabit the digestive tract of man. Of these six belong to Sarcodina, seven to Flagellata, and one each to Sporozoa and Cihata. Sarcodina The amoebae which inhabit the human body all belong to the group Amoebina, and are parasites of the digestive system. The tiophozoites absorb fluid nourishment and also engulf solid food particles. They multiply by binary fission. If the trophozoites leave by accident the human intestine, they perish within a short period of time. En- cystment takes place in four species. The cysts are capable of remaining alive outside the host body in the faeces, water, etc., for a considerable length of time and become the source of infection. When they are taken into the human mouth in contaminated water or food, the cysts pass through the stomach unharmed and excyst in the intestine. The emerged amoebae, if not uninucleate, divide to form uninucleate individuals and develop into tropho- zoites. The first three amoebae described in the following pages, have been in recent years designated by several writers as Endamoeba apparently in accordance with the opinion expressed by no. 99 of the International Commis- sion on Zoological Nomenclature ( 1928). This manual does 8 PROTOZOA IN THE DIGESTIVE TRACT 9 not follow this opinion, since the structures of the resting nuclei in Endamoeba, Entamoeba, lodamoeba, Endoli- max, and Dientamoeba, are distinctly different from one another. Unless all five genera are combined under one, these genera should be considered as distinctive. There- fore, the generic name. Entamoeba, is retained here. 1. Entamoeba histolytica Schaudiiin 1903 Synonym: Endmnoeha histolytica (Schaudinn) This is the pathogenic amoeba of man and commonly referred to as the "dysentery amoeba." The amoeba lives in the lumen and tissues of the colon wall, producing a typical ulceration and, in case of acute infection, it brings about dysentery, although in chronic infection there may not be any svmptoms at all. It frequently enters the liver by way of portal veins, and produces abscess in it. Other internal organs, such as the lung, brain, etc., have also been known to be invaded by this amoeba. The tropho- zoites are found in dvsenteric or diarrhoeic faeces, and cysts usually in formed faeces. Trophozoites 1. Living specimens. When seen in freshly obtained dysenteric or diarrhoeic faeces, the trophozoite (Fig. 1, l) is a typical amoeboid organism with the well differentiated cytoplasm. When undergoing progressive movements, it changes its body form little by forming a single pseudo- podium as broad as the body itself. Others may be seen changing their body forms continuously by actively form- ing lobose pseudopodia in an eruptive manner in which the hyaline ectoplasm plays a leading part, followed by 10 MANUAL OF HUMAN PROTOZOA a rapid flowing-out of the grayish granulated endoplasm. The amoebae vary in diameter 15-35ij. The monopodal individuals may be longer. On a warm stage, the activity may continue for hours if the cover-glass is sealed to the slide by vaseline or paraffin. But sooner or later, the amoeba will become less and less active and assumes a more or less rounded form. The formation of the pseudopodia and change of body form may continue for some time. Finally the organism becomes immobile. With the decrease in activity, the ectoplasm and endoplasm may often become more clearly difterentiated in many individuals. The ecto- plasm may be more voluminous than before, while the endoplasm becomes alveolated. In the endoplasm are found a nucleus and food particles. The nucleus is seldom seen in actively moving amoebae, but may be faintly seen as a ring of small granules, about 5m in diameter, in slug- gish individuals. The food consists of host tissue cells and tissue and body fluids which are absorbed through the body surface. The amoeba takes in erythrocytes and frag- ments of tissue cells, which give the organism a charac- teristic appearance. The number of erythrocytes found in an individual varies a great deal and not all individuals contain them. Bacteria and other microorganisms which are freely taken in by other species of Entamoeba men- tioned in the following pages, are only seldom engulfed by E. histolijtica. 2. Stained specimens. When fixed and stained (p. 58), the trophozoites (Fig. 1, ^-4) appear more or less rounded and their cytoplasm is reticulated. In some individuals the cytoplasm may be differentiated into the clearer ectoplasm and denser endoplasm (Fig. 1, 3). The erythrocytes pres- PROTOZOA IN THE DIGESTIVE TRACT 11 ent in the endoplasm are usually at various stages of disintegration (Fig. 1, --4). The nucleus is spherical, cir- cular in outline, and measures 4-7m in diameter. It is hicrhlv o 9:0 *C 0CJ V 6 a^ o ' 7 9 Fig. 1. Entamoeba histoh/tica. X 1150. (original) 1. A lixing trophozoite. 2-4. Stained amoebae, highly spread out. 5. A fresh cyst. 6-9. Stained cysts. vesicular. There is a centrally located endosome which may be surrounded by an ill-defined achromatic reticulum. The chromatin granules are e\enh distributed o\'er the inner surface of the nuclear membrane, and often referred 12 MANUAL OF HUMAN PROTOZOA to as the peripheral chromatin granules. These granules are smaller than those of E. coli (p. 16-17). The most com- monly observed specimens are 20-30|j in diameter. The trophozoites are usually found only in dysenteric or diarrhoeic faeces and occasionally in the mucus appar- ently eroded from amoebic ulcers voided in formed stools. While the majority of the amoebae are uninucleate, there may be a few binucleate ones which are undergoing divi- sion that ordinarily takes place in the tissues of the colon. Precystic forms The trophozoites become transformed into cysts. The transition form is known as the precystic form. Some ob- servers believe that the trophozoites which occupy the superficial position in the lesions of the intestine may be unable to grow after division, because of lack of fresh tissue cells or fluid, and remain small. Such amoebae secrete cyst walls and develop into cysts. The nucleus con- tains frequently larger chromatin granules and on this ac- count it is not possible to distinguish E. histolijtica from E. coli in this stage. Cysts 1. Living specimens. The cyst (Fig. 1, 5) is nearly spheri- cal in form, and its outline is almost always circular, though sometimes ovoid. It is highly refractile and appears some- what greenish, and therefore could be recognized with a little practice under a low power objective. The homogene- ous cyst wall is comparatively thin (about 0.5m thick), and the protoplasm fills the space within the wall. The nuclei vary in number from one to four, depending upon stages of development, and are difficult to make out, although PROTOZOA IN THE DIGESTIVE TRACT 13 sometimes recognized as indistinct rings. In addition, there may be seen a vacuole, and refractile, colorless rods which become deeph^ stained in permanent preparation and are known as chromatoid bodies. The c)'sts measure 5-20[j in diameter. When the cysts are stained with Lugol's solution (p. 57), the c\ toplasm becomes tinted in grayish l:)rown color and the nuclei are easily recognized. In many cysts there may be found one or more ill-defined masses which are stained reddish brown (the glycogen bodies), while the rod-like chromatoid bodies remain colorless. Thus Lugol's solution facilitates greatly the identification of the cyst. 2. Stained specimens. When the cysts (Fig. 1, 6-8) are fixed and stained (p. 58), the cvst wall remains unstained and is not noticeable as its index of refraction is nearly the same as that of the mounting medium, Canada balsam. The cytoplasm appears fineK^ reticulated and may show vacuolation especially in young uninucleate cysts (Fig. 1, 6). Such vacuoles as a rule contain glycogen. There are seen one to several rod-like bodies which stain intensely black. These are the so-called chromatoid bodies which in this species are almost always with rounded extremities. The nucleus in young uni- and bi-nucleate cysts may appear somewhat different in structure from that of the trophozoite described above, as in many cases the nuclei are preparing for, or completing, division (Fig. 1, 6). But when the nuclear divisions are completed, there are seen four small nuclei, about 2m in diameter, of the structure more closely re- sembling that of the trophozoite (Fig. 1, ^^ ^). Some of the four nuclei may divide once more on rare occasions so that cysts with six to eight nuclei may be seen. These are con- 14 MANUAL OF HUMAN PROTOZOA sidered abnormal and comparatively small in number. Tetranucleated mature cysts are typical of Entamoeba histolytica. In the faeces, the cysts do not undergo any further changes. The most commonly seen cysts measure about 7-16|j in diameter. Viability of cysts The dysentery amoeba is transmitted by viable cysts in contaminated food or water. The cyst-passers or carriers do often not show any symptoms of infection. According to Yorke and Adams (1926), cysts kept in faeces at 16-20°C. in the laboratory, begin to die rapidly and all are dead within about 10 days. Keeping the faeces in an ice box at 0°C. gives approximately the same result. Washed suspen- sions of the cysts live 17-21 days when kept at 0°C., and 10-11 days at 16-20°C. Chang and Fair (1941) state that the cysts are viable 90 days at freezing point, but only 30, 10 and 3 days at 50°, 68°, and 86°F. respectively. Yorke and Adams state that the cysts survive a tempera- ture of 45° C. for 30 minutes, but are killed within 5 minutes at 50°C. The lethal strengths of various chemicals and drugs when allowed to act upon the cysts for 30 minutes at 20- 25°C. and 37°C. are as follows: ' 20-25 °C 37 °C HCl 7.5% 5% NaOH 2.5% 2.5% CI (sat. aq. solution) 1/64 1/320 HgCL 1:2500 — Potassium permanganate 1% does not kill 1% does not kill Formaldehyde 0.5% 0.5% Carbolic acid 1% 1% Lysol 1% 0.5-1% Yatren 5% does not kill 5% does not kill Emetin HCl 5% does not kill 5% does not kill PROTOZOA IN THE DIGESTIVE TRACT 15 2. Entamoeba coli (Grassi 1879) Synonym: Endamoeha coli (Grassi) This is an amoeba which Hves in the kmien of the colon and is looked upon as a commensal. It is a very common amoeba in the human intestine and easily mistaken for E. histolytica because of the morphological similarity. Tro- phozoites and some cysts occur in fluid or semifluid faeces, but formed faeces contains only cysts. Trophozoites 1. Living specimens. This amoeba (Fig. 2, 1) resembles E. histolytica in size, but the ayerage indiyidual appears to be slightly larger than the latter. It measures 15-40m in the largest diameter, but the majority are about 20-35|j. Eyen in freshly obtained material, it is less actiye than E. histolytica. The cytoplasm is not well differentiated. The ectoplasm is thin and ill-defined and merges into the yo- luminous endoplasm. The endoplasm is usually yacuolated or alyeolated, and contains numerous bacteria, yeasts, other microorganisms of large size in addition to yarious debris that occur in the lumen of the colon. The erythro- cytes are normally not taken in by this amoeba, although instances of ingestion of erythrocytes by it especially in yitro haye been reported. For diagnostic purpose, there- fore, one can place a great emphasis on the presence or absence of the erythrocytes in the endoplasm as suggesti\ e of E. histolytica or E. coli. The nucleus is more easily seen than that of E. histolytica, especially in the specimens in which a comparatiyely small number of food particles are present. It appears as a ring of coarse refractile granules with a large granule located near, but not in, its center. 16 MANUAL OF HUMAN PROTOZOA 2. Stained specimens. In the majority of individuals (Fig. 2, 2-5)^ the body outhne is more or less romided and the cytoplasm is generally reticulated. Some individuals # ^» Q. .0 W^' S 6 8 g Fig. 2. Entamoeba coli. x 1150. (original) 1. A living amoeba. 2-5. Stained trophozoites. 3. A trophozoite infected by Sphaerita. 6. A precystic amoeba. 7. A fresh cyst. 8. A stained yomig cyst with a large glycogen vacuole. 9. A stained mature cyst. (Fig. 2, 2, 4) ii-,ay show the division between the ectoplasm and endoplasm. In the food vacuoles are found chiefly bac- teria, numerous inorganic faecal debris, and also micro- organisms which co-exist in the lumen of the colon. The PROTOZOA IN THE DIGESTIVE TRACT 17 nucleus is characterized by a thicker membrane than that of E. histolytica. The peripheral chromatin granules are coarser and often in block-form, and the endosome is a much larger body, about lu in diameter, and located usual- ly eccentrically. The clear ring around the endosome may be narrow or wide, and smaller chromatin granules may be lodged on the achromatic network present in the nu- cleoplasm. The nucleus is about 5-8m in diameter. Although the typical nucleus of this amoeba is quite different from that of E. histolytica, there occur invariably atypical or degenerating individuals with nuclei which may appear intemiediate between the two. If undecided, one must look for many typical individuals in which a positive identifica- tion can be made. Precystic forms Prior to encystment, the amoebae (Fig. 2, 6) become much smaller through division and transform themselves into precystic amoebae. These are usually rounded and more sluggish than the trophozoites. The cytoplasm does not contain food material. The amoeba resembles closely that of E. histolytica, but may be slightly larger. It is 8-20m in diameter. The nucleus shows a typical appearance, but may show a structure somewhat resembling that of £. histolytica. Therefore, it is quite difficult, as was stated already, to differentiate the two species of Entamoeba in precystic forms. Cysts 1. Living specimens. The cyst (Fig. 2, ") is usuallv spherical, but often ovoid in form. It measures 10-30m in 18 MANUAL OF HUMAN PROTOZOA the largest diameter. It is refractile and could be readily detected under a low power objective, although species identification is of course not possible under it. The cyst contents are granulated and often contain a large homo- geneous body particularly in uni- or bi-nucleate forms. It stains reddish brown with Lugol's solution and is con- sidered as a glycogen body. The nuclei are faintly visible. In uni- or bi-nucleate cysts, the nucleus may be seen as a ring pushed against the glycogen body. In older cysts with four or eight nuclei, not all of them may be seen. In addition, colorless needle-like bodies (chromatoid bodies in stained smears) may be present. When the cysts are treated with Lugol's solution, the nuclei become plainly visible, and the glycogen body stains reddish brown, while the filamentous bodies remain unstained. 2. Stained specimens. The cyst wall is not recognizable. The cytoplasm is finely reticulated, and in young cysts with one or two nuclei, there may be present a large well- defined vacuole in which glycogen body occurs. This vacuole may be very large in some cysts so that the cyto- plasm forms a narrow ring enclosing the nucleus (Fig. 2, 8). The chromatoid bodies are less abundant as compared with those of E. histolytica, and when present they are filamentous or irregularly shaped fragments with sharply pointed extremities (Fig. 2, 9). There are usually 1, 2, 4, or 8 nuclei, as in each of the second and third division, the nuclei divide simultaneously. Sometimes because of ir- regular divisions cysts containing 3, 5, or 7 nuclei of un- equal sizes may be seen. Very rarely cysts with more than eight nuclei may also be seen. All these cysts are more or PROTOZOA IN THE DIGESTIVE TRACT 19 less abnormal, and are insignificantly small in number as compared with tlie normal mature eight-nucleated cysts in routine work. The glycogen body appears to disappear before the cvst is mature, and may be entirely lacking even in vountT cvsts. 3. Entamoeba gingivalis (Gros 1849) Synonym: Endamoeba gingivalis (Gros) This is the amoeba of human mouth. It lives in carious teeth, in tartar and debris accumulated around the roots of teeth, and in abscesses of gum, tonsils, etc. Although held by some as the cause of pyorrhoea alveolaris, exact evi- dence for its pathogenicity is still lacking. It has been found in healthy gum and in false teeth, and is generally looked upon as a commensal. Only the trophic stage is known, cysts not having been observed up to the present time. It is of common occurrence. Trophozoites 1. Living specimens. As seen in fresh material in saliva under a sealed cover glass the amoeba (Fig. 3, i- 2) is as active as E. histolytica (p. 9). The cytoplasm is well differentiated. In some a broad single pseudopodium is formed at one end, which results in progressive movement, similar to the monopodal forms of E. histolytica or free- living limax amoebae; in others several pseudopodia com- posed chiefly of the ectoplasm are formed in quick suc- cession (Fig. 3, 1). The endoplasm is often vacuolated and contains a large number of food particles and a nucleus which is frequently seen as a ring about 3-4m in diameter. The food particles are almost always charac- 20 MANUAL OF HUMAN PROTOZOA teristically pale greenish and mostly rounded, each being enclosed in a food vacuole. They are probably the nuclei of leucocytes, pus cells, or other degenerating host cells found outside the gum tissues. Erythrocytes have however not been observed in this amoeba. Bacteria are also found in food vacuoles. The amoeba measures 8-30^ in diameter, i$4^' .A '1 4 5^7 Fig. 3. Entamoeba gingivalis. X 1150. (original) 1, 2. Living trophozoites. 3-7. Stained amoebae. but the most commonly seen individuals are 10-20|j in diameter. 2. Stained specimens. The amoeba (Fig. 3, 3-7) appears more rounded and somewhat contracted than in life, the majority having withdrawn their pseudopodia at the time of fixation. The cytoplasm shows a little differentiation into the ectoplasm and endoplasm. The whole is finely granu- lated or vacuolated and food vacuoles are very conspicu- PROTOZOA IN THE DIGESTIVE TRACT 21 ous. The various food particles seen in life are stained black to gray, in some of which reticulation is visible. Many bacteria occur also as food particles. The spherical nu- cleus is vesicular and measures about 2-4|j in diameter. There is a small endosome near the center, while the peripheral chromatin granules are small and compactly packed along the inner surface of the membrane so that the latter frequently appears as a uniformly thick ring. Depending on the degree of decolorization, there may be seen a clear zone around the endosome and the rest occupied bv an achromatic network. The stained speci- mens are a little smaller than the living ones. Although this amoeba is the very first parasitic amoeba seen by man and has been studied by numerous work- ers, encysted forms have, as stated already, not yet been observed. Therefore, the transmission appears to be carried on by the trophozoite. Koch (1927) found the effects of desiccation and varied temperatures upon the amoeba, as follows: The amoeba is killed at C. in 18 hours; 5^C. in 24 hours; 10°C. in 48 hours; 45°C. in 20 minutes; 50°C. in 15 minutes; 55 °C. in 2 minutes. At 40° C. survival is possible for an indefinite length of time. Complete desic- cation of the medium or immersion in water at 60° C. kills the amoeba. She considered that E. gingivalis may be disseminated both by direct contact and by an interme- diate contaminated article. 4. lodamoeha hiitschlii (Prowazek 1912) Synonym: /. williamsi (Prowazek 1912) This amoeba is intermediate in size between the two intestinal amoebae already described and the two smaller 22 MANUAL OF HUMAN PROTOZOA ones which follow, and is an inhabitant of the lumen of the colon. Like Entamoeba coli, it does not invade the tissues of the colon wall and live by absorbing fluid sub- stances and feeding on bacteria. Thus it is considered as a commensal. This amoeba is not as common as the amoebae already stated. The trophozoite and cyst are usually found together in diarrhoeic faeces, while the formed faeces contains cysts only. Trophozoites 1. Living specimens. This amoeba (Fig. 4, l) measures about 6-25m in its largest diameter, but the average indi- viduals are 8-15|j. It is a fairlv active amoeba, and when seen soon after being voided, it shows a monopodal pro- gressive locomotion, but presently rounds itself up and forms small pseudopodia, which reminds one of the slug- gish amoeboid form-change of Entamoeba coll. There is no sharp demarcation between the ectoplasm and endoplasm, but the ectoplasm is often well recognizable in pseudo- podia. The endoplasm is granulated and contains bacteria in food vacuoles. Large food particles which are commonly found in £. coli are ordinarily not present. The nucleus is usually not distinctly seen, but the large endosome may be seen surrounded by a clear circle around it in some individuals. 2. Attained specimens. The trophozoites (Fig. 4, --^) ap- pear as composed of reticulated or alveolated cytoplasm, depending upon the extent of degeneration (Fig. 4, 4, 5)^ in which are present bacteria taken in as food material. The vesicular nucleus measures about 3-4 m in diameter. The endosome which is about one-half the diameter of the PROTOZOA IN THE DIGESTIVE TRACT 23 nucleus, is typically surrounded by small spherules that do not take stains so that the achromatic interspherule substance may appear as a reticulum (Fig. 4, 3). There »|jwr^ \ 4 ■■> 6 7 9 10 Fig. 4. lodamoeba biitschlii. X 1150. (original) 1. A living amoeba. 2-5. Stained amoebae. 4, 5. Somewhat degenerated trophozoites. 6. A fresh cyst. 7-10. Stained cysts. may be small chromatin granules in this area. The endo- some is found in various parts of the nucleus, and may stain homogeneously or show a more deeply stained corti- cal layer. The well developed nuclear membrane is free from chromatin granules. Cysts 1. Living specimens. Although spherical cysts occur, the majority are ovoid, ellipsoid, triangular, pyriform, or square 24 MANUAL OF HUMAN PROTOZOA in form, which is a characteristic feature of this amoeba (Fig. 4, 6). Naturally the cysts vary a great deal in dimen- sions. More or less rounded cysts measure usually 6-15^ in diameter. The contents appear hyaline, but there is a conspicuous clear sharply outlined body which stains reddish brown with Lugol's solution and is considered as a glycogen body. It may be one-third to one-half the diameter of the cyst. Because of the presence of this glyco- gen body, the cysts had formerly been called "I cysts," before their relation to the trophozoite became known. Unlike the glycogen body present in the cysts of other amoebae already described, it persists in the majority of cysts in the present species, although it may become smaller when the cysts are kept in the faeces for several days. The nucleus is ordinarily faintly seen in a living cyst, but when treated with Lugol's solution, it may be recognized more clearly because of the characteristic endosome. 2. Stained specimens. The contents of the cyst (Fig. 4, 7-10) are reticulated and one (sometimes two) large clear vacuole is invariably noticed, which is the glycogen vacu- ole. The nucleus is usually situated close to the vacuole at one point. The endosome is often attached to the nuclear membrane and may be crescentic in shape. There is a single nucleus. Binucleate cysts are of rare occurrence and considered to be abnormal forms. 5. Endolunax nana (Wenyon and O'Connor 1917) This is one of the two smallest amoebae living in man and inhabits the lumen of the colon. It has not been seen to invade colon tissues and is therefore considered as a PROTOZOA IN THE DIGESTIVE TRACT 25 commensal. It is another vei)^ common amoeba and widely distributed. Trophozoites I. Living specimens. This amoeba (Fig. 5, l) is con- siderably smaller than the four species already described. It measures 6-15m in the largest diameter. The organism is a fairly active amoeba as seen in fresh material. It pro- s Fig. 5. Endolimax nana. X 1150. (original) 1. A living amoeba. 2-4. Stained amoebae, 5. A fresh cyst. 6. A stained cyst. gresses slowlv bv forming a broad pseudopodium in the direction of movement. When stationary, pseudopodia are formed at different points of the body surface. In these forms, the ectoplasm appears clearly differentiated from the endoplasm which is granulated and contains bacteria as solid food particles. The nucleus is not clearlv visible in living specimens because the endosome assumes varied appearances. 2. Stained specimens. The body is rounded (Fig. 5, --i). The reticulated cytoplasm contains bacteria, and is some- times infected bv Sphaerita (p. 69). There is a vesicular nucleus which measures about 1.5-3u in diameter. The 26 MANUAL OF HUMAN PROTOZOA nuclear membrane is delicate and a few peripheral chro- matin granules are attached to it. The size, form, and loca- tion of the endosome vary a great deal among different individuals. In the majority, the endosome is of triangular, square, or irregularly angular in shape, and may be in the center, toward one side, or attached to the membrane of the nucleus. In the latter case, a strand mav be seen con- necting it with a smaller chromatin mass on the opposite side of the nucleus. The endosome in well differentiated specimens may be seen as composed of a less deeplv staining matrix in which are imbedded 3-5 chromatin granules. Cysts 1. Living specimens. Usually ovoid in form, the cyst (Fig. 5, 5) appears as a hyaline and homogeneous body with a few small granules. In some there may be seen an irregularly shaped vacuole which stains red brown with LugoFs solution (glycogen body) which however disap- pears as the cyst matures. The nuclei are rarely seen in living cysts, but may be observed in Lugol-treated speci- mens. The cysts measure 5-12m in diameter, the majority being about 7-10|j. 2. Stained specimens. The cyst (Fig. 5, ^^) is finely reticulated. The nuclei vary in number from 1 to 4. Their structure varies as in the nucleus of the trophozoite, but appears to be characterized by an angular endosome and its variable location within the nucleus. 6. Dientarnoeba fragilis Jepps and Dobell 1918 This small amoeba appears to be another inhabitant of the lumen of the colon and considered as a commensal. PROTOZOA IN THE DIGESTIVE TRACT 27 It is widely distril)uted, ])ut iisuallv of rare occurrence, aldiough in certain areas the infection rate seems to he high. It has been seen only in the trophic stage in dysen- teric or diarrhoeic faeces, and encysted forms haye not yet been found. Trophozoites 1. Living specimens. This actiye amoeba (Fig. 6, !> -) undergoes progressive moyement by forming a few broad / 2 3 4 5 Fig. 6. Dientamoeba jragilis. X 1150. (original) 1, 2. Living trophozoites. 3. A stained uninucleate amoeba. 4, 5. Stained binucleate amoebae. and thin pseudopodia. The cytoplasm is well differentiated in actiyely moving individuals. The endoplasm is highl\' granulated and contains cocci or bacilli in food vacuoles. The nucleus is ordinarily faintly visible. The amoeba meas- ures 4-18m in diameter, but the majority are 5-12^ in diameter. 2. Stained specimens. The body is elongate round. The cytoplasm is reticulate and bacteria occur in food vacuoles. The amoebae (Fig. 6, 3-5) possess one or two nuclei. The ratio of uni- and bi-nucleate forms varies among different faecal specimens. In some binucleate forms may be 80% or more, while in others uninucleate forms may be pre- dominant. The nucleus is a small vesicle and about 1-2. 5m 28 MANUAL OF HUMAN PROTOZOA in diameter. There is a delicate membrane. The endosome is comparatively large and more than one-half the diameter of the nucleus itself. The ground mass of the endosome stains less deeply and is made up of plastin material. On its periphery are arranged 4-8 chromatin granules and occasionally a central granule. Degenerating trophozoites often develop vacuoles which coalesce into a large one and may resemble Blastocijstis hominis (p. 69-70). Transmission is apparently by the tro- phozoite. According to Wenrich (1940), the amoeba, if kept in faeces, remains viable up to 48 hours at room tempera- ture, but disappears, apparently by disintegration, in 2 hours at 3.5 °C. Keys to the genera and species of human amoebae A. The trophozoites in fresh and stained smears 1(10) Active trophozoite in dysenteric or diarrhoeic faeces 2 2(5) Trophozoites large, 20-30ii in diameter 3 3(4) Trophozoites contain often erythrocytes; nucleus with a small central endosome and comparatixely small pe- ripheral chromatin granules; more actively amoeboid. . . Entamoeba histolytica (p. 9-14) 4(3) Trophozoites do not ingest erythrocytes; nucleus with a large eccentric endosome and large peripheral chromatin granules; less active Entamoeba coli (p. 15-19) 5(2) Trophozoites smaller, average less than 20!ii in diameter 6 6(7) Trophozoites with 1 or 2 nuclei; endosome central, com- posed of plastin matrix and coarse chromatin granules; nuclear membrane without chromatin Dientamoeba fragilis (p. 26-28) 7(6) Trophozoite with only one nucleus 8 8(9) Trophozoite sluggish, 6-25|.i in diameter; nucleus with a large endosome, surrounded by a reticulum lodamoeba biitschlii (p. 21-24) 9(8) Trophozoite actively amoeboid, 6-18!ii in diameter; nucleus with an endosome of varied forms and location Endolimax nana (p. 24-26) PROTOZOA IN THE DIGESTIVE TRACT 29 10(1) Troplio/oitc in tlu' material aroiiiul Icclli K)ilmnoeha (^iuf^ivaVis (p. 19-21) B. Tlic c\sts ill fresh and stained smears Of the six known human amoebae, Entamoeba gingivalis and Dien- tamoeba fragilis have not yet been seen in eneysted condition 1(2) Mature cysts uninucleate and of various shapes; nucleus with a large endosome often attaclied to membrane; cyto- plasm witli a large glvcogen body lodamoeba butschlii (p. 21-24) 2(1) Mature cysts with more than one nucleus 3 3(4) Mature cyst with 8 nuclei; body spherical or o\al, about 10-30J.I in diameter; chromatoid bodies if present fila- mentous, acicular, or irregular with sharply pointed ends Entamoeba coll (p. 15-19) 4(3) Mature cvst with 4 nuclei 5 5(6) Mature cyst spherical, about 5-20|ii in diameter; chromatoid bodies numerous, rod-shaped with rounded ends; nuclei composed of a small central endosome and peripheral chromatin granules Entamoeba lu.stolytica (p. 9-14) 6(5) Mature cyst often ovoidal, small, about 5-121.1 in diameter; chromatoid body rarely found; nuclei with a large angu- lar endosome variable in position . . .Endolimux nana (p. 24-26) s 2 T 2 a li ll c 1 T n «o cS C % c 1 1 1 e s e s 1 1 1*3 2 ® 2 ph a II |i II a I 1. I s CS 1 "3 s Ill it III ^'a s 1 1 CO ~a I S ■ ~ c II 1 11 o.a cS 1 a > 7 CO llll 111 i nil 1 t*3 1 1 "^ 03 O li S 21 1 .2' a; c '0 1 Hi llll -ill ^" £ fe J j 1 M llll III PI 11 G "3 J;; 'o "cS .ii 4) m "O 5 C c Ill ®'-S 3 a ■p ill E- c a o J2 3 1 Z 1^ s Is ■|l r« cS 3 1 c a 1 1 a cS Q ■2 li ^1 <5< S 1 Chapter 3 Protozoa parasitic in the digestive tract (continued) Flagellata THE FLAGELLATES wliicli inhabit the digestive tract of man are all minute organisms. They live in the lumen of the intestine or in the mouth, by absorbing fluid sub- stances as well as by engulfing solid food particles. The trophozoites are able to move about only in fluid or semi- fluid contents of the intestine, and under favorable cir- cumstances multiply in large numbers. There is however no definite evidence to suppose that any of these flagellates is the cause of diarrhoeic condition. When the faeces be- come normal, the flagellates disappear, and the cysts ap- pear in their stead. As in Sarcodina, the trophozoite encysts. The cvsts are voided in the faeces and able to live outside the human host for variable lengths of time. Infection begins when viable cysts enter the mouth of a person in contaminated food or water. In the species in which the encystment does not take place, the trophozoite appears to be able to bring about new infection. 1. Retortamonas intestinalis (Wenyon and O'Connor 1917) Synonym: EmbacJomonas intesthmlis (Wenyon and O'Connor) This flagellate appears to inhabit the lumen of the 31 32 MANUAL OF HUMAN PROTOZOA intestine. It is widely distributed, but of comparatively rare occurrence. Fluid faeces contain both the active trophozoites and cysts, while formed faeces cysts only. Trophozoites 1. Living specimens. The flagellate (Fig. 7, 1-3) is highly plastic, and therefore polymorphic. It is 4-9|j long by 3-4n f I « '^ ::©' J 6 7 8 Fig, 7. Retortamonas intestinaUs. X 1150 1-3. Living trophozoites. 4, 5. Stained trophozoites. 6. A fresh cyst. 7, 8. Stained cysts. (1-4, modified after Wenyon and O'Connor; 5, after Dobell and O'Connor; 7, modified after Jepps; 6, 8, original.) broad. It undergoes continued jerky movements. The body is often pyriform or ovoid. The anterior end is broadly rounded, and the posterior end is either attenuated or rounded. The body length is usually about twice (or sometimes three times) the body width. Near the anterior end, there is seen a clear area which is the cytostome. The cytoplasm is granulated or slightly reticulated. There are PROTOZOA IN THE DIGESTIVE TRACT 33 two flagella on each individual; ])ut to see them clearly a dark field illumination is needed. When treated with Lu- gol's solution, the flagella can l)e seen. One flagellum which \ ibrates more actively is directed forward, and about as long as the bod\% while the second flagellum is shorter, but thicker and lies above or in the cytostome. 2. Stained specimens. The cytoplasm is finely reticulated or alveolated, and contains as a rule bacteria in food vacu- oles (Fig. 7, -^'5). The nucleus is located near the anterior end. It is spherical and shows the central endosome clearly. Very close to the anterior margin of the nucleus, may be seen the insertion points of the two flagella. At one side of the nucleus, there is the cytostome. The ridge surround- ing the mouth is marked by a delicate fibril. Mingled anions numerous uninucleate individuals, binucleate forms with four flagella may be seen. They are dividing indi- viduals. Cysts 1. Living specimens. The cyst (Fig. 7, 6) is a colorless refractile pyriform body. It measures 4.5-7m long by 3-4.5m broad. The contents are granulated and details cannot be made out because of the presence of a comparatively thick cyst wall. 2. Stained specimens. The cyst (Fig. 7, 7, 8) surrounded by a distinct membrane, appears granulated and contains an ellipsoidal area which is marked bv a looped fibril. The nucleus lies near the center of the cyst. In general appear- ance, the cyst resembles that of Chilomastix mesnili (p. 36), but is much smaller than the latter. 34 MANUAL OF HUMAN PROTOZOA 2. Enteromonas hominis da Fonseca 1915 This is a small flagellate of comparatively rare occur- rence. It probably inhabits the lumen of the intestine. Trophozoites 1. Living specimens. A small rounded or ovoid flagellate moves about actively with its three flagella. The body is ^ 9 € ^ 9 8 27 8 9 10 Fig. 8. 1, 2. Stained trophozoites of enteromonas Jwminis. X 1150 (modi- fied after da Fonseca). 3-10. Tricercomonas intestinalis. X 1150 (modified after Wenyon and O'Connor). 3-6. Living trophozoites. 7, 8. Stained trophozoites. 9, 10. Stained yomig and mature cysts. hyaline, but at times granulated, and contains a few gran- ules. The flagellate is about 4-6 u in diameter. 2. Stained specimens. The flagellate (Fig. 8, l^ 2) appears more or less spherical. The reticulated cytoplasm contains granules. The rounded nucleus is located close to the anterior end. Its nuclear membrane is moderately thick, and the endosome central and very large. The three flagella PROTOZOA IN THE DIGESTIVE TRACT 35 originate in the blepharoplasts attaelied to the anterior margin of the nuclear membrane. Encysted individuals have not yet been observed. Not all protozoologists agree on the validity of the species. Dobell (1935) examined da Fonseca's preparations and observed 4 flagella as well as encysted forms; and considered this flagellate and Tricercomonas intestinalis identical; while Wen\'on (1926) is inclined to think that this flagellate is a small form of Chilomastix mesnili (p. 36). 3. Tricercomonas intestinalis Wenyon and O'Connor 1917 This is also a minute flagellate which has been found in diarrhoeic faeces and appears to inhabit the lumen of the intestine. Though widely distributed, it is of rare occur- rence. The trophozoites and cysts occur in fluid or semi- fluid faeces, but in formed faeces only cysts are found. Trophozoites 1. Living specimens. The flagellate (Fig. 8, 3-6) is highly plastic, but usually pyriform in shape. The anterior end is broadly rounded, while the posterior end is drawn out. The cytoplasm is granulated and contains vacuoles. In a freshly made preparation, the flagella lash very actively and therefore it is difficult to observe them. There are three anterior flagella which produce jerky movements of the organism. The fourth flagellum runs along the flattened body surface and extends out freely at the posterior tip of the body. The flagellate measures 4-10m long by 3-6ij broad, but the majoritv are about 7 or 8[\ long. 2. Stained specimens. The stained trophozoites (Fig. 8, 36 MANUAL OF HUMAN PROTOZOA '^' 8) are more rounded than living ones. The cytoplasm is finely reticulated. The nucleus located close to the anterior margin is spherical or pyriform, and composed of a large endosome and a sharply defined membrane. The four flagella take their origin in the anterior border of the nucleus. Cysts 1. Living specimens. The cyst is a small ovoid body, sur- rounded by a distinct cyst wall. Its cytoplasm is homo- geneous, and contains small granules. There are 1, 2, or 4 nuclei which are not easily seen in life. The cyst measures 6-8m by 4-6m. 2. Stained specimens. The cyst wall is recognizable in stained specimens. The nuclei become clearly visible. Young cysts contain 1 or 2 nuclei (Fig. 8, ^), but mature cysts show 4 nuclei (Fig. 8, 10). The nuclei are spherical or ellipsoidal and highly vesicular, each with an endosome. The refractile granules seen in life are somewhat eosin- ophile. Some authors hold that this flagellate is the same as EntevGmonas hominis in which the fourth flagellum was overlooked. 4. Chiloinastix niesnili (Wenyoii 1910) This is a somewhat larger flagellate which inhabits the lumen of the colon and caecum. Some hold that it also inhabits the lower small intestine. Its solid food is exclu- sively bacteria and other microorganisms, and the organ- ism is considered a commensal. The trophozoites and cysts occur in diarrhoeic faeces, and the formed faeces contains cysts only. PROTOZOA IN THE DIGESTIVE TRACT 37 Trophozoites 1. Living specimens. The body (Fig. 9, l) is oval or pyriform in oudine, and measures about 5-20m long. The commonly seen individuals are about 10-15n in length. The anterior end is invariably bluntly rounded, while the posterior end is usually drawn out into a tapering process. The flagellate moves about in jerky fashion by means of A Fig. 9. Chilomastix mesnili. x 1150. (original) 1. A living trophozoite. 2-4. Stained trophozoites. 5. A fresh cyst. 6. A stained cyst. the three lashing flagella which are hard to be seen in life. The organism is less plastic than Trichomonas hominis (p. 38) which it superficially resembles. In the anterior one-third to one-half of the body is found a conspicuous cytostomal cleft which is somewhat spirally twisted and in which is located a short flagellum. The bodv itself is also frequently twisted. 2. Stained specimens. The body form is pyriform, with the bluntly rounded anterior and the Ions; drawn-out posterior end (Fig. 9, 2-4). The cytoplasm is reticulated and contains bacteria in food vacuoles. A spherical vesicular 38 MANUAL OF HUMAN PROTOZOA nucleus is located very close to the anterior tip. It is char- acterized by a distinct membrane to which chromatin granules are attached. The three flagella arise from a point anterior to the nucleus, and are of the same length (about 7-10m long). The fourth flagellum is about one-half the length of the other flagella and lies in the cytostomal cleft. The ridge of the cytostome is supported by two fibrils of which one on the right side is usually longer and makes a loop at the posterior margin. Cysts 1. Living specimens. The cyst (Fig. 9, 5) usually occurs with the trophozoite. It is pyriform and resembles a melon seed. It is about 7-10|j long. Its contents appear homo- geneous except a few coarse granules. In general appear- ance, it is similar to the cyst of RetoHamonas intestinalis (p. 31), but noticeably larger than the latter. 2. Stained specimens. The uniformly thin, but distinc- tive cyst wall is clearly recognized (Fig. 9, 6). The reticu- lated cytoplasm contains a spherical to ovoid nucleus located near the narrow end. The chromatin material is usually concentrated on a portion of the nuclear mem- brane. Surrounding or near the nucleus, may be seen the two cytostomal fibrils and the short flagellum. 5. Trichomonas hotninis (Davaine 1860) This is one of the commonest flagellates of the human intestine. It appears to inhabit the lumina of the colon and ileum. The organism frequently appears in large numbers in diarrhoeic faeces, but its presence is considered as a PROTOZOA IN THE DIGESTIVE TRACT 39 result and not the cause of the diarrhoea. It is widely dis- tributed, but more common in the tropics and subtropics. The trophozoite only is known. Trophozoites 1. Living specimens. This is an actively motile flagellate (Fig. 10, 1) which shows a jerky or spinning movement. It measures 5-20n in length. The organism is highly plastic, and assumes various body forms in life. It is however gen- erally ovoid or pyriform in body outline. Its anterior end is usually rounded, while the posterior end often tapers into a point through which the axostyle protrudes. A cyto- stome is located close to the anterior tip. On the opposite side of the body is seen the undulating membrane which extends slightly spirally towards the posterior end. The axostyle and undulating membrane are structures which distinguish this flagellate from Chiloniastix mesnili (p. 36) in life. Over a dark field condensor, the flagella become clearlv visible. There are tvpically five flagella in all, inserted at the anterior end of the body. Of these four are equally long, directed anteriorly, and lash freely, while the fifth flagellum makes the outer border of the undulating membrane. After being on the slide for some time, or when held stationary by debris in the preparation, the organism may become rounded and the undulating mem- brane becomes spread along the margin of the body and continues to undulate. Such an individual may simulate an amoeba. 2. Stained specimens. The majoritv of the stained speci- mens are more or less pyriform in shape, with the rounded 40 MANUAL OF HUMAN PROTOZOA anterior and the pointed posterior end (Fig. 10, 2, 3). A large ovoid vesicular nucleus is located near the anterior end. It is characterized by a distinct membrane which sur- rounds an endosome and reticulated achromatic substance in which chromatin granules are suspended. Four flagella arise from the blepharoplasts present near the anterior mar- # Fig. 10. Trichomonas hominis. X 1150. (original) 1. A living trophozoite. 2, 3. Stained trophozoites. gin of the nucleus. The fifth flagellum bordering the undu- lating membrane also takes its origin in a blepharoplast. The line of attachment of the undulating membrane is marked by a fibril which is known as costa. Unlike the flagellates already described, Trichomoims hominis has an endoskeleton, the axostyle, which is colorless or only faintly stained so that it appears a clear median line. The pos- terior portion of the axostyle usually extends beyond the body surface. The cvtostome is a clear crescentic area along the side of the nucleus opposite the undulating mem- brane. The body cytoplasm is granulated or alveolated, and often contains bacteria in food vacuoles. Some ob- servers noticed erythrocytes in this flagellate in bloody PROTOZOA IN THE DIGESTIVE TRACT 41 faeces or in culture media, but there is no evidence to show that the flagellate ingests erythrocytes still in tissues. This flagellate has not yet been observed in encysted condition. What were considered cvsts vears ago, are now known to be Blastocijstis hoininis (p. 69). Transmission is obviouslv by the trophozoites, some of which have been shown to withstand the gastric digestion and pass into the small intestine alive. 6. Trichomonas elongata Steinberg 1862 Svnonvm: T. huccalis Goodev 1917 T. tenax (O. F. Miiller) This flagellate is found in the human mouth, especially in tartar, and in pyorrhoeic sockets. It has also been ob- served in the sputum and pus of tonsils. Morphologically it is similar to T. hominis, and therefore description given for the latter will apply for the present species also. Whether these two species and T. vaginalis (p. 108) are identical or not, is still unknown, althoudi some of the recent observations seem to indicate that T. hominis and T. vaginalis are physiologically distinct species. As in T. Jwminis, T. elongata is known only in the trophic stage. Transmission is apparentlv bv the trophozoites which are transferred in food or water or by direct contact to another person. 7. Giardia intestinalis (Lanibl 1859) Synonym: G. lamhlia Stiles 1916 This is the most conspicuous flagellate of the human intestine and seems to be the commonest one. It li\ es in the duodenum and other parts of the small intestine. In the 42 MANUAL OF HUMAN PROTOZOA diarrhoeic faeces, both the trophozoite and cyst occur, but in formed faeces only cysts are present. In severe cases of infection with this flagellate, an enormous number of trophozoites appear to lie attached to the mucous mem- brane of the duodenum so that there may occur disturb- ances in the affected portion of the intestine. In some cases, the flagellate has been reported from the gall blad- der. But there is no definite evidence that it destroys the intestinal epithelium. Trophozoites 1. Living specimens. While the flagella lash actively, the organism progresses a little forward with a sidewise rocking motion. It is broadly pyriform (Fig. 11, 1), and about 9-20m long by 5-10|j broad. The anterior end is broadly rounded and the posterior part tapers into a usually upturned sharp point. Seen from side, one (dorsal) side is convex, while the other (ventral) side is flat or slightly concave (Fig. 11, 2). The anterior half of the ventral side is concave and acts as a sucking disc for attachment of the organism to the intestinal epithelium. The pellicle appears delicate, but the body form changes little, except the contraction and expansion of the sucking disc and bending of the posterior prolongation. The body cytoplasm is hyaline and seldom contains any solid food particles. The four pairs of flagella which in actively moving individuals cannot be seen without a dark field condensor, lash continuously. They may however be seen in less active individuals. Lugol's solution brings them out fairly distinctly and at the same time the two nuclei may PROTOZOA IN THE DIGESTIVE TRACT 43 ]:)ecome clearly noticeable near the center of the sucking disc. 2. Stained specimens. The bilateral symmetry of the flagellate is clearly revealed (Fig. 11, ^5, 4). Along the me- dian line of body there are seen two parallel rods rmming f- m S 6 7 8 Fig. 11. Giardia intestinalis. X 1150. (original) 1, 2. Li\ing trophozoites in front and side views. 3, 4. Stained trophozoites in front and side views. 5. A fresh cyst. 6, 7. Stained cysts. 8. A stained cyst in an end view. lengthwise. These are axostyles. Near the anterior end of the axostyles, there is an ovoid nucleus on either side. The two nuclei are similar in size and for the most part in structure. The nuclear membrane is comparatively thick and there is ordinarily one endosome in the center of the nucleus. There is sometimes present a deeply staining rod- 44 MANUAL OF HUMAN PROTOZOA like body lying across the axostyles near the posterior third of the body. The four pairs of flagella are usually arranged in the following manner: The anterior pair are inserted in the blepharoplasts located near the anterior end of the axostyles, cross each other near the anterior tip of body, follow the antero-lateral margin of the sucking disc, and become free flagella. The second pair originate in the an- terior part of the axostyles, travel backward along the latter structure, and become divergent by following part- ly the inner posterior margin of the sucking disc, leaving the body about one-third from the posterior end. The third (ventral) pair are thicker than others, and originate in the axostyles at points behind the disc, remaining free through- out. The fourth (caudal) pair arise from the posterior tips of the axostyles. Cysts 1. Lwing specimens. The cyst (Fig. 11, 5) is ovoid in form and refractile. It measures 8-14m by 6-10^. The cyst wall is thin, but distinctive, and there is ordinarily a nar- row space between it and the inner protoplasmic mass. Two or four rings and fibrils may be faintly seen. They are the nuclei, axostyles, fibrils of sucking disc, and some of the flagella. With Lugol's, these structures are more clear- ly visible. 2. Stained specimens. In stained cysts (Fig. 11, 6-8)^ the cyst wall is distinctly visible. Separated from it, is a granu- lated ovoid body of the flagellate. A young cyst contains two nuclei, each of which divides once, so that an older and mature cyst is tetranucleate. The axostyles, fibrils, flagella, are usually stained conspicuously. PROTOZOA IN THE DIGESTIVE TRACT 45 Keys to the genera and species of flagellates living in the digestive tract A. The trophozoites in fresh and stained smears 1(2) In the tartar of teeth Trichomomis elongata (p. 41) 2(1) In dysenterie or diarrhoeic faeces 3 3(4) The trophozoite l^ilaterallv symmetrical; witli two nuclei; eight flagella Giardia intcstinaJis (p. 41-44) 4(3) The trophozoite not bilaterally symmetrical 5 5(6) Witli an unduhiting membrane and axostyle Trichomonas hominis (p. 38-41) 6(5) Without undulating membrane 7 7(10) With a cytostome near the anterior end 8 8(9) With a large cytostome in which a short flagellum occurs; 3 anterior flagella; body 10-15|x long. .Chilomastix mesnili (p. 36) 9(8) With a small cytostome; two flagella; body 4-9|ii long. . . . Retortamomis intestinalis (p. 31-33) 10(7) Without a visible cytostome 11 11(12) Body more or less rounded; 3 anterior flagella; body about 4-6!lI in diameter Enteromonas hominis (p. 34-35) 12(11) Body more or less pyriform; 3 anterior flagella and one pos- terior flagellum; body about 4-101.1 long Tricercomonas intestinalis (p. 35-36) B. The cysts in fresh and stained smears Enteromonas hominis, Trichomonas hominis, and T. ehmgata ha\e not been seen in encysted condition 1(4) Cysts pyriform 2 2(3) Cysts small, 4-5-7|ii ^^^iig Retortamonas intestinalis (p. 31) 3(2) Cysts large, 7-10^1 long Chilomastix mesnili (p. 36) 4(1) Cvsts ovoid with nearly equally rounded ends 5 5(6) Cysts 6-8ti by 4-6!.i; 1, 2, or 4 nuclei Tricercomonas intestinalis (p. 35) 6(5) Cysts 8-14|Li by 6-10ii; 2 or 4 nuclei; with se\'eral fibrils Giardia intestinalis (p. 41) •2 i 1 00 Pyriform; 9-^20m long; 2 nuclei; 2 axostyles; without bacteria in food vacuoles Ovoid; H-UfM long; faint nuclear rings il ill, III! filial nil" £-j-i nil -2 c 3 ' il a &- Si II mw ilill 1^ 11 c ei II 1-1 -Si 3 C c 3 a^ C ■? 3 o 3 C 1:1 P t3 1 80 il 111 1 -14 c 3 1 Sec 2 = S c 1- 1 s £1 4> O 111 It — c n Chapter 4 Protozoa parasitic in the digestive tract (continued) Sporozoa 1. isospora honiinis (Rivolta 1878) THIS IS the sole coccidian parasite of man, and occurs in the faeces in the oocyst stage. Judging from the developmental cycle of other species parasitic in various mammals, it is assumed that the organism undergoes schizogony as well as sporogony in the epithelial cells of the small intestine, thus destroying host's intestinal cells. Oocysts The oocyst (Fig. 12, 1-4) is fusiform in general outline, but as a rule asymmetricallv drawn out at the two extremi- ties. One end mav be bluntly pointed, while the other often truncated. It is 20-33m long by 10-16m wide. The wall is composed of two membranes and is remarkably re- sistant to fixatives and stains, and therefore it can be far more satisfactorily studied in fresh or unstained conditions than in a stained smear. When freshly passed, the contents either fill up the oocyst completely (Fig. 12, 1) or more often appear as a spherical mass whose diameter coincides with the inner width of the cyst wall (Fig. 12, 2). The protoplasm is filled with refractile granules of various dimensions, among which the nucleus mav appear as a clear granule-free area. If a portion of the faeces contain- 47 48 MANUAL OF HUMAN PROTOZOA ing such oocysts is kept in a covered container at room temperature, the spore formation is completed within the oocyst in about 48 hours. At the end of the first day, the oocyst contents divide into two sporoblasts (Fig. 12, 3) and by the end of another day each sporoblast develops into a spore, measuring 10-16m long by 7-10m broad (Fig. 12, 4). Each spore contains four sporozoites and residual r\ /^, ^- ^"^' Fig. 12. Isospora hominis, unstained. X 1150. (original) 1. A young oocyst. 2. An oocyst in whfch the protoplasmic mass has contracted. 3. An oocyst with two sporoblasts, one of which shows the division of contents. 4. A mature oocyst with two tetrazoic spores. masses. Further changes would take place when the oocyst finds its way into the human intestine in contaminated food or water. This coccidian has been observed in Europe, North and South America, Africa, and Asia, but appears not to be of common occurrence. It is a cytozoic parasite in the intestinal epithelium and may be considered as pathogenic. However, no definite information on the effect (coccidio- sis) of this organism upon human host is available except PROTOZOA IN THE DIGESTIVE TRACT 49 that of Connal (1922) who described the course of an accidental oral infection by viable mature oocysts, as fol- lows: The incubation period was about six days, the onset sudden, and the duration over a month. The cure was spontaneous. The symptoms were diarrhoea, abdominal discomfort, flatulence, lassitude, and loss of weight. During the first three weeks of the illness no oocysts were found, but then oocysts appeared in the faeces for nine days. On the 10th, they were not seen, but reappeared on the 11th and 12th days, after which they were not found again. The acute signs of illness abated within one week of the finding of the oocysts. The faeces contained a large amount of undigested material, particularly fat which gave it a thick oily consistency, showing signs of slow gaseous formation. Although Isospora hominls is considered pathogenic to man in some cases, it appears to bring about no lasting disturbances in the majority of cases. Ciliata 1. Balantidium coli (Malmsteii 1857) This ciliate lives in the lumen as well as the wall of the colon and caecum, and is a pathogenic parasite. It has a wide geographical distribution, having been reported from Europe, Asia, Africa, and South and North America. In the United States a number of infections have been reported in recent years. But in the Philippine Islands more cases perhaps have been reported than anywhere else. Balanti- dium coli occurs more often in persons who come in con- tact with the pig in which it is a common parasite. Chim- panzee is also liost to this ciliate. A heavy infection with this organism brings about a chronic dysentery. Dysenteric 50 MANUAL OF HUMAN PROTOZOA or diarrhoeic faeces contains the trophozoites and occa- sionally cysts, but the formed faeces cysts only. Man becomes infected with the ciliate by taking in viable cysts through mouth. Trophozoites 1. Living specimens. The large protozoan (Fig. 13, l) is ovoid in form. The entire body is covered by numerous slightly obliquely longitudinal rows of cilia by means of which the organism swims about actively. Its size varies considerably, but the average specimens are 48-80n long by 30-60m broad. In slowly moving specimens, a short narrow peristome may be seen near the anterior end, which is lined by somewhat coarser cilia. The cytostome is located at its posterior end and is continuous with the cytopharynx through which solid food particles are taken in. The food particles are of various kinds, including the host's intestinal cells, erythrocytes, and leucocytes. Faecal debris of various types which occur abundantly in the gut lumen, are also ingested. The food vacuoles are found in various parts of the bodv. The cytoplasm is granulated. Near the posterior tip of the body, there is a narrow slit, the cytopyge, through which indigestible solid matter is thrown out. Unlike the other groups of protozoa already mentioned, this ciliate possesses contractile vacuoles, one near the center of the body and the other near the cytopyge. There are two nuclei, of which the large macronucleus is usually visible as a refractile sausage-shaped body. 2. Stained specimens. In the stained individuals (Fig. 13, 2) the structures seen indistinctly in life, may be clearly visible. The macronucleus is stained black and conspicu- PROTOZOA IN THE DIGESTIVE TRACT 51 ^xA ^ ? -^^^ Fig. 13. Balantidium coli. x 530. (original) 1. A lix'ing trophozoite. 2. A stained trophozoite. 3. A fresh cyst. 4. A stained cyst, ous. The micronucleus may also be seen. Food particles of different kinds are stained in different tones. Cysts 1. Living specimens. The cysts (Fig. 13, ^) are rounded or ovoid and appear slightly yellowish or greenish in color. They are about 40-60n in diameter. The wall is composed of two membranes. In a newly formed cyst, the ciliate may be seen actively revohing inside the wall, with the posterior contractile vacuole contracting rh\ thmi- 52 MANUAL OF HUMAN PROTOZOA cally. In older cysts, the movement ceases. The protoplasm of the cvst is hyaline, but granulated and does not usually contain any solid food particles. The macronucleus, peri- stome and a contractile vacuole are often noticeable in the cyst. 2. Stained specimens. Not much advantage is obtained by staining cysts (Fig. 13, 4). The macronucleus appears to be the most conspicuous structure. Two individuals may occasionally encyst together. Chapter 5 Technique for detection and identification of protozoa parasitic in the digestive tract T HE DETECTION and identification of the protozoa in- habiting the digestive tract are done under ordinary circumstances by examination of the faecal matter. Collection of material Natural movement should be collected. Do not use oily purgatives in obtaining faecal specimens, as they make the microscopical examination extremely difficult by the presence of numerous oil droplets in the microscopic field. The receptacle must be thoroughly cleaned and dry, and provided with a cover. The urine or water must be ex- cluded completelv. Avoid also using antiseptics for clean- inor the container. And if thev are used, the container should be cleaned and dried completely. The faeces must be examined as soon as possible, since the active tropho- zoites degenerate quicklv once outside the human intes- tine. If dysenteric or diarrhoeic faeces are to be examined, thev must not be older than one hour or two. In case this is not possible, wrap the container with a woolen cloth while transporting and keep it in an incubator at 37 °C. The organisms may live for several hours. Care should however be exercised during the microscopical examina- tion, since there unavoidably will be present a large num- 53 54 MANUAL OF HUMAN PROTOZOA ber of sluggishly moving individuals as well as degenerat- ing ones. The faecal specimen should be quickly examined with the naked eye. If it is formed, it should be examined mainly for the encysted form. If mucus, pus, or blood is present in the specimen, examine it for active trophozoites. In diarrhoeic or dysenteric faeces, one should look for trophozoites and occasional cysts. Microscopical examination Microscopical examination of the faecal matter is con- ducted as far as possible on fresh material, but in making out certain body structures which enable one to identify the protozoan under observation, permanent preparations are also necessary. The microscopic slides of standard size, 3'' by V\ should be of white glass and preferably thin, about 0.75 mm. thick (No. 1). For dark field illumination thin slides are es- sential. No. 1 cover-glasses should only be used for both fresh and permanent preparations. They are about 0.13- 0.17 mm. thick. The most convenient size is about Is' square covers, which are preferred to circular ones. The slides and cover-glasses must be cleaned thoroughly before being used. Immerse them in a concentric mineral acid (nitric acid is best fitted) for about 10 minutes. Pour off the acid, wash the slides and covers for about 10 minutes in running water, rinse in distilled water, and keep them in 90-95% alcohol. When needed they are dried one by one with clean cheesecloth. Handle slides and covers with a pair of forceps. If thumb and fingers are used, hold them by edges or ends. DETECTION AND IDENTIFICATION 55 Fresh preparation If the faeces is cl\ seiiteric, a small portion is placed by a toothpick or platinum loop on a slide and covered with a cover-glass. Before placing the cover, all large particles must be removed so that the smear preparation will be uniformh' thin. If the faeces is diarrhoeic, then the smear is made in a similar manner. But if the faecal specimen is semiformed or formed, a small drop of warm (37 C.) 0.85^ sodium chloride solution which has been boiled be- forehand, is first placed on the slide, and by means of a toothpick, a small portion of the faeces, particularly mu- cus, pus, or blood, is emulsified in it and a cover is placed o\'er the whole. The smear should not be too thick or too thin for a satisfactorv observation. If the smear is too thick, it will be impossible to distinguish objects clearly, and on the other hand, if it is too thin, there will unavoid- ablv be a great deal of time lost in detecting widely scat- ered protozoa. The optimum thickness of the smear is one through which the print on this page can be read. Place the preparation on the stage of the microscope and examine with a moderately low power objective, by mo\ ing the slide svstematically by hand or by a mechanical stage. Recognize the active tiophozoites bv the refractility and the change in bod\' form or movement, and in case of cysts by their form and size. The determination of the size of a microscopical object can be done quickly and easily, if ocular micrometer divisions have been calculated in combination with different objectives. When trophozoites or cvsts are recognized, examine them one h\ one under a high dry objective, and identify them. If necessar\ , an 56 MANUAL OF HUMAN PROTOZOA oil immersion objective should be used to make out finer details. The objectives used depend upon the training and ex- perience of the one who carries on the examination. A low power objective must be used as far as possible. The lower the magnification, the brighter and the larger the field. The success of examination depends almost entirely on continued practice, since the faecal matter contains myri- ads of objects which may resemble protozoa that them- selves may quite often be at various stages of degeneration and disintegration. Experienced observers can recognize most of the protozoa described in the preceding pages easily in a low power objective field. It is important to examine the fresh preparations care- fully and thoroughly. If no protozoa here mentioned are found, make several more preparations from different parts of the specimen and examine. Often it is necessary to obtain and examine specimens on several successive days from a person, before a positive or negative diagnosis can clearly be established. Remember demonstration of either the trophozoite or the cyst is absolutely necessary for a positive diagnosis of a protozoan parasite. The flagella of the actively moving flagellates are difficult to recognize in an ordinary bright field, but the peculiar movement in combination with the finding of certain struc- tures such as cytostome, undulating membrane, etc., will suggest that the organism is flagellated. In order to see actively moving flagella, a dark field condensor is neces- sary. Rut when this is not possible, treat the specimens with LugoFs solution (p. 57). The cysts are as a rule distributed throughout the formed DETECTION AND IDENTIFICATION 57 faeces, and therefore are difficult to detect in small por- tions of the naturally voided faeces. Flecks of mucus in the fluid faeces obtained by use of a saline purge may contain more cvsts than naturally passed one. In the ordinary formed faeces, the following concentration method is frequently advantageous in revealing more cysts. Emulsifv thoroughly a small mass of faeces, about the size of a lump of sugar in a mortar by adding a small amount of once-boiled tap water. Add to it about 500 cc. of water and pour the whole emulsion into a glass cylinder and let it stand for about 15 minutes. Remove the scum floating on the surface and draw oft the turbid fluid into another cylinder, leaving the sediment and a little fluid above it untouched. The majority of cysts are suspended in the drawn-out part of the emulsion. If a centrifuge is avaflable, centrifugalize the fluid, pour ofl the supernatant fluid and add water. Centrifugalize again. Repeat this three or four times until the supernatant fluid is clear. The deposit will contain many cysts which now can be examined and identi- fied. If no centrifuge is on hand, let the glass cylinder stand for about 20 hours and examine the sediment for cysts. The cysts will be more numerous than in untreated specimen. The cysts are frequently more satisfactorily identified if one or two drops of Lugol's solution is well mixed with the faecal matter on a slide. This solution is composed of potassium iodide 1.5 grams, water 25 cc, and iodine 1 gram. As the solution deteriorates easily, fresh solution should be prepared about every two weeks. Lugol's solu- tion of course kills all protozoa in the preparation. After about 5 minutes, examine the smear. The flagella become 58 MANUAL OF HUMAN PROTOZOA stained, the nuclei are much more clearly visible, and glycogen bodies are stained reddish brown, while the chromatoid body remains unstained. Permanent preparation Permanent preparations are employed to aid in identifi- cation of living protozoa, and not as substitutes for fresh preparations. Smears are made on cover-glasses, and not on slides as in fresh preparations; mark with India ink, wax pencil, etc., the unsmeared side of the covers. Instead of trying to place all data, simply write the number, and enter on a record card all necessary data, such as the name of the person, date, condition of the specimen, fixative, stain, and any other remarks. The smears should be left horizontallv with the smeared side up for a short while. Place a jar above them to exclude dust, flies, etc. The purpose of leaving the slide for a few minutes is not to dry the smear, but to allow the active trophozoites to become attached to the cover by pseudo- podia or flagella and at the same time to allow the fluid to evaporate a little. Smears made from dvsenteric or fluid faeces should be fixed almost immediatelv, and those made from diarrhoeic or formed faeces emulsified in warm salt solution should be left for a few minutes. In any case, do not let the smear dry, except a narrow peripheral zone. The smears are next to be fixed. For fixation, Schaudinn's fixative is most widely used and advocated here exclu- sively, though any good histological or cytological fixatives will do. Schaudinn's fluid is made up as follows: Mercuric chloride (HgCl, or corrosive sublimate) 6-7% (cold satu- rated) aqueous solution 6 cc, 95% or absolute alcohol 3 cc. DETECTION AND IDENTIFICATION 59 and glacial acetic acid about 4 drops. The first two can l)e kept mixed without deterioration, but the glacial acetic acid should be added just before fixation. Fix at room temperature. The fixative is put in a Petri (preferably square) dish or a wide stendor dish. The smear is gentlv dropped on the fixati\e with the smeared surface facing downward. With a little practice, air bubbles can be a\'oided and make the smear float on the surface of the fixative. After about one minute, turn the smear around and let it stav on the bottom of the container for 5 more minutes. If the smear is too thick, it will not float on the fixation. A thin coat of \ aseline on the unsmeared side will allow the co\ er to float. About six co\'er-glass smears can be fixed in an ordinarv Petri dish simultaneously. The cover-glasses are now transferred into a coplin jar or better Columbia staining jar for cover-glasses, containing 50% alcohol for 10 minutes, and then two changes for 10 minutes each. Transfer next the smears into a jar containing 30^ alcohol for 5 minutes, and then into a jar with water which is now placed under gentlv running tap water for 15 minutes. Rinse the smears in distilled water. They are ready for staining. The most dependable staining which is wideh^ used for staining intestinal protozoa is Heidenhain's iron haema- toxylin. It requires a mordant, ammonio-ferric sulphate (iron alum) and a dve, haematoxylin. Crystals of iron alum be- come vellow and opaque verv easily; select clear \iolet crvstals and prepare 2% waterv solution which will keep for a long time. 0.5-1^/c haematoxvlin must be well "ripe." The most convenient way of preparing it is to make 10% absolute alcohol solution. B\ diluting this stock solution 60 MANUAL OF HUMAN PROTOZOA with distilled water, prepare 0.5-1% slightly alcoholic solu- tion which will be readv for immediate use and which can be used repeatedly. The smears are now placed vertically in iron alum solu- tion and left in it for 1-3 hours. Wash with running water for 5 minutes and rinse in distilled water. Immerse the smears in haematoxylin solution for 1-3 hours. Smears are now washed for 5 minutes in running water and placed face up in a Petii dish containing a weak (0.25%) iron alum solution for decolorization which must be controlled under a compound microscope. If all smears are of uniformly the same thickness and meant for the same pro- tozoa, one of the smears may be watched continuously. If the smear contains, for example, the trophozoites of En- tamoeba histolytica, the whole amoeba will look black at the beginning, but the cytoplasm becomes gradually de- colorized, leaving the nucleus and the cytoplasmic inclu- sions still dark. As the process is continued, further de- colorization takes place, and finally the nucleus may ap- pear as a ring. Often it is necessary to control the last stage of the process under a high dry objective. For this purpose, flood the well in a depression slide with the iron alum solu- tion and place a representative smear over it with the smeared side facing downward. Such a smear could be observed with a high dry objective and will be better con- trolled. Optimum staining will be attained by repeated practice. When the decolorization is completed, the smears are washed gently in running water for about 15 minutes in order to insure the complete washing of the iron alum. Rinse in distilled water and transfer to 30%, 50%, and 70% DETECTION AND IDENTIFICATION 61 alcohol in order for 5 minutes each. Then if counter- staining is desired, dip in 1% eosin in 95% alcohol for a few seconds, and then transfer the smears to two changes of plain 95% alcohol, two changes of absolute alcohol, and finally two changes of xylol. The smears can now be mounted one bv one in a drop of Canada balsam-xylol which is placed in the center of a slide. The technique involved is simple and with a little practice, satisfactory smears can be prepared. Before examining the slides under the microscope they should be placed in a drying oven at about 60 °C. for a little while. As in the case of fresh smears, microscopical examination of permanent prep- aration should be done as far as possible under low mag- nifications. Chapter 6 Coprozoic protozoa and objects present in the faeces Coprozoic protozoa NUMEROUS free-living protozoa which inhabit waters containing abundance of organic matter or decompos- ing organic matter, mav sometimes be found in stale faeces. These have been collectively called coprozoic protozoa. With food or water, the cysts of these protozoa may enter the human mouth and pass through the intestine un- harmed. In the old faeces, they exc\'st and the trophozoites may develop in a larger number. Furthermore, the tropho- zoites or cysts of certain free-living protozoa which live in fresh, brackish, or salt water, may be introduced into the faeces after it has been voided and if conditions are favor- able, numerous trophozoites may appear in it. The copro- zoic protozoa are, of course, not seen in the trophic stage in freshly voided faeces, but develop in old specimens. There- fore, if old faeces contains actively moving protozoa which were not present in the specimen when examined fresh, they are most certainly coprozoic and not parasitic proto- zoa. But when the faecal material is not examined fresh, and examined a few to several days later, one cannot tell without careful examination if the protozoan observed is parasitic or coprozoic. Coprozoic protozoa belong to all of the three major groups, Sarcodina, Flagellata, and Ciliata. Here a few forms will be mentioned. 62 COPROZOIC PROTOZOA IN THE FAECES 63 1. Hartinannella hyalina ( Danjifeard 1900) Trophozoites. It is an actively amoeboid organism with flattened pseudopodia (Fig. 14, i), and is 5-20m in diameter. The cytoplasm is well difl^erentiated. The endoplasm con- tains a contractile vacuole and bacteria taken in as food. When stained, the nucleus, .3-4n in diameter, ])ecomes \ isible. It is composed of a distinct membrane, a large endosome, and chromatin granules distributed in the nu- cleoplasm. Cysts. Spherical; 10-15m in diameter (Fig. 14, 2). The inner cyst wall is thin, but the outer one is thick, brownish, and much wrinkled. The cvst wall has no pores. It contains a single nucleus. 2. Dimastigamoeba gruberi (Schardinger 1899) This amoeba has both amoeboid and flagellate stages (diphasic). It is ordinarily seen in amoeboid form, but when the medium becomes more fluid, it changes into flauellate form, Cvsts also occur. Trophozoites. 1. Amoeboid stage. The active amoeba (Fig. 14, ^^) forms a few broad lobopodia which are com- posed mostlv of ectoplasm. In the endoplasm of a living amoeba can be seen a vesicular nucleus with a large endo- some, a contractile vacuole, and bacteria taken in as food. The amoeba measures about 10-20n in length. When stained, the nucleus (about 3-4m in diameter) shows a central endosome and scattered chromatin granules be- tween it and the membrane. 2. Flagellate stage. When distilled water is added to the medium in which amoeboid forms occur, the latter become flagellated (Fig. 14, ^). The body is ovoid to pyri- 64 MANUAL OF HUMAN PROTOZOA form, and measures 10-30|j long. There are two equally long flagella at the narrow end, near which is located a nucleus. A contractile vacuole is present near the posterior end. Stained individuals show that the flagella arise from points just in front of the nucleus. Cysts. The cyst (Fig. 14, 5) is spherical and about 7-14|j in diameter. The cyst wall may show 3-8 small pores. The nucleus may be faintly seen near the center. When stained, the cyst wall is seen double-layered. The pores are sur- rounded by slight thickenings of the wall. The majority of cvsts are uninucleate. 3, Bodo caudatus (Dujardin 1841) This is a small biflagellated organism which is very common in stagnant water and moist soil. Trophozoites. Actively motile; body polymorphic, but usually elongate. About 10-20m long (Fig. 14, 6, 7). Its anterior end is bluntly pointed, while its posterior end is attenuated. The body is somewhat compressed. There are a vesicular nucleus with a large endosome near the center of body, and bacteria in food vacuoles. Two flagella arise in the anterior end. The anterior flagellum is short, but the trailing flagellum is long. A small slit, the cytostome, and a small contractile vacuole are located near the flagel- lated end. When fixed and stained, the body becomes more rounded. The nucleus contains a large central endosome and there are seen radiating strands between the latter and the membrane. The anterior flagellum is about the body length, but the posterior one is about twice the body length. Near the blepharoplasts there is a large rounded parabasal body. COPROZOIC PROTOZOA IN THE FAECES 65 ^^^' "" / :^4 - " ■ ■ .' .-•^ • io%^V\' *.♦-' "m 4- ^ 5 \ I Q 7 8 12 10 13 II Fig. 14. Coprozoic protozoa. X 1000 (modified after Dobell and O'Con- nor). 1, 2. Hartmannella hijalina. 1. A stained trophozoite. 2. A stained cyst. 3-5. Dimastigamoeha gruheri. 3. A stained amoeboid form. 4. A stained flagellate fomi. 5. A stained cyst. 6-8. Bodo caudatus. 6. A living trophozoite. 7. A stained trophozoite. 8. A stained cyst. 9. A stained trophozoite of Bodo cdax. 10-12. Cercomonas longicauda. 10. A li\ing trophozoite. 11. A stained trophozoite. 12. A fresh cyst. 13. A stained trophozoite of Copromonas subtilis. Cysts. The cyst (Fig. 14, 8) is oval, surrounded b\- a thin wall, and about 5-7m in diameter. In the granulated cytoplasm are found a nucleus and a parabasal body. 66 MANUAL OF HUMAN PROTOZOA Somewhat similar to this is Socio edax Klebs 1892 (Fig. 14, ^J) which is less frequently seen in faeces. The tropho- zoite is more stumpy and a little smaller, being about 6-15p long. The body is much more rounded in cross-section. The two flagella are nearly equal in length and both are longer than body. Cysts resemble closely those of B. catidatus. 4. Cercomonas longicauda Dujarclin 1841 Trophozoites. Body plastic and amoeboid, engulfing bacteria by pseudopodia (Fig. 14, 10). About 5-10m by 5-7m. The nucleus is located near the flagellated anterior end, pyriform in shape, and contains a central endosome. Of the two flagella, the anterior one is about 3-4 times the body length, but the posterior flagellum adheres to body surface and extends a little bevond the body (Fig. 14, H). Cysts. Spherical, about 4-6m in diameter. Uninucleate (Fig. 14, 1-). Coarse granules are often grouped around the nucleus. 5. Copromonas suhtilis Dobell 1908 Trophozoites. Body elongated pyriform (Fig. 14, 1-3), the pellicle is comparatively thick so that there is little change of body form. It is 7-20|j long. The anterior end is bluntlv pointed, while the posterior end is rounded. A single flagellum takes its origin in the anterior end, near which are seen a contractile vacuole and a reservoir. The vesicular nucleus with a large endosome is near the center of body. At the anterior end, the cytostome opens and is continuous with a long tubular cytopharynx. The cytoplasm usually contains bacteria. COPROZOIC PROTOZOA IN THE FAECES 67 Cysts. ThiiiK- enveloped cvsts are rounded or oval in form, and measure 7-8^ in diameter. Thev are uninucleate. Objects present in the faeces An enormous number of different kinds of objects occur in the faecal matter, some of which mav resemble super- ficially intestinal protozoa and thus may be mistaken for them. These objects are derived from foods, microorgan- isms present in drinking or cooking w^ater, or the digestive tract itself. To deal with all of them is bevond the scope of the present manual. The best information will be ob- tained bv frequent examinations of one's own faeces. Here only a few examples will be mentioned. Neutral fats. Fats often occur as spherical bodies of vari- ous sizes. Thev are highly refractile and mav resemble superficiallv cvsts of amoebae. However, thev do not have any wall and do not show the differentiation of the con- tents of a cyst. A little care will easily allow one to realize the real nature of fats. They stain with sudan HL In stained smears, the fat droplets are usually dissolved and do not appear. Coccidia. Coccidia are common parasites of animals that are important sources of human foods, and consequenth^ are often found in the faeces. The oocysts of Eimeria sardinae Thelohan (Fig. 15, l) parasitic in the testis of sardines, mackerel, herrings, etc., and of E. clupearum Thelohan (Fig. 15, 2), a parasite of the liver of mackerel, herrings, etc., are sometimes passed in human faeces. Both oocvsts are spherical, but the former are 33-55m in diameter and contain four fusiform spores, while the latter measme about 20n in diameter and contain four oval spores. The 68 MANUAL OF HUMAN PROTOZOA digestive tracts of various birds are often infected by Ei- meria and Isospora. English sparrows are frequently hosts for Isospora lacazei Labbe (Fig. 15, 3). The oocysts are ovoid and measure 18.5-30n long. They contain two spores when mature as in the case of /. hominis (p. 47). Drinking 1 4 m/i\ y Vv Fig. 15. Coccidian oocysts found in the stool. X 1150 (1, 2, modified after Thomson and Robertson; 3, original). 1. A fresh mature oocyst of Eimeria sardinae. 2. A fresh mature oocyst of E. clupearum. 3. A fresh mature oocyst of Isospora lacazei. water contaminated with the droppings of infected birds may introduce the occysts in the faeces. Yeasts and molds. These are exceedingly common in the faeces. Yeasts are spherical, ovoid or ellipsoidal bodies and measure about 5-15u in diameter. Surrounded by a distinct membrane, the cytoplasm contains one or more refractile granules and a vacuole. Yeasts must not be con- fused with the cysts of Endolimax nana. Mold spores some- times simulate also protozoan cysts under low power ob- COPROZOIC PROTOZOA IN THE FAECES 69 jective, but the walls are colored and sculptured in various ways. Sphaerita and Nticleophaga. These vegetable organisms are occasionalK^ found parasitic in intestinal amoebae, especiallv coelozoic forms such as Entamoeba coli, loda- jjioelm biitschlii, and Endolimax nana. Sphaerita (Dan- geard 1895) occurs in the host's cytoplasm as small spheri- cal bodies which are made up of compactlv packed spores (Figs. 2, 3 and 16, l). Because of high refractility, they are very conspicuously noticed in living amoebae. The sphe- roidal spores are 0.5-1 n in diameter. When stained with Heidenhain's iron haematoxylin, thev are stained black, but when differentiated, the spores appear to be made up of a deeply staining cortical layer and a less deeply staining core. The spherical group of spores is sometimes about the size of the nucleus of the infected amoeba. Nucleophaga (Dangeard, 1895) is a very closely related form, but apparently develops in the nucleus of the host amoeba (Fig, 16, -). The spores appear as dark-stained granules and therefore have occasionallv been mistaken for chromatin granules of the host nucleus. When heavilv infected, the host nucleus becomes hvpertrophied and completelv disintegrates. Blastocijstis Jwminis. This is an extremeh^ common plant organism, considered to be a fungus, occurring in human and various animal excrements. It is as a rule spherical in shape (Fig. 16, '^-^) and is about 5-25m in diameter, the commonest forms being about 8-12^. \\^ithin a very thin membrane, there is a narrow peripheral c\ to- plasmic layer in which 1-2 nuclei and several refractile granules are present. This c\toplasmic ring encloses a 70 MANUAL OF HUMAN PROTOZOA large homogeneous body which is not iodinophilous, but when fixed and stained is more or less eosinophilous. In some individuals, the cvtoplasmic ring may be thick and the enclosed body very small. This organism has often been confused with the cysts of intestinal protozoa. Divid- # O 1^5^ ') I J I J CD J 4 ^ ^ 8 Fig. 16. 1. Sphaerita in a stained trophozoite of Entamoeba coli. 2. Nucleophaga in a stained trophozoite of lodamoeba biitschlii. 3, 4. Fresh specimens of Blastocystis hominis. 5, 6. Stained specimens of Blastocystis hominis. 7. An epithehal cell found in faeces. 8. A polymorphonuclear leucocyte with three ingested erythro- cytes. All X 1150 (1, after Noller; 2, after Brug; 3-8, original). ing forms may appear peanut-shaped (Fig. 16, 6). It is considered as a harmless organism. Helminth eggs. In faeces of persons infected by various helminth parasites, eggs of the worms occur commonly. Some of these eggs, especially of nematodes, may resemble superficially the oocysts of Isospora hominis (p. 47-49) in general form and appearance, but can easily be distin- COPROZOIC PROTOZOA IN THE FAECES 71 guished, since the worm eggs are much larger than the coccidian oocyst. Eggs of the nematodes, Hetewphyes heterophyes and Clonorchis sinensis measure about 30m long, thus approximating the size of the oocyst of Isospora hominis. But these eggs are flask-shaped, the shell is thick and yellowish, and the truncate end is operculated. Algae. Some of the algae which abound in fresh, brack- ish, and marine water, are occasionally found in the faeces. They are covered by a conspicuous siliceous shell which is variously sculptured and the body is usually flattened. Cellular elements of the intestine. In the faeces there occur an enormous number of various cells derived from the intestinal wall of the host. As seen in fresh smear, there may occur squamous epithelial cells in various stages of degeneration, sometimes with an irregular "amoeboid" outline and a vesicular nucleus located near the center (Fig. 16, 7). But these cells are flat as seen edgewise and the nucleus is relatively small. It should be borne in mind that no matter how closely the object may resemble an amoeba, if there is no movement, it is most probably not an amoeba. Polymorphonuclear leucocytes may contain erythrocytes and simulate Entamoeba histolytica (Fig. 16, 8). Do not become confused with them. Free-living protozoa. If salt solution or distilled water used for making fresh or permanent preparations of semi- fluid or formed faeces are contaminated with free-living protozoa, the preparation will naturally show them and confusion may result. Therefore, the solution or water must be renewed frequently with boiled ones. Chapter 7 Protozoa parasitic in the circulatory system Flagellata Trypanosoma TRYPANOSOMES are slender flagellates with tapering ex- tremities. There is a vesicular nucleus located usually near the middle of the body. The posterior end is less attenu- ated than the anterior end. Near the posterior end is pres- ent a small refractile body, the blepharoplast, which stains deep red with Giemsa's stain. Originating in this or a small granule located near it, there is a flagellum which follows the outer border of an undulating membrane to the opposite end and projects beyond freely. The organism nourishes itself by absorption of liquid nourishment through its body surface. It multiplies bv binary fission in the human blood and gives rise to polymorphic individuals. When a blood-sucking insect (Glossina, Triatoma, etc.) sucks the blood of a person infected with Trypanosoma, the latter undergoes a series of change and multiplication in its digestive tract. During this development, the blepharo- plast may shift its position toward the anterior end, and the body may assume crithidia form. Finally in Glossina engorged with the blood containing Trypanosoma gam- biense or T. rhodesiense, the flagellates enter into and continue to multiply in the salivary gland. These flies are now infectious and when they feed on a human victim, the trypanosomes enter the blood. In Triatoma infected by 72 PROTOZOA IN THE CIRCULATORY SYSTEM 73 T. cnizi, the trypanosomes appear, after undergoing a series of divisions and form changes, in small trypanosome forms in the rectum of the bug, and are voided in the faeces at the time when the latter feeds on a human host. It is believed that infection in man is established by scratching the site of the insect bite and introducing the organisms into the subcutaneous tissue. 1. Trypanosoma gamhiense Duttoii 1902 This trvpanosome is confined to Africa and especially to its equatorial zone. It is the causative organism of the Gambian or Central African sleeping sickness or trypano- somiasis. It is found in the blood, Ivmph, and cerebro- spinal fluid of the human host, and of various domestic and wild animals (reservoir hosts). The flagellate is transmitted by biting flies belonging to Glossina, especially G. palpalis, in the digestive tube of which it undergoes certain de- velopmental changes and in the salivary glands of which the infective metacvclic trvpanosomes become finally lodged for inoculation into a new host individual. The trypanosome occurs in the human bodv as active flagellates only. Trypanosoma gamhiense (Fig. 17) is an exceedingly ac- tive flagellate, and is hardly seen in life, although in fresh blood preparation its presence may be seen indirectly by the movement and agitation among ervthrocvtes under a low power objective. In routine detection, stained blood smears (p. 97-100) are used. The body is elongate and tapers towards both ends. It is usually sinuous in general outline. There is a nucleus in the approximate center of body. Near the less attenuated 74 MANUAL OF HUMAN PROTOZOA posterior end, is a blepharoplast, a small granule, in which originates a flagellum. The flagellum runs forward along the outer border of the undulating membrane which is a somewhat spiral thin expansion of the pellicle and which arises near the blepharoplast and extends to the anterior end. The active waving movements of the undulating mem- Fig. 17. Trypanosoma gambiense in a stained blood smear of an inocu- lated rat. Two individuals are in the process of division. X 1150. (original) brane aid the organism in moving about in the blood plasma. The flagellum extends beyond the membrane at the anterior end and becomes a free flagellum. The bodv is 15-30m long by l-3p broad. Some are long and slender, others short and broad. Between these extremes are found intermediate forms. The long forms are those in which binary fission is taking place, while the short individuals are thought to be recently divided forms. Thick and thin smears of peripheral blood (p. 97) should be examined. In the majoritv of cases, the trypanosome is very scanty and may not be found until after several smears have been examined. Some advocate the examination of PROTOZOA IN THE CIRCULATORY SYSTEM 75 fresh blood preparation (under a co\ er-glass) with a low power dry objective and look for the disturbance among the erythrocytes, which may suggest the presence of the trypan- osome. But absolute identification must be made on stained smears under a high power objective. Others advo- cate centrifugalization of the citrated blood. If lymphatic glands are enlarged, gland-puncture should be examined in stained smears. In the advanced stage of infection and also in cases where an early nervous symptom is present, cerebro-spinal fluid should be drawn out, centrifugalized and the sediments examined in stained smears. If this ex- amination is also negative, some portion of the sediment may be inoculated intra-peritonealh^ into a rat or a monkey, the blood of which will reveal the trypanosomes if periodic examinations are made. 2. Trypanosoma rhodesiense Stephens and Fantham 1910 This trypanosome is considered the causative organism of Rhodesian or East African sleeping sickness which is more virulent than Gambian form and runs a course of only a few months. It is confined in its distribution to the southeastern coastal region of Africa. Tnjpanosoma rhodesiense (Fig. 18) is indistinguishable from T. gamhiense in human blood smears. But when inoculated into rat, the position of the nucleus shifts in a certain proportion of the individuals (usually less than 5^), toward the posterior end, near or behind the blepharo- plast, together with the shortening of body. Some investigators maintain that T . rhodesiense is the human strain of T. bntcei (of cattle and game animals), 76 MANUAL OF HUMAN PROTOZOA others hold that it may be a more virulent race of T. gam- hiense or transmitted by different species of Glossina. In nature, this trypanosome appears to be transmitted chiefly bv Glossina morsitans. i .9- f J Fig. 18. Trypanosoma rhodesiense in a stained blood film of an inoculated rat. X 1150. (original) g The method of detecting the flagellate is similar to that iven for T. gamhiense. 3. Trypanosoma cruzi Chagas 1909 Synonyms: Schizotrypamim cruzi Chagas 1909 Trypanosoma escomeli Yorke 1920 This trypanosome is the cause of Chagas' disease or South American trypanosomiasis which is mainly a chil- dren's disease, and which is widely distributed in Brazil, Argentina, Uruguay, Peru, Venezuela, Colombia, Panama, Salvador, Guatemala, and Mexico. The trypanosomes are found in the blood and the leishmania-forms (p. 78) in various tissue cells. The organism is transmitted by re- duviid bugs, Triatoma megista and T. infestans, etc., in the digestive tract of which it undergoes developmental changes. PROTOZOA IN THE CIRCULATORY SYSTEM 77 The trypanosome as seen in a stained smear of the peripheral ]:)l()()d, is a curved (in the form of the letter C or U) spindle-shaped organism with a sharply pointed posterior end, near which a conspicuously large ovoid blepharoplast is present (Fig. 19, 1). The organism measures ^Pk^ 2 i i / Fig. 19. Trypanosoma cruzi. X 1150. (original) 1. Fi\'e trypanosomes seen in a very thin stained blood film of an inoculated rat. 2. Leishmania forms found in skeletal muscle cells of a patient. 3. Flagellating individuals seen in a host cell. about 20|j in length. The nucleus is located in the middle of the body. The undulating membrane is narrow and only slightly convoluted. Both slender and short-broad forms occur. Dividing forms are usually not found in the human blood. The trypanosome enters the tissue cell: muscles, central nervous system, bone marrow, lungs, th\roid, etc., and transforms into an ovoid bodv, the leishmania form (Fig. 19, 2)^ by discarding the flagellum and undulating mem- brane. Here, it undergoes repeated binary fission, and increases in number. The host cell becomes distended. 78 MANUAL OF HUMAN PROTOZOA Sooner or later, each individual elongates and develops a flagellum (Fig. 19, ^) and later an undulating membrane. These young trypanosomes are set free in the blood. Trypanosoma crtizi should be looked for in thick and thin stained blood films. In acute febrile stage, it is pres- ent in the peripheral blood. Sometimes it is necessary to centrifugalize the citrated blood and sediment to be exam- ined in stained smears. Although the trypanosomes are ordinarily not found in the lymphatic glands, it has been reported to occur in some cases in the spinal fluid. If all results are negative, 5-10 c.c. of the blood may be inoculated into guinea pigs in which after about two weeks the trypa- nosomes will appear in the blood. Examination of excised muscle for leishmania forms of the trypanosome has also been used by some. Brumpt advocates to allow laboratory- bred (therefore free from T. crtizi) Triatoma to feed on the suspected subject and examining in about two weeks the digestive tract of the bugs for the flagellate. Leishmania The three species of Leishmania are morphologically alike. In human host, they are represented by small ovoid leishmania bodies. They invade various leucocytes of the reticulo-endothelial system. Each trophozoite is a small protoplasmic mass in which are found a nucleus, a blepha- roplast, and sometimes a rhizoplast. All indications point to the species of sandflies (Phlebotomus) as the vectors. In the flies as well as in culture tubes, the leishmania bodies become enlarged and elongate, and the blepharoplast gives rise to a long flagellum, the whole assuming a leptomonad form. In the mid-gut of the sandfly, the flagellate forms PROTOZOA IN THE CIRCULATORY SYSTEM 79 increase in number by division, and the mass population shifts to the anterior portion of the gut. By about the tenth dav after feeding on the infected person, the flagellates are often lodged in the proboscis of the fly. It is assumed that these are introduced into man when the fly feeds again. Adler and Ber (1941) succeeded in producing leish- maniasis in 5 out of 8 human volunteers by bites of labora- torv-bred Phlebotoimis papatasi which were infected arti- ficially with Leishmauia tropica. 1. Leishniania donovani (Laveran and Mesnil 1903) Svnonym: L. infantum Nicolle 1908 This is the causative organism of Kala-azar or visceral leishmaniasis which is widely distributed in parts of Eu- rope, Africa, and Asia. In Europe it occurs in the southern reeion which borders the Mediterranean Sea, such as Portugal, Spain, Italy, Malta, Greece, and southern Russia. In Africa it has been reported from Morocco, Algeria, Tunisia, Libva, Abvssinia, Sudan, Northern Kenya, Ni- geria, while in Asia south-eastern area of India, north of Yangtze River in China, Turkestan, etc. The leishniania body occurs in the macrophages, mononuclear leucocytes, polymorphonuclears, etc., of the reticulo-endothelial sys- tem of various organs such as the spleen, liver, bone mar- row, intestinal mucosa, lymphatic glands, etc. When the in- fected host cells are destroyed, the organisms will become free in the blood plasma (Fig. 20, 2). The organism as seen in stained smears of spleen punc- ture, is a rounded (1-3m in diameter) or ovoid (2-4m by 1.5-2.5|j) bodv (Fig. 20, ^-'^). Its cytoplasm is homogeneous except one or more minute vacuoles. The nucleus is a com- 80 MANUAL OF HUMAN PROTOZOA paratively large rounded body, stained red with Giemsa's stain. But in ordinary films, the body may be flattened to various extents, and the nucleus also becomes flattened and is of various shapes. The blepharoplast is stained more deeply, and is an oval or elongate body, much smaller \ Fig. 20, Leishmania donovani. x 1150. (original) 1-3. Forms found in a stained spleen puncture film. 1. An infected polymorphonuclear leucocyte. 2. Leishmania bodies scattered in the blood plasma. 3. A large endothelial cell heavily infected by the organism. 4-6. Flagellate forms which developed in the first five days of cultivation in blood-agar medium. than the nucleus. When the smear is deeply stained, there may be seen a short thread extending between the blepha- roplast and the periphery. This is the rhizoplast. The num- ber of parasites in a host cell varies greatly. A newly invaded host cell may show a single parasite, while host cells with 200 or more parasites are sometimes seen, this being a result of repeated binary fission. No flagellate forms occur in the human host. Dogs suffer also infection by this organism. PROTOZOA IN THE CIRCULATORY SYSTEM 81 When citrated l)lo()d or culture tube with blood-agar (p. 10) is inoculated with Leishmania and incubated at 20-24°C., the leishmania l:)odies become elongate and the rhizoplast develops into a flagellum in about 48 hours (Fig. 20, 4-6). Thus Leishmania develops into leptomonad flagellate. The transmission is most probably carried on by Phle- hotomus spp., sandflies, in the digestive tube of which flagellates identical with the cultural forms, develop after ingestion of the blood of a suspected subject. To detect Leishmania donovani, smears of peripheral blood should be made and examined (p. 97). The number of the organisms in the blood is ordinarily very small, but it is the simplest method and should be the first step to be taken. Look for the parasites in large mononuclear leucocvtes. The best result is of course obtained by exam- ining stained smears of spleen puncture. The organisms are far more numerous there than in the blood smear and the detection is easily done. 2. Leishmania tropica (Wright 1903) This is the causal organism of the Oriental sore or cu- taneous leishmaniasis. It has been reported from Africa (mainly regions bordering the Mediterranean Sea), Europe (Spain, Italy, France, and Greece), Asia (Syria, Palestine, Armenia, southern Russia, Iraq, Iran, Arabia, Turkestan, India, Indo-China, and China), and Australia (northern Queensland). The organism occurs in the endothelial cells in and around the cutaneous lesions, located on hands, feet, legs, face, etc. Leishmania tropica is morphologically indistinguishable 82 MANUAL OF HUMAN PROTOZOA from L. donovani, but some believe that it shows a wider range of form and size than the latter. In addition to rounded or ovoid forms, elongate forms are often found, and even leptomonad forms have been reported from the scrapings of lesions. This organism is also transmitted by sandflies (p. 79). For detection of the organism, smears should be made from the lesions. If the lesion is not ulcerated, the fluid taken from it by puncture may be examined in stained smears for the intracellular parasites. If the lesion is ulcer- ated, material should be obtained by puncturing the margin of the ulcer, as the superficial part of the ulcer itself contains pus cells, bacteria, and abnormal forms of Leishmania. In case the parasites are very scanty, culti- vation in blood-agar may aid the detection of the organism. 3. Leishmania hrasiUensis Vianna 1911 This causes Espundia or South American or naso-oral leishmaniasis, which seems to be confined to South and Central America. It has been reported from Brazil, Peru, Paraguay, Argentina, Uruguay, Bolivia, Venezuela, Ecua- dor, Colombia, Panama, Costa Rica, and Mexico. Its morphological characteristics are identical with those of L. tropica, and a number of investigators combine the two species into one. However, the lesions are mainly in the mucous membrane of the nasal passage, mouth, and other parts. Transmission is also probably carried on by sandflies as is the case with the other two species. For detecting the organism use the method given for L. tropica. Chapter 8 Protozoa parasitic in the circulatory system (continued) Sporozoa Plasmodium THE SPOROZOAN parasites of the human blood are all malarial organisms and placed in the genus Plas- modium. Although there are certain morphological dif- ferences which characterize the different species, the gen- eral cycle of development is nearly identical. The malarial organisms of man are transmitted by female mosquitoes belonging to the genus Anopheles. Asexual and sexual re- production takes place in man and mosquito respectively. When an infected anopheline mosquito feeds on man sporozoites are introduced (Fig. 21, 1). The immediate change the sporozoites undergo is unknown. But during about six days after the bite of an infected mosquito, no parasites can be seen in the peripheral blood and quinine has no effect. Judging by what has been observed in early phase of Plasmodium infection in ])irds, it is now supposed that there probably occurs exoerythrocvtic development of the organism. Sooner or later, however, the organisms enter erythrocvtes (Fig. 21, 2) and become schizonts (Fig. 21, 3). At the beginning the schizonts are ring-form. They grow in size and finally divide into 12-24 or more mero- zoites (Fig. 21, 4, 5) which are finallv set free in the plasma (Fig. 21, 6). This schizogonv takes place in 48 to 72 hours. 83 84 MANUAL OF HUMAN PROTOZOA Fig. 21, A diagram showing the life-cycle of Plasmodium vivax (from Kudo). (See next page for description) PROTOZOA IN THE CIRCULATORY SYSTEM 85 These merozoites will, if not ingested by leucocytes, enter again and repeat the schizogony in erythrocytes. After re- peated and simultaneous schizogony in geometric progres- sion, a large number of infected erythrocytes will be destroyed, setting free an ever-increasing amount of toxic substance. The onset of the malarial chill and fever corre- sponds with the time of liberation of merozoites. Some of the merozoites develop into gametocytes instead of schizonts in ervthrocvtes (Fig. 21, '^-l^). When fully formed thev can be differentiated into macro- and micro- gametocytes, but remain as such while in the human blood. When a female anopheline mosquito sucks blood contain- ing the gametocytes, microgametocytes develop into micro- gametes (Fig. 21, 10, 11) and macrogametocytes into macro- gametes (Fig. 21, 8, 12) ii^ the stomach. An ookinete is formed when a microgamete fuses with a macrogamete (Fig. 21, 1-. 13). The ookinetes are mobile, penetrate through the stomach wall, and become rounded into Description of Fig. 21 1. A sporozoite entering human blood. 2. Sporozoite entering an erythrocyte. 3. Young schizont. 4-6. Schizogony. 7, 8. Macrogametocytes. 9, 10. Microgametocytes. 11. Microgamete-formation in the stomach of a mosquito. 12. Union of gametes. 13. Zygote or ookinete. 14. Young oocyst. 15. Oocyst in which sporozoites are developing. 16. Mature oocyst from which sporozoites are emerging. 17. Sporozoites entering the salivary gland. 86 MANUAL OF HUMAN PROTOZOA oocysts between the base of the epithehum and outer mem- brane of the stomach (Fig. 21, 14), Withm the oocysts, repeated nuclear divisions produce many sporozoites (Fig. 21, 15), When fully mature, the oocysts rupture and the sporozoites are set free in the haemolymph through which they migrate to the salivary glands (Fig. 21, 16, IV). The sporozoites finally make their way through the gland epi- thelium and enter the duct of hypopharynx. They are now ready to infect a human victim when the mosquito goes after another blood meal. The development in the mos- quito is completed in about 8-14 days. 1. Plasmodium vivax (Grassi and Feletti 1890) This organism is the cause of the benign tertian malaria. It is seldom fatal. The schizogony is completed in 48 hours. Thus chill and fever appear every third day. It is the most widely distributed species in the tropical and subtropical regions as well as in the temperate zone. It has been reported as far north as the Great Lakes region in North America, England, southern Sweden and northern Russia in Europe, and as far south as Argentina, Australia, and Natal in Africa. Generally speaking this species predomi- nates in the spring and early summer more than the other species of Plasmodium. In routine detection and identifica- tion, stained blood films are examined for the organisms. Schizonts 1. Ring forms. Usually found soon after the characteris- tic malarial attack has passed ofl:. The young merozoite which has entered an erythrocyte recently is a ring form PROTOZOA IN THE CIRCULATORY SYSTEM 87 (Fig. 22, 1) which is about one-fourth to one-third the diameter of the ervthrocyte. Tlie unevenly narrow periph- eral portion is stained blue and encloses a clear vacuole. A small compact nucleus is stained dark red and appears .J\^. -^ Vf 9 10 II 12 Fig. 22. Plasmodium vivax. X 1150. (original) 1. A young ring fonn. 2, 3. Growing schizonts. 4. Two schizonts in an erythrocyte. 5, 6. Large schizonts. 7-9. Schizogonic stages. 10. Fully formed merozoites. 11. A macrogametocyte. 12. A microgametocyte. prominently. The infected erythrocvte at this stage does not differ appreciably from uninfected cells, measuring about 7.5|j in diameter. 2. Growth period. In about six hours the ring-form has now become larger and often assumes irregular amoeboid 88 MANUAL OF HUMAN PROTOZOA forms (Fig. 22, 2). In life, amoeboid movement is clearly visible. The infected cells may be a bit larger, stain slightly paler, and begin to show fine red dots known as Schliffner's dots (Fig. 22, 3, 4). 3. Grown schizonts. In about another 20 hours, the schizonts (Fig. 22, 5, 6) are considerably larger and occupy about two-thirds of the erythrocytes which may now be as much as 12n in diameter and distinctly paler than un- infected erythrocytes. Schliffner's dots appear to increase. The schizonts are of irregular outline, enclosing one or more large vacuoles and in addition pigment (haemozoin) granules appear in the cvtoplasm. The haemozoin granules are brownish in color in this species. A compact large nucleus is easilv recognized, often located at a point in periphery. 4. Schizogonic or segmentation stage. The schizont nu- cleus soon afterwards begins to divide (Fig. 22, 7) and the division is repeated. Often some 16 daughter nuclei are produced. The division of some of the nuclei are somewhat irregular (Fig. 22, 8) so that there may be 12-24 nuclei in a schizont (Fig. 22, 9). These multinucleate schizonts meas- ure about 8-9|j in diameter. Presentlv around each of the nuclei becomes difl^^erentiated a small cytoplasmic mass, and thus many young schizonts or merozoites are produced (Fig. 22, 10). The haemozoin granules present in the schiz- ont assume a loose mass and are completely excluded from the merozoites. The merozoites are somewhat irregu- larly rounded and measure about 1.5|j in diameter. These changes take place between 40 to 48 hours after the ma- larial attack. The infected erythrocytes now rupture and the merozoites are set free in the blood plasma. PROTOZOA IN THE CIRCULATORY SYSTEM 89 Ganietocytes Young ganietocvtes which originate in ring forms are difficult to identify, but thev are less amoeboid, and more rounded. Mature ganietocytes can easily be identified. Thev mav appear during earh^ phase of the malarial at- tacks. The development of ring forms to mature ganieto- cvtes has been estimated as requiring some four days. Thev are smoothlv rounded and occupy almost the whole of the enlarged erythrocytes which may show Schiiffner's dots. Brown haemozoin granules are numerous. The dif- ferences between the mature macro- and micro-gameto- cvtes are as follows: M acrogametocy tes Microgametocy tes (Fig. 22, 11) (Fig. 22, 12) Size Large, 9-10 |.i in diameter Small, 7-8li in diameter Cytoplasm Stains more deeply Stains less deeply Nucleus Compact and smaller. Large, composed of many staining dark red chromatin granules which stain less deeply No further change takes place in human ery throe vtes. If fresh blood containing microgametocvtes, is kept at room temperature on a slide under a sealed cover-glass, formation of microgametes mav be seen. 2. Plasmodium mnlariae (Laveran 1881) This species is the cause of the quartan malaria in which the attacks of fever occur at intervals of 72 hours or ever\' fourth dav which is the time necessary for completion of schizogonic cycle. It is distributed in the tropics and sub- tropics, though it is the rarest of the three common species of malarial parasites. As a rule in an area where all three species of Plasmodium occur, Plasmodium malariae seems 90 MANUAL OF HUMAN PROTOZOA to appear later in the year than the other two. The entire schizogony can be seen in the peripheral blood. Schizonts 1. Ring forms. The ring form is similar to that of P. vivax. Large ring forms may be about one-third the diam- V '^ 9 10 II 12 Fig. 23. Plasmodium malariae. X 1150. (original) I. Ring fomi. 2-5 Band-form schizonts. 6-8. Schizogonic stages. 9, 10. Merozoite formation. II. A macrogametocyte. 12. A microgametocyte. eter of erythrocytes (Fig, 23, 1). The cytoplasm stains more deeply than that of F. vivax. 2. Growth period. The schizont is less amoeboid and, as a rule, rounded in outline in stained smears, although somewhat irregular forms may occur. In about 6-10 hours, haemozoin granules begin to appear in the cytoplasm. PROTOZOA IN THE CIRCULATORY SYSTEM 91 These are coarse granules and of much darker color than those of P. vivax. For about 24 hours, the growth of the schizonts is relatively slow; now may measure about one- half the diameter of the host cell which remains un- changed, except sometimes they may be slightly smaller and stain more deeply than uninfected ones. As the growth continues, the schizonts tend to be stretched into band- form across the erythrocytes. The younger schizonts are narrow bands (Fig. 23, ^), while the older are broad bands (Fig. 23, ^-^). This "band-form" is one of the characteristics of this species and possesses a high diagnostic value. Dots comparable with Schiiffner's dots do not occur ordinarily in this species. 3. Mature schizonts and schizogony. In about 48 hours, the schizont nearly fills the erythrocyte. It is rounded and haemozoin granules first scattered, begin to collect into a mass. At about this time, the nucleus divides into 6-12 daughter nuclei by repeated binary fission (Fig. 23, 6-8). These nuclei are often arranged in a circle, and soon a ring of 6-12 merozoites are formed with the pigment mass in the center. These merozoites (Fig. 23, 9, 10) appear to be the largest of the three species. A little before the attack of fever, the infected erythrocytes rupture and set the mero- zoites free in the blood plasma. Gametocytes The development of gametocytes takes place in the peripheral blood, but it is difficult to distinguish them from growing schizonts, since the latter are more or less com- pact and rounded in this species. The mature gametoc\ tes are spherical and fill the erythrocytes which are normal in 92 MANUAL OF HUMAN PROTOZOA dimensions. They are uninucleate and contain haemozoin granules. The two kinds of gametocytes can be distin- guished from each other as follows: Macrogametocytes Microgametocytes (Fig. 23, 11) (Fig. 23, i^) Cytoplasm Derser, stains more Stains less deep blue deeply Nucleus Small, compact; more Large, elongate; less deeply stained deeply stained Haemozoin Coarser Finer; abundant granules 3. Plasmodium falciparum (Welch 1897) Synonyms: Laverania malariae Grassi and Feletti 1890 Plasmodium temie Stephens 1914 This is the causative organism of the most malignant malaria, known as subtertian, malignant tertian or aestivo- autumnal fever. It is widely distributed in the tropics. In the subtropical region, it is more prevalent in late summer or early autumn. It is, however, relatively rare in the tem- perate zone. The chill and fever appear every 36 to 48 hours, the schizogonic cycle being somewhat irregularly periodic. Schizoiits 1. Ring forms. Young ring forms are much smaller than those of the other species (Fig. 24, l). They measure about 1.5n in diameter. Quite often marginal forms are en- countered, and also multiple (2-6) infection (Fig. 24, 1) often occurs in this species. The nucleus of the ring form appears as a short rod or is divided into two granules (Fig. 24, 1). In deeply stained smears, the infected host cells may show red-stained granules (Fig. 24, 2-^), the PROTOZOA IN THE CIRCULATORY SYSTEM 93 Maiirer's dots, which are coarser in size and fewer in num- ])er dian Schiiffner's dots found in the erythrocyte infected bv P. vivax. In aliout 12 hours after the attack of fever, these schizonts disappear from the periplieral blood. 2. Growth and schizogonic stages. These are found only '^ at- o o t f Q 7 8 S fO Fig. 24. Plasmodium falciparum. X 1150. (original) 1. Three ring forms in an erythrocyte. 2. A somewhat grown schizont in an erythrocyte with Maurer's dots. 3-6. Growth and schizogonic stages. 7, 8. Merozoite formation. 9. A macrogametocyte. 10. A microgametocyte. in the capillaries of various internal organs As the schiz- onts grow, the cytoplasm becomes denser. Haemozoin granules which are much darker than those in other spe- cies, become grouped into a compact mass (in contrast to the scattered granules in the other two species). When the schizont reaches the size about two-thirds of the infected erythrocytes (about 5m), nuclear division begins and finally 8-24 or more nuclei are produced (Fig. 24, 5, «). Thus 8-24 small merozoites are formed. When these merozoites are 94 MANUAL OF HUMAN PROTOZOA liberated into the blood plasma and enter erythrocytes, the ring forms reappear in the peripheral blood. Gametocytes The gametocytes develop from the ring form in the internal organs, especially the spleen and bone marrow. Mature gametocytes which appear in the peripheral blood and therefore are present in ordinary blood smears, are characteristically crescentic or sausage form ("crescents") and have an important diagnostic value. They are about 10-12m by 2-3n. The macro- and micro-gametocytes may be distinguished as follows: Macrogametocytes Microgametocytes (Fig. 24, 9) (Fig. 24, 10) Cytoplasm Stains deeper blue Hyaline, stains less deeply or reddish Nucleus Pigment Small, compact; deep red Coarser, around the nu- cleus Large, chromatin closely set; less deeply stained Less coarse, more widely scattered 4. Plasmodium ovale Stevens 1922 This is considered as the cause of mild tertian or Ovale tertian fever which appears to be confined to Africa and Asia (Philippine Islands and India). The validity of the species is still disputed among malariologists. Its schizog- ony is completed in about 48 hours and its morphological characters resemble both P. vivax and P. malariae. The whole schizogony and gametocyte formation occur in the peripheral blood. Schizonts 1. Ring forms. They are similar to those of P. vivax and P. malariae. Schiiffner's dots appear early (Fig. 25, l). PROTOZOA IN THE CIRCULATORY SYSTEiM 95 2. Growth period. The infected erythrocytes are more or less oval with irregular fimbriated margins or outline (Fig. 25, 2-4). There seems to be a slight increase in size also. The schizonts are not actively amoeboid, and some- times seen in band form with brownish -black haemozoin •«|^' Fig. 25. Plasmodium ovale. X 1150. (original) 1. A ring fomi. 2, 3. Growing schizonts. 4-6. Schizogonic stages. 7. A macrogametocyte. 8. A microgametocyte. granules, resembling those of P. malariae. Schiiffner's dots are usually abundant. 3. Schizogonic stage. The fully grown schizont is smaller than the erythrocyte (Fig. 25, 5), and produces 6-12 mero- zoites (Fig. 25, ^), which resemble those of P. malariae. Schiiffner's dots appear abundantly. Gametocytes The gametocytes do not fill the erythrocytes, and re- semble closely those of P. malariae. But in P. ovale, the host cells show Schiiffner's dots and seem to be slightly enlarged (Fig. 25, "'^ ^). 96 MANUAL OF HUMAN PROTOZOA DIFFP:RENTIAL diagnosis of the three common species of PLASMODIUM AS SEEN IN GIEMSA-STAINED THIN BLOOD FILMS P. vivax 1 P. malariae P. falciparum Ring forms About J diameter of erythrocytes; marginal forms rarely seen Similar to P. vivax but cytoplasm slightly denser Smaller. i~i diameter of erythrocytes; marginal forms frequent; multiple infection common Infected erythrocytes Larger, up to lln, in size and paler in color than normal erythro- cytes (7.5/i in diameter); SchlifTner's dots Not enlarged; sometimes slightly smaller than normal ones; usually no dots Normal; some contracted and distorted in later schizogonic stage; Mau- rer's dots Growing schizonts Irregularly amoeboid; vacuolated; pale blue; brown haemozoin gran- ules small Not amoeboid; oval, rounded, band-form, rarely irregular; less vacuolated cytoplasm stains deeper blue; dark brown pigment numer- ous Partly grown ring forms often with a rod-shaped nucleus or "2 compact nuclei. Further develop- ment only in inner organs F'ully grown schizonts Irregular form; § the enlarged erythrocytes; vacuolated; brown hae- mozoin granules Nearly fill erythrocytes; less irregular, rounded; cytoplasm deeper blue; dark brown pigment In internal organs. | of erythrocytes; dark hae- mozoin granules com- pactly massed Schizogonic stages 12-24 nuclei; later mero- zoites; irregularly ar- ranged; brown haemo- zoin granules 6-1 2 nuclei, later mero- zoites which are largest of all, typically arranged in a circle with pigment mass in center In internal organs. 8-24 or more nuclei, later merozoites which are smallest of all; irregu- larly arranged; dark pig- ment mass eccentric Ganietocytes Almost fill enlarged erythrocytes; rounded or ovoid; brown pig- ment Fill normal-sized eryth- rocytes; round or ovoid, much smaller; dark brown pigment Crescentic or sausage- shaped Chapter 9 Technique for detection and identification of protozoa parasitic in the circulatory system TJiin film. The finger tip or ear-lo]:)e is cleaned with 70% alcohol. Prick it with an aseptic blood lancet or a steri- lized needle. Wipe off the first drop with gauze and receive the second drop on a thoroughly cleaned (p. 54) slide about half an inch from an end (Fig. 26, 1). Use care not to let the slide touch the linger or ear-lobe itself. Quickly bring a second slide, one corner of which had been cut away, to the inner margin of the blood drop (Fig. 26, i), and let the blood spread along the edge of the second slide. Next push the second slide over the surface of the first slide at an angle of about 45 towards the other end (Fig. 26, ^). Thus a thin film of blood is spread over the slide (Fig. 26, 3). Let the smear lie horizontallv and dry. It should he covered with a glass cover to prevent dust particles from falling on it and to exclude flies or other insects. If properly made, the film is made up of a single laver of blood cells with smoothlv rounded natural outlines. Thick film. Often parasites are so few that to find them in a thin film involves a great deal of time. In such cases, a thick film is advocated. For this 4-6 drops of blood are placed in the central area of about /2 square inch, and with a needle or a corner of slide, spread them into an even layer. Let the smear drv, protecting it from dust or flies. 97 98 MANUAL OF HUMAN PROTOZOA With a little practice, a satisfactory thick smear without crackling or peeling of the blood film can be made. It will take two hours or more to dry. Do not dry by heat, but Fig. 26. Diagrams showing how a thin blood film is made. placing it in an incubator at 37°C. will hasten the drying. When it is thoroughly dry, immerse it in water and de- haemoglobinize it. Air dry again. Thin and thick film. Sometimes it is time-saving if thin and thick films are made on a single slide. Place a single drop DETECTION AND IDENTIFICATION 99 of blood near the center and make a thin fihn of it toward one end of the shde. About one-fourth from the other end, place drops of blood in a small area and make a thick film. Dry. When thoroughly dry, immerse the thick film part in distilled water and dehaemoglobinize it. Let the slide dry. The blood films must be stained as soon as possible to insure a proper staining, as lapse of time or summer heat will often result in poor staining especially of thick films. Staining. Of numerous blood stains, Giemsa's and Wright's stains are used here. For staining with Giemsa's solution, the thin film is fixed after thorough drying in absolute methyl alcohol for 5 minutes. Wash the film in running water and rinse well in neutral distilled water. After shaking the stock Giemsa's stain (obtained from re- liable makers) well, dilute it with neutral distilled water in a ratio of one drop of stain to 1-2 c.c. of water. Mix the solution and the film is placed in it for one-half to 2 hours or longer if desired. It is now placed in neutral distilled water and excess of stain washed off the slide. Rinse it in fresh distilled water and then wipe off water with a tissue paper from the underside and edges of the slide. Let the slide stand on end to dry. When thoroughly dry, place a drop of xylol and a drop of cedar wood oil (used for im- mersion objectives) and cover with a cover-glass. For thick film the square cover-glass wall cover the entire film, but for a thin film use two such cover-glasses side by side if a rectangular one (40 bv 20 mm.) is not available. The mounting medium should be absolutely neutral. Do not use Canada balsam for mounting, as acid in it promptly spoils the staining. For Wright's staining, fixation is not necessarv. With a 100 MANUAL OF HUMAN PROTOZOA medicine dropper, cover the dried blood film widi drops of undiluted Wright's stain, and let the film stand hori- zontally for 3-5 minutes; then the same number of drops of neutral distilled water is added to the stain and the whole is left for 10-30 minutes. The stain is then poured off and the film is rinsed in neutral distilled water. Drv. Mount in xylol and cedar wood oils as stated for Giemsa's stain. Use of cover-glasses on a stained blood film is advocated, since a cedar wood oil mounted slide allows the use of dry objectives which in the hand of an experienced worker would give enough magnification for species determina- tion of Plasmodium, and which will very clearly reveal any trypanosome present in the film. Furthermore the film is protected against scratches and contamination by many ob- jects which may bring about confusion in detecting pro- tozoa. The splenic pulp smears for Leishmania and Trypano- soma are also similarly treated and prepared. When the parasites are very few in the blood, the thick film is highly useful for detection of ring forms and gameto- cytes of Plasmodium and of trypanosomes, as the organ- isms are much concentrated, and the haemoglobin is com- pletely absent. On the other hand, the thick film brings about a great deal of abnormalities in the organism so that many of the specimens may be variously distorted. There- fore, species identification is usually difficult. Thick films should be used for detection of organisms and identifica- tion should be made on thin films. In diagnosis of Trypanosoma and Leishmania, it is neces- sary often to use culture method. The oldest medium is DETECTION AND IDENTIFICATION 101 that first advocated by Novy, MacNeal and Nicolle (or N N N). This is prepared as follows: Dissolve 7 grams of agar-agar and 3 grams of NaCl in 450 c.c. of distilled water b\ heating. Place 5 c.c. of the mixture while still hot into each test tube and apply a cotton plug. Autoclave the tubes for 30 minutes under 15 lbs. pressure. Let the tubes cool down to about 50° C, and add to each tube 15-20 drops of rabbit's blood with care to prevent bacterial con- tamination. The tubes are rapidly evolved to bring about mixture without forming bubbles, and sloped. When the medium is solid, place rubber caps over the cotton plugs and incubate the tubes at 37 °C. for 24 hours. The testing blood is inoculated into the condensation water. Chapter 10 Objects which may be confused with blood-inhabiting protozoa in stained films CERTAIN artifacts and extraneous objects occur fre- quently in stained blood films. In spite of the precau- tions taken during the preparation, there will be seen a number of objects not present in the blood originally, because of chance contaminations of tue blood film by various animal or plant organisms, dust particles, particles suspended in reagents and stains, etc. Free-living protozoa. Thev may occur in long-standing distilled water, especially in warmer climate, and if it is used for dilution of the stain, some of them may adhere to the blood film and be stained. If flies or other insects drop faecal matter on the drying blood film, intestinal flagellates and other microorganisms may contaminate it. Dust particles. Dust particles from air or suspended in water, stain, etc., may adhere to the film and simulate malarial pigment. Scratches or breaks in the slide may take up the stain, and resemble trypanosomes. Plant organisms. Yeasts, fungi, and bacteria may be found in the film, often originating in the skin or air. Blood films made post-mortem, may contain them as they may have invaded the blood shortly before or just after death. They may be confused with young stages in Plasmodium, Erythrocytes. Normal erythrocytes are circular in outline 102 'U • O ' 10 i 4I II 13 ^ 18 12 16 17 Fig. 27. Various blood cells found in stained blood films. X 1150. (original) 1. A nonnal erythrocyte. 2. A crenated erythrocyte. 3. A macrocyte. 4. A microcyte. 5. A nomioblast. 6. A basophilic erythrocyte. 7. An erythrocyte with chromatin dots. 8. 9. Erythrocytes with Cabot's rings. 10. Semi-lunar erythrocyte. 11. Blood platelets. 12. A thrombocyte superimposed on an erythrocyte. 13. A neutrophilous polymorphonuclear leucocyte. 14. An eosinophile leucocyte. 15. A basophile leucocyte. 16. A small lymphocyte. 17. A large lymphocyte. 18. A large mononuclear leucocyte. 104 MANUAL OF HUMAN PROTOZOA and bi-concave, and stain homogeneously, except the central area which is lighter in color (Fig. 27, l). They are about 7.5n in diameter. Some erythrocytes show small knob-like projections from their surface. These are cre- nated ervthrocvtes (Fig. 27, ^), and appear in drawn-out normal blood. In addition to the normal erythrocytes, macrocytes, about 10-14n in diameter (Fig. 27, 3) and microcytes, 2-6m in diameter (Fig. 27, 4) may be present in the blood of persons suffering from anaemia including subtertian malaria. Some erythrocytes may be nucleated (Fig. 27, 5); they are called normoblasts. Some erythrocytes may show basophilic dots (Fig. 27, ^), others chromatin dots (Fig. 27, "), and still others Cabot's rings, either ir- regularly rounded (Fig. 27, 8) or in form of an 8 (Fig. 27, ^). Sometimes in the blood of tertian malaria patients may appear large erythrocytes with a yery large eccenti'i- cally placed yacuole, resembling half moon in shape with a somewhat undulating outline, which simulates a trypano- some (Fig. 27, 10). Blood platelets. Thrombocytes are rounded disc-shaped bodies with indistinct margins and are 1-3m in diameter. They may be found singly in clusters, or in chains (Fig. 27, 11). They stain bluish with red-stained granules in center. Occasionally filamentous processes may be seen. When an isolated platelet is attached to an erythrocyte, it may simulate a young schizont of Plasmodium, but there is always a clear zone around the platelet by which it can be identified as such (Fig. 27, 12). Leucocytes. There are many kinds. The main types are as follows: Neutrophile polymorphonuclear leucocytes (Fig. 27, i"^). EXTRANEOUS OBJECTS IN STAINED FILMS 105 Normally 60-70% of leucocytes . When rounded as is usually seen in a stained blood film, the cell is about 10^ in di- ameter. The nucleus is of irregular shape and often tri- lobed. The cytoplasm is stippled with fine neutrophilic granules. Eosinophile leucocytes (Fig. 27, i"*). Normally 2-4'/ of leucocytes. These cells are about 12-14|j in diameter. The nucleus is l:)i- or tri-lobed. The c\toplasm is filled with coarse red-stained granules. These cells increase in num- ber in cases of helminth infection. Basophile leucocytes (Fig. 27, 15). Normally about 0.5%. These cells are about 10m in diameter and similar to neu- trophile, but the granules are stained purplish or blue. Lymphocijtes. NormalK^ 20-30% of leucoc\'tes. The nu- cleus is large and compact, and stains deep dark red. The c\ toplasm is comparatively scanty and stains blue. Small lymphocytes (Fig. 27, 16) are about 5-8^ in diameter, while larger ones (Fig. 27, 1'^) measure 8-10m in diameter. Large mononuclear leucocytes (Fig. 27, ^^). Normally 3-6%. They are the largest blood cells and measure 12-20m in diameter. The nucleus is rounded, elongate, or reniform, and chromatin material is loosely packed so that the stained nucleus is much lighter than that of the lymphocytes. The cytoplasm is yoluminous and stains a clear pale-blue. In malaria and trypanosomiasis these leucocytes increase in number and in the former they may show ingested haemo- zoin granules. Chapter 11 Protozoa parasitic in the muscle and the reproductive organ In the muscle 1. Sarcocystis lindemanni (Rivoha 1878) THIS sporozoan is responsible for the sarcosporidiosis in man which is very rare, and has been reported from the tropical regions. It invades the muscle cells of man. Information on the species which attack the human body is very incomplete. The questions such as what species occur in man and how man is infected, etc., are unanswered at present. The published records indicate that there is a wide morphological variation among the organisms ob- served by different workers. The organism is oval to spindle form and imbedded in the muscle cell of larynx, biceps, tongue, heart, chest, etc. The infected muscles seem to become distended owing to the growth of the parasite within. The infected muscles may be seen white-streaked to the naked eye. Seen in sec- tions, the body of the organism (Fig. 28) may be divided into numerous compartments. The size of the organisms varies a great deal according to different observers: 16 mm. by 170|j, 5.3 cm. by 320m, 84m by 27m. Near the peripheral zone are found rounded sporoblasts and in the central area crescentic or banana-shaped spores occur usually in abundance. The size of the spores also differs among dif- ferent cases: 8-9m long; over 10m long; 8.33m by 1.6m; 4.25m 106 PROTOZOA IN THE MUSCLE 107 by 1.75n. The spores are rounded at both ends or more often one end is more rounded than the other. There is a nucleus near the broader end, and the cytoplasm near the Ml: ^ % 7^?^ Fig. 28. Sacrocystis lindemanni in human niusclae. X 1150 (modified from the drawings and description of Baraban and St. Remy). other end stains darkly and contains several granules. The detection of the organism must be carried on fresh preparation of excised muscle cells or sectioned permanent preparation of muscle tissue. 108 MANUAL OF HUMAN PROTOZOA In the reproductive organ 1. Trichomonas vaginalis Donne 1837 This flagellate occurs in the vagina in which the reaction of the secretion has changed from alkaline to acid. There are some investigators who consider it as the causative organism of vaginitis, but definite information to support this view is still missing. As in the other two species of human Trichomonas, encysted forms have not yet been found. Trophozoites 1. Livings specimens. The organism (Fig. 29, l) resembles r. hominis. General body form is broadly pyriform or fusi- / 2 3 4 Fig. 29. Trichomomis vaginalis. X 1150. (original) 1. Living trophozoite. 2. A degenerating amoeboid trophozoite in hfe. 3. 4. Stained trophozoites. form, but may be varied, as it is extremely plastic. It measures 10-30m long by 10-20m broad. When the prepa- ration is a few hours old, many degenerating forms appear (Fig. 29, 2). In such forms active amoeboid form-change together with the continued movement of flagella and PROTOZOA IN THE REPRODUCTIVE ORGAN 109 undulating membrane will be noticed. Further degener- ated forms are rounded and are with or without fiagella. The undulating membrane in larger individuals, does not usuallv reach the posterior end of body. 2. Stained specimens. The general morphological fea- ture (Fig. 29, 3, 4) is similar to that of T. hominis (p. 38). But the costa is less conspicuous in the present form. The nucleus is p\ riform or oval. The cvtoplasm contains bac- teria and numerous granules. The detection of the flagellate can be done easily by examining fresh smears of vaginal secretions in bright and dark field under the microscope. For permanent prepara- tion, use either Heidenhain's iron haematoxylin in the way the faecal smears are handled (p. 59) or Giemsa's stain as given for blood-inhabiting protozoa (p. 99). Reference Books For further information concerning protozoa parasitic in man not dealt with in the present work, the reader is re- ferred to the following works. Craig, C. F. 1934 Amebiasis and amebic dysentery. Springfield, Illinois; Charles C Thomas. Craig, C. F. 1942 Laboratory diagnosis of protozoan diseases. Philadelphia; Lea & Febiger. Dobell, C. and F. W. O'Connor 1921 The intestinal pro- tozoa of man. London: John Bale, Sons and Danielsson, Ltd. Kudo, R. R. 1939 Protozoology. Springfield, Illinois; Charles C Thomas. Manson-Bahr, P. 1940 Manson's tropical diseases. To- ronto: Cassell & Co. Strong, R. P. 1942 Stitt's diagnosis, prevention and treatment of tropical diseases. 6th edition. 2 vols. Philadelphia; The Blakiston Co. Wenyon, C. M. 1926 Protozoology. 2 vols. London: Bailliere, Tindall k Cox 110 Index Algae in faeces, 71 Amoeba, dysentery, 5, 9-14, 28, 29, 30 Amoebae, coprozoic, 63-64 free-living, 19, 63-64 intestinal, 5, 9-19, 21-30 oral, 19-21 Amoebiasis, 5, 9 Amoebic dysentery, 5, 9 Amoebina,'8-30, 63-64 Anopheles, 83, 85 Axostyle, 39, 40, 43, 44, 108 Bacteria as food of Protozoa, 6, 15, 19, 22, 25, 27, 30, 33, 37, 39, 41, 108 Balantidial dysentery, 5, 49 Balantidhim coli, 3, 4, 5, 49-52 Balantidiosis, 5, 49 Bandform schizont, 90, 91, 95 Basophile leucocyte, 105 Binary fission, 3, 4 Blastocijstis hominis, 28, 41, 69-70 Blepharoplast, 72, 74, 75, 77 Blood agar culture medium, 81, 101 Blood films, 74, 97-100 making of, 97-99 staining of, 99-100 thick, 97-98, 100 thick and thin, 78, 98-99 thin, 97, 100 Blood platelets, 104 111 112 MANUAL OF HUMAN PROTOZOA Blood-sucking insects, 5, 72, 73, 76, 78, 79, 81, 83, 84, 85, 86 Budo caudatus, 64-65, 66 edax, 65, 66 Cells in blood films, 102-105 faecal smears, 70, 71 Centrifuge, 57, 75, 78 Cercomonas longicauda, 65, 66 Chagas' disease, 5, 76 Chill and fever, 85, 89, 92, 93 Chilomastix mesnili, 33, 35, 36-38, 39, 45, 46 Chromatin, 3, 9, 17 Chromatoid body, 13, 18 Cilia, 4, 50 Ciliata, 3, 8, 49-52, 62 Cleaning of cover-glasses, 54 slides, 54 Clonorchis sinensis, 71 Coccidia in birds 68 intestine, 6, 47-49 faeces, 6, 47-49, 67, 68 fishes, 67 sparrow, 68 Coccidiosis, 6, 47-49 Collection of blood, 97 faeces, 53-54 Commensals, 6, 15, 19, 22, 25, 26, 31, 36 Conjugation, 5 Contractile vacuole, 4, 50, 51 Copromonas suhtilis, 65, 66-67 Coprozoic Protozoa, 62-67 Costa, 40, 109 Cover-glasses, 54, 99, 100 INDEX 113 Crescents, 94, 96 Crithidia form, 72 Cyst, 4, 8, 31, 56, 57 carriers, 14 chemicals on, 14 concentration of, 57 drugs on, 14 formation of, 4, 12 of Balantidiiim coli, 51-52 Cercomonas longicauda, 66 Chilomastix mesnili, 33, 38, 45, 46 Copromonas subtilis, 67 Diniastigamoeba griiberi, 64 EndoUmax nana, 26, 29, 30 Entamoeba coli, 17-19, 29, 30 histolytica, 12-14, 29, 30 Giardia intestinalis, 44, 45, 46 HartmanneUa hyalina, 63 lodamoeba biitschlii, 23-24, 29, 30 Retortamonas intestinalis, 33, 38, 45, 46 Tricerconwnas intestinalis, 36, 45, 46 passers, 14 temperature on, 14 \'iability of, 8, 14 Cytopharynx, 50 Cytoplasm, 3 Cytopyge, 50 Cytostome, 4, 32, 37, 38, 40, 50 Darkfield illumination, 33, 39, 42, 54, 56, 109 Dehaemoelobinization, 98, 99 Detection of haematozoa, 74-75, 76, 78, 81, 82, 86 114 MANUAL OF HUMAN PROTOZOA Detection of (continued) intestinal protozoa, 53-61 Sarcocystis, 107 Trichomonas vaginalis, 109 Dientamoeba, 9 Dientarrioeba fragilis, 26-28, 29, 30 Differential diagnosis of intestinal amoebae, 30 flagellates, 46 Plasmodium, 96 Dimastigamoeha gruheri, 63-64, 65 Diseases caused by protozoa, 5, 6, 9, 48, 49, 73, 75, 76, 79, 81, 82, 86, 89, 92, 94, 106 Dust particles on blood films, 102 Dysentery, 5, 9, 49 Dysentery amoeba, 9-14 Ectoplasm, 3, 9, 10 Eimeria clupearum, 67 sardinae, 67 Ernbadomonas intestinalis, 31 Encystment, 4, 8, 31 Endamoeba, 8, 9 Endamoeba coli, 15 gingivalis, 19 histolytica, 9 Endolimax, 9 Endolimax nana, 24-26, 28, 29, 30, 69 Endoplasm, 3, 10, 11 Endosome, 3 Entamoeba, 9 Entamoeba coli, 12, 15-19, 22, 28, 29, 30, 69 gingivalis, 19-21, 29 histolytica, 5, 9-14, 15, 17, 19, 28, 29, 30, 71 INDEX 115 Enteromonas ho minis, 34-35, 36, 45, 46 Eosin, 61 Eosinophile leucocytes, 105 Epithelial cells in faeces, 70, 71 Erythrocytes, 102-104 crenated, 104 dots in, 104 in protozoa, 10, 40, 41, 50 macrocytes, 104 microcytes, 104 normal, 102-104 normoblast, 104 semilunar, 104 with basophilic dots, 104 Cabot's ring, 104 chromatin dots, 104 Espundia, 6, 82 Examination of slides, 55-57, 61 Exoerythrocytic development, 83 Excystment, 4, 8 Faeces, blood in, 54, 55 collection of, 53-54 fresh preparation of, 55-58 microscopical examination of, 54-61 mucus in, 12, 54, 55, 57 objects present in, 67-71 permanent preparation of, 58-61 pus in, 54, 55 Fixation of blood films, 69 faecal smears, 58-59 Flagella, 4, 42, 56, 62 Flagellata, 3, 4, 5, 8, 31-46, 72-82, 108-109 blood-inhabiting, 72-96 116 MANUAL OF HUMAN PROTOZOA Flagellata (continued) coprozoic, 64-67 free-living, 64-67 intestinal, 31-46 oral, 41 vaginal, 108-109 Food substances, 4, 8 Free-living protozoa, 19, 62-67, 71, 102 Fungus, 69-70, 102 Gametocytes, 5, 85, 89, 91-92, 94, 95, 96, 100 Giardia inte.stmalis, 6, 41-44, 45, 46 lamblia, 41 Giemsa's stain, 72, 80, 99, 109 Glossina, 72, 73, 76 Glossina morsitans, 76 pal pedis, 73 Glycogen body, 13, 18, 19, 24, 26, 58 Haematoxylin, 59-60 Haemozoin granules, 88, 89, 90-92, 93, 95, 105 HartmanneUa hijalina, 63, 65 Heidenhain's iron haematoxylin, 59-60, 109 Helminth eggs, 70-71 Heterophyes heterophijes, 71 I cysts, 24 Illustrations of Balantidiiim coli, 51 Blastocystis horninis, 16, 70 Blood cells, 70, 103 Blood film, making of, 98 Bodo caudatus, 65 edax, 65 INDEX 117 Illustrations of (continued) Cercomonas loufj^^icaiida, 65 Chilomastix mesnili, 37 chromatoid bodies, 11, 16 cilia, 51 compact nucleus, 51 Copromonus siibtilis, 65 Coprozoic protozoa, 65 Dientamoeha jragilis, 27 Dimastigamoeha gruheri, 65 Endolimax nana, 25 Eimeria clupeanim, 68 sardinae, 68 Endosome, 11, 16, 20 Entamoeba coli, 16, 70 gingival is, 20 histoli/tica, 11 Enteromonas hominis, 34 Epithelial cell, 70 Giardia intestinalis, 43 Hartmannella hyalina, 65 lodamoeha hiitschlii, 23, 70 Isospora hominis, 48 lacazei, 68 Leishmania donovani, 80 Leucocytes, 103 Nucleophaga, 70 Nucleus, vesicular, 11, 16, 51, 108 Plasmodium, life cycle of, 84 Plasmodium falcipariitn, 93 malariae, 90 ovale, 95 vivax, 87 Retortamonas intestinalis, 32 118 MANUAL OF HUMAN PROTOZOA Illustrations of (continued) Sarcocystis lindemanni, 107 Sphaerita, 16, 70 Tricercomonas intestiruiUs, 34 Trichomonas honiinis, 40 vaghialis, 108 Trypanosoma cruzi, 77 gamhiense, 74 rJiodesiense, 76 Intestinal amoebae, 9-19, 21-30 flagellates, 31-46 lodamoeba, 9 lodamoeba butschlii, 21-24, 28, 29, 30, 69 williamsi, 21 Iodine, 57 Iodine solution, 57 Iron alum, 59 Isospora hominis, 6, 47-49, 68, 70, 71 lacazei, 68 Kala azar, 6, 79 Keys for genera and species of intestinal amoebae, 28-29 flagellates, 46 Laverania malariae, 92 Leishmania, 4, 78-82, 100 Leishmania brasiliensis, 6, 82 donovani, 6, 79-81 infantum, 79 tropica, 6, 79, 81-82 Leishmania forms, 76, 77, 78 Leishmaniasis, cutaneous, 6, 81 naso-oral, 6, 82 INDEX 119 Leishmaniasis (continued) South American, (S2 visceral, 6, 79 Leptomonad form, 78, 81 Leucocytes, 104-105 basophile, 105 eosinophile, 105 lymphocytes, 105 neutrophile, 104-105 mononuclear, 79, 105 polymorphonuclear, 71, 79, 81 Life cycle of intestinal amoebae, 8 flagellates, 31 Leishmania, 78-79 Plasmodium, 83-86 Trypanosoma, 72-73 Lugol's solution,' 13, 18, 24, 26, 33, 42, 57 Lymphocytes, 105 Macrocytes, 104 Macrogametes, 5, 85 Macrogametocytes, 85, 89, 92, 94, 95 Macronucleus, 3, 50, 52 Macrophages, 79 Malaria, 83-96, 105 aestivo-autumnal, 92 benign tertian, 6, 86-89 malignant tertian, 6, 92 mild tertian, 6, 94 Ovale tertian, 6, 94 quartan, 6, 89 subtertian, 6, 92, 104 tertian, 86-89 120 MANUAL OF HUMAN PROTOZOA Material, collection of, 53-54, 97 Maurer's dots, 93 Mercuric chloride, 14, 58 Merozoites, 83, 85, 88, 91, 93 Methyl alcohol, 99 Microcytes, 104 Microgametes, 5, 85 Microgametocytes, 85, 89, 92, 94, 95 Micronucleus, 51 Microscopic cover-glasses, 54, 99, 100 slides, 54 Microscopical examination of blood, 97-105 faeces, 54-61 Molds in blood films, 102 faeces, 68 Mononuclear leucocytes, 79, 105 Mordant, 59 Mosquito, anopheline, 5, 83, 85 Mounting media, 61, 99 Canada balsam, 61 cedar wood oil, 99 Mucus in faeces, 12, 54, 55, 57 Multiple division, 4, 5, 83-85, 88, 91, 93, 95, 96 N N N medium, 101 Neutral fat, 67 Neutrophile leucocytes, 104-105 Nitric acid, 54 Nucleophaga, 69, 70 Nucleoplasm, 3 Nucleus, 3, 10, 11, 15 Objectives, microscopic, 55, 56 INDEX 121 Objects in blood films, 102-105 faecal smears, 56, 62-71 Oocyst of Eimeria clupeantm, 67 sardinae, 67 Isospora hominis, 47-48 hicazei, 68 Plasmodium, 5, 86 Ookinete, 85 Oriental sore, 6, 81 Parasites of protozoa, 25, 69, 70 Peripheral chromatin granules, 12, 17 Peristome, 52 ' Permanent preparation, 58-61 Phlebotomus, 78, 81, 82 Phlebotomus papatasi, 79 Plasmodium, 4, 5, 83-96, 100, 104 Plasmodium falciparum, 6, 92-94, 96 malariae, 6, 89-92, 94, 95, 96 ovale, 6, 94-95 tenue, 92 vivax, 6, 84, 86-89, 90, 94, 96 Polymorphonuclear leucocytes, 71 Potassium iodide, 57 Precystic amoebae, 12, 17 Protozoa, free-living, 19, 62-67, 71, 102 Protozoa in blood, 9, 72-96 bone marrow, 79, 94 brain, 9, 73, 75, 76 cerebro-spinal fluid, 73, 78 circulatory system, 9, 72-96 cutaneous tissues, 81, 82 digestive tract, 8-52 122 MANUAL OF HUMAN PROTOZOA Protozoa in (continued) duodenum, 6, 41, 42 gall bladder, 42 gum, 19-21, 41 intestinal wall, 9, 49, 79 intestine, 9-19, 21-52 liver, 9, 79 lung, 9 lymph, 73, 79 mouth, 19, 41, 82 muscles, 106 reproductive organ, 108 spleen, 79, 94 sputum, 41 tartar, 41 tonsile, 41 vagina, 108 Pseudopodia, 4, 9, 10, 19 Pus, 54, 55 Pyorrhoea alveolaris, 19 Reproduct, asexual, 3, 4, 5 sexual, 5, 84-86 Reservoir hosts, 49, 73, 75, 80 Reticulo-endothelial system, 78, 79, 81 Retortamonas intestinalis, 31-33, 45, 46 Rhizoplast, 78, 80 Ring forms, 83, 86-87, 90, 100 Salt sokition, 55 Sand flies, 78, 81, 82 Sarcocijstis lindemanni, 6, 106-107 Sarcodina, 3, 5, 8, 9-30, 62 INDEX 123 Sarcosporidiosis, 6, 106 Schaudinn's fixative, 58 Schizogony, 4, 5, 83-85, 88, 91, 93, 95, 96 Schizonts, 4, 5, 83-85, 86-88, 90-91, 92-95, 96 Schizotrypanum criizi, 76 Schiiftner's dots, 88, 89, 93, 94, 95 Sleeping sickness, Central African, 5, 73 East African, 5, 75 Gambian, 73 Rhodesian, 75 Slides, microscopic, 54 Smears, blood, 97-101 faecal, 55-61 Sodium chloride, 55 Sphaerita, 25, 69, 70 Spleen puncture, 79, 81, 100 Spores of Eimeria cliipearum, 67 sardinae, 67 Isospora hominis, 48 lacazei, 68 Sarcoci/stis lindemanni, 106-107 Sporoblasts of Isospora hominis, 48 Sarcocystis lindemanni, 106 Sporozoa, 8, 47-49, 83-96 Sporozoites, 48, 68, 83, 86 Staining of blood films, 99-100 faecal smears, 59-61 Trichomonas vaginalis, 109 Sucking disc, 42, 44 Sudan III, 67 Technique for protozoa in blood, 97-101 124 MANUAL OF HUMAN PROTOZOA Trypanosoma hriicei, 75 faeces, 53-61 muscles, 107 vagina, 109 Thick blood films, 97-98 Thin blood films, 97 Thrombocytes, 104 Toxic substances, 85 Trichomonas huccalis, 41 elongata, 41, 45 hominis, 37, 38-41, 45, 46, 108, 109 Tenax, 41 vaginalis, 41, 108-109 Trophozoites, 3, 4, 8 Trypanosoma, 4, 72-79, 100 Trypanosoma hrucei, 75 cruzi, 5, 73, 76-78 escomeli, 76 oambiense, 5, 72-75, 76 rhodesiense, 5, 72, 75-76 Trypanosomiasis, 5, 72-79, 105 Tricercomonas intestinalis, 35-36, 45, 46 Triatoma, 72, 78 Triatoma infestans, 76 megista, 76 Ulcers, 9, 12 Undulating membrane, 39, 74, 77, 78, 109 Use of cover on blood smears, 100 Vaginitis, 108 Vegetative stage, 3, 4 Vesicular nucleus, 3 INDEX 125 Viability of cysts, 14 trophozoites, 8, 21, 28 Wright's stain, 99-100 Yeasts in blood films, 102 faecal smears, 68 Zygotes, 5 This Book ff/anuat of HUMAN PROTOZOA By RICHARD R. 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