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WleHr This is to certify that the thesis entitled lAJuCIlIb’l Ofl fic/jilo’l‘l‘ah b)’ 2X0“)! {#2me M at dfz‘lw “CAIN.“ presented by Jig, Alzbaf. 3a kaKaIF-IQJ'OLJ has been accepted towards fulfillment of the requirements for D. 1”"? degree in 2 ° °\°{:l)/ %%/£W( Major professor 5:/ W30 0-7639 I LIBRAR Y Michigan State . "Q OVERDUE FINES: tryih“. .. 4" 25¢ per d” pep-1m "‘H ”3““ ‘ momma LIBRARY MATERIALS: 1; c.3333!” _'11 Place In book return to remove charge from circulation records ‘ INDUCTION OF ENCYSTATION BY AXENIC ENTAMOEBA IN A DEFINED MEDIUM BY ALI, A. JAHANGIR NEJAD A THESIS SUBMITTED TO MICHIGAN STATE UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ZOOLOGY 1980 In the Name of " ALLAH ", the Compassionate, the Merciful. ABSTRACT INDUCTION 0F ENCYSTAIION BY AXENIC ENTAMOEEA IN A DEFINED MEDIUM BY ALI, A, JAHANGIR NEJAD The objective was to identify the encystation requirements of Entamoeba invadens, strain IP-l. Oxygen was excluded and 0.05% cysteine was added to the encystation medium. Growth and encystment both require CO but at different concentrations. For this strain of amoeba at 23°C, 2 302 CO2 was optimum for growth; while this amount of C02 inhibited encystation. Maximum encystation took place with 1% to 102 C0 The 2. optimum encystation temperature ( 23°C ), was less than that used for the optimum growth ( 29.500 ). Encystation was induced between pH 5 and 7, in media with osmotic pressures between 56 and 300 mOsm. encystation was observed in Diamond's TP-S-l culture medium without glucose or phosphate and in other complex media. Glucose inhibited encystation in all media tested. Encystation was induced in Trypticase ( BBL ) and in vitamin-free salt free, casein ( acid ) hydrolysate ( NBCo ). Mixtures of amino acids resembling casein were used in deletion experiments. An amino acid mixture that supported encystation was : alanine, aspartate, glycine, isoleucine, leucine, phenylalanine, proline, serine, valine and cysteine Ali, A. Jahangir Nejad. However single deletions from this mixture did not completely inhibit encystation, except aspartate. Encystation was not observed without aspartate. Encystation media that supported 1002 encystation, also support up to a 50% increase in vegetative amoebae. Some encystation media supported greater than 1002 encystation and a corresponding increase in the number of vegetative amoebae. Thus encystation of Entamoeba, may be cell cycle related or the physiological state of encysting amoebae may not be too much different from that of the dividing organism. Exogenous divalent ions were not required for encystation by Entamoeba. To my Mother. To my Father. To my Wife. ii AKNOWLEDGMENTS I would like to express my sincere appreciation to my major advisor, professor Dr R. Neal, Band, for his invaluable guidance and encouragement; and for his critical suggestions durind the course of this research work. I am also very grateful to my other professors, Dr Herbert. W. Cox ( Microbiology ); Dr Ralph. A. Fax ( Zoology ); and Dr Donald. A. Twohy ( Microbiology ), for serving as membres in my committee and for their enjoyable and fruitful discussions. iii .TABLE OF CONTENTS TITLE PAGE LIST OF FIGURES. o o o o o o o o o o o o o o o o o o o o o o o o 0 vi LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . vii INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Terminology of different stages of the life cycle . . . . . . 2 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . 2 Trophozoites . . . . . . . . . . . . . . . . . . . . . . 3 Cysts . . .,. . . . . . . . . . . . . . . . . . . . . . 3 Encystation media . . . . . . . . . . . . . . . . . . . . . . 5 Culture media . . . . . . . . . . . . . . . . . . . . . . . . 7 Metacystic development. . . . . . . . . . . . . . . . . . . . 11 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . 14 Encystation-media . . . . . . . . . . . . . . . . . . . . . . 15 Gassing . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 General procedures . . . . . . . . . . . . . . . . . . . . . l6 Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Components of the encystation media . . . . . . . . . . . . . 17 Measurement of osmotic pressure . . . . . . . . . . . . . . . 18 RESIJLTS O C O O O O O O O O O O O O O O O O O O O O O O O O O O O O 1 9 Growth of Entamoeba invadens in TP-S-l culture medium. . 19 Encystation of §fi_ invadens in different encystation media. 19 Encystation and growth of §h_ invadens in different concentrations of Trypticase . . . . . . . . . . . . . . . . 23 iv TITLE £4.62 Encystation of §L_invadens versus time, in 4% Trypticase, , , 27 Effects of adding TP-S-I culture medium components to Trypticase-cysteine solution on the encystation of fig. invadens. . . . . . . . . . . . . . . . . . . . . . . . . 36 Deletion of TP-S-l components . . . . . . . . . . . . . . . 36 Encystation in dialysed Trypticase ( against distilled water ) 40 Effect of tyrosine. . . . . . . . . . . . . . . . . . . . . . 42 Molecular size and encystation. . . . . . . . . . . . . . . . 42 Effect of dilution of TP-S-l culture medium on encystation. . 43 Effect of osmotic pressure on encystation . . . . . . . . . . 46 Effect of temperature on encystation. . . . . . . . . . . . . 46 Induction of encystation of ‘§;_ invadens in the mixture of amino acids resembling casein . . . . . . . . . . . . . . . . 53 Effect of CO2 on encystation. . . . . . . . . . . . . . . . . 55 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Effect of CO2 on encystation of §;_ invadens . . . . . . . . 70 Effect of temperature . . . . . . . . . . . . . . . . . . . . 71 Effect of pH. . . . . . . . . . . . . . . . . . . . . . . . . 71 Mechanism of encystation of E5_ invadens . . . . . . . . . . 71 Cell cycle and encystation. . . . . . . . . . . . . . . . . . 74 ADDENDUM :. . . . . . . . . . . . . . . . . . . . . . . . . . 74 SUMMARY AND CONCLUSION ... . . . . . . . . . . . . . . . . . . . . 75 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . 77 LIST OF FIGURES FIGURE PAGE ( 1 ) - Growth of Entamoeba invadens in TP-S-l culture medium, versus time. 21 ( 2 ) - Growth of §;_ invadens in different concentrations of Trypticase without and with 0.052 cysteine. 26 ( 3 ) - Encystation of §;_ invadens in different concentrations of Trypticase without and with 0.05% cysteine. 29 ( 4 ) - Encystation and growth of §;. invadens in different concentrations of Trypticase without cysteine. 31 ( 5 ) - Encystation and growth of Ea. invadens in different concentrations of Trypticase with 0.05% cysteine. 33 ( 6 ) - Encystation of §h_ invadens versus time in 42 Trypticase with 0.05% cysteine. 35 ( 7 ) - Effect of adding components of TP-S-l culture medium to 42 Trypticase on encystation of E;- invadens. 38 ( 8 ) - Effect of osmotic pressure of different concentrations of Trypticase on encystation of §fi_ invadens. 48 ( 9 ) - Effect of CO2 on growth and encystation of §;. invadens. 57 ( 10 ) - The most common way of metacystic development of Entamoeba histolytica. 59 ( 11 ) - The five ways of metacystic development of E;_ histolytica in decreasing order of occurence frequency. 61 ( 12 ) - Speculative mechanism of encystation of §b_ invadens. 63 ( 13 ) - Speculative mechanism of encystation of §;_ invadens. 65 vi LIST OF TABLES Encytation of Entamoeba invadens in some of the media. Encystation and growth of ‘§;_ invadens in different concentrations of Trypticase. Effect of deletion of TP-S-l culture medium components on encystation of §fi_ invadens. Effect of components added to inside dialysate of 4% Trypticase ( dialysed against distilled water ), on encystation of §:_ invadens. Molecular size and encystation of EL. invadens. Effect of dilution of TP-S-l culture medium on encystation and growth of ‘§;_ invadens. Effect of cysteine on encystation of §;_ invadens. Encystation of §;- invadens in dried and reconstitutioned solution of outside dialysate of dialysed 4% Trypticase ( against distilled water ) with 0.05% cysteine at two different temperatures. Effect of cysteine and temperature on the encystation of §:_ invadens using 42 Trypticase as encystation medium. (-10 ) - Amino acids of casein and their concentrations. vii. PAGE 22 24 39 41 44 45 49 51 52 54 INTRODUCTION film‘V Parasitic amoeba have.been described in all phyla of animals. For example in Zelleriella an astomatous ciliate of’ amphibia (Stabler 1933; Carini and Reichenow, 1935). Prior to 1933, Entamoeba histolytica, the dysentery amoeba of man and other mammals, was the only known parasitic amoeba. Although Ilowaisky (1923) described a case of amoebic hepatitis from the frog, Rana temporaria , he did not present sufficient pathological evidence for his observation. Ratcliffe and Geiman (1933, 1934 ) , observed amoebiasis in reptiles that was essentially comparable to that of man and the’causative organism, Entamoeba sp. , was strikingly similar to Entamoeba histolytica. Rodhain (1934) described Entamoeba invadens , parasite of snakes which was more likely the same species that Ratcliffe and Geiman found in their reptiles. Many aspects of E; invadens , such as morphology, size, movement, location in the host's body, nutritional requirements, pathogenicity, are strikingly similar to those of E,_ histolytica, and it was because of this close relationship that Geiman and Ratcliffe(l936) discussed the possibility of their synonymity.. Since the study of E3_ invadens , helps in understanding and unravelling the problems of human amoebiasis ; and §;. invadens is more resistant or less fragile than. §fi_ histolytica to changes in almost all of environmental factors, it has been used as a target by a considerable number of investigators. TERMHNOLOGY OF DIFFERENT STAGES OF THE LIFE CYCLE: The life cycle of §fi_ invadens is arbitrarily divided into eight stages (Barker and Svihla, 1964). l'- Recently germinated amoeba with four nuclei; or premetacystic amoeba (Dobell). when the amoeba encysted, the nucleus of the cyst divides two times and gives rise to four nuclei; at this time encystation is complete. These nuclei are called, cystic or "N" nuclei. 2 - Primary division or metacystic amoeba which are defined as all amoebae resulting from the first division of the recently gerninated or premetacystic amoeba. 3 - Young trophozoites, including amoebae from the final division of the primary division, or metacystic amoeba. 4 - Late trophozoites, which are found in cultures approaching encystation. S - Precystic amoeba, which is smaller than late trophozoites. 6 - Early cyst, which contains the classical rod or bar-shaped chromatoid bodies, a single nucleus and the glycogen mass. 7 - Maturing cyst, characterised by 2 to 4 nuclei and less amount of glycogen and reducing chromatoid bodies. 8 - Mature cyst, containing little or no glycogen and chromatoid bodies. MOROHOLOGY: Morphology of the trophozoites and cysts of §£_ invadens are identical with that of §h_ histolytica. Morohology of E. histolytica has been described in detail by Dobell (1919, 1928). Geiman and Ratcliffe (1936), described the morphology of §;_ invadens. Trophozoites: Trophozoites have typical amoeboid shape, and pseudopodial movement. CytOplasmic streaming can be easily seen by a light microscope. There are many membrane bound food vacuoles in the cytoplasm and there is a central endosome in the nucleus. Ray £5 31, (1955), after staining E;_ histolytica with Janus green, described the presence of mitochondria in this organism. Deutsch and Zaman(1959), however did not observe mitochondria in E;_ histolytica or §fi_ invadens, with the electron microscope. Cysts: Cysts of §;. invadens are spherical or oval in shape. The cyst wall consists of a thin electron dense layer (30 -40 nm), on each side of which there is a thick electron transparent layer (250-300nm). (Deutsch and Zaman, 1959). There is no visible pore in the cyst wall (Mo Connachie, 1969). There are numerous chromatin particles in the cytoplasm of trophozoites. These particles, in the cytoplasm of the cysts, aggregate to form relatively large chromatoid bodies which then disaggregate into smaller fragments. They are called chromatoid bodies because they stain with basic dyes such as haematoxylin . Several ideas about the formation, function , and the origin of the chromatoid bodies have been suggested. Dobell (1919) believed that they are food sores. Hartmann (1912) said that they originate from the nucleus. Hakansson (1936) stated that they originate from the cytOplasm and have no relation to nuclear chromatin. Jones (1946) also reported that they originate from cytoplasm by condensation of smaller fragments. Hopkins and Warner (1946) suggested that they were formed by the coalescence of clear vacuoles. Deutsch and Zaman (1959) believed that chromatoid material distributed in the cytOplasm of trophophozoites, in association with food vacuoles and the nucleus; aggregation of these materials in the cyst makes chromatoid bodies. Barker and Svihla (1964), showed that in the cytoplasm of late trophozoites, ribonucleoprotein is synthesized very actively. At the end of the encystment, large amounts of ribonucleoprotein are concentrated to make chromatoid bodies. Morgan gt 21_(1968) showed that ribosomes isolated from cysts of §L_ invadens, are sensitive to hydrolysis by ribonuclease and were most likely derived from chromatoid bodies. The late trophozoite, the cyst and the primary division amoeba are actually three different stages of Entamoeba differentiation. This single cell differentiation, that is expressed by the process of encystation, is comparable to the early development of higher eukaryote organisms. In the late teophozoite precystic state, excess DNA, nutritional energy stores and ribosomes are synthesized, as occurs in the oocyte Barker (1976). Using DNA synthesized during precystic stage, two nuclear divisions occur; and for rapid growth and production of new cells, major cytOplasmic reorganisations take place (Barker 1976). On excystation one nuclear division takes place in the premetacystic state, before the eight nucleate cell divides to give rise to the primary division amoeba. This process is comparable to early cleavage (Barker 1976 ). Cyst ribosomes are completely inactive in protein synthesis. Ribosomes stored in the cyst as microcrystals are used in excystation to provide for growth of the amoeba. The cyst ribosome still can be traced after two generations, until a cyst gives rise to 32 trophozoites (Barker, 1976). Another inclusion of the (immature) cyst is a "glycogen mass" or "glycogen vacuole". It is named so, because in some preparations of cysts the space that was in earlier stages occupied by glycogen, looks like a clear vacuole and stains brown with the solution of iodine in potassium iodide. Cytochemical tests indicate the presence of glycogen in the cysts of most entamoebae. In late trophozoites glycogen is accumulated in preparation for encystment. glycogen has also been shown in the cytoplasm of trophic amoebae of several Species growing in gitrg_ (Hallman gt 21 , 1955; Bullock, 1966). The glycogen mass usually appears in the cytoplasm at the time when the cyst wall is being formed but Ray and Sengupta (1954), observed the glycogen mass in the precystic stage of ‘EL. histolytica. The glycogen mass is large in uninucleate and binucleate cysts, and it occupies a large space pushing the nucleus aside. It's size decreases as the age of the cyst increases. Finally when the cyst is mature and tetranucleate, the glycogen mass disappears. ENCYSTATION MEDIA: One of the problems in the biology of Entamoeba, that has been studied by many research workers, is the physiology of encystation in this organism. In nature, encystation of £5. invadens occurs in the lumen of the alimentary tract of snakes, usually in the colon.(Mc Connachie, 1969). Although cysts of §,invadens have been found in the liver and in the intestinal lesions of snakes, probably it has been due to encystation after the host had died (Neal, 1957). Usually tissue invading entamoebae do not encyst in the tissues of the living host. Dobell and Laidlaw (1926), were first to induce encystation of IE,. histolytica by deplition of the essential nutrient (rice starch) from their biphasic culture medium called " R,E.S ”, (_Ringer-Serum overlay on egg slant ). Cleaveland and Sanders, (1930), induced encystation of E;_ histolytica‘ by addition of rice.starch in their biphasic media. Meerowitch, (1958), induced encystation of §L_ ivvadens in media containing mixed bacterial flora, but he.failed to induce cysts in bacteria-free medium. Meerowitch.confirmed Chang's (1946), and Everitt‘s (1950) conclusion about the effects of some factors on encystation and considered three factors-necessary for encystation of §5_ invadens as follows: 1 — Consumption of particulate-material (rice starch) or mucopolysaccaride (mucin) by .Ea. invadens. . 2 a Presence of a factor (3) produced by living bacteria in order to breack down polysaccaride.to glycogen and to support growth of the amoeba. 3 — Presence of an intrinsic factor (s) in the culture, that probably is produced by overpopulation of 3;; invadens, Meerowitch then stated that " It was clear therefore that bacteria and starch or mucin were two extrinsic factors, without which, mass encystation could not take place ". Balamuth, (1963), induced encystation of ‘E; invadens in multibacterial media . He also used insoluble rice starch in his media and confirmed three above mentioned " principles " for encystation. Richards 35 31 (1946) observed encystation of §;_ invadens in association with bacteria and nutrient particles and in diluted medium but they did not give good enough details about their observation. Mc Connachie (1969, 1970) also induced encystation of §;_ invadens, by diluting the culture medium in axenic cultures of this organism growing in a medium based on liver-extract- saline but she also did not give too much detail about her work. Ghosh, (1970), induced encystation of .E; invadens in a modified biphasic medium of Dobell. Thepsuparungsikul and Bailey (1971), were able to induce encystation of §;_ invadens, in an axenic medium containing per liter NaCl, 43; Difco Yeast Extract, Zg; KHZPO4’ 1.6g; K2 was adjusted to pH, 7 and was diluted with one-half volume of filter HPOa, Bg. This medium sterilized horse serum or calf serum. This medium was preconditioned with Proteus morganii; Bacillus subtilis; Clostridium perfringens; or Escherichia coli. They also induced encystation of §;_ invadens, in monoxenic encystation medium, without changing the components of that medium. In this case, they incubated amoebae and bacteria simultaneousely in the medium. Rengpien and Bailey (1975), induced encystation of §;_ invadens, in an axenic encystation medium ( AEM ), without preconditioning with bacteria. This medium contained per liter, Trypticase, 5g; yeast extract, 53; RH PO 0.25g; K HPO 2 4’ 2 4’ 0'553‘ dialysed horse serum, 50 ml. CULTURE MEDIA: Since the discovery of Endamoeba histolytica, Loseh, 1873, and Entamoeba histolytica, Schaudinn, 1903, the causative organism of human amoebiasis, numerous workers have concentrated their efforts to find an artificial growth medium for it. walker and Sellard (1913) concluded that Entamoeba histolytica, could not be cultured in any up to that time, available methods. They stated that free living, non-parasitic amoebae cuold be easily grown in artificial media. This conclusion impressed many protozoologists and physicians to use this feature ( ability to grow in an artificial medium ), as a basis for diagnosis and separation of parasitic versus non-parasitic or free-living amoeba. Cutler (1918) described two media; one of these media was actually Dean and Mouat's (1916) and consisted of suspension of egg in distilled water (1 egg in 300 ml distilled water). This suspension should be heated to the boiling point in the water bath for 30 minutes. Cutler suggested adding to this medium. a few drops of human blood. Cutler's other medium was called " Blood clot medium ". This medium was a suspension of human blood in water ( 500 m1 human blood clot in one liter of water to be boiled for one hour ). To this suspension he added 25 sodium chloride and 21 peptone and a few drops of fresh blood before use. Cutler's animal inoculation experiments and his illustrations of the organism, indicated that he was probably dealing with Entamoeba histolytica. He claimed that the cultured cells were growing good at 28 - 30°C in this medium. This work however has been criticized by many workers including Dobell (1919). The first successful cultivation of Entamoeba could be credited to Barret and Smith (1923 - 1924) who described a medium applicable to in 31552. cultivation of §;_ barroti and ‘§;_ ranarum. Boecck and Drbohlav (1925) described a method for cultivation of §k_ histolytica. Their media were called LES (Locke - Egg - Serum) and LEA (Locke - Egg - Albumin). Modifications of these media have been used until the early 60's. All the media mentined above, were polybacterial, allowing the flora of the intestine present in the feces, to grow along with the amoebae. Phillips and Rees (1950) described a medium for monoxenic culture of §£_ histolytica with Trypanosoma cruzi and with other hemoflagellates. Diamond (1968 a) devised an improved method for monoxenic cultivation of E;_ histolytica and histolytica - like entamoebae, by using trypanosomids. Miller (1951, 1953) and Lamy (1948 a & b) were the first and the second in claiming methods for axenic growth of §;_ invadens. Shaffer §£_al_(1953) reported a method for cultivation of §fi_ histolytica without using living organisms as a component of their culture medium. This method has been successfully used by Rees .EEHE£.(1953)' Since in these media minced chicken embryo or liver cells were used, according to the definition of Daugherty (1953, 1959) , axenity of these media is questionable. Diamond (1960) gives credit for the first axenic medium to Stoll (1957). Stoll's medium contained particulate materials instead of living cells. Mc Connachie (1956) presented a method for elimination of the bacterial flora in EE- invadens cultures. Most of the media described above, ( for example Boeck and Drbohlav, 1924, Dobell and Laidlaw, 1926' Diamond, 1961 ), were called " biphasic " or " diphasic " media. These media consisted of two parts or " phases ": 1 - a solid nutrient slant; 2 - a liquid overlay. Modifications of these media have been reported through the decades. Because of the numerous disadvatages of these media, investigators tried to develop and improve liquid, axenic media, for this organism. 10 For the first time Snyder and Meleny (1943), reported an entirely fluid medium, by infusing coagulated whole egg in 0.08% sodium chloride. Modification of this medium was later reported by Balamuth and Snandza (1944); Balamuth, (1946) and other workers. Diamond (1961), presented an axenic biphasic medium for Entamoeba histolytica. Mc Connachie (1962), was the first to claim an axenic, liqid medium for E;_ invadens. Jackson and Stoll (1964), devised . an axenic liquid medium containg liver infusion broth, mucin, Trypticase and sodium chloride, for E;. histolytica. Diamond (1968 b), reported an axenic liquid medium for cultivation of §L_ histolytica . and E. histolytica - like amoebae, Called TP-S-l. Diamond (1978), modified the TP-S-l medium, by replacing Panmede with Yeast-extract, supplemented with 3 iron, vitamin B 2, thioctic acid and Tween 80. This medium is 1 called TYI-S-33. To date, TP-S-l medium is the best available culture medium. Modification of this medium (Band and Cirrito, 1979), was used in this research work. Excystation of §fi_ invadens occurs by incubating the cysts in the culture medium. In some amoebae like Hartmannella, encystment is not a reproductive stage of the life cycle (Band, 1963), and is just a response to an unsuitable environment. In Entamoeba however, encystation is also a reproductive stage of the life cycle, and each cyst produces eight trophozoites. These trophozoites will encyst in a suitable encystation medium; or grow and devide mitotically-in the culture medium Dobell, (1928), described encystation of E;_ histolytica, and to date this is the best description available. Neal (1966) in a review, 11 summarized Dobell's description. Since the mechanism of encystation, and all stages of the life cycle of E;_ histolytica are identical to that of .EL invadens, here I briefly mention the topics of this event. Actively growing and feeding trophic amoebae become round and less physically active. Their cytoplasm become free of all food inclusions but usually contain glycogen in diffuse form and occationally contain chromatin granules. This stage is called precystic stage. Precystic amoebae are usually smaller than trophozoites. Their nucleus is peculiar and has a more or less concentric karyosome and occasionally chromatin granules. The time between the active precystic stage and the beginning of the encystment is about two hours. Precystic amoebae usually contain variable amounts of glycogen and chromatoid bodies. AS the age of the cysts increases, the amount and the size of these inclusions decreases. In Dobell's terminology the four nucleate amoeba that emerges from the cyst, is called the metacystic amoeba. This name is also given to any form of the amoeba that results from the division of the recently germinated amoeba. The nuclei of the metacystic amoebae are called cystic nuclei or " N ", and the nuclei resulting from the division of the cystic nuclei are called daughter nuclei and are symboled by " n ". METACYSTIC UDEVELOPMENT : Geiman and Ratcliffe, (1936), studied the life cycle of §fi_ invadens in detail, which is identical to that of §L_ histolytica, described by Dobell (1928). After four nucleate amoeba was hatched through a small pore, Dobell,(1928), or through a slit,('ranabe, 12 Swartzwelder, 1939; Hegner ‘gt 31, 1932), it starts feeding and undergoes a very complicated series of cell divisions. All of these stages have been observed and described by Dobell (l928)in detail. The nuclear pattern of the recently germinated (newly hatched) metacystic amoeba with four cystic nuclei is " NNNN " and is octagenic. There are five different ways of development of the metacystic amoeba. The most frequent method of dividing is that one of the " N " nuclei divides to two " n " nuclei. The nuclear pattern of the amoeba in this stage is octagenic " NNNnn ". This amoeba divides to two cells, trigenic " Nn". and pentagenic " NNn Then " Nn " gives rise to nnn ", by the division of the " N " nucleus, and this cell finally divides to two " n " and " nn " cells. The later cell " nn " then divides to two " n‘" cells. One of the " N " nuclei of the pentagenic " NNn " amoeba, divides and gives rise to a " Nnnn " cell. This cell divides to a digenic nn ", and a trigenic " Nn " cell. The " nn " cell divides to two " n " cells.. The " N " nucleus of the " Nn " cell divides to two n nuclei, resulting an amoeba with " nnn " nuclear pattern. This amoeba then divides to three " n " nucleus amoebae. Each of these amoebae with " n " nucleus grows and becomes a trophic amoeba. There are four other ways of metacystic development that have been described by Dobell (1928) and occur less frequent than the above mentioned method, and I will mention them diagramatically. The order number of the methods is in the order of decreasing frequency of their occurence ( figures 10 & 11 ). The main purpose of this research priject was: 1 - To find the 13 essential requirements for encystation of Entamoeba invadens, and to speculate on the trigger(s) of encystation; 2 - to study the effect of some environmental factors on encystation which relates to the first part. Since the answers to these questions are more accurate by using simple and defined encystation media, first I focussed my efforts to find that kind of medium. For this purpose several deletions of the axenic encystation medium, " AEM " (Rengpien and Bailey, 1975), and of the TP-S-l culture medium (Diamond, 1968 b), were planned. With these experiments, I was able to induce encystation of §fi_ invadens, axenically in a medium containing just Trypticase. However the presence of a low concentration of cysteine, ( 0.052 ), improved encystation. It was also found out that other single component media such as Panmede, or horse serum, supported encystation of this organism as well. Since Trypticase is a very complex compound (pancreatic digest of casein), I tried another product of casein which is simpler. This compound ( hydrochloric acid - hydrolysate of casein ), is salt-free, vitamin-free, and tryptophan— free . Encystayion in this medium also was successful. A simpler medium that supported encystation of this organism, was a mixture of amino acids mimicking casein. Effects of other environmental factors on encystation of gfi_ invadens also was studied. The hope is that these findings or modifications of them, could be used as a model, in future studies of human amoebiasis. 14 MATERIALS AND METHODS Axenically grown Entamoeba invadens strain lP-l, were obtained from Dr L.S. Diamond, National Institute of Health. These cells have been cultivated in Dr R.N. Band's laboratory since 1975. The culture medium is the axenic TP—S—l (Diamond, 1968 b), that has been modified (Band and Cirrito, 1979). The difference between the modified medium and the original medium is omission of ascorbic acid and addition of fresh, filter sterilized glucose-cysteine solution. before incubation of amoebae into the medium. This solution (glucose- cysteine), is added to the preautoclaved part (first part) of the medium. This part of the medium " first part ", contains : Trypticase Peptone, pancreatic digest of casein, BBL, 1 g Panmede, liver — extract, Paines & Byrne, 2 g potassium phosphate monobasic, KHZPO4’ 0.06 g potassium phosphate dibasic, KZHPO4’ 0.10 3 NaCl, 0.5 g distilled water, 85 ml After autoclaving, this solution is stored at 5°C. Just before inoculating amoebae into this solution, the following solutions are added: 1 - Glucoseecysteine solution: glucose, 1 8 cysteine 0.2 g distilled water, 10 ml Add 0.5 ml of this filter sterilized solution to 8.5 ml of the firsr part. 2 - Vitamin mixture was made according to Diamond (1968 b). Add 0.25 of this solution to 8.5 ml of the first part. 15 3 - Horse serum (heat inactivated). Add 1 m1 of horse serum to 8.5 ml of the first part. The subcultures were done once a week, and were incubated at 29.50C. Stock cultures were kept at 23°C. ENCYSTATION MEDIA : Encystation media, used, were several different media. All of these media, excluding " AEM " (Rengpien and Bailey, 1975), are being reported for the first time; 1 - Axenic Encystation Medium " AEM " (Rengpien and Bailey,1975). 2 - Trypticase, BBL. 3 - Casein Acid Hydrolysate" CAH "; salt-free, vitaminvfree, tryptophan- free, hydrochloric acid of casein, containing all amino acids of casein except tryptophan, (N B Co, ICN Nutritional Biochemicals). The pH of the media was adjusted to 6.8 by adding 1N sodium hydroxide , and were autoclaved for 30 minutes and stored at 5°C. Stored media were used for up to 30 days. Just before inoculating amoebae in the encystation media, 0.05% fresh, filter—sterilized cysteine was added. 4 - Amino acid mixtures mimicking casein were made up with desired combinations. The concentration of each mixture was equal to it's estimated concentration in 3% casein. 5 - Other compounds were also tried such as Panmede; dialysed horse serum; dialysed Trypticase; etc, which will be discussed later. GASSING : Gassing of the tubes was carried out by two methods; 1- The tubes containing cell suspension in the encystation media, ewre gassed 16 individually with desired amount of C0 and argon. The tubes were 2 capped with tight rubber stoppers. Gassing was repeated every 12 hours. 2 - Gassing was carried out, inside a gloved gas bag and the tubes were capped with loose (not quite tight) screw caps. Gassing was repeated every 12 hours. GENERAL PROCEDURES : In all experiments 16 X 125 mm test tubes with rubber stopper or screw caps were used. The volume of the solution in each tube was 10 ml. Amoebae from the cultures inoculated 72-96 hours earlier, were used for the encystation experiments. Cells were washed three times with the same expermental solution, and then 0.1 - 0.5 ml of washed , compact cells were inoculated into the test tubes containing encystation media. After 24 hours cells in each tube were counted with a haemacytometer. The final concentration of cysteine was usually 0.052. Double, glass distilled water was used in the preparation of all solutions. Amoebae were incubated at 29.50C for growth. For encystation experiments the cells were incubated either at 29.50C, or at 23°C. Viability of the cysts were estimated by observation with a Phase Contrast microscope and also by testing their ability to excyst after acid treatment. For this purpose the cyst- trophozoite suspension was incubated for one hour with 0.1N, HCl (Barker and Svihla, 1964). The HCl kills trophozoites but cysts are resistant to 0.1N HCl. Then cysts were washed three times with the TP-S-l culture medium and were incubated at 29.50C to excyst. DIALYSIS EXPERIMENTS : Dialysis, when necessary, was carroed out either against 17 distilled water or agaist 0.15M, NaCl solution for 24 hours at 4°C. Percentage of growth or encystation, was calculated based on the initial inoculum size ( or concentration ), in each experiment. C, cyst; _C_ I J]. I _11_ I v X 100 = Z C X 100 = Z V Dr G X 100 = Z T , vegetatives; G, growth; T, total (C-+ V), at the end of the experiment. All experiments were carried out under sterile conditions in a transfer chamber. 1975). COMPOSITION OF THE ENCYSTATION MEDIA : 1 - Axenic Encystation Medium " AEM ", Trypticase, yeast extract, potassium phosphate monobasic, KHZPO4, potassium phosphate dibasic, KZHPO4 dialysed horse serum, distilled water, raise the volume to 2 - PPG, Acanthamoeba culture medium : MgClz, 6 H o, 2 NaCl, CaClz, 4, 7 H20, KHZPO4, FeSO All the pipets and test tubes were autoclaved. (Rengpien and Bailey 0.25 g 0.55 g 50 ml 1000 ml 0.003 g 0.120 g 0.003 g 0.003 g 0.136 g 18 NazHP04 0.142 g proteose peptone, 10 g glucose, 18 g distilled water, 1000 ml 3 - PPGF, The composition of PPGF is the same as that of PPG but the concentration of glucose is 10 fold less than that of PPG. 4 - High salt solution, " HS ": MgSO4, 65 mg CaClz, 4 mg NaClz, 1.461 g distilled water, 100 m1 5 - Low salt solution, " LS ": M83049 65 mg CaClz, 4 mg NaCl 0.292 g distilled water, 100 ml 6 - Medium salt solution, " MS ": M3804, 65 g CaCl2 4 mg NaCl 0.88 g distilled water, 100 ml 7 - Amino acid mixtures : Amino acid mixtures resembling casein were made up based on their concentration in 3% casein (Table 2). MEASUREMENT OF OSMOTIC PRESSURE : Osmotic pressure of solutions were determined by " Precision Osmometer The mean of three measurements was considered as osmotic pressure of each sample. 19 R E S U L T S GROWTH OF ENTAMDEBA INVADENS IN TP-S-l CULTURE MEDIUM Trophozoites of §;. invadens were grown in TP-S-l culture medium. The initial inoculum size was 1.5 X 104 amoebae per ml of the medium. For each time interval, six replicas were used. To determine the related growth curve,contents of each tube were counted and then discarded. All tubes were incubated at 29.50C. The generation time or the time required for each doubling of the cells was about 30 hours. In the undisturbed tubes, maximum growth was attained 9 days or 216 hours after cultivation of the amoebae ( Fig 1 ). ENCYSTATION OF §fi_ INVADENS IN DIFFERENT ENCYSTATION MEDIA Encystation of §;_ invadens could be induced in a variety of media, at 29.5°C ( Table 1 ). This list excludes Trypticase and CAH ( casein acid hydrolysate ) and amino acid mixtures, which will be discussed iater in detail. Composition of the listed compounds is given in " Materials and Mehods ". The important points are : 1 - There is a low encystation ( 582 ) in PPGF medium.but no encystation occures in PPG; probably because the concentration of glucose is 10 fold less in the former medium. 3 - Encystation of .3; invadens in diluted culture medium indicates the effect of osmotic pressure and/or a negative effect of FIGURE ( 1 ) Growth of ‘E; invadens in TP-S-l culture medium, versus time. Generation time is about 30 hours. Incubation temperature in this experiment was 29.5°C. Mean of 6 replicas for each point appears in this figure. Variation is shown for each point. 21 20.. 9 11 13 15 17 19 21 as 93 x ”Zamora ‘5 7 3 DAYS Hmon A n v “ msowmnmneo: cm Mb. H= A ... v HchF mxwmxHZMZHm 3m>2 >mz cc n ma « o» u . mm >mz zwntocn nemnmwnm _ so u we u mm >m: + o.o~N nneonoorms ~o~u me a mu >m: + c.cmN nenomwso Npm” ~am « pea >mz tpnrocn women mxnnmon we « Nu ” uu >mz sunrocn «moon mannmnn + c.o~u nneonooros u— a uu « us rot sown mowcnwoa cu m em a aw new: noun mochuoa c u c n c Emanca sown moHanoa c u c u c H~ amass wanwcam o.OuN nwmnmhsm. coupon onrontpmo. soanpoaom. Arnhem dumpemmnm cm N.uu anowwmmm amneson anonuuuoa canon. AA ‘3 1- vv .0 23 glucose on encystation which will be discussed later . ENCYSTATION AND GROWTH OF §;_ INVADENS IN DIFFERENT CONCENTRATIONS OF TRYPTICASE The results of this experiment are given in ( Table 2 ). In this experiment 11 to 52 Trypticase solutions were used as encystation media. Experiments were carried out, in two groups. In one group, 0,05; cysteine was added to the medium; and in the other group no cysteine was used. All cells were incubated at 29.5°C and were cultured 72 to 96 hours before inoculation in the encystation medium. The initial inoculum was 5 X 104 amoebae per ml of the medium. Each observation was repeated 6 times . Cysts and trophozoites of each set of experiments were counted 24 hours after inoculation in the encystation medium. Gassing with CO2 was not done in this series of experiments. This experiment indicates that the presence of cysteine improves encystation, in all concentrations of Trypticase; and also shows that 32 and 42 Trypticase supports better encystation,and growth of §h_ invadens, compared with 11 and 52 Trypticase. Since 42 Trypticase with cysteine supports better encystation than 32 Trypticase ( 122.7 vs 119 ), and also supports better growth ( 128.2 vs 120.2 ), all other experiments in this research work (in the case of using Trypticase) were carried out in 42 solution. Figure ( 2 ) shows the growth of ‘§;. invadens in the different concentrations of Trypticase with and without cysteine shown in Table 2. The data show that cysteine generally supports better growth and the optimum concentration of Trypticase for the growth ofvthis organism is 4%. 24 Hooum A N v. waneanmnnos cm nnwonpnmnm. .ose «worn: on mansammwm uncommon. A: mnmmonosn nosomsnnsnpoae 7‘ .k m 5:5...” 8.23.6 5:. 934. 82:13 3 T. 3 P s I a N. 9.2 was s 32.9 was a 9.: w as n 93.2. in m: H - Z. 8 p; 2.“. flu are m.— N 3; Si. 8; 5L 3 5; 5m 3.» w Sh Sh Eu 5% to web Sch are a __o ~e.~ -u.m ~u.~ _-.e u~.u me.~ we.» m we; a; mo 8 :5 are _-.~ No Aav . .wmc. masseuse emHmo no one some enounmnmm Howevennoo om moowmnmneoa ow zmnH. fi OOZwOZNZHm lemla EMUHGZ m mzoemefiHoz 985.: n a e z a... o. a. < n. N ”we N was a + + + + + + + + o o New 2.... > + + + + l + + + mm N Nmo HA. a + + + n I +. + + 8o 2. So 3.» U + + + l + + + + O O umo No.u m . + + + n + u + + o o :8 Z a. + + + u u + + .. we a N: I. n + n .. u u + n + H3 5 Now a... m + + + .. u .. u + mm a Ba Sr. H + + + u I + u + 2. E u; 3G a + + + n .. n u u o o Sc Ne Asv. H. anwvnunmmmw w. monsoon” n.. «Honommu 0.. owmnmhom. z. zmaww w.. promormno nouns" =.. romeo memes» <. mnmn N9 rouse Zones s 9.2 in me s 92.9 N me n A monumen. anew~Cnma o o Mme o.# amepca v H\~ newcnpo: ku Hb.u Ham Hw.u u\wc newcneos mu 9.0 HHN uN 46 and/or depends on starvation due to dilution of nutrient materials. Dilution of glucose in diluted culture medium could be another trigger of encystation; These subjects however will be discussed later. EFFECT OF OSMOTIC PRESSURE 0N ENCYSTATION The osmotic pressure of Trypticase increases as the concentration increases. Figure (_8 ) shows the change in percent encystation of §£_ invadens in different osmotic pressures. In this figure, the osmotic pressure of different concentrations of Trypticase and the percent of encystation, are plotted. The data from this experiment indicates that the optimum osmotic pressure for encystation of §h_ invadens is about 230 mOsm. This osmotic pressure is the osmotic pressure of 42 Trypticase. EFFECT OF TEMPERATURE 0N ENCYSTATION‘ T - Outside dialysate of dialysed 42 Trypticase.(;against distilled water ), does not support encystation of §;_ invadens. This solution that has small molecules of Trypticase, was boiled directly on the hot plate until the volume of the solution was reduced ( by evaporation ) to the original volume of 42 Trypticase that was dialysed. This procedure increased the osmotic pressure to about 200 mOsm and also restored the nutrient concentration. Then this solution with and without cysteine was used as an encystation medium. The experiment was done at two incubation temperatures, 29.5°C and 23°C. The results shown in Table (,7 ) reveals that : In the presence of cysteine, at 23°C encystation is 75.52 and the increase in cell number is 2132. At 29.5°C the same solution 47 FIGURE ( 8 ) Effect of osmotic pressure of different concentrations of Trypticase, on encystation of §;. invadens. Incubation temperature, 23°C. 48 .. 350 OSMOTIC PRESSURE Z ENCYSTAI‘ION . — _ —. .300 .250 3.0! gang Dunn—baa 0 0 m u C O O . 100 .50 2 TRYPTICASB 14-0 .. 100. ' 20H adamrozu N 49 aways A u v. mmmmnn on nwmnmwso use noevmnmncnm o: osnwmnmnwos 0m.msnmaomam H=mnmn Ne roses >mnmn em rocno Hmavmnmncnm fl nEeno bongo om canoes use nsmwn nosomsnamneosm. 1‘ 1‘4!“ .4 11 ‘11!“ 2+. ‘ 1411114414 “.“‘.J “ >XHzo >0H5m mxpoom own zwmoma . >Hwawsm b.m . momenmnm y uh .m M owcnwaeo ween A_0chmsmnm v ~N.¢ m .m . remesm ~.~ m... m Bit—Ham u.o :Hmnpewsm u m deIlnAuiliAl‘J 55 These experiments showed that addition of aspartic acid to that solution supports about 802 ( t 18.5') encystation and about 1182 (if 7.5 1 increase in cell number. Omission of aspartic acid or substitution of aspartic acid for the other eight amino acids C of second 10 amino acids of casein ), did not support encystation of this organism. EFFECT OF CO 0N ENCYSIAIION 2 Trophozoites of Entamoeba invadens were inoculated in 32 CAH ( casein acid hydrolysate ), plus 0.052 cysteine, as encystation medium, The test tubes were capped with loose (,not quite tight 1 screw-caps. The experiment was carried out in a gloved gas bag, filled with desired amounts of CO2 and argon gasses C 0 to 1002 ). Gassing was repeated every 12 hours. After 24 hours cysts and trophozoites were counted. The result (,Figure 9 ) shows that with.02 C02, encystation is about 602. If 12 - 102 C02 1s introduced in the environment, about 952 encystation results. However it assumes that 302 C02 supports better encystation and growth of §;_ invadens. Applying 402 CO results in 502 inhibition of 2 encystation, and with 502 C02 encystation decreases to about 202. Incubation temperature in this-experiment was 23°C. 56 FIGURE(9) Effect of CO2 on growth and encystation of _E_. invadens. Incubation temperature, 23°C. Six replicas for each point. G. c. 57 150 ... 50... AW. mm C" I]! n'l I'll-Inf". @2233 :52. ..o . 58 FIGURE ( 10 ) The most common way of metacystic development of Entamoeba histolytica, showing a cyst giving rise to eight trophozoites. Digenic " N " nucleus divides and gives rise to two monogenic " n " nuclei. ( Modification of Dobell's, 1928 ). 60 FIGURE ( 11 ) The five ways of metacystic development of Entamoeba histolytica in decreasing order of occurence frequency. ( Modification of Dobell's, 1928 ). 62 FIGURE (,12 ) Speculative mechanism of encystation of Entamoeba invadens , 63 aoa>r excesses. _oo >zome>m. llllllllll II.I.II.I.II.I.II;IL$1.I.II.I-II.I.II.I:II|I:IIII. A poc ewow=ON0mHmm v. so >zcme>m mzaema eewmnaee. as >zcme>n c~<~em. 4“ as we ,Jx’r so nemam so»: ceasefire. . mm\mo. mzneme ~s\mo. enema. ecu am so. was on me. A ....Ssr. So Eons: v. + __a cease. ms ewcwzoNoHHMm. .TIIIIII 64 FIGURE (,13 I ' Speculative mechanism of encystation of Entamoeba invadens. 65 Hoe>h HZOOGfiCZ. woo >zcmw>m. so Biomass ozooso Boonie. so goose Bios Afr H6 ac ”‘ so sass some BEBE. szso. seams also. some. on. on so. sou ow so. A .83: So seesaw o. $ 5 583883... coo oases. lruuun. 66 1315031331911 To determine the trigger(s) or the induction agent(s) of encystation for Entamoeba invadens different encystation media and methods should be considered. Up to the early 70’s it was beleived that bacteria and/or bacterial metabolites were required for encystation. ( Meerovitch, 1962; Mc Connachie, 1969; Ghosh, 1970; Thepsuparungsikul and Bailey, 1971 ). The specificity of " this " requirement was ruled out after induction of encystation of E4_ invadens in in diluted culture medium ( Mc Connachie, 1969, 1970 ); and the description of an axenic encystation medium, " AEM " ( Rengpien and Bailey, 1975 ). This medium contained, Trypticase, yeast extract, phosphate salts and dialysed - horse serum. Because of the ability of this organism to encyst in media lacking starch or glucose, it is now obvious that deplition of starch by the organism ( Me Connachie, 1955; Balamuth, 1962; ghosh, 1970 ), also could not specifically be responsible for encystation. So, what could be the trigger of encystation?. To answer this question I tried to find a defined encystation medium. The media in which I induced encystation of §fi_ invadens, were : 1 - Trypticase 42. 2 - Panmede, 2.52. 3 - Dialysed horse serum. 4 - Vitamine free, salt free, tryptophan free, acid hydrolysate " CAH ". This compound contains 19 amino acids of casein ( out of casein of 20 amino acids ). These amino acids are : alanin, valine, leucine, isoleucine, proline, methionine, phenylalanine, tyrosine, glycine, serine, 67 threonine, cysteine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine. 5 - A more simple medium containing 11 amino acids including : alanine, leucine, isoleucine, phenylalanine, tyrosine, glycine, proline, serine, aspartic acid and cysteine. All of these media supported encystation successfully. Encystation of §;_ invadens begins 12 hours after incubation in the encystation medium ( 42 Trypticase and 0.052 cysteine ),,and reaches a peak after 24 hours. This is similar to it's behavior in "AEM"as reported by Rengpien and Bailey (1975). Induction of encystation in Trypticase indicates that whatever the requirement of encystation is, could be provided by Trypticase. Induction of encystation in the simpler medium, casein acid hydrolysate " CAH ", that presumably contains 19 amino acids, suggests that this requirement could be fulfilled by the presence of amino acids. This idea gained more reliability when I induced encystation in the mixture of 11 amino acids that I mentioned before. Of these amino acids the function of cysteine seems to be stimulation of encystation by reducing the oxidation - reduction potential. Presence of cysteine however improves the final yield of encystation in any media that I have used. Although " CAH-" is a tryptophan - free compound, addition of tryptophan to some‘encystation media( AEM ), improves encystation significantly. This effect may be explained by the presence of large amounts of tryptophan 1n the cysts of §;_ invadens (Morgan,gtugl, 1974). The role of aspartic acid in the encystation seems to be critical. The reason for this conclusion is the fact that deletion of aspartic acid from the mixture of 11 amino acids decreases encystation of §;_ invadens 68 drastically. Also substitution of aspartic acid for the other amino acids of casein does not support encystation, Another amino acid that has-a major role in the encystation of §;_ invadens 18 tyrosine. The reason is that in the case of using dialysed Trypticase as an encystation medium, inside dialysate, does not support encystation; but addition of 0.052 tyrosine results in encystation of g:_ invadens. substitution of tyrosine for the other amino acids does not induce encystation. The data in this research work indicates that " glucose " not only is not required for encystation of §L_ invadens, ( Rengpien and Bailey, 1975 ), but it has an inhibitory effect on encystation. Since the deletion of glucose from the TPesél"culture medium causes encystation, it seems that what inhibits §fi_ invadens from encystation in the culture medium, is " glucose ". Induction of 1152 encystation in 1/2 dilution of culture medium, may reveal that one of the triggers of encystation could be starvation of the amoebae, as it is so in the case of Acanthamoeba ( Band, 1963; Band and Mohrlok, 1969; Griffiths and Hughes, 1968, 1969; Neff 32 3;, 1964; Neff and Neff, 1969 ). Another possible trigger is osmotic pressure. The osmotic pressure of 1/2 dilution of TP-S-l culture medium is about 155 mOsm. The osmotic pressure o'fi undiluted culture medium is about two times this amount. So a hypotonic environment may tigger encystation. A combination of the effects of starvation and a low osmotic pressure also may be the case. In the more diluted culture medium ( 3/10 dilution ), encystation is poor ( 632 ) and no encystation takes place when the culture medium is diluted further ( 1/10 ). Considering that the osmotic pressure of 1/10 dilution of the culture medium is about 69 85 mOsm, it could be concluded that this osmotic pressure is lower than the optimum osmotic pressure for encystation. However according to the results of my experiments, encystation of §;_ invadens occurs within a broad range of osmotic pressure. I have observed induction of encystation in an osmotic pressure as low as 56 mOsm ( 12 Trypticase ) which is a very hypotonic medium; and in a medium with osmotic pressure as high as 300 mOsm ( dialysed horse serum ). The osmotic pressure of 72 Trypticase is about 400 mOsm and encystation at this cocentration is not observed. The optimum osmotic pressure for encystation of _§, invadens is about 230 mOsm. One of the factors resulting in poor encystation in some of the encystation media for example 12Trypticase, might be low osmotic pressure not defficiency of the Trypticase components in the medium. As shown in figure ( 6 ), encystation is proportionally related to the osmotic pressure. As the concentration of Trypticase increases, the osmotic pressure of the medium and consequently the encystation of the amoebae increases. In 12 Trypticase with about 50 mOsm osmotic pressure, encystation is about 402; and in 42 Trypyicase with about 230 mOsm osmotic pressure encystation is more than 1002. When the concentration of Trypticase is 72, the osmotic pressure is about 400 mOsm and no encystation takes place. On the other hand lowering the osmotic pressure in some cases may lead to encystation of §£_ invadens. This idea seems to be true in the case of 1/2 diluted TP-S-l culture medium which induces encystation of this amoeba. The osmotic pressure of this medium is about 155 mOsm. This factor may combine with the other factors such as dilution of nutrient materials~(,particularly glucose ) to trigger encystation. 70 A medium containing 12 Trypticase and 0.052 cysteine.with an osmotic pressure of 120 mOsm results in about 802 encystation. When 12 NaCl is added to this solution, the osmotic pressure would be 400 mOsm and no encystation occurs. Inhibition of encystation in this case could be the presence of " NaCl " as an inorganic salt, or it could be the result of high osmotic pressure or both, EFFECT OF C02 0N ENCYSTATION OF ‘E;. INVADENS : T f fifiw—f fiw j“ *v—r Organisms living in the gut generally require CO2 (-Fridovich, 1977 ). Band and Cirrito ( 1979 ), showed that presence of up to 202 C02, supports better growth of Entamoeba histolytica and that CO2 in high concentrations, inhibits growth of this organism. My experiments show that CO2 has the same effect on the growth of ‘§;_ invadens. The experiments carried out in the " gloved gas-bag ", indicated that up to 302 CO2 supports growth and up to 102 C02 is required for the encystation. Between 102 to 302 C0 better growth is obtained but encystation is 2’ inhibited. Inhibition of encystation in 402 C02 is about 502. Mbre than 302 C02, inhibits also growth of §é_ invadens. EFFECT OF TEMPERATURE : At 29.590 ‘§;' invadens grows faster than at 23°C; but the encystation of this amoeba is better at 23°C. The reason probably is that at lower temperature, metabolism is slower. At 29.500 encystation is poor and most of cells lyse with longer incubation in the encystation medium. 71 EFFECT OF PH : Encystation of IE;’ invadens occurs within a relatively wide range of pH, depending on the medium. The pH in which encystation could occur is as low as 5, in some mixtures of amino acids, and as high as 7, in the case of Trypticase solution. The mixture of 11 amino acids containing : alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptOphan, serine, cysteine and aspartic acid, with the pH of 5, induces about 502 encystation. In a medium with alkaline pH, ( 7.5 and higher ), amoebae aggregate in a flat disc shaped layer and die. In the media with acidic pH ( 4.5 and lower ), cells lyse within a short time ( about two hours ). MECHANISM OF ENCYSTATION 0F ENTAMOEBA INVADENS : What is the prerequisite of encystation ?. What are the intermediate stages between a trophozoite and a cyst. Is growth required for encystation or cell division ?. I try to answer these questions considering my data in this research work. The different stages between a trophic amoeba and a cyst include: young and late trophozoite; precystic amoeba; early cyst;maturing cyst and mature cyst. I have already mentioned the definition of these terms in detail. However young trophozoites including eight amoebae resulting from the final division of the metacystic amoebae, will grow and become larger. In this stage they are called late trophozoites. When the growth of these amoebae is maximum, they will divide and in a right encystation medium they will encyst. According to my data, cell division in most cases is required for encystation. This requirement ( cell division )for encystation even has 72 indirectly been mentioned by Dobell ( 1928 ). When he describes precystic amoeba, he says " ...... while the reduction in bodily diameter and the telophasic characteristics of the nucleus, indicate that the precystic amoeba is the product of a recent division. ". Considering the proportion of the cysts and total number of the cysts and trophozoites in my data, could be concluded that encystation in most media occurs after an increase in the total cell number. So what is required for encystation ?, growth or just cell division. In order to answer this question, this point should be mentioned : in the culture medium with all required nutrients available, growth rate or the time required for the doubling of the cell number, is about 30 hours. However in encystation media with the presence ofa limited nutrients, the doubling of the cells occurs in 24 hours or less. So this presumably is not a cell growth resulting in an increase in the " net weight " of the cells, but is just a cell division without growth. It assumes that cell division in almost all encystation media that I have used, is a pre-encystation phenomenon. I believe there are several patterns of cell differentiation that finally ends to encystation. Depending on the condition of the cells, composition of the encystation media, environmental variables, such as temperature, osmotic pressure, pH, etc; and other conditions of the experiment, the encystation patterns would be different. The probable pattern of encystation of §;_ invadens is as follows : Encystation of the amoeba in 42 Trypticase is shown in Fig ( 7 ). For 100 amoebae inoculated into the encystation medium, the yield after 24 hours is : 115 cysts; 20 trophozoites; 135 total . What happens in this case is that 602 to 702 of the amoebae are the result 73 of a recent cell division which has ocCured in the culture medium. Some of these cells are already in the precystic stage; and the other cells become precystic after a few-hours. After 18 to 20 hours 602 to 702 of the cells encyst. The growth of 302 to 402 of the cells is complete after a few hours and then divide, About 702 of these newly divided cells (_42- 56 cells ) become cysts and the remaining 302 ( 18—24 cells ) do not encyst and stay-as trophonites. The.result would be : 60 cells directly become cysts, 56 cells become cysts after division in the encystation medium, 24 cells remain trophozoites, The end result is : 116 cysts; 24 trophozoites; 140 total Fig (_12 ); or : ‘ ” 70 cells-become directly cysts, 42 cells become cysts after division in the encystation medium, 18 cells remain trophozoites. The end result is: 112 cysts; 18 trophozoites; 130 total,Fig (13). In some cases when an enriched medium is used for encystation, about 2002 encystation will be induced. The reason in this case is that due to the availibility of enough nutrients, cells grow and divide faster. Recently divided cells ( while they are in the culture medium ), divide once and the other cells which are fully grown, divide twice. Most of the cells encyst ( in 24 hours ) and some cells remain trophozoites. In some other cases it seems that the amoebae encyst without going through a cell division. For example in 52 Trypticase and no cysteine, the final yield is : 56.72 cysts and 802 ( of the initial inoculum ), total number of cysts and troohozoites ( Table 2 ). So here 74 no cell division has occured before encystation. This confirms Barker ".. a trophozoite can form a cyst without and Svihla's statement that-says going through a differentiational division. " ( Barker and Svihla, 1964 ). CELL CYCLE AND ENCYSTATION : The sequence between two sucvessive cell division is called the cell cycle. This period according to different lphysiological events occuring in the cell, is usually divided to four stages. The time, during which, mitosis (“M ) or division ( D ) occurs; the time between the end of cell division and the begining of the DNA synthesis, ( G1 ); the DNA synthesis period, (,3 ); and the time or " gap " between ( S ) and ( D ) period which is called (02 ). This cycle and it's sequences may be different in different organisms. ..- Considering the above mentioned hypothesis about the mechanism of differentiation of- §fi_ invadens, could be speculated that 602 to 702 of the cell cycle is spent at " G1 " ( 18 - 24 hours ); and 302-402 of the the cell cycle ( 9 - 12 hours ), is spent at the " G " period. This is 2 quite different with the cell cycle of other amoebae. For example in Amoeba proteus, there is no " G1 " ; about 802 of the 36 hours of the cell cycle is spent during " 62 " period, and 202 of the cell cycle is the time for " S " and " D " period ( Prescott and Stone, 1967 ). ADDENDUM : Following preparation of this thesis, a paper was published ( Das £5 51., 1980 ), which reported encystation in an inorganic medium. I was unable to repeat this observation; no cysts were formed and most amoebae died within a few hours. 75 SUMMARY AND CONCLUSION Encystation of Entamoeba invadens was induced in several different media including: dialysed horse serum; diluted TP—S-l culture medium; Panmede ( 2.52 ); Trypticase ( 12-52 ); acid hydrolysate of casein , which is a salt-free, vitamin-free and tryptophanwfree compound; mixture of 11 amino acids containing; alanine, valine, leucine, isoleucine, proline, methionone, phenylalanine, glycine, serine, cysteine and aspartic acid; and glucose—free TPPS—l culture medium. Encystation of £2; invadens in diluted culture medium could be triggered by starvation due to dilution of nutrient materials and a decrease of the osmotic pressure. Encystation in the glucose-free culture medium indicates that in this case the nutrient-rich medium provides growth; and absense of glucose which is the inhibitor of encystation, triggers encystation of the organism. Encystation in the salt-free, vitamin-free, tryptophan-free, acid hydrolysate of casein, indicates that the requirement of the encystation could be the presence of a few'amino acids. Some amino acids such as aspartic acid could be critical in encystation, while other amino acids are required to provide a suitable osmotic pressure or act as buffers, and their requirement could be nonspecific. However it could be concluded that there are two factors necessary for encystation of Entamoeba: requirement and trigger. When these factors are both present, encystation takes place. The requirements for encystation are nutrient materials ( amino acids ), optimum temperature ( 23o ), CO and a particular stage of the cell cycle. 2 The trigger could be the removal or absence of glucose and decrease of 76 osmotic pressure. However absence of glucose is critical for encystation of §;. invadens, because even if all other requirments are provided, the absence of glucose inhibits encystation. 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