Hymenolepis microstoma has been grown in vitro from the cysticercoid stage to the young tapeworm. A basal medium HM 67 has been established from stock solutions of Medium 115 (Berntzen and Mueller, 1964) and is composed of 6% component A, 28% component B:C:D:E:F (20:20:20:20: 5; v:v), 50% Earle's + 0.5% glucose, and 16% distilled water. With Medium HM 67 + 30% serum (pH 7.5) under air, worms grew from 0.160 mm to an average length of 0.950 mm. The addition of 1% whole hamster bile resulted in further growth to an average length of 4.75 mm. Terminal proglottids of cultured worms contained normal appearing testes, seminal vesicle, seminal receptacle, cirrus, and ovary. In vitro culture methods have been developed for two hymenolepidid cestodes. Berntzen (1961, 1962) cultured Hymenolepis dimintta and H. nana. A simplified method for cultivation of H. diminuta was developed by Schiller (1965). To date, there have been no reports on the in vitro cultivation of H. microstoma, a tapeworm naturally occurring in the bile ducts of mice. Techniques developed by Berntzen (1961, 1962) maintained H. microstoma for only very short periods of time. Therefore, different combinations of components of a basal medium used for the in vitro cultivation of Spirometra mansonoides by Berntzen and Mueller (1964) were screened for optimum growth of H. microstoma. MATERIALS AND METHODS Adult H. microstoma were maintained in male Swiss albino mice, and the cysticercoid stage in the flour beetle, Tribolium confusum. Cysticercoids dissected from beetles infected 20 days previously were freed of microorganisms and excysted in the following manner: 1) Cysts, free of host tissue, were transferred to an antibiotic solution containing 200,000 units penicillin, 20,000 units mycostatin, 200 mg streptomycin, 100 mg sulfasuxidine, and 66 mg neomycin per 100 ml distilled water for 30 min; Received for publication 13 September 1966. *Aspirant of the Belgian National Foundation for Scientific Research and C.R.B. Fellow of the Belgian American Educational Foundation at the time of this study. Present address: University of Gent, Instituut voor Dierkunde, Laboratorium voor Fysiologie, Ledeganckstraat, 35, Gent, Belgium. t Present address: Portland State College, Division of Science, Department of Biology, Portland, Oregon. 2) Transferred to a second antibiotic solution containing 200,000 units penicillin, 20,000 units mycostatin, and 200 mg streptomycin per 100 ml distilled water for 3 min; 3) Transferred with Pasteur pipettes to an acid-pepsin solution (1% 1:10,000 pepsin, 1% HC1 in Ringer's) for 10 min at 38 C; 4) After acid-pepsin, transferred to a 0.3% sodium glycotaurocholate, 0.5% trypsin (1:300) in Ringer's at pH 6.7, 38 C for 20 min; and 5) Excysted scolices from (4) were placed in a final antibiotic solution containing 200,000 units penicillin, 20,000 units mycostatin, 200 mg streptomycin in 22 ml Earle's balanced salt solution + 0.5% glucose, for 1 to 2 min, then inoculated into the culture tubes. The number of worms per culture ranged from 30 to 50. Cultures were maintained in 15.0 by 1.5 cm screw-cap tubes at 38 C. Sterility checks were carried out at the beginning and end of each experiment. In experiments to determine basal medium ratios, 5 ml of medium was used per tube. These cultures were tightly closed and neither the medium nor gas phase changed. When testing the effect of additives to the basic medium HM 67, 3 ml medium per tube was used; medium and gas phase were replaced daily. In evaluating the quality of a given basal medium the highest figure for per cent survival for a given time was considered decisive. In experiments where additives were incorporated into the basal medium, total length, survival, and stage of differentiation were used as criteria for the growth-promoting characteristics of the additives. To determine a basal medium for survival of H. microstoma in vitro, various ratios of Medium 115 (Berntzen and Mueller, 1964) stock solutions were tested. In addition Earle's + 0.5% glucose and distilled water were used. All experiments were run in duplicate.
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