A New Liquid Medium for Xenic Cultivation of Entamoeba histolytica and Other Lumen-Dwelling Protozoa

Abstract

A new liquid medium, TYSGM-9 (Trypticase, yeast extract, serum, gastric mucin), devised to facilitate the transition of Entamoeba histolytica from xenic to axenic conditions of growth has proved valuable for initiation and maintenance of cultures of the following protozoa inhabiting the intestine or mouth of man: E. histolytica, E. coli, E. gingivalis, and Dientamoeba fragilis. Its utility in cultivation of other protozoa from these sites has not been tested. Moreover, its usefulness as a diagnostic medium has yet to be established. In each case studied, microscopy had demonstrated the parasite(s) in the source material prior to culturing. An Entamoeba resembling E. gingivalis recovered from the genital tract of a user of an intrauterine device (deMoraesRuehsen et al., 1980, Acta Cytol. J. 24: 413420) and which could not be grown in LES medium was isolated in the new medium. The medium which has been in use for 2 years offers certain advantages over others commonly employed for xenic cultivation of these parasites. Its clarity permits examination of the protozoa in situ, obviating the necessity of sampling the cultures between transfers to monitor them as must be done with diphasic media, e.g., LES (Boeck and Drbohlav, 1925, Am. J. Hyg. 5: 371-407, modified by Reardon and Rees, 1939, J. Parasitol. 25(Suppl.): 13-14). Egg-yolk infusion, probably the most widely used liquid medium for isolation and culture maintenance, requires overnight to prepare and involves numerous procedural steps (Balamuth, 1946, Am. J. Clin. Pathol. 16: 380-384). In contrast, TYSGM-9 can be prepared within a few hours from ingredients commonly stocked in diagnostic microbiological laboratories. It has a storage life of a month or more at 4 C. In established cultures, yields of protozoa are consistently better than those obtained with LES and equal to or greater by twofold than those obtained with egg-yolk infusion. Inocula of 5,000 organisms/ml medium taken from established cultures yield the following fold increases in 48 hr: E. histolytica, 30 to 40; E. coli, 20 to 30; E. gingivalis, 15 to 25; and D. fragilis, 20 to 30. TYSGM-9 was derived from TYI-S-33, a medium developed for axenic cultivation of E. histolytica and related Entamoeba (Diamond et al., 1978, Trans. R. Soc. Trop. Med. Hyg. 72: 431-432). It is prepared as follows. Basal medium: Add and dissolve in 600 ml of glass-distilled water: potassium phosphate, dibasic, 2.8 g; potassium phosphate, monobasic, 0.4 g; sodium chloride, 7.5 g; Trypticase (BBL Microbiology Systems, Cockeysville, Maryland), 2.0 g; and yeast extract (BBL), 1.0 g. Bring total volume to 970 ml with distilled water. Place 200 mg gastric mucin (U.S. Biochemical Corp., Cleveland, Ohio, # 16025) in dry, sterile, screw-capped Erlenmeyer flasks (125-ml capacity). Add 97 ml of broth to each flask. Autoclave 15 min, 121 C, 15 lb to both solubilize the gastric mucin and sterilize the medium. Cool to room temperature. Aseptically add 3 ml of bovine serum (inactivated 30 min, 56 C), and 0.05 ml of Tween 80 prepared as a 10% solution (v/v) in absolute ethanol. Swirl vigorously to keep gastric mucin particulates in suspension and dispense in 8-ml portions to 16 x 125-mm screw-capped tubes. The pH of the medium is 7.2; the osmolality, 320 mosmol/kg. Biosate (BBL), 3 g, can substitute for Trypticase and yeast extract. Gastric mucin should be of the type prepared according to the New and Nonofficial Remedies (N.N.R.) 1953. Rice supplement: Purified rice starch is preferred, but unpolished rice flour can be used. The rice is placed in 500-mg amounts in 16 x 125-mm screw-capped tubes and sterilized with dry heat at 150 C, 2.5 hr. To assure sterilization the tubes are positioned horizontally with the rice dispersed in a thin layer and the screw cap loosened to allow free movement