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Attempts by investigators to cultivate the parasitic form of Coccidioides immitis in vitro have met with varying degrees of success (G. L. Gilardi, Bacteriol. Rev. 29:406, 1965). In their development of an efficacious spherule-endospore vaccine, H. B. Levine, J. M. Cobb, and C. E. Smith (Trans. N.Y. Acad. Sci. 22:436, 1960) employed modified Converse medium for the production of endosporulating spherules of C. immitis strain Silveira. However, purification procedures (Levine et al., Trans. N.Y. Acad. Sci. 22:436, 1960; H. B. Levine, Sabouraudia 1:112, 1961) were necessary to remove hyphae invariably present in the liquid medium. As shown in this report, this disadvantage was overcome by the addition of refined agar to modified Converse medium to permit colonization of spherules. To each 100 ml of modified Converse medium, 1.5 g of agar (Difco), 0.75 g of Ionagar No. 2 (Colab Laboratories, Inc., Chicago Heights, Ill.), or 0.75 g of Agarose (Bausch & Lomb Inc., Rochester, N.Y.) was added and heated to boiling before 10-min sterilization at 15 lb of pressure (121 C). Volumes of 5 ml were distributed in sterile disposable petri dishes (50 by 12 mm; Falcon Plastics, Los Angeles, Calif.). Prepared media were stored at 4 C and generally were used within 1 week. C. immitis strains Silveira, Mil, CI4, and CI-5 were used; the last two strains were isolated from patients with disseminated coccidioidomycosis hospitalized at our institution. Arthrospores were harvested from 2to 4-weekold cultures grown at 34 C on 1% glucose-yeast extract-agar. Inoculated agar media were incubated at 37 or 40 C. Carbon dioxide-treated cultures were placed in a pressure cooker modified with intake and exhaust shut-off valves. A mixture of 20% CO2 and 80% air was used to provide increased CO2 tension. Cultures were examined at 2to 3-day intervals for 2 weeks. Since A. M. Breslau and M. Y. Kubota (J.

Use of refined agar for the in vitro propagation of the spherule phase of Coccidioides immitis