”Oh—d“... 4'_ A STUDY ON THE iNHiBiTION OF BACTEREA BY ‘E'HE METABOLIC PRODUCTS OF TRECHOMONADS hesis for the Deg-rec of M. S. MECHIGAN STATE. COLLEGE Dcrothy jean Hitchcock: 3947 THESIS" . <11 1. 5-K! |.| This is to certify that the thesis entitled The Study of the Inhibition of Bacteria by the Metabolic Products of Trichomonads presented b1] Dorothy Hitchcock has been accepted towards fulfillment of the requirements for “Laws; .-degree ingar 351:1},01933’ am professor DateJdaLlS+ 4247: ,_ _- M-796 A 8101!! 03 TH! INHIBITION 01' BACTERIA B! m METABOLIC PROHJOTS 01' moaomnm By BORDER! Jill HITOHGOC 7' A TBSIB Submitted to the School of Graduate Studiee of maxim State college of Agriculture and. Applied. Science in pert-.131 fulfillment; of the requirements for the degree of WC? some: Department of Bacterioloa and Public Heelth 19"? THEE-M5 I. II. III. 7. VI. 71 1. VII 1. 3910 of context: Intro auction mil-tuxe- Nethede meme eten Results conclusion Acknowledges“ Referencee oi ted. 189106 I. Introduction Research has been extensively carried out to determine the inhibi- tion of bacteria by the metabolic products of bacteria and acids. A survey of the literature revealed the production of an antibiotic by Paramecium anrelia (Someborn. Jacobson and Dippell, 19%; Austin. 19%). be mtabolic and disintegration products of certain strains of g. aurelia when added to liquid cultures of other species of Parueciun produced toxic or lethal effects. the action of known antibiotics has been studied on protoeoa. In this study the Oxford cup plate assay nethod was used to determine the inhibition of bacteria by the metabolic prod- ucts of trioholonads. II. ailmres The pmtosoa studied consisted of bacteria-free cultures of m- mg m and Echo-ones M. the g. 3.9.9.912. cultures. Strains BR, 0 and 0. were obtained fron Dr. B. B. lbrgan. University of Viscou- sin. Madison. Visconsin. he 3. We culture. #1. was provided by m. G. Johnson, Ortho Research loundation. Baritan. New Jersey. Schneider's citrate nediun (Schneider, 1912) was used for cultur- ing and assaying the three strains of g. _f_o_e_t_u_s_. he nediun was sodi- fied by omitting the bron cresol purple. lach tube of nediun. consist- ing of 5 ill. of egg-blood nixture slanted. and 12 al. of overlay con- taining 6.5% bovine serun and egg shells. was mtoclaved at 121%. 15} for 30 min. the uninoculated nediun was stored in the refrigerator and incubated 3 m. at 3750 for sterility. ‘ the g. 3.9.23.2! cultures were subcultured once a week by transferring approxinately 1 ll. of overlay with a sterile opsonic pipette to a fresh tube of Schneider's citrate nediun. The subcultures were incubated the first two days at 37°0. and the remainder of the tine until assayed at 22%;. The 3. M culture. {1. was maintained and assayed in cysteine- peptone-liver-naltose nediun (0. P. L. K.) (Johnson and Erussel. 1938) nodified by omitting the methylene blue and agr. fhe base nediun was tubed in 9 n1. anounts and autoclaved at 121%. 15} for 30 sin. After the addition of 1 ml. of sterile horse serum to each tube. the nediun was incubated it days at 37‘0 and examined grossly for contanination. The 3. 2293.11. cultures were subcultured every 2 to 3 days by trans- ferring approxi-tely 0.5 ll. of the culture with a sterile opsonic pipette to a fresh tube of 6. P. L. I. aedium. ihe subcultures were incubated at 37‘0 until assayed. The bacterial cultures were kept in stock on slants of the base as- say agar (Schmidt and Meyer. 19%) and subcultured once a month. the seed inoculun bacterial cultures were submluired daily froa broth to broth (Schmidt and Itoyer. 191th) and incubated at 37'0. Ivory two weeks the broth bacterial cultures were started.fron.stock cultures. the bac- teria chosen for this study; tables 1 and.13. were ltaited.to those that would grow satisfactorily on the base agar. give even turbidity in the broth and.were not too prolific growers in the 22-hr. assay incubation period. III. lethods rho Oxford cup plate assay method used for 3. £229.! is described. followed by the nodifications used for _!_. m. 'i'he Petri plates of 20 al. of base agar (Schmidt and byer. 19%) were poured and left at rooa temperature 16-18 hours before seeding. The bacterial inoculum. consisting of 3 ml. of base agar seeded with a 1% 2h- hr. broth culture (Schmidt and layer. 19th) of a bacterium. was evenly distributed over the 20 ml. of base agar. When the seed inoculun was firm. five sterile porcelain Oxford cups. warned on a hot plate. were evenly arranged on each assay plate. he 3. £252; cultures to be tested for bacterial inhibition were enmined microscopically for nfficient growth of the trichononads, tested for bacterial sterility in broth. pH determined with nitrasine pape‘.‘ and centrifuged at 1150 r.p.n. for 10 minutes. he supernatant fluid was decanted. thus removing the tricho- monads from the protosoan culture naterial to be assayed. lbur drops of the supernatant of the centrifuged protosoal culture eaterial were placed in three of the cups by means of a sterile 1 ml. pipette. lbur drops of uninoculated control nediua were added to the control cup. to control was treated in the sane manner as the protozoal‘ cultures. except the pH was adhsted to 5.0-5.5 with 801. Elie pH adjustment was necessary as the 2. £229.! after incubation lowers the pH froa 7.2-7.lt to 5.0-5. 5. me cup, to which no fluid was added, served as a heat control. he as- say plates were covered with sterile porcelain lids and incubated 22 hrs. at 37'0. the sense of inhibition were observed by reading over a substage microscope lamp and measured in millineters with a ruler. One of the cones of inhibition from each plate was touched on the surface with a sterile wire and seeded to broth to determine if the inhibitory substance was bacteridcidal or bacteriostatic. lach assay on each tube of protozoal culture apinst one bacterium consisted of three plates with five Oxford cups per plate; three of these for the centrifuged protozoa]. culture material. one for the uninoculated, adjusted medium control and one for a control on the heat of the cup. A complete assay consisted of three protosoal cultures of the same strain and same age against three bacteria. requiring a total of 27 plates. it least two complete assays were done with 7. 10 and 12 day 3. £23.39... cul- tures. Strains 33. 0 and 0. against the bacteria listed in table 1. mo above procedure was modified with g. m by allowing the base agar plates to stand 12-114 hrs. at room temperature before seeding. centrifuging at 1150 r.p.m. for 15 min. ad assaying 2 to 3 day cultures aainst the bacteria listed in table 13. Three comlete assays were done with the g. vagi_nalis cultures against each bacterium. Assays were also done on 3. 32352;. Strain n. 10 day cultures grown in Schneider's citrate medium without the citrate and brom cresol purple. 2. £2533. Strain 33. was maintained 8 weeks on this modified Schneider's citrate medium. Veekly transfers were made and subcultures incubated the first two due at 37°0 and remainder of time until assayed at 22°C. Quntitative citric acid determinations (Pacher. Vickey and Leaven- worth. 193%) were done on pooled g. m. Strain 33. 10 day cultures showing inhibition against Salmonella Elam by the above-described assay method. in equal number of pooled tubes of uninoculated. unadjusted Schneider's citrate medium served as controls. he quantitative citric acid determinations were done on known volumes of the centrifuged tricho- monad cultures and controls as described in the cited reference except the filtration through Gooch crucibles was omitted and extractions were done in a Boxhlet apparatus using lhatman fat extraction thimbles. 33x91t -. the determinations were done on each sample of pooled protcsoal cul- ture material and on each pooled control. 117. Discussion Ten-day cultures of _r_. _f_g_o_t_u_s_. Strains BB. 0 and 0. produced inhibi- tion of g. aileron. Seven-day cultures of Strain 33 showed inhibition of Mebacterium £221.! and Salmonella schctt-ielleri. me other bac- teria listed in table 1 were not inhibited by 7. 10 or 12-day cultures of Strains 3h. 0 or 0. as determined by this assay method. fable 2 shows the percentage of 2. £9.21“: assay cups showing no in- hibition. fewer colonies or definite inhibition against _S_. allorum. with 7 and 10-day cultures of Strain BIB, lt2$ of the cups showed inhibition. he maxim percentage of cups showing inhibition with Strain 33 were 12-day cultures and with Strain 0. 10-day cultures. Iith Strain 0 the older the culture the higher the percentage of inhibition. Attention should 'be called to the fact that the least number of cups (75) were used with 12-day cultures of Strain 0 which had the highest percentage of in- hibition. figure 1 was prepared for clarification of the data pertaining to the parentage of cups showing inhibition in table 2. g. £23322. Strains O and 0. 7-day cultures. showed no inhibition against Monsterium regal: and Salmonella schottmelleri. Strain 33. 7-day cultures. produced 10% inhibition of 9. 3:13. table 3. and 9f inhibition of _S_. .srchottmuelleri. table It. here is need for further study before evaluating this data. Vith all strains of g. £21923 the largest sense of inhibition of g. pullorum, table 5. were with the oldest mltures. i.e.. 12 day. Strain 33 produced the maxim sense of inhibition of g. puller-um of the three strains for 7. 10 and 12-day cultures. . 0f the 95 cups showing inhibition of g. pullorum. table 6. 50% were shown to be bacteriostatic and 50$ bacteriycidal when seeded in broth. for 2. 5223230 Strain 33. assayed on Schneider's citrate medium. modi- fied by omitting the sodium citrate. the percentage of cups showing inhi- ' bition. table 7. increased with the age of the protcsoal culture. his data conpares favorably with the data in table 2 grown in Schneider's citrate medium containing the citrate. Of all the _r_. £93.22 cultures. gown in Schneider's citrate medium. inhibitory and non-inhibitory for g. Elem. the maxim percentage of them showed a pH of 5.5. table 8. with the exception of Strain 0. l2-day cultures. thich showed the highest percentage at a pH of 6.0. Table 8 was separated into tables 9 and 10. grouping the pH of cul- tures showing inhibition in table 9 and those non-inhibitory in table 10. no maximm percentage of the cultures. showing inhibition of g. agorum. had a pH of 5.5. table 9. he two exceptions were 7 and 12-day cultures of Strain 0. which showed the hidust percentage of cultures with a pH of 6.0. he maxim percentage of 2. £223.32. cultures. non-inhibitory for g. Elem. had a pH of 5.5. with the exception of Strain 0. 10 and 12-day cultures. table 10. i'he variation in pH of inhibitory and non-inhibitory cultures is not of great amend: significance to account for the inhibition of g. pullorum. ihe p3 of g. foetus. Strain 33. gross: in Schneider‘s citrate medium modified by omitting the sodium citrate. table 11. closely similates the pH of the same strain and same age found in table 8. 0n the possibility that the inhibition of _S_. pullorum was due to the pensitivity of this bacterium to citric acid. quantitative citric acid determinations. table 12. were done to determine the amount of citrate synthesised or utilised by g. m. Strain BB. 10-day cultures. he amount of variation in age. per ml. of citrate is negligible and within the accuracy of the method. 2. m. {-1, 2 and 3-day cultures. grown in S. P. 1.. to medium produced no inhibition of the bacteria listed in table 13 by the des- cribed assay method. Streptococcus Emma and Iberthella ms; were unsuitable hr assay by this method as a white precipitate formed around each assay cup and the control cup. Some mtbstance or substances in cultures of _!_. £252.! produced in- hibition of _S_. pullorum. g. 5222 and g. schottmuelleri. there was no attesxpt made to determine the chemical nature of this material or to concentrate it. However. there is substantial evidence which indicates that it is not alone associated with the pH of the culture. the sub- stance tends to increase in the medium on cultivation whereas the pH of the medium is relatively stable at a pH of about 5.5 for the 7. 10 and 12-day cultures. hrther there is no relation between the pH of the cul- ture and the production or non-production of the inhibitory substance. i‘he mode of action of the material is not known since in different tests it has been bacteriostatic and bacterifcidal. his would probably indi- cate a difference in concentration of the inhibitory substance. V. Results hble 1 Bacterial Inhibition Produced by l‘richomonas foetus Schneider‘s citrate 363nm """"" Bacteria 7-day cultures Strains BB 0 0 Salmonella ullorum + + + _S, t himr um - - .. g. schottmuefieri + - .- So ebacterium renale + - . filigglla £11m“ (14.8.0.) - . .- lo-day cultures Strains BB 0 0 Salmonella mllorum + + + g. ett - I- o g. E narum (U. of Kentucky) - - - is Chalemm. - u as Stmlococcus eureus 209 - - - Aerobacter aerogenes l2-day cultures Strains BR 0 O Salmonella 2110mm 4- + + 5.- 28m - - . g. snteriditis - - .. + Inhibition - lo inhibition ll fable 2 Percentage of‘g. foetus assay cups showing inhibition of gulls puIIorum. Schneider's citrate medium Es of cultures 7 days 10 days 12 days L Strains as o c on o o on o c o mun... 50$ 67$ 859‘ m 521% 75$ 38$ 69% 3i ewer colonies S 18 S 17 20 ll 7 12 9 ibition he 15 7 he 28 1h 55 19 as o. of cups 299 152 182 299 120 162 170 81 75 ‘0 deo OfiHflONm uOHHHUe-omfln HOFHHUOwUM e———-————. e——-——e 0—0—0 mammals or mm mm: 3! mmmmms mums ‘ mzm's cxmn mm 81811333 81131130 513.1130 f/ '1; ’5 ‘/ [If I . ”(I l/‘ 7.; \ , "’ \\. .r “M, kc“ ‘ o 10 DAYS ‘9] 01' 2. JOINS mime: 13 hble 3 Percentage of g. foetus assay cum shoving inhibition of Mbacterium renale. Schneider's citrate medium Age of cultures 7 days Strains BR 0 6 lb inhibition 80% o 0 lower colonies 10 O 0 Inhibition lo 0 0 No. of cups 150 N8 78 hble ll Percentage of g. foetus use: cups shoving inhibition of Salmonella Inhottmuefleri. Schneider's citrate medium Age of mltures 7 days Strains BR 0 0 no inhibition 67% o o lever colonies 2h 0 0 Inhibition ' 9 O 0 lo. of cups 5% 75 102 1h fable 5 Average zones of inhibition of _§. gullorun by g. foetus Schneider's citrate medium Age of Oilmres 7 days 10 days 12 days Strains 33 See m. 6.0 we 7e9 “a o in mg 6.9 c 5.0 Mt 5.8 Of comebacteriun____rsna1e by I. .f___oetus Schneider's citrate medium Age of 011ch 7 days Strains BR h.3 m. 0 O G 0 Of Salmonella schottmuelleri by I. _f_____oetus Schneider's citrate me Age of cultures '7 days Strains BR 2.5 mm. 0 o 0 o 15 fable 6 Inhibition of 5. Elam by g. 3.2.23.2... Bacterircidal or Bacteriostatic Action of Mtures 7 days 10 days 12 days Strains BR 0 $3 0 0 BB 0 Pactcrifcidal 7 2 2 9 s 5 2 u Bacteriostatic 11$ 0 O 9 3 5 O 3 Iotal cups 21 2 2 18 11 10 2 7 16 i'able 7 Percentage of I. foetus assay cups shoving inhibition of Salmonella llorum Schneider's citrate medium without the citrate Strain 33 Age of culture He inhibition Mar colonies Inhibition No. of cups 7 days 10 days 12 days 50.0% 50.0; 11.1; 33-3 13-7 37.0 16.6 36.2 51A st 2011 27 i?- fable 8 pH of Trichomonae foetus cultures Inhibitory and non-Inhibitory. mder's citrate medium e of cultures 7 days 10 We 12 days Strains 1m 0 c m o 0 an 0 0 pH 5.0 2.65 - - - - .. 8.75 .. :— 5.1 2.6 - - - - .. . .. - 5.2 13.1 11.1%! tun 23.9; 6.6“ 5.5% 13.1 .- - 5.3 5.2 - - - - - k3 - - 5A - - - - - .- h.‘3 - - 5.5 50.0 50.0 52.3 52.h 53.3 um. 52.1 25.0; 66'. 5.6 - - - - - - 153 .. - 5.7 15.7 5.5 19.0 1h.2 26.6 16.6 9.3 25.0 11.1 5.8 - - . - - - .. .. - 5.9 - - . - - .. .. .. - 6.0 5.2 33.3 1&2 9.5 13.3 16.6 3.2 50.0 22.2H 6.1 - - . - - . - .. .. 6.2 - - - - - - .. . .. 6.3 5.2 - - .. - .. .. .. - 6J4 - - - - - - - - - 6-5 - - 9.5 - - 5-5 - ~ - 7.0 - - - - - 11.1 - .. .. fatal mltures 36 18 21 21 15 13 23 8 9 l ~80 data 18 !able 9 pH of I. foetus cultures Inhibition of g. Schneider's citrate no 2222:. of cultures 7 days 10 days fil 12 days Strains 32 0 0 32 o 0 an o 0 pH 5.0 5.2% - - - - - 5.3; . - 5.1 5.2 - . - - - - - - 5.2 L263 111.221 .- 91.6; 11.15 20. 5.3 .- - 5.3 10.5 - - - - - 7.6 . - 5A - - .. - - - 7.6 - - 5.5 +7.3 28.5 90.4 50.0 77.7 80.0 .1 25.4 66.6fl 5.6 - - .. - -. .- 7.6 - .. 5.7 .-. - i70.0 - 11.1 .- .. 25.0 11.1 5.8 - - - - - - - .. - 5.9 - - .. - - .. .. 2 - 6.0 5.2 57.1 20.0 0.8 - . .- 50.0 22.2 “cilfiflz‘m’j 19 7 5 12 9 5 t3 h 9 ~10 data 19 fable 10 pH of g. foetus cultures Hon-inhibi tcry for g. Eullorua Schneider's citrate medium Age of Oiltures 7 days 10 days Strains an 0 c 32 0 1 1135.0 - - - .. - 5.1 - - - - - 5.2 - 9.0% 6.2% - - 5-3 - - - - - 5.1; - - - - - 5.5 117.0% 63.6 56.2 55.5% 16.6% 30.7% 5.6 - - . - - ' 5.7 35.2 9.0 12.5 33.3 50.0 23.0 4 5.8 - - - - - ' 5.9 - - - - - 6.0 5.8 18.1 12.5 11.1 33.3 23.0 f 5.1 - - - - - ' 6.2 - - - - - 6.3 11.6 - - - - 6.11 - - - - - 6.5 - - 12.5 .. - 7.0 - - - - - 15.3 . i'otal non- 2111::an 17 11 16 9 6 - No data 2O fable 11 pH of grichononas foetus cultures. Strain BB Inhibitory and non-inhibitory Schneider's citrate medium without the citrate Age of cultures 7 days 10 days pH 5.0 - 19% 5.1 - - 5.2 50% 23.8 5. 3 - - 5.9 - - 5.5 33.3 lV1.6 5.6 I - - 5.7 16.1 17,-, 5.8 - - 5.9 - - 6.0 - 11.7 and cultures 6 21 - No data .HiLs 3.0 .03 833.3» 9+5. 00.0 .03 SA GA GA flan S m 3.0 .2: $.n {an , RA no.0 00A 3 m 3830 0: 8A $.m «Tm 09m 3 0 «0.0 .03 SA 23m 0.1m R.» :mA 3 m «0.0 .2: 07m $.m 3% 6A 2 m 8.0 .08 5A RA 8% un.n 8A 3 H #8280 m 32.8 4 £23 .330 no N .250 H 0.00.8 .fifi... fl“. fix“. flu“. fin“. flaw. flaw. .33.... .2 Spade: 30.3.3 0783233 .IduoBll m.w. new ”3335 3.3013 310.." .3 593m .0300“ 03303.39 30303333 goo 0.330 05.033930 NH 3”de 22 Table 13 Bacterial inhibition produced by Trichononas vggnalis. #1 Ge Po In “a new 2-day cultures Bacteria Salmnella schottmuelleri -- 2- 12292.12 - g. gullorun - g. cholerasuis - g. Elinarum (U. of Kentucky) - 2. 2221212 - g. enteriditis '- ‘grobacter aero nes . - Pseudomonas aeruénosa - Escherichia gel; - 3-day cultures lococcus m 209 - .3322. £222 3 " filigella flinarum (I. S. O.) - 2. 229221 N - g. Euflsenteriae Boyd 88 - g. 2_ar_adzsenteriae Honor '7' «- - Ho inhibition VI. 23 conclusions 1. 2. 5. 6. By the described Oxford cup plats assay method 3. m. Strains 33. O and o. 7. 10 and 12-day culmres. produced inhibition of g. zu_110rum. g. m. Strain 33. 7-day cultures inhibited 9. £221.! and g. schottmelleri. Further study is needed as a limited number of cultures were assayed. , Sodium citrate is not necessary in the Schneider's citrate medium for the growth or for production of inhibition of 2- 22222»; 29,219.. simian- he pH of the g. m mltures was not responsible for the inhibition of 5. Elem in Schneider's citrate medium or in the medium modified by omitting the sodium citrate. citrate is not synthesised or utilised by g. 3.9.9.9220 Strain BB. lO-dw culmres. 3. we, #1. 2 and 3—day cultures did not prodice inhi- bition of bacteria by the described assay method. 21L VII. Acknowledgment The writer wishes to take this opportunity to express her apprecia- tion to Drs. Philip Hawkins. Garth Johnson. Banner Bill ucrgan. Nicholas Iattu. members of the Department of Bacteriolog and Michipn Department of Health for their assistance in this study. 25 VIII. References cited hstin. n. 1.. 1916 contributions toward an analysis of the killing ac- tion of variety ’4 killers in Paramecium anrelia. inat. Rec. 9525134. Johnson. 0.. and I'russell. B. 3. 19h} Experimental basis for the chemo- therapy of Erichomonas minalis infections I. Proc. Soc. 31p. Biol. and 14.0. 511228322119. . ' Pacher. G. In Victory. 3. 3.. and Loavenworth. 0. S. 193,4 Determina- tion of citric acid. Indust. and Engineer. wen. Analyt. Id. 6: 190-192. Schmidt. V. 3.. and lioyer. A. J. 19% Penicillin I. Methods of assay. Jour. Pact. 117:199-208. Schneider. N. D. 19152 A new thermostabile medium for the prolonged bacteria-free cultivation of grichomonas m. qur. Parasitol. 283128-1129. < Sonneborn. !. 1L. Jacobson. t. and Dippell. R. V. 19% Paramecin 51. an antibiotic produced by Paramecium anrelia. inat. Sec. 96353.n- 515-