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Angeles, Calif.), and Mike Y. Kubota. Continuous in vitro cultivation of spherules of Coccidioides immitis. J. Bacteriol. 87:468-472. 1964.-Spherules of Coccidioides immitis were grown in vitro in continuous culture for more than 4 years. Cultures were grown in modified Converse medium in a modified Lubarsky and Plunkett culture tube incubated at 40 C under increased CO2 tension. Spherules that had transformed from hyphae were freed from mycelial fragments, and were allowed to settle to the bottom of the tube. The mycelium which normally degenerated at 40 C was removed repeatedly with a syringe and needle until the culture was almost pure spherules. For continuous culture, 10 to 12 ml of old medium were remnoved, and were replaced by fresh medium previously bubbled with a mixture of 20% carbon dioxide and 80% air.

Spherules of C. immitis have been grown in vitro in modified Roessler's medium under COs tension and continuous cultures now maintained for over 18 months. Transformation of hyphae and development of the spherule form have been studied by thin section electron microscopy. Cells of organisms in the hyphal stage have thin (ca. 50 m#), apparently structureless walls and a cytoplasmic membrane. Many nuclei, elongated mitochrondria with both transverse and longitudinal cristae, and lipid particles are present. The hyphal wall thickens and the cell transforms into spherules. A large central accumulation of electrontransparent polysaccharide appears in the spherule. The peripheral cytoplasm contains nuclei, each enclosed in a double-layered membrane, mitochondria, and small dense particles. Prior to cleavage the polysaccharide droplets are lost, while mitochrondria become small and spherical. Endospores are formed and liberated when the spherule wall breaks. These begin to grow and repeat the cleavage cycle.