5 m — V“ -% M \. N ‘ ~— “ — a R K a .5 -\ “_ 5—. x “— m % a E 5— K R — _—_~‘ \, 5. a ‘ ‘— — m ‘— \ E .E x \— . 5 ‘— x m a 112 086 THS LLLLL GLQLL @L Li ‘E‘rfL :LQLLL {LESTQLWéEég "rt".'“ “mm w-r. - .: ¢ 1‘ f ' 9 {Lasts for 8.19 D mm c‘.‘ 8/1. 3). “<5 ' :92?- r9::1‘ Tr. tébVLLLb‘C-K{ ST‘IL 0.:- \tkf‘ IL 'L-mer Han L988 [J-ZIRRARY Michigan State Univcrsity PHYSIOLOGY OF ENTAMOEBA HISTOLYTICA BY Im-mer Hsin A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1968 '../"""? 65/7») ACKNOWLEDGMENTS I wish to express my sincere thanks to the members of my guidance committee, Dr. R. Neal Band, Dr. Ralph A. Fax, and Dr. Donald W. Twohy for their guidance and advice throughout this study. Particular appreciation is expressed to Dr. Band, my major professor, for his willingness, patience, enthu- siasm, and valuable assistance in my work and for sharing many of his ideas and much appreciated help whenever help was required. ii ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION CULTURE MEDIUM GROWTH BEHAVIOR PHYSICOCHEMICAL AND TABLE OF CONTENTS MULTIPLICATION RATE FACTORS O O O O O O O 0 MATERIALS AND METHODS . . . . . . . . . RESULTS . DISCUSSION SUMMARY . REFERENCES iii Page ii iv 12 15 19 31 39 41 LIST OF TABLES Table Page 1. Length of time to reach maximum yields by different inoculation size and the rela- tion between the maximum yields and the number of round forms . . . . . . . . . . 20 2. Growth rate of E, histolytica under different conditions . . . . . . . . . . 23 iv LIST OF FIGURES Figure Page 1. Normal growth curve . . . . . . . . . . . . 21 2. K9 strain E. histolytica shows the response to poly-D-lysine (0.3 ug/7.5 ml medium) . 28 3. 200 strain E. histolytica responds to poly-D-lysine (0.1 ug/7.5 ml medium) . . 29 INTRODUCTION On morphological evidence the species of Entamoeba fall into four groups according to the number of nuclei present in the mature cyst: 1 nucleus in E. polecki and E. bovis; 4 nuclei in E. histolytica, E. hartmanni, E. invadens, and E. moshkowskii; 8 nuclei in E. coli, and E. muris; no cyst formation in E. gingivalis. There are sufficient physiological differences, such as degree of pathogenicity, critical temperature for survival and growth, host-restriction, metacystic development, development of the newly hatched amoeba, etc., which could be used to characterize these species (Neal, 1966). Furthermore, each species may consist of a series of races adapted to a particular host. In Entamoeba histolytica, a small race and a large race have been observed. The small race is not pathogenic while the large race may or may not be. The small race was usually called E. hartmanni, (Levine, 1961). In it's virulent phase the large race invades the tissues; the trophozoites of this phase are large; in it's commensal phase it remains in the lumen of the intestine, feeding on bacteria, saprozoically; the trophozoites of this phase are small. Brumpt (1925) further divided the large race into pathogenic E. dysenteriae and non-pathogenic E. dispar. However, the question whether there actually are completely non-pathogenic strains of E. histolytica which cannot be induced to become pathogenic has not yet been answered satisfactorily (Levine, 1961). Concomitant bacteria, nutritional deficiency and other factors effect the pathogenicity of the amoebae. A lot of different strains of E. histolytica were discovered and named. "Laredo" strain has been maintained since 1958 at both 25°C and 37°C. Entner and Most (1965) isolated two more strains, "JA" and "AG," which are capable of growing at 25°C and 37°C. Huff strain has been main- tained in culture (BS-37°C) in several laboratories since about 1956 (Richards, gE_EE., 1965). They differ from E. histolytica K9 and 200 by their low virulence and ability to grow at room temperature. There are many other strains of E. histolytica. Two strains used in this experiment, K9 and 200, were kept by National Institutes of Health lab since 1951 and 1948. These amoebae grow well at 37°C (Richards, et a1., 1965). Since E. histolytica has been cultivated Ea vitro, many investigators studied its nutritional requirement, physical factors, and physiological characteristics. The relation between the associated bacteria and E. histolytica, their virulence and invasive ability, were established at the same time (Nakamura, 1953; 1954; Blumenthal EE_§l°I 1954; Benham and Isabella, 1958; Karlson, 1952; Cabrera, 1958; Phillips and Gorstein, 1966). The present studies were made on some physiological characteristics of two strains of E. histolytica. Special emphasis was placed on their surface requirement, because amoebae always move by attaching to a surface. It would be very interesting to find out whether, and how, growth and multiplication of E. histolytica would be effected if their chance of contacting a surface is disturbed. Other experiments, like change of pH value in the media used, temperature, horse serum requirement, and pinocytosis induction were also tested. CULTURE MEDIUM As early as 1916, Penfold et a1. (Nakamura, 1952) claimed to have cultivated E. histolytica in a medium con- taining nutrient broth and a pancreatic digest preparation. The first successful cultivation of the amoebae without great difficulty, upon special media was demonstrated by Boech and Drbohlav (1925). Their medium became known as the Locke-egg-serum medium (L. E. 8.). After the publication of Boech and Drbohlav appeared, several modifications of their medium became known. In England, Dobell and Laidlow (1926) modified the Boech-Drbohlav medium by adding starch to the culture without dextrose, stating that the starch particles could be ingested by the amoebae, thus supplying an abundant carbohydrate source. In view of the finding that the amoebae grew in the liver of an amebic patient, Cleveland and Collier (1930) used a liver infusion agar slant covered with serum and saline. Balamuth and Sandza (1944) prepared a standardized fluid culture medium from an infusion of coagulated yolk made up in a buffered salt solution. This medium was almost transparent and wholly liquid; when Wilson's liver concentrate powder was added, the growth of the amoebae was accelerated. These were advantages of this liquid medium over a diphasic medium. Since the primary site of invasion for E. histolytica in man is the mucous membrane of the intes- tinal tract, mucous might contain some elements which, in whole or in part, would support the growth of E. histolytica. Dalkart and Halpert (1958) developed a monophasic medium using powdered gastric mucin. Excellent growth of two strains of E. histolytica with bacterial associates was obtained in this medium. All the media described above contained bacteria together with the amoebae. It seemed to become an accepta- ble fact that the amoebae required the bacteria and that if the bacteria were eliminated the amoebae would not survive. Phillips and Rees (1950) first eliminated the bacteria from amoeba cultures and replaced them with Trypanosoma cruzi and found that this haemoflagellate could support growth of E. histolytica in this monophasic culture medium. Trypanosoma lewisi, Leishmania donovani, E. enriettii, E. brasiliensis, and Endotrypanum schaudinni were also found to support the growth of E. histolytica (Pan, 1960). The best condition to study the physiological characters of E. histolytica is to eliminate all the other living organisms in the culture. Many attempts were tried. The first advancement was made on development of monoxenic cultures by Jacobs (1947). He grew the amoebae in a medium in which bacteria were not obviously evident. The medium, the usual Locke's egg slant solution, had been conditioned by the cultivation of E. ggii_for 24 hours, after which bacteria were killed by heat. After heating, heat killed bacteria and rice powder were added. A relatively bacteria- free culture of E. histolytica was maintained in this medium for a period of several months. Some workers worked on antibiotics. Shaffer 22 EE. (1948) cultivated E. histolytica with a 24 hour culture of Streptobacillus sp. and an undefined bacteria, "t organism," in a fluid thioglycolate, glucose, and rice flour medium plus normal horse serum as a support. By adding penicillin, the growth of bacteria was inhibited. After his work, many authors claimed successful cultivation of amoebae under the action of penicillin (Reeves gE_EE., 1960; Karlson, 1952; Faust gE_EE., 1950; and Gleason 22.2lf' 1960). It was anticipated that certain desirable objectives might be more readily attained if it were possible to employ in the amoeba cultures bacterial cells which were incapable of continued vegetative growth and multiplication. An immediate advantage would be that all antibiotics could then be eliminated from the culture fluid; a further gain would be that the metabolic process in such inactivated bacterial cells must surely be less complicated, than in cells capable of employing the whole cycle of normal growth and multiplication. Numerous procedures for killing the vegetative growth capability of bacterial cells were investigated, and at the same time the effects of the treatment upon the ability of the cells to support amoeba growth were studied. The most successful results were obtained with radiation to inactivate the cells (Reeves g£_2l., 1960). This method produced bacterial cells which appeared not to be able to continue vegetative growth or multiplication, but did not lose their capability to support the growth and multiplication of E. histolytica. Shaffer and his colleagues (Shaffer and Siekiewicz, 1952; Shaffer EE.E£°I 1953) had reported that E. histolytica could be cultivated without other living organisms, in medium containing minced chick embryo, and this method had been used successfully by Rees g£_3l. (1953). Considerable effort has been made during recent years toward the goal of axenic cultivation. While there are many reports on the axenic culture of Entamoeba, the first consistent and con- firmed results were Stoll's (1957). The species was E. invadens from snake, and the medium used at room temperature was a monophasic liquid incorporating substances from natural sources. Subsequently, Diamond (1960) reported a clear monophasic medium which supported this amoeba as well as the related species E. terrapinae, which is also a parasite of reptiles. Diamond used a diphasic medium at 37°C for the cultivation of the human parasite E. histolytica, in 1961. Recently Diamond and Bargis (1965) reported a new monophasic transparent medium which was used in the present experiments. In this medium he elimi- nated tryptose and replaced yeast extract with liver extract, which were in the liquid portion of the previous diphasic medium. Other components were also varied in quantity. Jackson and Stoll (1964) tried to grow E. histolytica from diphasic cultures into the original monOphasic medium for E. invadens with the addition of heat in-activated serum agar and NCTC 107, only one or two subcultures could be obtained. Serum was found to be an intrinsic requirement of this human parasite. GROWTH BEHAVIOR AND MULTIPLICATION RATE The growth of E. histolytica follows the classical pattern of microorganisms which is divided into several phases: lag phase, log phase, stationary phase, and death phase. The duration of lag phase depends on various physi- cal conditions, size of inoculum, different types of medium, and different strains of amoebae, etc. Harinasuta and Harinasuta (1955) found that the lag phase in the early stages of amoebic growth ifl.XiE£2.Wa3 considerably short- ened if the medium was preconditioned by allowing growth of the associated bacteria to take place before the amoebae were inoculated. Growth of amoeba populations was found to be more prolific in such preconditioned medium, than in fresh medium. Boech and Drbohlav (1925) reported that the amoebae grew mainly at the bottom of the culture tubes in the bac- terial sediment. Faust EE_El° (1950) grew E. histolytica on Shaffer-Frye medium and found that the population in small Erlenmeyer flasks had almost ten times that in tubes which contained the same amount of medium and received the same size of inoculum. This observation suggested the possibility of investigating various physical factors 10 which might influence the p0pu1ation growth of cultures in Shaffer-Frye medium or other kind of media. They also set-up 24, 16 x 150 mm test tubes containing 5 ml of freshly prepared medium and 0.5 m1 of medium with strain "22" E. histolytica, and put glass tubes of 10 x 30 mm in 12 cultures to increase the vertical surface area. All tubes were sealed with a layer of melted petroleum to maintain the anaerobic condition. These tubes were divided into four groups: 1) tubes with glass and agitated, 2) tubes without glass and agitated, 3) tubes with glass but not agitated, 4) tubes without glass and not agitated. It was found that there was no significant difference in growth between them. They thus postulated that the failure to produce better growth, when the area was increased vertically, may be due to the action of the growth of the E. histolytica on the products in the media or of them- selves, particularly since the amoebae were not observed to migrate through the entire length of the inner vertical wall. Balamuth and Isabella (1958) also reported that in the use of Shaffer-Frye medium, in Koch flasks, the motile amoebae adhered to the glass. Reeves (1962) tested some surface active agents such as quaternary ammonium salts, cetyltrimethyl ammonium bromide, dimethyl ammonium bromide, cetylpyridium chloride, 11 cetyl benxyl dimethyl ammonium chloride and Tween 80 on E. histolytica growth. By increasing the concentration of these surfactants, both multiplication and surface tension diminished gradually until, the multiplication of amoebae ceased finally. The results of his work indicates that amoeba growth might be strongly affected by the pres- ence of surface active agents. However, the increased property of air-liquid surface tension of the cultures did not bear a simple relationship to amoeba growth. When a surfactant was added to the medium the relationship between the air-liquid surface tension and amoeba multiplication could be markedly altered and the result depended upon the type of surfactant. He concluded that it might be that the surface of amoebae contained sites which selectively bound cations, and when the cation was also a surfactant the amoebae were seriously impaired. Microsc0pic observa- tion indicated that all of the surfactants were capable of rupturing the outer membrane of the amoebae and caused them to disintegrate. A suitable concentration was re— quired. Low concentrations did not produce this effect. PHYSICOCHEMICAL FACTORS The various physicochemical factors that enter into the physiology of an organism appear to be especially critical for E. histolytica. 1) pH factor: The variation in pH has marked effect on the ability of E. histolytica to propagate. Shaffer and Sienkiewicz (1952) obtained 6.0 as the optimum pH. A value of 7.9 was observed by Baernstein g£_3l. (1957) as the Optimum value. Rees EE_El° (1960) got maximum average yields at a pH of 6.4 by using another medium. He concluded that the results would seem to suggest the Optimum pH for prOpagation of E. histolytica to be determined by the medium or other conditions rather than by some exact requirement of the organisms. 2) Temperature: Entamoeba coli has a survival time of less than a day at 3°C and 18°C and disappear within 8 to 10 hours at these temperatures. Entamoeba gel; is very sensitive to cold while E. histolytica was less easily killed by lower temperatures. Entamoeba coli is more resistant to higher temperatures (40-45°C) than the histolytica group. At 33°C all the strains of E. histolytica and E. coli under investigation by Cabrera 12 13 and Porter (1958) gave a luxuriant growth, and at 32°C all the histolytica strains with the exception of the F-22 and SB strains under observation had a longer survival time than at 37°C. Several strains of E. histolytica were found to be able to adapt to lower temperatures by periodic lowering of the temperature, always less than 0.5°C each time. These strains could keep growing at 29°C for longer than 18 months. Adaptation of these organisms to high temperatures was also accomplished (Cabrera, 1958). All three strains of E. histolytica (UC, 201,202) grew at a little above 42°C, but when the temperature was raised to 43°C, all adapted strains failed to grow. Siddiqui (1963) studied the effect of temperature on E. histolytica (DSB and Laredo strains), E. moshkovskii (DSR strain), and E. invadens (IP strain) and found the DSB strain was very sensitive to temperatures and DSR strain of E. moshkovskii and Laredo strain of E. histolytica were similar in their range of temperature tolerance. Individual trophozoites from the 30°C-adapted strain grown at 37°C. Amoebae of the IP strain of E. invandens adapted to 35°C were smaller than those of the original strain grown at 30°C. 3) Anaerobiosis: Many workers had concluded that the pressure of free oxygen was definitely harmful to E. histolytica (Balamuth and Howard, 1946; Shaffer EE_El°I 1948). Shaffer et a1. (1948) were not sure whether the 14 oxygen was directly harmful to the amoebae or whether oxidation destroyed some essential oxygen-labile factor needed by the amoebae. A petroleum seal had been used to eliminate atmospheric oxygen. Jacobs (1950) thought that there was not enough evidence to conclude that the complete removal of oxygen was necessary for the growth of E. histolytica. Information currently available indicated that an anaerobic condition was necessary for optimum growth and metabolism, but small amounts of oxygen did not hurt the growth (Jackson and Stoll, 1964). 4) Other factors: E. histolytica can tolerate considerable changes of tonicity (Dobell and Laidlow, 1926), a medium with a salt concentration of 0.94% NaCl was shown to be Optimal. Nelson and Jones (1964) showed prompt and vigorous growth of E. histolytica by adding sodium bicarbonate to the culture medium. Sadun et a1. (1950) found that a single irradiation exposure of the E. histolytica to 30,000 roentgen units did not effect their growth. Exposure to 6,000 and 120,000 r inhibited the amoebae, although viable amoebae were ob- served for 16 transfers after irradiation with 120,000 r. Reeves EE_El° (1960) showed that dosages of X—ray irradia- tion up to 120,000 r's did not greatly harm amoebae, nor did they cause the E. histolytica to lose its infectivity for guinea pigs. The factors that influence amoebic growth seem to be varied with various conditions as described above. MATERIALS AND METHODS The medium (Diamond and Bartis, 1965) consists of a nutrient broth supplemented with horse serum and vitamins. The nutrient broth (TP) has the following composition: Trypticase 1 gm Panmede, liver digest 2 gm Glucose 0.5 gm L-cysteine hydrochloride 0.1 gm Ascorbic acid 0.02 gm Sodium chloride 0.5 gm Potassium phosphate, monobasic 0.06 gm Potassium phosphate, dibasic 0.1 gm Distilled Water 90 m1 To prepare TP broth, the ingredients were dissolved one by one in water in the order presented, the pH adjusted to 7.0 with l N sodium hydroxide, and the final mixture passed through #1 Whatman filter paper in a funnel. It was then autoclaved for 30 minutes, 15 1b., 121°C. The complete medium (TPS-l) was prepared by com- bining under aseptic conditions 90 m1 Of TP borth, 10 ml of inactivated horse serum and 2.5 m1 of vitamin mixture, 15 16 NCTC 107. The vitamin mixture was prepared from five primary stock solutions which were made as follows: 1. Water-soluble B. vitamins: a. 62.5 mg niacin and 125 mg p-aminobenzoic acid were dissolved in boiling, glass distilled water and brought to a total volume of 150 ml. b. 62.5 mg niacinamide, 62.5 mg pyridoxine hydrochloride, 25 mg thiamine hydrochloride, 25 mg calcium pantothenate, 125 mg i-inositol, and 1250 mg choline chlo- ride were dissolved in glass distilled water and brought to a total volume of 150 m1. c. 25 mg riboflavin was added to 75 m1 of glass distilled water and dissolved with the aid of 0.1 N sodium hydroxide added drop by drop. The total volume was then brought to 100 m1. Solutions a, b, and c, were then combined and the total volume brought to 500 ml with glass distilled water. 2. Biotin solution: 30 mg D—biotin was dissolved in 200 m1 glass dis- tilled water with aid of 0.1 N sodium hydroxide and the total volume brought to 300 m1. 3. Folic acid solution: 30 mg folic acid was dissolved in 200 ml glass distilled water with 0.1 N sodium hydroxide and the total volume brought to 300 m1. l7 4. Lipid—soluble vitamins A, D, and K: a. 300 mg vitamin D2, calciferol, was dissolved in 63 m1 of 95 percent (v/v) ethyl alcohol. To this, 300 mg vitamin A, crystalline alcohol was added and dis- solved. b. 60 mg vitamin K, menadione sodium bisulfite, was dissolved in 300 m1 Of 5 percent (v/v) aqueous solu- tion of Tween 80. Solution b was then combined with solution "a" and the total volume brought to 3000 ml with glass dis- tilled water. 5. Vitamin E solution: 25 mg vitamin E, alpha tOCOpherol acetate, was dissolved in 250 m1 glass distilled water. The working mixture of vitamins was prepared by combining the five primary stock solutions in the following prOportions: 500 ml water-soluble B vitamins, 250 m1 biotin solution, 250 ml folic acid solution, 2500 m1 lipid-soluble vitamins A, D, and K, and 250 ml vitamin E solution. Sterilization was accomplished by passing the mixture through a millipore filter (HA, pore size 0.45 u). The medium was distributed in 7.5 m1 portions to 9 m1 screw-capped tubes as stock cultures. The amoebae used in these experiments were obtained from Park Davis and Company. Subcultures were made at 18 intervals of 72 or 96 hours alternatively. Cultures were chilled in the refrigerator for five minutes till cool, inverted several times to loosen the amoebae from the walls of the tube and centrifuged for 10 minutes at 230 X g. After this, the supernatant fluid was aspired from each tube and the remaining sediment of amoebae was transferred to fresh medium. During the experiments, some tubes were silicone- coated immediately before the fresh medium was distributed into them. In the agar test, 0.02% (w/v) of Ionagar #2 (Oxoid) were added before autoclaving. The number of amoebae was determined from an average of 3 culture tubes every 24 hours. After thorough mixing with a sterile pipette a small amount of the chilled fluid medium was withdrawn and placed in the counting chamber of a haemocytometer (A0 Spencer). The amoebae within the 9 large ruled squares of the chamber were counted. Most of the experiments were run more than twice. The results obtained were more or less consistent. RESULTS The growth and multiplication of E. histolytica was similar to that of other microorganisms except that no lag phase was obtained, in the medium used. The length of log phase differed with the size of inoculum. A small inoculum took more time to reach the peak than the larger one did (Table 1). The inoculation size did not determine the maximum yields which were almost consistent with different initial inoculations used, except with a very small inoculum. For instance, with less than 1,000 amoebae per millimeter, it was hard to get the normal log phase of growth. This was true for both strains. The growth rate of the two strains was different, K9 amoebae grew more rapidly than the 200 strain. With the same inoculum, for instance, 17,700 amoebae per ml, the K9 strain took 72 hours to get to the peak while 200 amoebae needed 96 hours in the same medium (Table 1). During active growth, round forms were found. They were round, non-motile, and did not exclude dyes. Round amoebae were most commonly found during and after the stationary phase (Table 1, Fig. 1). The stain taking character indicated that they were dead amoebae. These 19 20 o.m N.o mv m.mH no.5 v.v m.o mv o.ON v¢.m m.H H.o mw 5.mN mm.m N.N H.o mv H.mN 55.m N.N N.o N5 N.5N NN.¢ N.N N.o N5 5.mN mm.m 5.N N.o N5 5.mN mm.N m.o H.o N5 m.5H NN.N oom H.H H.o mm N.mN mm.H m.N N.o mm m.HN 55.H N.N H.o mm 0.5 mm.o m.o H.o mm o.5 mm.o m.H H.o ONH H.mH 55.0 o.w 5.0 wN N.mN 55.m m.m H.o mv m.ma mm.m o.m H.o mv 5.0m mm.¢ v.m m.o mv m.0N mm.¢ o.N H.o mv m.mH fiv.v o.m N.o mv N.mN mm.m H.H H.o mv N.0N ww.m N.N N.o mv m.mN mm.m mm ¢.N H.o mv v.5a NN.m m.N w.o mv 5.mN mm.N o.m N.o N5 5.¢H 55.H N.N m.o mm H.ma mm.o ¢.5 v.0 ONH m.mH fiv.o mm.N H.o ONH ¢.5H mm.o Aaa\voa xv AHE\voa xv Ansonv AHE\¢OH xv AHE\voa xv xmmm EOEmeE mam: um Esawxmfi HOQEOO muflm mcflmuum um maamo mHHmO Ommc no # nomou Op Esaflxmz COAHMHOOOOH pump mo * :mxmu mafia mEHOM @250“ m0 Hwnada may cam mcamflm EOEmeE map smo3umn :oflumamu oz» can Oman soflumasoosfl uGOHOMMHU ma mbamflm EOEmeE sommu Op OEHu mo numcmq .H mqm¢a '21 u. E ,. 8 ’fi\\ I/n O ’9" ' \ IE - Joel" I, /\ dead cells a) 80 / . ‘s a live cells Q ’l / . \\ '——- K9 strain a) 60 .0 ,F \ —— 200 strain 8 / \ ‘ (D 40. '/ \ 0 a \ E *’ ,I \ “3 I ., \\ ,_ \ O \ o ‘. 8 ‘o 0) ‘5 _0_. . _ Fig.1 Normal .growth curve . 4» ‘ ‘ ' fL— E 'r E . I; E ' o 24 4s 72 ,- 96 120 134 T ime ( hours) 22 dead organisms decreased in size in the following days and finally disintegrated. By direct observation, all the amoebae were seen to adhere to the bottom of the test tubes. Amoebae tended to pile up and form small clumps as the number increased near maximum and the available space for their attachment became limited. In order to test whether multiplication of E. histolytica required a surface, 9 groups of tests were set up: 1) control tubes having the normal TP broth medium 2) medium with 0.02% Ionagar added 3) silicone—coated tubes with normal medium 4) silicone-coated tubes with 0.02% Ionagar in the medium These tubes were put in slanted position. The other 4 groups (5-8) had the same constitution as groups 1-4 but were put in a rotator with a speed of 1/5 rpm. 9) Vertical tubes with normal medium The results were shown in Table 2. Growth rate was calculated as: log N-log No Time in which, N0 = initial NO. N = No. of amoebae after a certain time later. 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