The ultrastructure of Entamoeba histolytica

Bull. Org. mond. Santh 1970, 43, 301-308 Bull. Wld Hlth Org. J The Ultrastructure of Entamoeba histolytica J. LUDVfK I & A. C. SHIPSTONE' The ultrastructure of the trophozoite of Entamoeba histolytica cultivated in vitro on artificial media is described from electron-microscope studies made by the authors and from other published accounts. The authors point out that the description does not present a complete picture of the trophozoite of this species ofamoeba since no detailed studies of the dysenteric form in infected host organs have yet been made and that nothing is known about the ultrastructure of cysts and the process of cyst formation, maturation and germin- ation. The cytoplasm is confined by a 3-layered cytoplasmic membrane and contains many different organelles: subpellicular dark bodies, ribosomes, helical aggregates of ribosomes, osmiophilic lipoid granules, short dark tubular bodies ofunknown origin andfunction, food vacuoles and some very small vacuoles thought to be lysosomes. Neither mitochrondria nor a Golgi apparatus could be found. In the nucleus, the chromatin material is accumulated around the periphery and the nucleolus is centrally situated. The nuclear envelope is a double-layered membrane with numerous pores. Some groups of button-like particles were seen within the nucleus and it is thought that they are probably symbiotic virus-like particles. Some comparisons are made with the ultrastructure of other c-.oebae, both parasitic and free-living, and some further lines of study are suggested. The ultrastructure of Entamoeba histolytica is known from electron-microscope studies of the vegetative forms. All authors who have dealt with this matter (Bird, 1956; Fletcher et al., 1962; Miller et al., 1961; Osada, 1959; Shipstone & Ludvik, 1970) made electron-optical investigations of tropho- zoites only and their studies, including the work of the present authors, are based on material obtained through the cultivation of amoebae in vitro on various kinds of media. The fine structure of tiophozoites cultivated in vitro is therefore rather well known, but it is not known how, and to what extent, the ultrastructure is differentiated during parasitic life in host organs. It must be realized that the character of some ultrastructural features may be influenced by the artificial environment in cultiv- ation. Various factors of cultivation must be taken into account, they include: the heterogeneity of food intake resulting in differences in the content 1 Laboratory of Electron-Microscopy, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague 4, Czechoslovakia. ' Biophysic Division, Central Drug Research Institute, Lucknow, India. of food vacuoles and, consequently, in the way the food is digested; the amount of, or even the absence of, oxygen in the environment, as well as the presence of micro-organisms; and the chemical properties of the environment which affect the rate and intensity of metabolism and, thus, the synthesis and growth of the cell. All these factors may influence the fine structure of cells. The present survey of the ultra- structure of E. histolytica, which presents a summary of present-day knowledge, does not present a com- plete picture of the trophozoite of this species; to do that, it would be necessary to obtain electron-opti- cal micrographs of trophozoites of the dysenteric form from infected host organs. MATERIALS AND METHODS The electron-microscope study was made with trophozoites of E. histolytica strains kept for a certain time in in vitro cultures (NRS-strain; see Miller et al., 1961) or of strains newly isolated from an acute case of human dysentery (STA-strain; see Shipstone & Ludvik, 1970). In most cases, the trophozoites were cultivated in Boeck & Drbohlav 2557 -301- J. LUDVIK & A. C. SHIPSTONE (1925) medium, Dobell & Laidlaw (1926) medium or in the medium modified according to Dutta & Rao (1966). The cultivation medium regularly contained rice starch and sometimes also bacterial monocultures. The morphological study was based on 24-hour or 48-hour cultures of trophozoites. The trophozoites were first concentrated by centrifug- ation (at about 1500 rev/min) and the sediment was fixed by stirring with fixative solutions. Fletcher et al. (1962) washed the material in an isotonic phosphate buffer at pH 7.4 before fixation. Fixation The osmium fixation according to the original method of Palade (1952), a 1 % solution of osmium tetroxide in barbital acetate buffer at pH 7.2, was used by Bird (1956), Osada (1959) and Fletcher et al. (1962). The fixation period was 30-60 minutes. Some authors (Fletcher et al., 1962) fixed amoebae during 90 minutes in ice-cold osmium tetroxide solution and the fixative was changed after 15 minutes and 45 minutes. Miller et al. (1961) applied chromium-formol fixative (Low & Freeman, 1956) and potassium permanganate fixative (Luft, 1956). In the study made by the present authors, mainly the osmium fixative according to Caulfield (1957), chromium- osmic fixative according to Dalton (1955) and glutaraldehyde fixation according to Sabatini et al. (1963), followed by osmium postfixation, were used-always at 4°C. Bhowmick & Wohlfarth-Bottermann (1965) made very thorough studies of various types of fixation of amoebae for electron microscopy, the species Amoeba proteus and Chaos chaos being chosen as models. The authors tested different concentrations of fixatives, the rate of fixation according to the concentration of osmium tetroxide, the presence of potassium dichromate and the temperature, the duration of fixation and the pH. It was found that the maximal rate of fixation is attained mainly through the concentration of osmium tetroxide solution at room temperature at which there is a reduced fixation time of 8-10 minutes. For optimal fixation, it was considered that quick fixation of cells is obtained in a 9.5% solution of osmium tetroxide in a 4.5-% solution of potassium dichro- mate at pH 7.2, the actual fixation not exceeding a period of 8-10 minutes. By means of this method of fixation, the basic cytoplasm and all the membranous components are perfectly visible. The authors proved that too long a fixation time, especially in low concentrations of osmium tetroxide, results in the basic cytoplasmic matrix being washed out. In the present studies, after dehydration in a graded series of ethanol or acetone solutions, the material was embedded in either methacrylates, epoxyresin or polyester Vestopal W. To afford con- trast in the ultra-thin sections, lead citrate (Reynolds, 1963) and 5% uranyl acetate were applied; this appeared to be the most convenient method. SURVEY OF MORPHOLOGY AND ULTRASTRUCTURE OF TROPHOZOITE CELLS Size of trophozoites The size of trophozoites differs somewhat from one type of amoeba to another and depends mainly on the number of food vacuoles in the cytoplasm and on the size of phagocytosed starch grains. On the basis of a great number of serial sections, it was found that the average size of a cell is 15 ,t- 30 ju (Fig. 15, 16); this measurement corresponds to the dimensions obtained by measuring living amoebae under a light microscopy. Similar data have been given by Fletcher et al. (1962). Cytoplasm The cytoplasmic membrane is in the form of a typical unit-membrane consisting of three layers: two thinner, osmiophilic, electron-dense layers, 35 A-4o A thick, and a wider, electron-transparent, middle layer, 45 A-50 A thick (Fig. 1, 2). Close under the cytoplasmic membrane, or some- times directly adjoining the interior osmiophilic layer, subpellicular dark bodies, 160 A wide and 300 A-400 A long with a lens-shaped profile, could be observed. They were scattered under the whole cytoplasmic membrane, being particularly well discerned after chromium-osmium fixation (Fig. 1, 2, 3). At present, nothing can be said about the significance or function of these subpellicular bodies. In some cells they were very few in number or completely lacking; in others, they were absent over a large part of the cell surface, being accumul- ated in certain places only. It may be assumed that these bodies are closely connected with the new synthesis of cell membrane or represent directional chemotactic centres. Certainly, they do not seem to be an artefact due to fixation or to coagulation of the contrasting substance. The dark bodies were never seen in the cytoplasm and they were totally different from the dark particles observed by Pappas (1959) in A. proteus and classified as artefacts 302 THE ULTRASTRUCTURE OF ENTAMOEBA HISTOLYTICA of fixation. No difference could be found between the ectoplasm and endoplasm in any of the tropho- zoites examined, either in the density of cytoplasm or in the location of cytoplasmic components and organelles. The cytoplasm contains a great number of ribosomes and polysomes (Fig. 3, 4, 14, 15). In addition, groups of osmiophilic particles are scat- tered everywhere in the cytoplasm, except in the region of the endoplasmic reticulum. The particles, as could be seen at higher magnifications, were composed of dense, adjoining, ribosome-like bodies and seemed to be a kind of giant polysome; they were observed in all sections of amoebae fixed by osmium tetroxide and were particularly prominent after chromium-osmium fixation (Fig. 1, 3, 4, 14, 15), averaging about 80 m,u in size. In addition to these giant polysomes, a number of compact osmiophilic granules of the same size, or a little larger, were present in the cytoplasm. The endoplasmic reticulum is located in the cytoplasm, but only in some places, it consists of fine tubules and small longitudinal lacunae (Fig. 7). These tubules and vesicles are surrounded by a number of small ribosomes (Fig. 7, 14). In the cytoplasm of some trophozoites could be seen a number of helical bodies of lengths ranging from 0.3 ,u to 1 ,u and 40 m,u-50 m,u in thickness (Fig. 5). These bodies were fairly regularly distrib- uted over the cytoplasm and were absent only in the areas of endoplasmic reticulum; they were never seen inside the nucleus. Some authors (Osada, 1959; Behnke, 1963; Waddington & Perry, 1963; Maniloff et al., 1965; Echlin, 1965) observed and described helical forms in the cytoplasm of different cells and tried to explain them as aggregates of ribosomes having a helical configuration. Pappas (1956, pp. 221-222) described clusters of helices in A. pro- teus but saw them only inside the nucleus. Stevens & Prescott (1965) found the same helical forms in A. proteus, not only in the nucleus but also in the cytoplasm surrounding the nucleus, and they supposed that " these helices might represent some sort of packaged messenger RNA ". We are inclined to accept the view that the helical bodies found in large numbers over the whole cytoplasm of E. histolytica trophozoites represent an arrangement of ribosomes. In one preparation, a polycrystalline body was seen in the cytoplasm of a trophozoite (Fig. Sa). In size, lamellar arrangement and helical substructure, the formation corresponds to the helical ribonucleo- protein (RNP) bodies described in E. invadens by Siddiqui & Rudzinska (1963; 1965). Furthermore, the cytoplasm of the trophozoites contained several short, osmiophilic tubular bodies. These short electron-dense tubules were irregularly scattered over the whole cell; in size they were 240 m,u-250 mu long, 70 mp, in exterior diameter and 35 m,u-37 m,u in interior diameter (Fig. 6, 10, 16). The nature and function of the tubules are not known. Very similar tubular structures were observed in the cytoplasm of the same species of amoeba by Avakyan & Sarkissian (1964). In the cytoplasm of E. histolytica trophozoites neither mitochondria nor any kind of mitochondria- like organelles have ever been observed. Similarly, a typical Golgi apparatus could never be identified. It is, moreover, very difficult to identify the Golgi zone; perhaps some of the groups of small vacuoles appearing in some sections might be characterized as a Golgi-like zone (Fig. 3). The most morphologically varied organelles in the cytoplasm of trophozoites are the different kinds of vacuoles and in the cytoplasm the food vacuoles (Fig. 4, 9, 14, 16, 17) are the most frequent; their size varies from the small vacuoles 0.5 it in diameter to giant ones 12 , in diameter and they are filled with more-or-less digested food, usually bacteria (Fig. 8, 10, 14, 16) or starch grains (Fig. 1, 17). The largest food vacuoles usually contain one large grain of starch up to 10 ,u in diameter (Fig. 1, 17). Some amoebae had only a single large vacuole containing a big starch grain. In some sections, the process of enzymatic digestion could be observed. It was interesting to see that, frequently, the outer part of starch particles was intact while the inner portion was digested (Fig. 17). The enzymes pro- bably penetrate into the starch grain through a break; apparently, the inner part of starch grain is more easily hydrolysed than the outer part. The limiting membrane of all vacuoles has the same fine structure as the cytoplasmic membrane (Fig. 14). In food vacuoles this is to be expected since such vacuoles are formed when food is engulfed by invagination of the plasma membrane. Occasion- ally, some remaining subpellicular bodies can be seen on the surface of vacuoles (Fig. 2, 15). Inside the vacuoles, there may even be a few concentric unit-membranes representing " myelin-like forms " which are well-known and frequent in phagocytic cells. Such inclusions have been described in other amoebae (Vickerman, 1962); they are probably phospholipids and appear to be produced in the 303 3. LUDVIK & A. C. SHIPSTONE course of digestion of bacteria. The number of membranous layers observed in food vacuoles of trophozoites never exceeded 5 or 6; far fewer than those observed in certain other species of amoeba and described as myelin forms (Fig. 4, 9). Some larger vacuoles contained also a number of minute vacuoles but they were not very common and were probably the remains of food vacuoles. The digested material in single vacuoles usually consisted of either bacteria or starch grains. Both types of food in the same vacuole were seen only exceptionally. It is probable that the intracellular digestion of different foods is carried out in separate vacuoles, i.e., that different physico-chemical con- ditions are created in individual vacuoles for the hydrolysis of specific substrates. Fletcher et al. (1962) supposed that one of the functions of food vacuoles might be to enable the organism to create and maintain optimum conditions of pH, ionic concentration, etc., within the vacuole for enzyme activity related to a specific food substance. It is probable that the enzymes are activated in, or at the surface of, vacuoles either by prosthetic groups or by conversion from enzymatically inert precur- sors; alternatively, inhibitors may be present in the cytoplasm proper. Similar considerations pro- bably apply to the secretion of enzymes across the plasma membrane. Fletcher et al. (1962) considered it possible that maximal extracellular activity of enzymes of parasitic amoebae could be achieved only at certain times, when the external environ- mental conditions approximate to the optimum. This consideration may well have an important bearing on the invasiveness of particular organisms. In addition to the food vacuoles described above, there are also in the cytoplasm some very small or minute vacuoles, the size of which varies from 50 mit to 200 m,u (Fig. 3, 9, 10, 16). These vacuoles are thought to be lysosomes or a kind of spherosome and they occur either singly, scattered among the food vacuoles, or in groups. In the groups, 2 or more small vacuoles may unite with each other to form elongated or otherwise irregular lacunae (Fig. 3, 9). The cytoplasm of trophozoites further contains a few dark, mostly oval, micro-bodies, 0.3 u-0.9 u in size. Since they consist of electron-dense material it may be supposed that they are of lipid origin (Fig. 1, 7, 9, 17). Some cytoplasmic regions are characterized by the lack of any structure and by the absence of ribosomes, such regions cannot be contrasted in any way (Fig. 8, 16) and this part of the cytoplasm probably contains reserve food material of polysaccharide origin. In ultra-thin sections of a trophozoite cell, a structure, which may be considered to be a pseudo- podium (lobopodium), in course of formation (Fig. 15) was seen. If the interpretation of the electron-micrograph is correct, the pseudopodium is first created by very small cytoplasmic extensions following the presumed direction of movement; these extensions unite successively into the larger lobopodium proper, leaving a number of small closed vacuoles in the cytoplasm. The small vacuoles mark the original cell boundary and probably disappear later. On the surface of the small vacuoles some remaining subpellicular bodies could be observed. Nucleus The nucleus of E. histolytica trophozoites is generally a regular oval measuring 3.5 ,u by 5 u-6 u and the nucleoplasm is more electron-dense than the surrounding cytoplasm. The nuclear envelope is composed of a double membrane with numerous pores (Fig. 12, 13, 15). Each membrane is a typical triple-layered unit-membrane, 120 it thick. The number of pores in the nuclear envelope is rather high and Miller et al. (1961) stated that " the interstices of such a pore system may compose as much as 50 per cent. of the area of nuclear membrane". The pores are in most cases 50 m,u in diameter. The chromatin material is usually deposited in the peripheral part of the nucleus under the nuclear membrane and forms irregular clusters correspond- ing to the "beaded" areas within the nuclear membrane of E. histolytica seen in light-microscopy. The karyosome, usually situated at the centre of the nucleus, has an irregular shape (Fig. 12, 13) and is often composed of small electron-dense clusters. In the nucleus, particular dark bodies having a distinct organization with a button-like structure (Fig. 10, 11, 13) were very often observed. These formations, present after all types of fixation, have never been observed in the cytoplasm; they were extremely osmiophilic and in ultra-thin sections, at a greater magnification, their ultrastructure could be observed. In the interior there were either irregular electron-light areas and small vacuoles (Fig. 10, 11, 15) or an inner concentric core (Fig. 11). The shape of the button-like bodies was oval or round; their size range was between 0.2 ,u and 0.5 ,u. The number of dark bodies observed in the nucleus ranged from 304 THE ULTRASTRUCTURE OF EN-TAMOEBA HISTOLYTICA 2 to 30 in one section. Similar formations in the nucleus were described also by Miller et al. (1961) who called them " nuclear aggregates ". Vicker- man (1962) found similar bodies in the cytoplasm of Acanthamoeba and characterized them as virus-like particles, suggesting that they might be symbionts. Although nothing positive about the origin and function of the button-like organisms in the nucleus of trophozoites is known at present, it seems likely that they are a kind of nuclear virus-like parasite or symbiont of E. histolytica. DISCUSSION If we compare the fine structure of trophozoites of E. histolytica with that of other species of Ent- amoeba no important morphological differences are found; E. invadens, particularly, has been studied by electron-optics (Siddiqui & Rudzinska, 1963 and 1965; Deutsch & Zaman, 1959; Zaman, 1961, 1962). Siddiqui & Rudzinska (1965) recognized two types of particles in the cytoplasm of E. invadens, namely-smaller, less-dense particles 200 A-300 A in diameter, probably representing Palade's particles containing ribonucleoprotein; and much more numerous larger, high-density particles, 400 A- 700 A in diameter, which are probably glycogen particles comparable to those described in electron- micrographs of liver cells (Revel et al., 1960). The smaller particles probably correspond to those we have designated ribosomes; the larger ones, having regard to their size and number, might be identical to E. histolytica polysomes. Several authors (Deutsch & Zaman, 1959; Fletcher et al., 1962; Miller et al., 1961 ; Osada, 1959; Siddiqui & Rudzinska, 1965) have characterized small circular vesicles scattered in the cytoplasm as a poorly developed endoplasmic reticulum. Zaman (1962) studied starch ingestion by E. invadens, describing, in the cytoplasm surrounding the nucleus and in the proximity of starch particles, digestive spherules which, through coalescence, form a food vacuole around the engulfed starch grain. In an ultra-thin section of E. invadens, Zaman (1961) successfully photographed a very interesting posterior part of a moving amoeba, the so-called uroid or tail end "; its function remains unknown. In the electron-micrograph, the uroid appears as a narrow, elongated cytoplasmic extension surrounded by an irregular clump of electron-dense material. The cell membrane on the surface of the uroid is continuous, forming numerous small convex swellings which are largely separated from the cell body. A number of small vacuoles could be observed, not only inside the cytoplasm of the uroid but also exteriorly in the surrounding electron-dense material. In all pro- bability, this mucoid substance is constantly being formed during the movement of the amoeba and seems to contain non-digested food evacuated from the vacuoles. The vacuoles are emptied by a sudden bursting of the surface or by the expulsion of entire vacuoles into the swellings on the surface of a uroid and the electron-micrographs seem to prove the excretory function of the uroid. The origin of the mucoid substance surrounding the uroid is not yet clear; in the opinion of most observers, it is either secreted on the surface of the amoeba and accumul- ated at the posterior end of the cell body during move- ment or it is formed directly from the contents of emptied vacuoles and from whole vacuoles pinched off together with their contents. The uroid has never been observed in trophozoites of E. histolytica. Particular attention should be paid to two ultra- structures seen in E. invadens by Siddiqui & Rud- zinska (1963). The first, a cortical honeycomb layer on the surface of the nucleus, was also observed in E. blattae (Beams, 1959). This structure has a typical regular hexagonal pattern in cross-sections, each hexagon being 100 ,um in diameter; it was previously described on the nuclear surface of A. proteus (Pappas, 1956, pp. 431-434). The honey- comb layer can be seen only in oblique sections and close to the surface of the nucleus. In electron- microscope studies of E. invadens cultures with concomitant bacteria no such structure was re- ported (Deutsch & Zaman, 1959). The second very interesting structure observed in the cytoplasm of E. invadens (Siddiqui & Rudzinska, 1963, 1965) is the helical ribonucleoprotein body which corre- sponds to the organelles called " chromatoid bodies," well known from light-microscopy. These bodies in E. invadens were studied cytochemically and electron- optically by Barker (1963) and Barker & Deutsch (1958) and were described as polycrystalline masses in precystic and cystic stages. Siddiqui & Rud- zinska (1963) found them also in axenically grown trophozoites which did not encyst. In a single trophozoite of E. invadens, as many as 18 chromatoid bodies of sizes varying from 1 it to 3 ,u may be seen. They are scattered throughout the cytoplasm and in electron-micrographs appear to be composed of parallel bands or lamellae. At higher magnification, the lamellae appear to be composed of coiled fibrils which seem to form a closely packed helix. In trophozoites of E. histolytica, the compactly formed 7 305 J. LUDVIK & A. C. SHIPSTONE FIG. 1-20 ELECTRON-PHOTOMICROGRAPHS ULTRA-THIN SECTIONS OF E. HISTOLYTICA Fig. 1, 2 & 3. Marginal parts of amoeba. Small lens-shaped subpellicular bodies are situated under the cytoplasmic membrane; the cytoplasm contains many ribosomes and large dark polysomes. Dalton's fixative. Fig. 4. Central part of cytoplasm with food vacuoles filled with concentric membranes. Dalton's fixative. Fig. 5. Central part of cytoplasm with helically arranged ribosomes. Fig. 5a (inset). Helical ribonucleoprotein (chromatoid) body. Sabatini's fixative. Fig. 6. Part of amoeba cell with short dark tubular bodies. Caulfield's fixative. Fig. 7. Endoplasmic reticulum surrounded by ribosomes. Dalton's fixative. Fig. 8. Part of cell with reserve material. Food vacuole with bacteria. Caulfield's fixative. Fig. 9. Small vacuoles and vesicles (lysosomes) in cytoplasm. To the right, osmiophilic electron-dense microbody. Caulfield's fixative. Fig. 10. Button-like bodies in the nucleus and a food vacuole with a group of bacteria in the cytoplasm. Dalton's fixative. Fig. 11. Detail of button-like bodies. Sabatini's fixative. Fig. 12 & 13. Sections of nucleus. Chromatin is located in clusters peripherally under the nuclear membrane. The double-layered nuclear membrane has numerous pores. In the nucleus in Fig. 13 are dark button-like bodies. Dalton's fixative. Fig. 14. Detail of cytoplasm with numerous polysomes. In the food vacuole is a cross-sectioned bacterium. The limiting membrane of the vacuole Is structurally identical with cytoplasmic membrane. Dalton's fixative. Fig. 15. Section of pseudopodium, probably in course of formation; below and to the right, part of the nucleus with button-like bodies and clearly visible pores in the nuclear membrane. Dalton's fixative. Fig. 16. Section of a whole trophozoite of E. histolytica. The cytoplasm contains numerous food vacuoles of varying size. Small electron-transparent particles are probably reserve material (polysaccharides). In the middle of the very large food vacuole are engulfed bacteria. Caulfield's fixative. Fig. 17. Section of a whole trophozoite of E. histolytica. In the centre of the cell body is a large starch grain which is internally partly digested. In the surrounding cytoplasm are small food vacuoles containing bacteria. Dalton's fixative. ULTRA-THIN SECTIONS OF E. MOSHKOVSKII Fig. 18 & 19. Section of nucleus and the surrounding cytoplasm of E. moshkovskii. Perinuclear cloud of electron-dense material lies around the nucleus. In the proximity of the nucleus are osmiophilic dark bodies. Small button-like bodies are situated in the nucleus among peripherally located chromatin material. Sabatini's fixative. Fig. 20. Part of cell body of an E. moshkovskii trophozoite. 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As previously mentioned, the trophozoites of E. histolytica typically contain a number of free, isolated helices, situated in the cytoplasm, which appear to be an orderly arrangement of free ribo- somes. When present knowledge of the occurrence of helical arrangements of ribosomes in the cyto- plasm of cells is reviewed (protozoa: Shipstone & Ludvik, 1970; Siddiqui & Rudzinska, 1963, 1965; mammals: Behnke, 1963), an interesting feature common to all cells, also noticed by Siddiqui & Rudzinska (1965), is seen, namely-all are in a phase of active protein synthesis and growth and their endoplasmic reticulum is very poorly developed. From these results, it can be deduced that in cells actively synthetizing protein but having little or no endoplasmic reticulum the free ribosomes aggregate to form either polyribosomes or helices (Shipstone & Ludvik, 1970; Siddiqui & Rudzinska, 1965). Ludvik (1970) recently studied the trophozoite of free-living E. moshkovskii (Fig. 18, 19, 20) and found that the structure did not differ much from that of E. histolytica and E. invadens. All the ex- pected organelles were present; namely-a double nuclear envelope, subpellicular bodies, food vacuoles of various sizes and having various contents (bac- teria, starch grains), small vacuoles often fusing into long, narrow cisternae, electron-dense oval lipoid bodies (Fig. 18, 20), a number of free ribo- somes as well as helical bodies, i.e., helically arranged polyribosomes (Fig. 19, 20) and short tubular formations (Fig. 18). Only the nucleus appeared to have a rather different morphological structure, being more electron-dense, of irregular shape and frequently showing cytoplasmic engulf- ments. Between the peripherally situated chromatin material and the nuclear membrane, a lighter zone was seen. The centrally situated endosome had an irregular shape and was more electron-dense in the centre. Button-like particles were also observed. Typical of E. moshkovskii was a perinuclear cloud situated around the nucleus, consisting of material more electron-dense and clearly distinguished from the surrounding cytoplasm (Fig. 18). In conclusion, it may be pointed out once more that, in spite of very interesting new contributions, it has been possible to gain further insight only into the structure of the cell body of intensively metabolizing Entamoeba trophozoites. A detailed ultrastructural study of the cysts and of the whole process of cyst formation, maturation and germina- tion is lacking. In future, highly interesting results may be expected from the electron-microscopic investigation of parasitic Entamoeba within the host and the ultrastructural study of interactions with host cells and tissues should yield important results. Cytochemical studies on the ultrastructural level, especially after enzymatic treatment, are also suggested as areas of investigation. oUME ULTRASTRUCTU RE D'ENTA MOEBA HISTOL YTICA La microscopie dlectronique permet depreciser certains details de la structure des trophozoites d'Entamoeba histolytica cultives in vitro sur milieux artificiels. La membrane cytoplasmique, d'une epaisseur totale de 120 A-130 A, comprend une couche interne et une couche exteme, opaques aux electrons, separees par une couche interm6diaire transparente. A proximitd immediate de la couche interne, on distingue des corps sombres, plus ou moins nombreux, dont le r6le est encore inconnu. Le cytoplasme renferme un grand nombre de ribosomes et de polysomes, ainsi que de nombreuses particules osmiophiles de grande taille, for- mees par des amas de corps ressemblant a des ribosomes. D'autres structures, helicoidales, semblent resulter d'un arrangement defini de ribosomes. Des corps osmio- philes, de forme generalement ovale, sont consideres comme des granules lipoidiques. Enfin, on observe frequemment des corps tubulaires courts et opaques, aux fonctions non identifiees. On ne decele ni mitochon- dries, ou organites similaires, ni appareil de Golgi typique. La membrane limitante des vacuoles nutritives pre- sente la meme structure que la membrane cytoplasmique. Les vacuoles contiennent habituellement des vestiges bacteriens ou des grains d'amidon. En outre, on y trouve tres frequemment une serie de membranes concentriques et parfois un certain nombre de formations vesiculaires. Les tres petites vacuoles dispersees dans le cytoplasme sont supposees etre des lysosomes. Le noyau d'E. histolytica, generalement ovale, peut atteindre 3,5 sur 5-6 ,u. La chromatine est dispos6e a la peripherie, pres de la membrane nucleaire. Le 307 308 J. LUDVIK & A. C. SHIPSTONE nucleole, de forme irregulire, est le plus souvent situe au centre du noyau. 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