118 STUDIES OF Tritrichomonas batrachorum. I.

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J . PKOTOZOOL., 4, 118-119 (1957)

Replacement of Serum for In Vitro Cultivation of Tritt*ichomonas f o e t z d

MARVIN SANDERS Haskins Laboratories, N e w York City

SUMMARY. The combination Tween 80 and cholesterol replaced blood serum as a requirement for the cultivation of Tritrichornonas joetits. Choline + potassium glycerophosphate enhanced growth in the presence of Tween 80 4- cholesterol. A method for ensuring adequate cholesterol suspension-an essential factor for consistent growth-is described.

____ .- _____

OSVESTIONAL MEDIA for growing pure cul- C tures of trichomonads include blood serum. The efficacy of serum as a nontoxic source of fatty factors

*This investigation was aided by- grants from the Rocke- feller Foundation, and the American Cancer Society on the recommendation of the Committee on Growth of the National Research Council.

1 I wish to thank Dr. H . Sprince, then at the Ortho Founda- tion. for the pure strain B2 of Tritrichomonas foetiis.

in microbial nutrition has been noted(3). Though both cholesterol( 1 ) and additional fatty factors(3) have long been recognized as growth requirements for trichomonads, previous efforts to replace serum were unsuccessful. The discontinuance of our experiments prompts a description of how serum was replaced for Tritrichomonas f0etus.l

SERUM REPLACEMENT FI

MATERIALS AND METHODS T . foetus was grown in modifications of the basal

medium of Sprince and Kupferberg( 5 ) henceforth called “TC” (Table I ) . Agar was not essential for growth but it stabilized the suspension of cholestero1 and helped maintain anaerobiosis. Choline dihydro- gen citrate and potassium glycerophosphate, each a t 0.1 mg. %, improved growth but could not substitute for Tween 80 + cholesterol.

The degree of dispersion of the water-insoluble cholesterol markedly affected growth. The following method of preparation for each transfer permitted good dispersion :

Fresh basal medium was prepared from the dry components. Dilutions of Tween 80 (v/v) were freshly prepared in 70% ethanol (aqueous solutions become inactive on standing). The tubed basal me- dium was heated in a boiling water bath to dissolve the agar, then cooled to 5 5 ” C. Cholesterol (from a stock solution in 95% ethanol) was then added to the warm, tubed final medium just before autoclaving. Controls contained 5.0 m1/100 of serum added asep- tically.

The basal medium contains Trypticase, an enzy- matic digest of casein, as a source of water-soluble factors. The effectiveness of Trypticase remains to be explained.

TAI:J,E I. Basal iiiecliuni “TC.”*

A~noiiiits in 11)O nil. of fiiinl base Agar (l)ifcSo “Xoble”) 0.1 g. Mctliyleiic !due 0.4 nig. Met:il inis S o . 44t 0.5 1111. MgSO, * iH,O I l .Oi5 g. &PO, 0.05 g. Clioliiic Ililiprlrogeii citrate 0.1 mg.

ophosphate 0.1 nig. Full strength

\-it:iiiiiii niix: Full strength RiboSc iiuclcic- :icid (sodiuni salt) 0.02 g. Sucrose' 1.5 g. Pot:isaiuiii tlrioiiialate$ 0.4 g.

1.0 g. 0.35 g.

* The nieiliiini is niljusted to pH i . G with KOEI. t Nctnl niis S o . 44: Etl i~lr~iedi~u~ii ie te t raacct ic acid, 0.25

mg., Zii ( : I S SO, I , 2.5 nig., Fe (as SO,), 1.0 nig., Mn (as SO,), 0.5 uig., Cn (:is S O , ) , 0.1 nig., Co (as SO,:), 0.05 nig., R (:is ITJ30:j), (1.02 nig. These nietals are dissolved in dis- tilled watrr to yicl(l the above final c.oncent~r:itio~ix in 100 nil.

$ This is the niiz clkscribed in refcrcncc ( 4 ) . $ T1iioni:ilic :iciil is dissolved in distilled water and the p N

is acljustcd to 7.11 with KOH. Thc appropri:ite amount of this freshly prc*lmrvcl solution is added to each tube of inediuiii just lwfurc. autoclaving to obtain m:~siinnl reduc- tioii.

-. ~ ~ ~ __

)I This c . o i i i ~ ~ o i i ~ i ~ l w:is used as a buffer.

DR Tritrichotnonas foetus 119

Growth of T . foe tus in the absence of serum.

Tween 80 (nig. % i n finalmedium)

TABLE 11.

Basal nirtliiiiii ‘ ‘ TC ’ ’ i - 0 0.4 1.0 2.0

Cholesterol (iiig. in filial mediuni)

0 0 0 0 0 (1.25 200” 450 450 100 0.5 400 600 800 200 1.0 300 600 800 200 2.5 300 600 800 1000 5.0t 300 800 800 800

8el.ulll 5.0 m1./100 1400

Af t r r six sc+al tr:insfers: t Serum 5.0 m1./100 1350 C,holrsterol 2.5 mg. % +

Tmec.n 80 1.5 nig. % 950

* All vnlucs are expressed as cells/mm? 2 25. t At this conccntrat,ion, cholesterol began to precipitate. $ Ciiltures grown in niediuni “TC” + cholesterol +

Twer11 80 were harvested after 96-120 hours ; cultures grown in inediuni “TC” + serum were harvested in 48-72 hours. Masinial growth in medium “TC” + serum reached a den- sit,y of I600 cells/nim.a; in medium “TC” + cholesterol + Tween 80, ~iinxinial growth reached a density of 1050 cells/ 1n111.3.

RESULTS Such poorly defined fatty materials as ox bile, leci-

thin, or sodium taurocholate replaced serum in the presence of cholesterol. As fatty acids and their glycerol derivatives by themselves were inactive. it was thought that the crude preparations also embodied necessary dispersing agents. Therefore, synthetic com- pounds which were both dispersing agents and con- tained fatty acids were tested. Medium modification “TC” lacked serum but supported growth in the presence of cholesterol + Tween 8.0 (Table 11).

Tween 20, 40, and 60, (respectively the monolaur- ate, monopalrnitate, and monostearate of polyethylene sorbitan) were inferior to Tween 80 (the monooleate) in maintaining growth through several transfers.

~. _ _ _ ~

REFERENCES

1. Cailleau, R. (1938). Le cholestkrol et l’acide ascorbique -facteurs de croissance pour le flagell6 tktramitidk Tricho- monas foetus Riedmuller. Conzpt. r m d . soc. biol., 127, 861-863.

2 . Cowperthwaite, J., Weber, M. M., Packer, L., and Hut- ner, S. H. (1953). Nutrition of Herpetomonas (Strigomonas) czilicidaruin. Ann. N . Y . Acad. Sci., 56, 972-981.

3. Rodwell, A. (1956). The role of serum in the nutrition of Asterococcus mycoides. Australian J . Biol. Sci., 9, 105-116.

4. Sprince, H. (1947). An improved procedure for separating human blood serum into two fractions essential for the sus- tained growth of Trichonzonas vaginalis. J . Bacteriol., 53, 441-447.

5 . Sprince, H. & Kupferberg, A. B. (1947). A simplified medium for the investigation of unknown factors in blood serum essential for the sustained growth of Trichomonas vaginalis. J . Bacteriol., 53, 435-439.