IWWIIHI llHIHHIWH l ‘. I 129 144 THS we: EFFECT <32: BACTAMA ON m: :mwm 5:: A32“ TATA wommms 2+:: Am: Thai: for the: Emma 05 M. S. JCT’AQAN STATE UK? ‘ :‘ERSITY EMT A. Tacks: T962 ‘ LIBRARY Michigan State University ABSTRACT THE EFFECT OF BACTERIA ON THE GROWTH OF PENTATRICHOMONAS HOMINIS by Paul A. Tucker The effect of bacteria on trichomonad populations both'ig_vivo and LEM has been observed by many investigators, but the nature of the effect has remained largely unexplored. The purpose of this research was to observe whether bacteria affect Pentatrichomonas hominis adversely through the production of toxins or whether they have a beneficial effect as a source of nutrients for the trichomonads. Two strains of E, hominis were used; strain Huf-Z which had been in culture for six years and strain 5M5 which had been recently isolated. In order to observe whether bacteria exerted a toxic effect on the trichomonads, strain Huf-Z was cultured in modified Diamond's medium, a complete trichomonad medium, either with living bacteria or bacterial filtrates of Escherichia coli, Streptococcus jgecalis, Proteus vulgaris, Pseudomonas aeruginosa, Aerobacter aerogenes, Clostridium sporogeneg, and £1, perfrinqens. Except where the bacteria over-grew the cultures in bacteria-trichomonad cultures, trichomonad populations were equal or nearly equal to those of the bacteria-free controls. When bacteria-free filtrates of bacterial cultures were added to trichomonad cultures, growth of trichomonads was equal or higher than that of the controls. There were no observable toxins produced by either the bacteria or their filtrates. Any inhibition noticed could be explained as competi- tion for available nutrients or to overpopulation by the bacteria resulting in unfavorable physical conditions for the trichomonads. In comparing trichomonad growth at different bacterial populations it became evident that a range of bacteria of about l09 per ml gave optimum trichomonad growth and that in future research of this nature some concern should be given the number of bacteria involved. The ability of bacteria to supply trichomonads with various nutri- tional factors was first tested by raising both strains of.£, hominis with EA £5fll_in modified Diamond's medium from which the essential components (trypticase, yeast extract, glucose, and serum) had been singly deleted. fl. hominis, strain 5M5, was able to utilize g. 291; to replace trypticase, yeast extract, glucose, and serum; whereas, strain Huf-Z could only use the bacteria to replace glucose. It seemed that strain Huf-Z through extensive culturing had probably lost its major ability to utilize bacteria. In order to better delineate the nutritional factors the bacteria were supplying toufl. hominis, strain 5M5, a glucose-balanced salt solution enriched with a metals mix was used as the basic medium. This medium did not support the growth of trichomonads. To this deficient medium was added varying combinations of trypticase, alkali-hydrolyzed yeast RNA, vitamin mix, cholesterol, and TEM AT (a source of long chained fatty acids) to test the minimum essential components in the presence of g. _9_9_l__§_. The trichomonads were found to be able to utilize‘g,‘ggll as a source of vitamins, fatty acids, and RNA. Trypticase and serum had a common unknown growth factor or factors which did not permit the simultaneous deletion of both from the medium. ‘E, coli also failed to supply the cholesterol required by the trichomonads. THE EFFECT OF BACTERIA ON THE GROWTH OF PENTATRICHOMONAS HOMINIS By: ,2 Ti" PAUL A2 TUCKER A THESIS Submitted to Michigan State University in partial fulfillment of the requirements ' for the degree of MASTER OF SCIENCE. Department of Microbiology and Public Health 1962 ACKNOHLEDGMENTS The author wishes to take this opportunity to express his sincere thanks to his major advisor, Dr. Donald H. Twohy, under whose teaching the author first became interested in parasitology and under whose guidance and encouragement the author began and completed the present study. The author also wishes to thank Dr. Twohy for supporting this research through his National Institutes of Health research grant. Special thanks goes to the author's wife, Fern T. Tucker, for her help in preparing the manuscript and for her patience and encouragement over the past four years. TABLE OF CONTENTS PAGE INTRODUCT'ON C O O O O O O O O O O O O O O 0 O O O O O O O O O I REV | E" OF THE L 'TERATURE O O O O O O O O O O O O O O O O O O O 2 GENERAL METHODS AND MATERIALS . . . . . . . . . . . . . . . . 9 EXPERIMENTS AND OBSERVATIONS O O O O O O O O O O C O O O O O 0 '2 I. The effect of bacteria on the growth of Pentatri- chomonas hominis, strain Huf-2, in modified Diamond's Medium 0 O O O O O O O O O O O O O O O O '2 II. The effect of bacterial filtrates on the growth of.fi. hominis, strain Huf-Z, in modified Diamond's medium l5 Ill. 2The growth of £5 hominis, strain Huf-Z, in varied population levels of.§. coli in a glucose-salts synthetic medium . . . . . . . . . . . . . . . . . 17 IV. The constituents of modified Diamond's medium essential for the growth of two strains of.fi. hmIinis With E. COIi O O O O O O O O O O O O O O O '9 V. The growth of.g, hominis, strain 5M5, with.§. coli in glucose-salts synthetic medium enriched with supplemental nutrients . . . . . . . . . . . . . . 22 VI. Sustained growth of.fl. hominis, strain 5M5, with.§. coli in a minimum medium . . . . . . . . . . . . . 29 GENERAL DISCUSSION AND CONCLUSIONS. . . . . . . . . . . . . . 33 SUMMRY00000000000000.000oooooooeo 38 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . A0 TABLE IV. VI. LIST OF TABLES is, strain Huf-Z, in modified Growth of g. homin Diamond's medium A comparison of the growth of two strains of.fl. hominis containing l0% bacterial filtrates . with.§. coli in the partial components of modified Diamond's medium The effect of supplemental nutrients on the growth of l_’_. hominis, stra in SMS, with g9 coli in a glucose- salts synthetic medium plus serum . . . . . . . . . . The effect of supplemental nutrients on the growth of g. hominis, stra in EMS, with.§. coli in an enriched glucose-salts synthetic medium . . . . . . . . . . . The substitution of various nutrients for serum in the growth of g, hominis, strain SMS, with.§. coli in an 'enriched glucose-salts synthetic medium . . . . . . . Sustained growth of.fi. hominis, strain SMS, in a minimum medium PAGE l6 2] 26 27 28 32 LIST OF FIGURES FIGURE l. Growth of g. hominis, strain Huf-Z, in Diamond's medium with various bacterial species . . . . . . . 2. Growth of‘g, hominis, strain Huf-Z, In four population levels of E} coli in glucose-salts synthetic medium PAGE lh l8 INTRODUCTION The host-parasite relationship is an interesting biological phenomenon which is receiving the increased attention of parasitolo- -gists and others in allied biological fields, according to Cole (l955) and Stauber (l959). One aspect of this relationship - the interactions of symbionts within the host - was the general basis of this present study. The purpose of this investigation was to determine: (l) whether bacteria plus their secretions can be utilized by trichomonads for food; (2) whether certain bacteria or their secretions inhibit tri- chomonad growth; (3) and whether either of the above effects are influenced by different bacterial population levels. Amongst the many families of intestinal flagellates the choice of an organism for study was directed toward the Trichomonadidae, the family best known with respect to their nutrition and physiology. Hithin the family, Pentatrichomonas (=Trichomonas) hominis was chosen as the best known representative of mammalian intestinal inhabitants and one which could also be easily isolated and grown in axenic culture. This study was undertaken in the hope that more information on the bacteria-trichomonads interaction in the host would lead to a better understanding of the trichomonad-host relationship. REVIEW OF THE LITERATURE Davaine (l860) described and named the first flagellate found in the intestine of man as Cercomonas hominis vars.‘A and Q. For a number of years this name, as well as other names, was used to de- scribe this organism until Kofoid (l920), after reviewing the litera- ture, proposed that Cercomonag hominis var..§ be given the name Trichomonas hominis (Davaine, l860). After a detailed study of the structure of this trichomonad Kirby (l945) suggested that due to its constant five flagellar pattern it should be raised to a new genus denominated as Rentatrichomonas (Mesnll, l9l4). Hith the advent of axenic culturing of trichomonads, many investi- gators turned their attention to the problems of trichomonad nutrition and physiology. Cailleau (l936a, l938) using a peptone-serum bouillon ' medium demonstrated the necessity of adding cholesterol and ascorbic acid for the growth of Trichomonas‘jgg;g§.and Trichomonas columbae. In determining whether cholesterol was a specific requirement, Gailleau (1936b, I937) used 66 different sterols and sterol derivatives as additives to the medium and found that l8 of them supported growth of‘I} columbae. In I939 Cailieau demonstrated that egg albumin, dead Shigella sp., and even unidentified bacterial contaminants could not support‘I. bactrgchorum in the absence of cholesterol. Johnson (l9h7) found that CPLM medium, modified to contain only 0.375% serum and 50% of the normal liver infusion complement, sup- plements of ascorbic acid, glutamic acid, and choline stimulated the growth of mm Mo Sprince and Kupferberg (l947a) devised a complex medium containing serum, trypticase and a series of possible chemically defined growth -2- -3- factors. In trying to simplify their basic medium they found that trypticase (BBL) gave better and more consistent growth with;1. vagigglis than Difco peptone. Trypticase, a pancreatic digest of casein, contained no detectable carbohydrates and appeared to be a purer source of nitrogenous products than most peptones. Sprince and Kupferberg (l9h7b) later separated human blood serum into two fractions, an ether-soluble fraction and an ether-insoluble fraction. They demonstrated that both fractions were necessary for the growth of.l. vaginalis in the above medium minus serum. Linoleic acid and an unknown heat-stable dialyzable factor were the essential nutri- ents in the ether-soluble fraction. Serum albumin was demonstrated to be one of the active nutrients in the ether-insoluble fraction. It is interesting to note that cholesterol was not defined as an essential nutrient of the ether-soluble fraction. Kupferberg, Johnson, and Sprince (l9h8) felt that serum and trypti- case had the effect of masking the importance of some of the added known growth factors in the highly complex medium of Sprince and Kupferberg (l9h7a). It was found that the serum concentration could be reduced from 5% to 0.5% if calcium pantothenate was added to the medium. Trypticase could be reduced from 2% to 0.25% if 9% serum was present. If 0.2#% trypticase, 0.5% serum, and calcium pantothenate were used together no growth was noticed unless a 0.027% phosphate buffer was added. Thus for.I..x§glggll§ they reported calcium pantothenate and phosphate to be essential growth factors in limiting amounts of trypticase and serum and that some unknown factors were common to both serum and trypticase. -4- Fourteen amino acids were found by Weiss and Ball (I947) to be essential for I,‘£Qg§g§ in a serum-free medium. They also found that _I..£Qg£gg responds well to proteins which have been partially digested. As proteins neared the amino acid stage their effectiveness rapidly decreased. This was interpreted by Weiss and Ball as a possible indication that I} foetus utilizes strepogenin or at least unknown peptides as a growth factor. Sprince, Goldberg, Kucker, and Lowy (1953) reported the effects of ribonucleic acid and its nitrogenous constituents on the growth of I. vaginalis in the complex medium of Sprince and Kupferberg (l947a). They found that RNA could partially replace trypticase up to an optimal concentration of 0.l% RNA, but a higher concentration was inhibitory. Acid hydrolysis of RNA destroyed its growth-promoting activity; but alkaline hydrolysis had no affect on the activity. DNA under no condi- tion was capable of promoting growth. A mixture of adenylic, guanylic, and uridylic acid substituted for O.l% RNA supported good growth of the trichomonads. Nucleosides could not be demonstrated to appreciably sustain growth; and in media low in peptides and/or amino acids, ribo- nucleosides were inhibitory. The purine and pyrimidine bases of the above nucleic acids supported as much growth as the ribonucleotides. The authors hypothesized that the essential ribonucleotides could be formed by I, vaginalis from free purine and pyrimidine bases.and the inactivity of the nucleosides might be due to the inability of the organism to phosphorylate nucleosides. Sanders (l957).in the culture of.I..£g§ggg, confirmed the importance of cholesterol (Cailleau, l938) by replacing serum with cholesterol and -5- Tween 80 in a medium where the primary ingredients were small quantities of trypticase, ribonucleic acid, a metal mix, vitamins, and amino acids. Shorb and Lund (1959) in using a complex medium for the cultiva- tion of.1, gallinae demonstrated the presence of both an unknown trypticase and an unknown ribonucleic acid factor. No growth was seen when RNA was replaced with #0 different purines, pyrimidines, nucleo- tides and nucleosides. The active RNA factor was found to be in the contaminating protein of the RNA hydrolized yeast. It was found to be nondialyzable and amino acids alone or in mixtures could not replace this factor. The nondialyzable trypticase factor was analyzed using two-dimensional chromatography and was found to possibly be a large peptide but different from the RNA factor. They replaced serum by using cholesterol and a mixture of unsaturated and saturated fatty acids. Lee and Pierce (1960) working with Hypotrichomonas acosta replaced serum with cholesterol and TEM #T as a source of fatty acids. Alkali- hydrolized yeast RNA was replaced by a RNA-nucleotide mix. The trypticase was reduced as low as 0.29% in a medium whose major nutri- tional components were glucose, a metal mix, a nucleotide mix, cholesterol, TEN AT, a vitamin mix, and a complex amino acid mixture. Trichomonads have been shown to use a wide range of carbohydrates and the variation encountered among species was partially prevalent even among strains of the same species (Read, l957; Lee and Pierce, 1960; Twohy, I959) . As far as nutritional studies have progressed in the last twenty years, there are still many problems yet unsolved and much work remains -6- to be done on the interactions of many of the various nutritional factors. As von Brand (I952) concluded, “Obviously a multitude of possibilities exists in such a complex situation and, in most instances, it is at present impossible to decide definitely whether a certain effect was due to a direct or indirect action of a given nutritional component of the diet.” Pentatrichomonas hominis infects a wide range of animals. The natural hosts are the dog, cat, rat, mouse, golden hampster, man and thirteen other primates. Guinea pigs, ground squirrels, and chickens have served as experimental hosts (Levine, I96I). Hegner and others studied the influence of diet upon populations of trichomonads in the intestine. It was observed that a diet heavy in protein caused trichomonad populations to diminish markedly and that high carbohydrate diets with a minimum of protein produced a fluorishing population of trichomonads (Hegner, I923, I92h, I927, I933; Hegner and Eskridge, I935; Uantland, I95A). Hegner and Eskridge (I937) noted the similar reactions of amoebae in rats to high carbohydrate and protein diets. They attributed the change in trichomonad populations to the altered composition of the bacterial flora resulting from change of the diets. Proteolytic anaerobic bacteria seemed to be associated with the decrease in the trichomonad populations and acidophilic bacteria favored the increase of trichomonads (Ratcliffe, I928; Hegner, I932, I937) . Although trichomonads were isolated and grown.ig‘vlg£g with bacteria in the early thirties, the study of the specific effects of different bacteria on the.ig vitro growth of trichomonads was not really begun -7- until the l9h0's. Johansson,.gt.§1. (l9h7) studied the effect of various bacteria upon the growth of Trichomonas figgtgg, They employed a modification of Schneider's (I9h2) medium consisting of an egg slant and overlay. Serum, glucose, and whole egg were the primary sources of nutrients which supported adequate growth in the absence of bacteria. There was no attempt to control or determine bacterial populations. The bacteria found to be inhibitory were Corxnebgcterium diphtheriae, g. Exogenes, g. xerosis, Staphylococcus §_t_l_l'_e_l._l_5_, m. M, Strepto- mm (five strains), £922.. faecalis (two strains), £13.32- §alixarlu§,‘§5;gg, hemolxticus, Shigelia dysentariae, Shlg, page: dysenterlae, Escherichia‘ggll, and Aerobacger‘ggggggggg. Bacteria that had no appreciable effect were Mm Neisseria catarrhal is, M. pyogenes, 5;. hofmannii, g. _r_e_n_a_l_g, Bacillus subtilis, Psegdomonas aeruginosa, Sarclna sp., and nine unknown cultures of diphtheroids. .Q..§ggl,alone prolonged the survival of the trichomonads beyond that of the bacteria-free controls. Johansson,‘g;.gl. (l9h7) also used bacterial filtrates and heat-killed organisms in‘lgigltgg cultures of trichomonads which produced similar effects on the growth of the trichomonads to those noticed with the living bacteria. He explained the results which ranged from accelerated growth to In- hibition as varied: (l) hydrogen ion concentrations of the culture, (2) production of toxins and (3) recruitment of additional trichomonad nutrients by bacterial enzymatic action on the medium. Hitchcock (l9h8) demonstrated an inhibiting effect of trichomonad secretions upon bacteria. Trichomonas foetus, strains BR, 0, and C, inhibited Salmgnella pullorum. Strain BR Inhibited Corxnebacterium -3- £33.12 and Salmonella schottmuelleri. I. vaginalis had no effect on any of ten species of bacteria tested. Pray (I952) used the medium of Sprince and Kupferberg (I9A7a) consisting of serum, trypticase, maltose, and cysteine, plus vitamin, purine and pyrimidine supplements to study.I. vaginalis. This medium supported excellent growth in the bacteria-free.1. vaginalis controls. Bacterial populations were not controlled or counted. The bacteria which curtailed multiplication of the flagellate and the life of the cultures were Escherichia coli, Aerobacter aero enes, Pseudomopas aerugiposg, Salmonellp schottmuelleri,.§, paratxphi, and Ppoteus mirpbllis. Brucella suis, Streptococcus lgctis, fig, fluorescens, Alcaligepes faecalis, Sarcina Iutea, and Bacillus subtilis caused a moderate inhibition of trichomonad growth. Staphylococcus gp;gp§_and ,§;§ph,‘§lpp§_prolonged the life of the cultures although they did not increase the total number of the trichomonads over that of the control. Bacterial filtrates produced no detectible inhibition on the tri- chomonads. Recently Isolated trichomonads and the older cultural forms were the same In their reactions to the various bacteria. Pray concluded that the limiting factor in the trichomonad growth with bacteria was the low amount of available carbohydrate as a result of the competition with bacteria for the carbohydrate present in the medium. In the presence of both abundant sugar and bacteria tri- chomonad populations may be higher than in the bacteria-free controls. GENERAL MATERIALS AND METHODS Two strains of Pentgtrichomonas hominis were used in this study. .3. hominis, strain Huf-Z,l was used through the early part of this study and.£. hominis, strain 5M5,2 was used in the latter part of the research. The bacteria3 used were Escherichia gpLL, Proteus vul aris, Pseudomonas aeruginosa, Aerobacter aerogenes, Streptococcus faecalis, Clostridium sporogenes, and £1, perfringens. All of the bacteria were without strain designation or known history. Routine cultures of trichomonads were maintained in a modification of Diamond's (I957) medium consisting of 2% trypticase, l%.yeast extract, 0.5% maltose or glucose, 0.I% L-cysteine hydrochloride, 0.I% agar and 5% Inactivated calf serum maintained at about pH 6.6 In 0.0llM dibasic sodium phosphate. This medium produced optimum growth with both strains ofufi. hominis. The minimum medium for the maintenance of.§..ppll was a glucose- salts synthetic medium (Pelczar and Reid, I958) consisting of 0.5% glucose, 0.5% sodium chloride, 0.02% magnesium sulfate, 0.I%. ammonium acid phosphate, 0.I%.dibasic potassium phosphate, 0.I%.agar and brom cresol purple as a pH indicator. This medium would not support the growth of.£. hpmipis. Uhen effects of pH on the trichomonad-bacteria association were investigated, all values were determined with a Beckman zeromatic pH meter. Later when It was found that variations of less than one pH I Isolated by Dr. Louis Diamond from human feces on March 26, I956. 2 Isolated from dog feces on October 4, I960, at Michigan State University. 3 Kindly supplied by Miss Lisa Neu, Department of Microbiology and Public Health, Michigan State University. 9 .]0- unit were not affecting the results, brom cresol purple indicator was used in the medium to follow the changes of pH. Trichomonads to be used for inoculation were cultured for 36-98 hours in a 25 x l50 mm screwcap test tube containing 20 or #0 ml of modified Diamond's medium. The cultures were centrifuged and the supernatent discarded. The organisms were either re-suspended in a basic portion of the experimental medium or were washed with a Kreb's- Ringer's phosphate solution and recentrifuged before adding the experi- mental medium. In all experiments the inoculum of trichomonads was adjusted to give a final concentration In growth tubes of approximately I x l05 organisms per ml. The inoculum of bacteria was 0.l ml of a twenty-four culture per 5 ml of medium. All organisms were cultured at 37°C and the time of culturing varied with the particular experiment. The final volume, Including the inoculum, was 5 ml of experimental medium In each growth tube except as otherwise noted. In most experi- ments four or five growth tubes were used to test each nutritional component in order that experimental variation could be evaluated. The organisms were killed and preserved for counting by using a final concentration of approximately l%.formalin in each tube. The formalin-killed trichomonads were mixed thoroughly, and samples for counting were withdrawn in a Pasteur capillary pipette and plated under a Levy hemacytometer with an improved double Neubauer ruling. The population levels of the bacteria were determined by serial dilutions of a non-formalized culture. Each of the dilutions were inoculated into nutrient agar using a standard pour plate technique. -11- Various concentrations of a penicillin-streptomycin antibiotic solution were used In each experiment in order to hold the bacteria at favorable population levels. Without the antibiotics It was found that the bacteria outgrew the trichomonads in the experimental tubes to the detriment of the trichomonads. Effective concentrations had to vary with different media. Routine Inoculations were made of bacteria-free trichomonad cultures into brain-heart infusion broth In order to test for bacterial contamination. All possible care was taken against contamination with foreign organisms or materials. The F-test was used in all tests of significance and the standard error of the mean is given with each average growth value. EXPERIMENTS AND OBSERVATIONS I. The Effect of Bacteria on the Growth of Pentatrichomonas hominis, Strain Huf-2, in Modified Diamond's Medium. Since It has been reported that certain species of bacteria Influence the growth of trichomonads in complete media (Johansson, gfi_tfl,, I947; and Pray, I952), an attempt was made to test the effects of five enteric bacteria upon the growth of 2, hominis. Materialsqug.Methods. Due to the great number of experimental culture tubes necessary If all five bacteria were tested at one time, Experiment I was divided into two series which were run at different times. In series I Escherichiaugpll,‘Streptococcpg faecalis, and Proteus vulgaris were used as test bacteria, and in series 2 Pseudomonas aeruginosa and Clostridium sporogenes. Sterile modified Diamond's medium was prepared in IOO ml quantities In each of h flasks for series I and in each of 3 flasks for series 2. Each of the test bacteria was inoculated into a separate flask and an equal amount of sterile water was added to one flask of each series to serve as a bacteria-free control. Six hours later for series I and 8 hours later for series 2, a penicillin-streptomycin solution was added to each flask to give a final concentration of 6000 units of each antibiotic per ml. A series of h ml tubes were prepared from each flask. A I.O ml inoculum of trichomonads suspended in fresh Diamond's medium was added to each tube. At given intervals of time, designated by the points in Fig. I, a pH reading was taken and the remaining trichomonads were killed and counted. The value for each point represents the average count of 3 tubes. Results. As seen in Fig. l maximum growth of the trichomonads -12- -13- with four of the bacterial species was attained at about #0 hours. The exception was'§. faecalis where maximum growth occurred in 29 hours. Only the trichomonads grown with.fig. aeruginosa had populations equal to that of the bacteria-free controls, but trichomonads still achieved relatively heavy populations with all other bacteria except S, faecalis. The F-test comparing maximum trichomonad populations showed a signifi- cant difference between the trichomonad population grown with all bacteria except‘fig. aeruginosa and that of the controls at the l% level of probability. The pH of the E. £9115 fl. vulgaris-, £3. aeruginosa-, and £1. spotpgenes-trichomonad cultures was similar or higher than that of the controls over the range of the experiment (Fig. l). The very low growth of trichomonads with StLQp, faecalis probably resulted from bacterial overgrowth which rapidly depleted the nutrients and lowered the pH prematurely for optimum trichomonad growth. Discussionlppg_ConcIusions. The results of culturing various bacteria with.fi, hominis in complete medium has confirmed the effects noticed by Johansson, _e_t__a_l_. (I947) with Trichomonas m and Pray (I952) with Trichomonas gaginalis in which moderate to heavy inhibition of trichomonad growth was observed with most of the test bacteria. Although the magnitude of the effects was different, this is probably due to the differences in trichomonad species, the medium used by the different authors, and the concentration of bacteria present. In this experiment the reduced populations of the trichomonads grown with.§. .ppli,.fi. vulgaris, and.§l. sporogenes most probably resulted from bacterial-trichomonad competition for the available nutrients rather per ml Average number 01" lrlcnomonaas K IU’ 50-— 30- 20'- 44- Q pH o—-0 Control 3%? 1% 4t___,¢ E‘HEQLL 6.] 5.6 H 35.93%. faecalis 5.I 5.2 H L vulgaris 6.l 5.7 22.1»; mg. x—-—x Control 6.] 5.2 H32; aeruginosa 6.l 5.1} +-—-+ EL, sporogenes 6.9 6.0 15 25 35 Ad '56 Hou rs Fig. l. Growth of L hominis, Strain Huf-2, In Diamond's Medium with Various Bacterial Species. -15- than the production of a bacterial toxic factor. II. The Effect of Bacterial Filtrates on the Growth of‘fl. hominis, Strain Huf-Z, in Modified Diamond's Medium. It was felt that filtrates from a high concentration of bacteria would show the presence of any toxic factors by inhibiting the growth of,£, hominis in a medium suitable for the growth of trichomonads. Materials gpg_Methods. The bacteria from which the filtrates were obtained were _E_. ggLL, LL. W, 1:. fllgaris, and Aerobacter aerogenes. The filtrates were obtained from bacteria cultured in 20 or 40 ml lots of modified Diamond's medium for #8 hours. The cultures were adjusted to pH 7.0 with l N NaOH and centrifuged to remove some of the bacteria and other large particles. The super- natant fluid was filtered through a Seitz bacteriological filter to remove the remaining bacteria. The filtrates were added to tubes of modified Diamond's medium in sufficient quantity to make a l0%.con- 'centration by volume before the trichomonads were inoculated into the medium and cultured for no hours. Controls were grown in equal volumes of modified Diamond's medium. Results. The results in Table I indicate that the filtrates of _E_. £9_LI_ and _Cj_. perftingens enhanced growth above that attained by the filtrate-free controls and that none of the filtrates inhibited growth. Di§cu§sion‘gpg,§pnclusions. The use of bacterial filtrates showed no evidence of toxins or toxic materials being secreted by bacteria. Bacteria-free preparations from bacterial cultures either had no effect or slightly stimulated the growth of.fi. hominis. The slight increase in growth from added.fl..ggll,and.§1..perfripgens filtrates (Table I) -15- .>u___nonoLa mo _o>o_ x. 059 um oucau_m_cm_m a ._E can mo. x muocoeo:u_cu *0 Logan: ommca>< a mo; 3. H 3.? a mu. m 8.3 a a .d N SA 8. H 3.? e an. H .36: a g a... N $58 mm. H $.22 : mm. H 3.? a a .m _ «~m.mm mm. A m:._: a me. « mo.~: : __oo .m. _‘ o:_m> a a.u.mflouotu__a guess.) manna o.u.mnmosmtu__a cu_: moans n_tobuam .axm co .oz eo .oz «ounce—.m _a_couuom .3. 9.53:8 233: 3.2233 8:28: E .93: £23... £2.52. .a .6 £55 ._ 39:. -17- could be due to the presence of a bacterial metabolite which enhances growth. Previous observations on the-E. hominis-bacterial association (Fig. I) showed no evidence of the bacteria producing strong toxins or even a marked inhibition of trichomonads. Ill. The Growth of.£. hominis, Strain Huf-2, in Varied Population Levels of,§..gplj in a Glucose-Salts Synthetic Medium. Previous experiments Indicated that the growth of.£. hominis, strain Huf-2, was only moderately affected by most of the bacteria. However, this was in a nutritionally complete medium for.fi. hominis. To test the ability of _E_. 9.91.1 to support the growth of g. hominis, a glucose-salts synthetic medium was used which would by itself support the growth of the bacteria but not the trichomonads. Materials gm Methods. The inoculant culture of _E_. ggLi was grown for 2h hours in the glucose-salts synthetic medium. An inoculum of 0.] ml was Introduced into each tube of A series of h tubes each containing glucose-salts synthetic medium. Tubes in the first series contained one unit; tubes in the second series, 5 units; tubes in the third series, I0 units; and tubes in the fourth series, 20 units per ml of both penicillin and streptomycin. A fifth series of tubes received no antibiotics or bacteria. The bacteria were given two hours to adjust to the medium and then a suspension of.£..hgmipj§ In glucose- salts synthetic medium was inoculated into the growth tubes. The experiment was terminated at 3A hours by taking a pH reading and counting the bacterial and trichomonad populations. Results. As seen from Fig. 2, the antibiotics allowed varying levels of bacteria to develop throughout the experiment. The tri- I. .E:_vaz umuocuc>m mu_mmiomou:_o c_ __ou .m we m_o>oa co_um_:qom Laou :— .Nimaz c_mcum .m_c_eo; .m.mo nuzoLo .N .o_u .E can __oo .m mo cease: m_o_ ~_o_ __o_ o_o_ mo. mo. no. mo. mo. :o. no. «o. .o. o — p p 1_ T N [m Jad 501 x Speuowoqong jo JaqwnN aBEJaAv -19- chomonads appeared to grow best with about I09.§,lpptj per ml. With an E. c_o_i_i_ population of IO8 organisms per ml, the number of tri- chomonads was significantly different from the bacteria-free control at the 5% level of probability and with bacterial levels of IO9 and lO'3/ml they were significant at the l%,level. Discussion‘gpg_Conclusions. The results indicated that an optimum concentration of bacteria did support growth slightly higher than the bacteria-free control. It was difficult to imagine that this minimum growth over the controls, no more than a 2-fold increase in numbers, would sustain the trichomonads over several cultural transfers. It seemed doubtful if the bacteria supplied all the essential nutrients needed by B. hominis. IV. The Constituents of Modified Diamond's Medium Essential for the Growth of Two Strains of g. hominis with t. 2911. Since.fi. hominis showed very poor growth with.§..gpll in a deficient medium, an attempt was made to determine what components of modified Diamond's medium were necessary for growth in a trichomonad- t; cofli association. In order to accomplish this, major components of modified Diamond's medium were eliminated singly from the medium. Mpterlals gpg.Methods. The general procedures used in experiments I, 2, and 3 were similar. Separate series of tubes of media were pre- pared. The media of each series lacked one of the four major components of Diamond's medium (trypticase, yeast extract, glucose, or serum). Within each series A to 5 subseries of A to 5 tubes each were prepared without antibiotics as a bacteria-free control. The inoculum culture of E. coli was grown in glucose-salts synthetic medium for 2A hours. -20- The trichomonads were suspended in Kreb's Ringer's solution which was added in l ml quantities to each tube to give a final concentration of I.O x l05 trichomonads per ml. The time for terminating the three experiments varied. Experi- ments I, 2, and 3 were terminated at 2A, 30, and 31 hours, respectively. Bacterial counts were only made on the first experiment. The pH was taken before the tubes were killed and the trichomonads counted. In each series with different levels of antibiotics, the bacteria- trichomonad subseries with the highest trichomonad population was chosen to compare with bacteria-free controls. Results. The results are given In Table II. Throughout the experiments the pH did not seem to cause any noticeable effect on trichomonad numbers. The difference between the two strains was brought out in this experiment by their different abilities to utilize .§%_gplj in place of the missing medium component. Strain 5M5 produced significantly higher populations with bacteria than without bacteria when trypticase, yeast extract, glucose, or serum was deleted from modified Diamond's medium, while strain Huf-2 showed no higher popula- tion with bacteria when trypticase or serum was deleted. The strain Huf-Z was able to utilize the bacteria for part of its glucose require- ment, but even here strain 5M5 was able to uselg,‘gpli to a much greater extent than strain Huf-Z. Discusgion.ppg_Conclusions. At optimum growth withig..pplt the SMS strain of‘fi, homipis was able to utilize the bacteria to replace trypticase, yeast extract, or serum. 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H mm.~ : o._ o.~ _E\m_o_ m #0. .I... whoo .J O.— I —E\N—O—IO—O— m mo. H .m.: a I o.~ _E\o_o_INo_ m :o. H mm.o : I I _e\:o_ m N N u_e\mo_ x manna Humcuxm ummm> ammu_uaxch _m>04 m_cauumm .axm mvmcoeo£u_ch mo .02 .E:_voz u_uo:uc>m mu_amIowoo:_a nuo:o_ccm ea z. __oo .m.nu_3 .mZm c.0LHm 0.258,. .M Co 5320 2: 8 35:32 3055390 Co 33: 2: .>_ 0.55 -23- ._E\mvmcoeo;o.cu mo_xmm.o :H_z voum_:uoc_ u: .QXm ._E\mvmcoso:u_cu mo_xmm.o sum: cope—300:. “N .axm e .AN00. ..HN.HH...0=00=V N. .02 x.z c.emu.> u .N as 00 . 00~>.0L0>; I__mx_m vcm “N me m.m .Aoom_ .ouco_m ocm 004v om .02 x_: m_oHoz mo co_u_vvm 050 ca.) 0 .mtg 0N 0< a NN. H 0..N 0 0.. 0.0 I 0.N 00.00 0 0 ... H 00.0. 0 I 0.0 0.N 0.N 00.00 0 0 00. H N0.0 0 0.. I 0.N 0.N 00.00 0 0 00. H 00.0 0 I I 0.N 0.N 00.00 0 0 0.. H 00.0 0 0.. 0.0 0.N 0.N 00.0: 0 0 N0. H N0.0 0 0.. 0.0 0.N 0.N 00.00 0 N 00. H 0N.. 0 0.. 0.0 0.N I 00.00 0 N N 0... N 0... 00.:5 N 0.e\00. x 0000. H 20H .otoumm.o;0 ox.z .0.> 0000.00>LH .e\.00L00I.000 .axm mvmcoeocu_ch mo .02 mace—EH32 .oucaea_ 3m .Ea_cox u.Hoch>m 0H_mmIomoo:_o vocu.ccm ea c. __ou .m.:u_3 .mZm c_acum .m_c_wmc .M.mo azuzoco ezu c_ Escom com mucm_cuaz m:o_cm> mo eo.u:H_Hnn:m web .> mHmch NonN new "cm .02 x_z m_mHoz N we m.m .E:.ooz u_uonuc>m 0H_mmIomou:_u mo once a mu: E:_voe E:E_c.z a mm.o +m I 0.0 0:009... N o 0 cm I Neomocm N o o I I «apnoea N o o I I . Heomocm _ 0 0 00 0.0 0:000< N 0N. _ +m om m.o H.800...— N Nm._ . +5 om m.o Hammocm _ N.me N.mE Hcoe_caqxu mo co_uoc.ELoH «Laemcoch .awmmoouam _ocoumo_o;u __ou .m. .axu 00 .s\ 0. x muocoso;o.th .0 .oz .0>;I..ax_< m.E:_ooz E:E_c_z a 2. .mZm c_mcum _m_m_eo: mm.mo cuzocu coc_oum:m ._> m4m_l_i_ as a source of RNA. LITERAIURE CITED. Cailleau, R. I936a Le cholesterol, facteur de croissance pour Ie Flagelle' Trich mnas columbae. Compt. Rend. Soc. Biol., l2l, A2A-A25. . 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