CONTINUOUS IN VITRO CULTIVATION OF SPHERULES OFCOCCIDIOIDES IMMITIS

Abstract

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.

Proper Care and Feeding of Coccidioides: A Laboratorian's Guide to Cultivating the Dimorphic Stages of C. immitis and C. posadasii

“…For in-depth investigations of this critical stage in disease establishment, see several prior publications (Cole & Hung, 2001;Cole, Seshan, et al, 1988;Drutz & Huppert, 1983;Hector & Pappagianis, 1983;Mandel, Galgiani, Kroken, & Orbach, 2006;Mead, Roe, et al, 2020;Viriyakosol, Singhania, et al, 2013;Whiston et al, 2012;Wise, Hung, Whiston, Taylor, & Cole, 2013). Morphogenesis is triggered by exposure to high temperatures (>37°C), gas concentrations (5% to 15% CO 2 ), and other factors that have not been precisely defined (Breslau & Kubota, 1964;Klotz, Drutz, Huppert, Sun, & DeMarsh, 1984). In vitro, spherules can be obtained using either chemically defined Converse medium (Converse, 1955) or RPMI 1640 supplemented with 10% fetal bovine serum (FBS; RPMI-sph medium) (Petkus, Baum, Ellis, Stern, & Danley, 1985).…”

“…Endospores are discussed in the literature; however, methods for encouraging endosporulation are limited. One study suggested that removing hyphal fragments, replacing the medium, and maintaining 10% CO 2 produces a continuous culture of spherules, and therefore endospores (Breslau & Kubota, 1964). We based our laboratory methodology and Alternate Protocol 4 on this publication.…”

Nutrition of systemic and subcutaneous pathogenic fungi.

“…Increased temperature (between 34 and 40 C) induces spherulation (10,17,18,19,20,46,48,72), but Converse and Besemer (20) stated it is not the only factor influencing development. The studies by Breslau and Kubota (10) also indicated that temperature is a critical factor, but not the only factor for spherulation.…”

“…Investigations have demonstrated the favorable effects of metabolic or bubbled CO2 on the growth of the parasitic form (10,45,48). Lubarsky and Plunkett (48) were able to obtain spherules in tissue culture media supplemented with various animal sera aerated periodically with 23% 02 and 77% CO2.…”

Pure spherules of Coccidioides immitis in continuous culture

“…The most successful medium for the continuous growth of spherules is modified Converse medium which is a mixture of salts, glucose, and the detergent N-Tamol (Rohm & Haas Co., Philadelphia, Pa.). Spherule-endospore cultures have been grown over long periods in modified or unmodified Converse medium (2,15); unfortunately, the medium is not suitable for mammalian cells, and cultures routinely contain some hyphae. Teramura (M.S.…”