SYNOPSIS. Several strains of particle‐bearing and particle‐free Paramecium aurelia have been cultivated in an axenic medium composed of proteose peptone, trypticase, yeast nucleic acid, MgSO4.7H2O, TEM‐4T (diacetyl tartaric acid esters of tallow monoglycerides), stigmasterol and a mixture of vitamins. The “yeast fraction,” an indispensable component of previous media used for the cultivation of these ciliates has been replaced by a mixture of trypticase, yeast nucleic acid and TEM‐4T. Particle‐bearing animals of stock 299 lambda, 138 mu, and 139 pi maintain their particles when cultivated in the medium, whereas particle‐bearing animals of stock 51 kappa, 225 kappa and 114 signia do not. With the exception of stock 92 (syngen 3) the medium appears to be selective in its ability to support the growth of animals of the even‐ but not odd‐numbered syngens of P. aurelia. Maintenance of the particles was dependent only to a small degree upon environmental conditions brought about by changes in pH and temperature. Division of the particles was found to be comparable with the division of the protozoan. Methods for the growth, maintenance and mass cultivation of particle‐bearing P. aurelia are given in detail.

SYNOPSIS. Paramecium aurelia, stock 299 (symbiote‐free) was cultivated in a synthetic medium consisting of amino acids, vitamins, purine and pyrimidine derivatives, fatty acids, stigmasterol, sodium acetate and salts. The medium supported the continued growth of this stock in serial subculture. Populations up to 17,000 organisms/ml were obtained in 9 or 10 days in the medium supplemented with a phospholipid. Synthetic 1‐oleoyl‐2‐stearoyl‐dl‐phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine and cephalin were comparable in growth‐promoting activity. The nutritional need for each of the components of the medium was examined. The following were determined to be essential nutrilites for P. aurelia: arginine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, valine, folic acid, nicotinamide, calcium pantothenate, pyridoxal, riboflavin, thiamine, DL‐6‐thioctic acid, guanosine, uridine (or cytidine), oleic acid, stigmasterol, calcium and magnesium. Serine replaced glycine for growth in the presence of thymidine. In the absence of thymidine, comparatively high levels of folic acid were required for optimal growth. Sodium acetate did not replace DL‐6‐thioctic acid. Populations were reduced in the absence of the non‐essential amino acids, alanine, asparagine, aspartic acid and glutamic acid. These were restored to optimal levels by the addition of sodium acetate to the medium. Pyruvate was about as effective as acetate in this respect; glucose and certain other carbohydrates were not.

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SUMMARY:Paramecium awelia, var. 4, stock 51.7 (s) requires a steroid as a growth factor ; p-and y-sitosterol, fucosterol, brassicasterol, stigmasterol and ,541 22-stigmastadienone are active in supporting its growth. Esterification of the 3-hydroxyl group decreases the activity. The presence of more than one double bond in the ring system inactivates the molecule, as do the more drastic changes of the side chain, as found in diosgenin, digoxigenin, progesterone, estrone and methyl cholate. Oxidation of the ring system destroys activity. The specificity, of these requirements suggests that the steroid functions as an essential metabolite for this organism.Pararnecium aureZia, var 4, stock 51.7 (s) has been established in axenic culture in a medium consisting of a yeast extract and proteose peptone, provided a steroid of plant origin is added (van Wagtendonk, Conner, Miller & Rao, 1953). This steroid, identical with either @-or y-sitosterol or with a mixture of both sterols, was subsequently isolated from lemon juice . The observation that no difference between the growth-promoting activities of ,&sitosterol and its optical isomer y-sitosterol could be detected, led to a study of the molecular configuration of the steroid molecule, necessary for biological activity for P . aurelia. A preliminary report has been published . METHODSStock cultures of Paramecium aurelia, var. 4, stock 51.7 (s) were maintained at 27" in a medium consisting of a yeast extract, proteose peptone and lemon juice. The preparation of the yeast extract, and the preparation of the steroid solutions for assay, as well as the assay technique have been previously described (Conner et al. 1953). It should be pointed out here that P . aurelia was able to grow at a slow rate in the yeast extract prepared from a few lots of Fleischmann's bakers' yeast (Standard Brands Inc,) ; in these extracts the active steroids were only stimulatory. The majority of the batches of this yeast yielded extracts in which P . aurelia did not grow unless an active steroid was added. Only these batches of yeast extract were used in the survey of the various steroids.The following steroids were tested in effective concentrations of 800 mpg., 400 mpg., 200 mpg., 100 mpg. and 50 mpg./ml.: cholesterol (U.S.P., Eli Lilly and Co., Indianapolis, Indiana) ; cholesteryl acetate (Eli Lilly and Co.);