“THE RELAJ)“: N BETWEEN THE CRTHCD Q? STF’RILMAWON .5 CCLIMAH’AL CN LN!) GROEE' C“? CHILO MONKS ”IRAMECLH 3‘2’ “‘4‘“ " SOLJ‘VCNS flask or iém Deg?“— af M. S. gimmifififi $333373 aoLLEGB ‘ ”saw“ Li.i2 Wig Bcuwmam .3? 12347 This is to certify that the thesis entitled The Relation Between The Method Of Sterilization, Acclimatization, And Growth Of Chilomonae garamecipg In Swthetic Solutions. presented by Frederick L. Bowman has been accepted towards fulfillment of the requirements for _H.§._degree in_z9°_19fl Major professor Date November 7, 19h] m mirror 3m 1-33 man or STERILIZA‘HOI, AchmnurIoa m GROUT! or cmoaons amount at sumac sum-Ions by Frederick Ludwig Eur-an , Jr. A mars Submitted to the School of Graduate Studies of lichigan State College of Agriculture and Applied Science in partial fulfillment of the requireaenta for the degree or EASTER 0]? source Depart-eat of Zoology 19W THESIS ACUWS Sincere expressions of appreciation are extended to Dr. R. A. Pennell, Professor of Zoology, for invaluable assistance, advice, and encouragenent during the course of the experiaental work and preparation of the manuscript , for the use of facilities in his office, and for’aany'other courtesies. Acknowledgaent is made to Dr. H. 3. Hunt, Head of the Departaent of Zoology, for providing the necessary equipment, reading the aanuscript and for occasional words of encourageaent. I Acknowledgnent is also nade for advice received frca Dr. D. I. Kayne, Assistant Professor of Research. Thanks are expressed to Dr. R. Lewis who read.the manuscript, and to mw'wife, Gloria, and.Hiss Cecilia Dragisity for aid in preparation of the manuscript. Appreciation is extended to Kiss Bernadette McCarthy'(lrs. Dale Henderson) for her many favors and suggestions. 193310 /. I. II. III. V. CONTENTS Introduction............................................ n‘terial. and Methods...".............................. Acclimatizaticn and Growth of Chilomonas paramecium.in m'h ma Conditioned ”Gdi‘eeeeeeeeeeeeeeeeeeeeeeeeeeeee Acclimatization and growth of organisms cultured in Sluce..'pepton9 nediul............................... Acclimatization and growth of organisms cultured in adxtures of glucose-peptone and acetate media........ Influence of Concentration of Acetate on Growth and Acclimatization of Chilomonas paranecium................ Growth and reproduction of Chilomonas.paramecium.cul- * tured in acetate medium»............................. Growth and reproduction of Chilomonas arameciun.cul- tured in a mixture of glucose-peptone and acetate media................................................ Growth of Chilomonas araaecium in Autoclaved and Arnold Sterilised IcetaEe SqutIon............................. marmnt One....................................... Expcrllent “000eeeeeeoeeeeeeeeeeeeeeeeeeeeeeeeeeeeee mermnt Three..................................... Growth of Chilomonas aramecium in Acetate Solutions in Which the Sodium.Acetate was Sterilised Separately...... Growth of Chile-ones paramecium.in Pasteurised and Auto- CICVCd ACCE‘E. '03Itll................................... Starch and Pat in Specimens of Chilomonas parameeium 0‘11th 13 Acet‘t. Solutioni........................... D1.°u.'1on000000000000OOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.... 3my................................................. BibliographyOOOOOOOOOOOOOOOO0.0....OOOOOOOOOOOOOOOOOOOOO Page 1 l3 13 ll 18 18 21 24 27 29 32 34 38 39 OOIiQOm-emesmea-eceaegllooome IUQ‘ODINFOIOIQCQRQICOVI..'I300‘. seeoaelaanem'Iohele-OIQCDIOOOOO ILLUSTRATIONS Figure I.......................................follows page 20 Figure II......................................follows page 23 Figure III.....................................follows page 26 Plate I........................................follows page 33 Plate II.......................................fellows page 33 Pl‘t. IIIOOOOOOO00.00.000.00....00..OOOOOOOOOOOf°11°" p“. 33 I . Introduction last and Pace (1933) found that the colorless flagellate, (milestones parameciun, could synthesise fats and starch in a bacteria-free medium made with sodium ace— tate, anouium chloride, potassium phosphate, and magnesium sulfate. Other workers Lwoff and Dual (1931b) and Hall and Loefer (1936) contended that Hast's ammonium-acetate medium would not maintain growth and reproduction in this species of organisms. Although, both groups . of workers used the same formula for preparation of media the methods of sterilization differed. Blast and Pace's culture solutions were pastuer- ized whereas those used by Hall and Loefer were autoclaved. The objective of the present study is to ascertain whether or not method of sterilization of the media influences growth and reproduction of Chilomonas memecium. II. Materials and Methods Chilomonas paramecium, a colorless cryptomonad flagellate , was employed exclusively as the experimental organism. This species is widely distributed in nature and specimens do not contain chlorophyll or chloroplasts. They are ellipsoidal and vary in length from 30 to 35 microns. The anterior end shows an oblique notch with two anteriorly directed flagella. Organisms grown in organic media show an abundance of fat and starch in the cytoplasm. Specimens of Chilomonas parahecium were freed from bacteria in essentially the same manner used by last and Pennell (1938). The required number of measuring pipettes, micro-pipettes, and Petri dishes containing depression slides were sterilised in an oven maintained at 160 degrees for two hours. A drop of rice infusion culture of chilomenads was put into 0.05 cc. of sterile glucose (0.5%) peptcne (15) solution in a sterile depression slide in a sterile Petri dish with a sterile micro-pipette. The organisms were left in this solution for six hours to .11.. them to become accli- mated to the medium. By means of a micro-pipette about ten were carefully picked out of the depression with a minimum amount of medium and transfered to .05 cc. of glucose-peptcne solution in a sterile depression slide in a sterile Petri dish. Five minutes later the chilomonads were transferred to another .05 cc. of glucose-peptcne solution in a sterile depression slide in another sterile Petri dish. This procedure was repeated twelve times; then one organism was placed in each of three depression slides containing glucose-peptcne solution in sterile Petri dishes: after 36 hourstwo of the depression slides were bacteria-free and chilomonads were abundant. The bacteria-free chilomcnads were transferred to three 125 cc. Erlenmeyer flasks each containing 50 cc. of sterile peptcne medium. After the flagellates became abun- dant additional stock cultures were inoculated by transferring 5 cc. of the culture to other 125 cc. .flashs containing 50 cc. of glucose-peptcne solution. Experimental cultures were grown in the same type of flash or in depression slides in Petri dishes and were incubated at 26° c. lass cultures of chilcmonads were transferred with sterile 5 or 10 cc. pipettes and organisms in isolation cultures in depression slides were transferred with sterile micro-pipettes. ’ Growth rates of chilcmenads were ascertained by the following method. A sample was withdrawn with each in- culum and measured into five 0.01 cc. drops. The organisms in each drop were counted under a binocular dissecting micro- scope, and the five counts were averaged and multiplied by 100. The resulting figure was the number of flagellates per cc. when chilomonads were abundant 0.1 cc. of the culture was withdrawn, diluted 1:10, 1:20, or 1:50 with culture medium and the regular counting procedure followed. Growth rates for the cultures are expressed by the formula G - x/xo: G 8 growth rate 3 x0 :- the number of organisms in the culture inediately after inoculation (number of organisms per co. in inoculum x volume of inoculum) 3 x a the number of organisms at termination of growth period (1 , 2, 3, or 5 days). Uni- form distribution of organisms in the culture was attained by vigorously shaking the flask before withdrawing the cul- ture medium to be transferred. Throughout the procedure great care was taken to prevent bacterial contamination. All chemicals used were analytical reagent grade. Hater redistilled through Pyrex glass was used for preparing all solutions and media. All flasks, and pipettes and de- pression slides were cleaned by leaving them overnight in potassium dichrcmate-sulfuric acid cleaning solution. The dichromate solution was removed from the glassware by rinsing it fifteen times in tapwater and three times in ordinary distilled water. All glass apparatus to be sterilized was put into an oven and maintained at 160° c . for two hours . Hydrogen ion concentration was measured with a quinhydrone electrode. All drawings of cellular fat and starch were made with a 15x ocular, a 90x objective and a Spencer camera lucida. Pat globules in chilcmcnads were stained with Sudan III and starch with Lugol's solution. ’ ' ’ III. Acclimatisaticn and Growth of Chilomonas paramecium in Fresh , and Conditioned Hedia Hall and Loafer (1936) ' contended that Chilomonas w did not grow and reproduce in last ' s monium- acetate medium. They suggested that last and Pace were using a special strain of this species. In order to test this hypoth- esis last and Pace (1936) successfully cultured in acetate medium organisms obtained from Loefer, and also from three other localities in the eastern U. S. A. last and Pace trans- ferred chilomonads to a mixture of glucose-peptcne and acetate - media (505 of each) and left them for 12 hours before putting them into acetate media. They found that acclimatisaticn in mixed media and a sufficient quantity of acetate in the acetate medium were important factors involved in maintaining growth. Hall (19klb) in his review of the literature on growth of flagellates found that co; content of the medium, oxygen tension, concentration of inorganic salts were important factors in growth. Robertson (1922, 1923) , Yocum (1928) , Peterson (1929) , last and Pace (1938), and Hall (1941b) found that protozoa produced allelocatalytic substances which facili- tated growth and reproduction. A medium with allelocatalysts is usually described as being biologically conditioned. The object of the following experiments was to study the acclima- tisation, growth and reproduction of Chilomonas paramecium in conditioned media. The ”fresh" culture medium used in the following experiments was Hast's acetate medium, prepared according to the following formulaxl wa(c2n502) 150 Is- IB‘C]. 50 lg. IZHPOg 20 lg. 38304 ' 12 mg. n20 100 cc. Conditioned acetate media was prepared from cultures in which the number of organisms had been at a maximum but at. the time of experimentation the majority of chilemonads were dead. Such cultures were maintained at 60 degrees for 30 minutes and then centrifuged at a moderate speed for about 15 minutes. The supernatant fluid was removed with a sterile pipette and put into a sterile flask. . All organisms used in the following experiment were originally cultured in glucose-peptcne media. Chilomonads were freed from peptcne by washing them one time in fresh acetate solution. One of the organisms freed from peptcne“ in this manner was transferred to .05 cc. of conditioned ace- tate medium in a sterile depression slide in a sterile Petri dish. The number of organisms in the solution was counted after 72 hours and then one organism was selected to inocu- late another .05 cc. of conditioned media in a sterile de- pression slide in a sterile Petri dish. Transfers were made at 2‘ hour intervals for 3. successive days. Essentially the llast and Pace (1933) Solution n. same procedure was used for testing acclimatisation, growth, and reproduction of chilomonads in mixtures of fresh and con- ditionod media, and in fresh media. The experiment was re- peated and the results obtained are given in Table I as the average growth rates obtained in the two experiments. The figures represent the growth rates of the cultures expressed as x/ko values, i.e. , the ratio of the number of organisms put into the culture to the naber of organisms in the culture after growth (incubation). ‘ It is evident from the results su-arised in Table I that the I division rate of chilomonads was highest in condi- tioned acetate media, lower in the mixture of fresh and con- ditioned media and negligible in fresh acetate media. The data presented indicate that conditioning of the media was an important factor in acclimatisation and survival under the conditions of this experiment . . Table I Acclimatisation and Growth of Chilomonas paramecium.in Fresh.Medium, Conditioned Medium, and Mixtures of Fresh and Conditioned Medium Q Fresh Conditioned § Conditioned Fresh Mediu! Media Media 111:0 x to tho X/xo Hours 72 27.0 #.5 1.5 2‘ 2 o 5 3 .0 0 oo 2‘ ' 1 o 5 0 o 5 2‘ 1 o 5 o 0 5 l. x, number of organisms per cc. at end of experiment; x9, number of organisms per cc. after inoculation. Table I Acclimatisation and Growth of Chilomonas paramecia in Fresh Medium, Conditioned Media, and Mixtures of Fresh and Conditioned Media § Fresh Conditioned 4} Conditioned Fresh Mediu! Media Media T11: . x x0 x/xo x/xo Hours 72 27.0 #.5 1.5 2‘ 2.5 3.0 0.0 2" ‘ 1 o 5 o 0 5 2h 1 .5 0 .5 l. x, number of organisms per cc. at end of experiment; x0, number of organisms per cc. after inoculation. Acclimatisation and my; _o_f_ Chilomonas paramecium M left in. _a mixture _o_f_ glucose-peptcne _a_.gd acetate _f_o_l; _2}. 3193!! preceding sacrimentation. Essentially the same pro- cedure was used in this experiment as is described in the preceding section. The flagellates were obtained from the same stock:cu1ture and the media were from the same lots as used in the preceding experiment. Organisms used for inocula- tion were transferred from.glucose-peptone to a mixture of glucose-peptcne and acetate solutions (50 per cent of each) and left for 2* hours. The chilomonads were then transferred into the experimental media. This experiment also was repeated. The results obtained are presented in Table II in which the figures represent the growth.rates expressed as values obtained from.thc ratio x/xo (x0 : number of organisms introduced into culture at inoculation, x 8 number of organisms in culture after growth). The figures given are the averages of the x/xo values of two experiments. It is evident in Table II that under the conditions of this experiment growth in conditioned acetate medium greatly exceeded growth in ”fresh,” or in a.mixture\of 'fresh' and conditioned.media. The effects of acclimatisation in mixtures of glucose- peptono and acetate media can be ascertained by comparing Table I with Table II. Greater growth rates were obtained if chilo- monads were left for 2* hours in mixtures of glucose-peptcne and acetate media before transfer into the experimental media. The increased growth is particularly evident in conditioned media. 10 A repetition of the Mast and Pace procedure for the acclimatisation of chilomonads to acetate solution demonstrated that it was not practical. Mast and Pace took one organism from glucose-peptcne solution and put it into a mixture of glucose-peptcne and acetate media. In this study it was found necessary to transfer 50 to 100 organisms as the majority of them.died after two hours in the mixed media. Further it was found impossible to transfer chilomonads directly from.glucose- peptcne to acetate as the majority of such died within three hours after being transferred, and none lived after the second transfer in acetate media. Further, these results suggest that its concentration of acetate (75 mg. per 100 co.) in glucose-peptcne acetate mixtures was sufficient to acclimatize.§. paramecium.to undi- luted acetate medium. The results of the experiments with conditioned media concur in general with those presented by Mast and Pace (1938) who found that g. paramecium in acetate cultures pro- duced a heat labile substance which accelerated division rate. If the conditioned.mediumwwas heated at 100° 0. before use, growth rates were comparable to those in ”fresh" medium. Further check for the presence of growth (allele? catalytic) substances in solutions in which chilomonads had lived was made at the end of the experimental period. The unused conditioned medium was heated on a boiling water bath for 20 minutes. Two chilomonads were taken from.poptone cultures and washed in ”fresh" acetate medium. One was trans- ‘ferred to a depression slide containing boiled conditioned 11 medium and the other was put into a depression slide of ”fresh" media. This procedure was repeated five times and in no case was growth greater in the heated conditioned media than in ”fresh” acetate media. Mast and Pace also found that conditioned media prepared from old cultures which had supported large nabers . of organisms had an inhibitory effect on the division rate of chilomonads . They concluded that the growth substance pro- duced by g. paramecium stimulated fission in low concentrations but inhibited it in high concentrations. Similar results were obtained by Kidder (19“) who studied the growth of the ciliate Tctr na 591.31.; in conditioned media. He found that the organism produced tw0 heat labile substances: one accelerated growth and the other inhibited growth. The former could be removed from culture media with a Borkfeld or Seits filter. 12 Table II Growth in Fresh scam, Conditioned, Media and in Mixtures of Fresh and Conditioned Medium of Chile-ones paramecium Time in Hours 72 2t 2t 24 Acclimated in Glucose-peptcne-acetate Medium i Fresh Conditioned f Conditioned Fresh Medipm. Medium. Medium x/xo X/Io 2/2., 100.0 7.5 L5 1 .5 n 0.5 5 ‘ 0 . 5 l. x, number of organisms per cc. at end of expermment; x0, number of organisms per cc. after inoculation. 13 IV. Influence of Concentration of Acetate 0n Growth and Acclimatization of Chilomonas paramecium m _a_n_d reproduction _9_f_ Chilomonas paramecium cultured _i_l_i_ acetate _m_e_d_i_t_l_m_. Mast and Pace (1936) found that the concentration of the acetate used in the media was an ' important factor in the rate of growth of Chilomonas. Four concentrations of acetate were used in the following experi- ments, i.e., 100 mg., 115 mg., 130 mg., and 145 mg. per 100 cc. of media. The other salts, mol, MgSOa, and EgHPO), were used in the same concentration as described in the pre- ceding section. All organisms used were taken from a stock culture in acetate media. Stock solutions of Mast's medium were made with the concentrations of acetate described in the foregoing paragraph. Five one-hundredths cc. of each solution was put into a sterile depression slide in a sterile Petri dish. Then each 0.05 cc. of media was inoculated with two chilomonads which were first washed in media made with 100 mg. of acetate. Examination after 2! hours showed two or more active flagellates in every culture. Twenty-four hours later 20 to 60 active cells were counted in. each. Four days after inoculation no motile flag- ellates were seen in the solution containing 100 mg. of acetate, but in media in which the concentration of acetate was 115 mg. , 130 mg., and 145 mg. , respectively, about 100 cells were found in each .05 cc. of media. After 6 days of growth the 115 mg. and 130 mg. cultures contained more non-motile than active chilomonads , and clumping was evident in the latter. In the 11} culture containing 145 mg. of acetate the majority of the organisms were active. At the end of the seventh day of growth estimations were made of the naber of organisms in each cul— ture. The 115 mg. and 130 mg. solutions had no motile organ- isms. The former contained 100 to 150 and the latter about 200 non-motile cells. Approximately 100 motile and an equal naber of inactive flagellates were seen in the culture con- taining 145 mg. of acetate. The results obtained indicate that Chilomonas Lagg- _m_e_ci__;__wm grew in acetate media containing sodia acetate in concentrations varying from 100 mg. to 135 mg. per 100 cc. Previous observations made in both mass and depression slide cultures showed that chilomonads grew and reproduced in ace- tate media containing 170 mg. of sodia acetate per 100 cc. It is evident from the results that increased concentration of acetate increased longevity of the culture and the maximum population of g. paramecia. Phelps (1936) found that a similar relationship existed between food concentration and growth of Glaucoma miformis. Difco yeast extract when increased from 0.15 to 3} and various concentrations of liquid yeast autolysate ' (2-15 per cent) increased the maximum yield of organisms per cc.‘ of media. M £93 reproduction if g. mamecium cultured in _a. mixture 33: glucose-peptcne 55d acetate 35%. ~ All organisms used in this experiment were transferred with a pipette from glucose-peptcne media into a mixture of glucose-peptcne and acetate media (50 per cent of each) and left for 2# hours. 15 Then one organism was put into .05 cc. of acetate media (100 mg. per 100 cc.) in a sterile depression slide in a sterile Petri dish and left for 5 mdnutes. It was then trans- ferred to another .05 cc. portion in a sterile depression slide in a sterile Petri dish. Portyaeight hours later the chilomonads were counted and one was transferred to another 0.05 cc. of the medium in'a sterile depression slide in a sterile Petri dish. Essentially the same procedure was re- peated at 2# hour intervals until the experiment was closed. The same procedure was also followed for cultures in.medium prepared with 130 mg. and 145 mg. of sodium acetate per 100 cc. The results obtained in this experiment are tabulated as x/xo values in Table III. ' It is evident in Table III that the rate of growth for 2# hour periods is essentially the same in solutions made with 100, 130, and 145 mg. of sodium.acetate. Further the results show that acelimatixation in acetate media was inde- pendent of the salt concentration. It is evident in the preceding subsection that chilomonads which had maintained themselves in acetate medium for fourteen days continued to grow and reproduce when cultured in media in which the concentration of sodium acetate was increased from 100 mg. to 170 mg. per 100 cc. It is also evident that increased concentration of acetate was primarily eoncerned with supporting life and a maximum.population over a greater length of time. Further it is evident in Table III that chilomonads which had been previously cultured in glucose- peptcne, left in a mixture of glucose-peptcne and acetate for 16 24 hours and put into acetate media prepared with the afore- mentioned concentrations of sodium acetate grew and reproduced in all concentrations of acetate that were used. Thus it may be concluded that in depression slide cultures the cultural history of the organisms before experimentation was not an important factor in growth and reproduction of chilomonads . The results given in Table III do not agree with last and Pace who contended that concentration of acetate was an import- ant factor in acclimatization. Hall and Loefer, who maintained that chilomonads would not grow and. reproduce in last's ace- tate media, counted their mass cultures after 7 to 15 days of growth. It is possible that the concentration used by these authors was hardly sufficient to support life . 0n the other hand, last and Pace ascertained growth and reproduction of chilomonads in depression slide cultures which were counted at 2" hour intervals. Under the conditions of their experi- ments it is possible that the concentration was sufficient to maintain the cultures . 17 Table III Acclimatisation and Growth of Chilomonas paramecium In‘Various Concentrations of Sodium.Acetate Concentration of sodium acetate mg. per ce. 23:. 1‘2. :22. :92. #8 10 1» 7 2n 1 1 5 2h 2 2 3 2t ' 3 2 2 18 V. Growth of Chilomonas paramecium in Autoclaved and Arnold Sterilized Synthetic Solution Growth and reproduction of g. paramecia was studied in autoclaved acetate media and Arnold sterilized acetate media. The various media used for experimentation were pre- pared by following the same formula and procedure as is given above in Section III. The autoclaved media was maintained at 15 lbs. steam pressure for l5minutes. Arnold sterilised med- ia was left in the sterilizer for one hour. The experiment was done three times with the procedure varying somewhat in each. Eermnt 9_t_1_e_ . Ten cc. of glucose-peptcne solution in which the chilomonads were abundant were transferred to 100 cc. of autoclaved acetate media. Forty-eight hours later this culture was used for inoculating autoclaved and Arnold steri- lised media. One cc. of culture was added to each of three flasks, each containing 50 cc. of autoclaved media. The num- ber of chilomonads added was found by use of the method described in the section on materials and methods. The cultures were left for five days. at 26 degrees and then the naber of chilo- monads per co. in each of them was determined. The culture with the greatest naber of flagellates was used. to ascertain the growth rate and also for the inoculation of three subcul- tures. This procedure was repeated after another five day growth period and every five days thereafter. The same pro- cedure was employed for the cultures in Arnold sterilised solution. The growth rates of the organisms in autoclaved and Arnold sterilised media are given in Table IV. 19 It is evident from an examination of Table Iv and Figure I that autoclaved acetate solution supported greater growth and reproduction of _q. paramecia than the Arnold sterilized media. The chilomonads did not continue to live in the latter after the third transfer, but growth continued in autoclaved solutions until the cultures were discarded after thirteen transfers. The results of this experiment sug- gest that autoc laved media was more favorable for the growth of chilomonads than Arnold sterilised solution. The results also suggest that _c_. paramecia can .be cultured indefinitely in hast's acetate solution. Figure I shows that the cultures were growing vigorously when the experiment was discontinued after 65 days. [1 20 Table IV - Growth of Chilomonas paramecium.in Autoclaved and Arnold Sterilized Media Arnold Concentration Sterilized Autoclaved of Time Medium Medium Peptone 1n x/x° x/xo f Days 5 4.79 71.t 1 x 10"1 5 0.03 0.1 2 x 10".3 5 76.0 480.0 # x 10".5 5 51.1 3 x 10-6 5 9.8 3 x 10‘5 5 0.5 5 252.0 5' 33A 5 16.1 5 1.7 5 155.0 5 53.0 5 k8 1000 100 Rate of Grout Aufcclaved and A Figure I h of Chile rncld Sferi monas paramecium in l'qed Acefai'e Solutions 1 O Growfh expressed 3.5 log x/xo ”Autoclaved solutions Arnold s+crili3cd solutions 55 60 65 45 50 21 merinont _T_w_o_. Lines of chilomonads were estab- lished in autoclaved and Arnold sterilised acetate media in the following manner: Ten cc. of glucose-peptcne solution in which the organisms were abundant were added to a flask which contained 50 cc. of autoclaved acetate media. The culture was left for 2! hours and then ten cc. were transferred to 50 cc. of autoclaved media in another sterile flask. This pro- cedure was repeated 2‘! hours later at which time the peptcne concentration was estimated to have decreased to 8 x 10"”. The culture of chilomonads was then considered sufficiently free of glucose-peptcne for experimental purposes. The na- ber of chilomonads in the culture was ascertained and then two 5 cc. portions were removed with pipettes and used to establish two lines of organisms in autoclaved media. Essen- tially the same procedure was used for establishing two lines of organisms in Arnold sterilised acetate solution. All sub- sequent transfers were made at #8 hour intervals with five cc. of culture. The results obtained for growth and reproduction in Arnold sterilized and autoclaved acetate media are presented in Table V. ' It is evident in Table v that the growth ratio in the autoclaved media was higher than it was in Arnold steri- lised solution. It is also evident that growth decreased to zero on the fourth transfer in Arnold sterilized media while growth in autoclaved media continued for #8 hours longer. A comparison of the results given here with those presented in Table IV shows that in both experiments the growth rate was higher in autoclaved solution than it was in Arnold sterilised 22 media. The discrepancy between the results presented in Tables IV and Y may be attributed to one or both of the fol- lowing factors: 1) It was shown in the preceding pages that acetate cultures in which chilomonads had lived and attained maxima growth contained materials which facilitated acclima- tisation, growth, and reproduction of chilomonads. Thus it was possible that 24 to 48 hour cultures contained less growth substance than cultures in which the organisms were left for five days . As a consequence growth and reproduction was prob- ably facilitated to a greater degree in the latter. 2) Another factor which may be operative in establishment and maintain- ancc of lines of g. paramecium in acetate solution is muta- bility of the organism. It has been found that when large nabers of organisms were transferred from a conditioned glucose-peptcne media to an unconditioned (fresh) acetate media only a few were left 24 hours later. Some of the sur- viving organisms under suitable conditions were maintained in acetate media while others died after one or two transfers. This suggests that there were inherent ' differences between organisms. Oakberg and Luria (1947) obtained similar results with Staphylococcus m in media in which the concentration of sodia sulfathiaxol varied from 1 x 10"5 n. to 1 x 10'2a. They presented evidence indicating that the organisms which survived and reproduced were mutants. It may be suggested that when chilomonads are transferred from glucose-peptcne to acetate mutants are produced which have varying degrees of adaptibility to growth in acetate media. 23 Table V Rate of Growth of Chilomonas paramecium.in Arnold Sterilixed and Autoclaved Acetate Medium Arnold Concentration Sterilized Autoclaved of Time Medium. Medium. Pcptone 1n the 1/10 5 Hours #8 5.6 0.2 8 x 10"6 #8 3.9 26.3 8 x 10"7 48 6.2 26.8 8 x 10"8 #8 0.0 0.07 8 x 10"9 48 0.0 0.0 30 25 b. u. '3 Growfh expressed as X/Xo 6 Figure I! Growth of Chllomonas paramecium an Aulocloved and Arnold .Srerllljed Soluhons MK g____h_-J_M.._-__ W”. -1l___#___ l 1% Joli //l\ _ /7 ':?”T /7 Ix ‘i / / . l \ / ,QAufoclaved soluh‘ons \ \ _ / r) \ J‘ 3’4 Arnold sferllned soluhons\ i 4!\ J ’ g \ o 2 4 e E :0 Time in days 24 merinont M. Essentially the same procedure was used in this experiment as was used for Experiments 1 and. 2. For further details in regard to methods used for the trans- fer and counting of organisms, and sterilization of media, con- sult those experiments . The growth rates obtained in Experiment 3 are presented in Table VI. . It is evident in Table VI that the chilomonads in Arnold sterilized media showed (a higher growth rate and that the cultures were maintained for a longer period than they were in autoclaved media. A comparison of the results obtained in Experiments 1 and 2 with those obtained in Experiment 3 will show that in the former (Experiments 1 and 2) the growth rate was higher and maintained for a longer period in autoclaved solution than in Arnold sterilised solution. Further a comparison will show that the results obtained in Experiment 3 contradict those ob- tained in the previous experiments for in Experiment 3 the growth rate was' higher and maintained for a longer period in Arnold sterilized solution. The discrepancy betwnn these results would seem to indicate that the differences in the growth rates obtained were not caused. by the method of steri- lization of the media, but that other factors were involved. Two possible factors that were operative were offered in Experiment 2, i.e. , concentration of growth substances and mutability of the organisms. The former can probably be ruled out since the concentration of growth substances was essen- tially the same until the second transfer in Arnold and auto- claved cultures; yet the organisms died out in the latter 25 after the third transfer. Further the results in Table VI substantiate those given in" an earlier section. This would indicate that growth in Arnold media was made possible be- cause the original organism was a mutant. 26 Table'VI Rate of Growth of Chilomonas paramecium.in Arnold Sterilized, Autoclaved Acetate Media Arnold Concentration Sterilixed Autoclaved of Time Medium. Medium Peptone in , x/xo x/xo fl Hours as 6.7 5.6 . 1.6 x 10"5 48 20.0 0.2 1.6 x 10"6 #8 2.5 0.; 0.6 x 10"7 ‘8 9e9 00° 48 3.5 48 0.95 48 0.0 G Fn19”? In row+h Of Chile and Ar- m°n95 P r 20 nold .Sferlli3e; ::::;::°$: ufi‘ch’aved _W_\ ‘Ons x l I I l H; / .' / / l , fl / :5 / l / _ / I O " l x / I ' V) I ' / o / I / ‘0 IO l I w . 3 l g / a I S / f i o L 0 / .5 l1: / // / / / / ‘A + ‘_ u ocloved soluh’ons \\ Arnold 5+erih3ed solufions 0 , l , + O >- 1 I \ . 2 \ 4 6 Q T' - 8 ’0 I2 27 VI. Growth of Chilomonas paramecia in Acetate Solutions in Mhich One Constituent of the Media was Sterilized Separately In this series of experiments the method used for sterilization of acetate medium differed from that given in the preceding sections. Magnesia sulfate, dibasic potassium phosphate and ammonia chloride in distilled water were auto- claved in one flask and sodia acetate in distilled water in another flask. At completion of sterilization the sodia acetate was pipetted into the flask containing the other salts. The concentrations of the salts used were the same as those given in Section III . The procedure for inoculating the experimental cultures, counting and transferring of the organ- isms, closely followed that employed for experiments described in the preceding section. The 'results are given as x/xg ex- pressions of the growth rates. It is evident in Table VII that chilomonads grew and reproduced in acetate solution sterilised as described in the preceding paragraph. Upon comparing the data in Table VII with that presented in the preceding section in Tables IV, ' V, t and VI , it is evident that growth and reproduction obtained in solutions in which the sodia acetate was sterilised sepa- rately were essentially like those obtained when the solution was sterilized with all salts present. Thus it may be con- cluded that there was no reaction, that affected growth of organisms, between sodia acetate , the organic constituent, and the mineral salts during sterilisation. This is additional evidence that method of sterilization has little if any effect on growth and reproduction. Table VII 28 Growth of Chilomonas paramecia in Acetate Media Prepared Time in Hours 48 48 48 48 48 48 48 Experiment naber With the Sodia Acetate Sterilised Separately I/io I/io Ila x/io Ilia 88.2 53.7 1n.n 28.0 33.0 5.3 0.07 2.0 20.0 12.0 2.2 8.2 1.5 2.1 3.6 0.6 1.2 0.0 3.5 2.0 13.2 5.0 53.0 32.0 0.0 0.0 1.# 0.7 0.0 0.0 29 VII. Growth of Chilomonas paramecia in Pasteurized and Autoclaved Acetate Media In this series of experiments the procedure followed for sterilization of the acetate media was the same as that described by Mast, who left the media at a temperature of 85 - degrees for one-half hour on three successive days. Growth and reproduction of chilomonads in acetate media sterilized in this manner were compared with growth and reproduction in autoclaved media. The chilomonads used were obtained from a stock culture in acetate media. All experimental cultures were grown in 0.05 cc. of solution in sterile depression slides. The methods used for transferring and counting the organisms were the same as those used in the experiments de- scribed in Section III. The experiment was repeated three times and the average growth rates are presented in Table VIII . The figures are the x/xo values obtained. 4 It is evident in Table VIII that two of ten lines of chilomonads established themselves in acetate solution. The table also shows that in Experiment 1 the growth rate of chilomonads was slightly greater in autoclaved media than it was in pasteurized media. In Experiment 2 the growth rate was higher in pasteurized media, but the organisms died out after the third transfer. As a consequence the results are difficult to interpret. It is also evident in the table that lines of organisms in Experiments 3, 4, and 5 died out after one transfer. The data presented in this table suggest that the discrepancies between the results of the various experiments 30 were due to differences in the organisms. It is suggested that some chilomonads mutated upon transfer from glucose- peptone to acetate solutions and as a consequence they grew and reproduced in acetate media. Other organisms remained unchanged and died after one or two transfers. It is suggested in view of the results obtained in these experiments that Hall and Loefer did not establish iso- lation cultures of chilomonads as they used relatively few organisms and as a consequence mutations did not occur. Pur- ther it is suggested that they left organisms in mass cultures for too long a period before making subcultures.- 31 .onsoaso on» ouch ooooHooonH one: mauamowno on» handnohm .ooahon noon am a :a can sanuon haaeoapoaoonp ea some nomads ad surname nodasaoud nu ma ow ao>o no osae> oM\M mu .H .sdooa oobedoopso neoonoo .< .sanofl nonahsopasn morocco .m o.m o.m #N O.N 0.: #N o.mm o.m m: o.N 0.0 mm o.mH o.m ma o.H o.NN wt Wo.om o.h am 0.0 0.0 mm 0.0 o.m m: o.H o.H w: m.&H o.N mt o.HH o.HH am .oam .ahm 1m .onm .I. .oam .ohm ox\H ox\w ox\w ox\w 0M\H oJ\H 0J\H ox\H oj\H 0M\K .4 .m .4 .m .4 .e .4 .m .< .a 5.33 2326 m e m m a person roofless have: ououoo< oo>eaoopsd one venunsepaom nu mnaoomonom,nonomoaamo no munono HHH> MAMdB 32 VIII. Starch and Pat in Specimens of Chilomonas paramecium Cultured in Acetate Solutions It was found during the course of these experiments that mass cultures of chilomonads would live in acetate solu- tion through five or six transfers and then die out. The organismm increased in numbers for several days although the concentration of peptcne was too low to be effective (8 x 10’75). The writer was led to believe that death was rather closely correlated with.loss of’cytoplasmic fat and starch. In order to test this hypothesis organisms were removed from acetate cultures which showed decreased numbers of chilomonads. They were stained with Lugol's solution for starch and with Sudan III for fat. Representative organisms were selected and are reproduced in Plates I, II, and III. It is evident in Plate I that chilomonads taken from cultures that supported small numbers of organisms (Figures 1 and 2) showed relatively small amounts of starch while organisms from cultures in which thef number reached 15,750 and 47,000 per cc. (Figures 3 and 4, respectively) showed an abundance of cytoplasmic starch. Plate 11 illustrates the starch content of an organimm taken from a culture that contained 25 chilomonads per cc. (Figure 1) come pared with that of an organism.from a culture that supported 12,500 per cc. (Figure 2). Further it is evident in Plate III that organisms taken from cultures that supported a popu- lation of 1,320 flagellates per cc. (Figures 1 and 2) contained relatively less fat than those taken from cultures in which the population reached 47,000 and 15,000 organisms per ec. (Figures 33 3 and 4, respectively). This would indicate that there is a rather close correlation between division rate and the abili- ty of an organism to synthesize starch and fat, and that death follows loss of cytoplasmic fat and starch. Plate I Specimens of Chilomonas paramecium grown in ac¢+a+e .solu'l'iOn stemmed with Luqol's solufion fa show cyfoplasmic starch. Figure I Figure 2 1.! Figure 3 Figure 4 Plate II Specimens of Chilomonas paramecium qrown in acefa+¢ soluh’on and Siaincd with Luqolls soluh‘on #0 show cyfoplasmic 5+arch. Flgure I Figure 2 IOAL Plate III Specimens of Chilomonas paramecium grown in acetate medium and stamed with Sudan [1] to ‘Show cytoplasmic fat Figure I Figure 2 Figure 3 Figurc4 10.14. 34 IX. Discussion last and Pace (1933), Burrows (1938), Hutchens (l9ilb), Bowen (l9#0), and Swith (1938, 1939, 1940) were able to aaintain the colorless flagellate Chilomonas paraneciun.in bacteria-free cultures in a medium that contained sodiun.acetate as the source of carbon, a-Ieniun.chloride as the nitrogen source, Iagnesiun.sulfate as the source of sulfur and dibasic potassium phosphate as the source of phosphate and as a buffer. last and Pace (1939) reported that magnesiun.was necessary for cell division in g. paraneeiun. The solution was isotonic to the organisn.and the hydrogen ion concentration was pH 7. last and Pace also were able to maintain chilomonads in solutions prepared with all of the aforementioned salts except sodiun acetate if the cultures were kept in a mixture of one part of carbon dioxide and five parts air and if the nediun.contained sodiun.silicate in a ccncentration of 0.0008 I. They contend that the silicate radical was equally effective in low salt concentrations (8 x 10"7 I) and in high salt concentrations (8 x 10"3 I). These authors suggested that this salt acted as a catalyst. Hall and Loefer (1936) stated that they were unable to Iaintaiu child-onads for'lere than two or three transfers in.nedia prepared according to nast's forlula. Further, they were unable to maintain depression slide cultures in a nediun prepared from sodium acetate, nagnesimn sulfate, dibasic potassiul.phosphate, and glycine (hast's solution with glycine substituted for alloniun.chloride), but they aaintained lass cultures of chilomonads in this Iediua through five transfers. 35 nwoff and Dusi (1937) grew from 50 to 90 chilomonads per cc. on a medium that consisted of inorganic salts, sodium.acetate, and asparagine. Upon addition of thiazol to their medium populations of chilomonads were increased to 2,000 per cc. They concluded that thiasol was necessary if chilomonads were to grow and reproduce in synthetic media. It was found during the course of this study that '.Q. paramecium could be maintained in.Hast's acetate medium.if the organisms were acclimated by'a step-bybstep addition of acetate to glucose peptcne solution. Thus it is possible that the discrepancy between the results of hast and Pace and those of Hall and Loefer may be attributed to Loefer's failure to acclimatise the organisms to acetate solution before ascertaining growth rates in sterile acetate media. It was also shown that another important factor in the cul- ture of chilomonads was acetate concentration. Hall in repeating the experiments of last and Pace left mass eultures in test tubes of acetate medium.for 7 days before establishing subcultures. 0n the other hand last and Pace made transfers after 24 or #8 hours; further, they suggested that concen- tration of acetate was a factor in acclimatisation and growth of chilomonas although no experimental data was submitted to support this view. The results.presented in the preceding sections show that both longevity and total growth in cultures of organisms is dependent on acetate concentration, e.g., cultures in media prepared with 100 mg. of sodium.acetate per 100 cc. had no active organisms at the end of n days, while cultures in which the concentration of acetate was 36 150 mg. per cc. chilomonads were abundant after 7 days. 'Another factor in the culture of g. paramecium in acetate medium is concentration of growth substances. It was shown in this work that cultures in which chilomonads had lived contained substances which facilitated acclimati- zation and growth. Further, heating of solutions which con- tained these fission-stimulating substances destroyed them. . Kidder (19M) and last and. Pace (1938) .1... identified these substances. The former maintained that _’1_' . _gg_l._e_i;:l_. produced two substances, one inhibited growth and the other facilitated growth. The latter authors contended that weak concentrations of growth substances accelerated growth while stronger concen- trations inhibited growth. Results obtained during the course of this work illustrate the importance of acclimatisation and of growth substances in establishing chilomonads in acetate ' medium. ' ‘ Another factor seems to be operative which involved the organism itself. It was shown that lines of chilomonads were established in acetate solution in a rather random nanner. ' It was shown that the majority of organisms put into acetate solutions die. These results would indicate that heiratable differences develop in some chilomonads when transferred into acetate media, i.e. , some chilomonads mutate, but the majority do not and soon die. It may be concluded that if the surviv- ing organisms were mutants the autation enabled them to utilise sodium acetate as a carbon supply and mm as a nitrogen sup- ply. The cther organisms were apparently poorly adapted to acetate media and as a consequence such lines died out after 37 'a few transfers. It is possible that the imperfectly adapted lines of organisms continued to live in acetate media until the supply of cytoplasmic fat and starch was exhausted. Another question arises as to the source of energy necessary for the synthesis of protoplasm carbohydrates, and fats in g. paramecium. Little in regard to this aspect of the problem has been done in the course of this investigation, but other authors have published results] of investigations con- cerning this question. last and Pace (1933) presented evidence that the energy requirements of the organism in acetate media were met by the oxidation of monium ions to nitrate ions . They ana- lyzed portions of media in which chilomonads had lived and obtained positive tests for nitrate using the diphenylamine method . With the starch iodide method they did not find any indication of l02. Burrows (1938) concluded that UK; was not oxidised to nitrate or nitrite ions. Hutchens (l9‘lla) found that no significant amounts of ancnium ions were oxidized. Iavanau (1947) contends that the energy required by micro- organisms in soil is obtaincdfrom small changes in temper- ature. At present no satisfactory explanation as to the source of energy for growth and reproduction of g. paramecium is to be had. 38 X. SUMARY 1) Lines (of g. paramecium were established and maintained in Hast's acetate medium. 2) Organisms must be acclimated to acetate medium by a step-by-step addition of acetate solution to peptcne medium. 3) Conditioned acetate medium increased the division and growth rates of chilomonads. 4) As the concentration of sodium acetate was in- creased from 100 mg. to 170 mg. per 100 co. the longevity and total population of the cultures increased. 5) The method of sterilization of acetate medium did not affect growth and reproduction of the organisms. 6) Results suggest that acetate solutions produce mutations in specimens of g. paramecium which enable them to synthesise protoplasm, fat and starch. 39 BIBLIOGRAPHY’ IBowen, Hm. J., 1940, The Effects of‘Vanadium, Copper, langancse, and Iron on the Synthesis of Protoplasm by Chilomonas Paramecium., Biol. 13:111., 79:11t-13o. 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