NUTRITION OF THE PARASITIC PHASE OF COCCIDIOIDES IMMITIS IN A CHEMICALLY DEFINED LIQUID MEDIUM

Abstract

In previous reports (Converse, 1955, 1956, 1957) a chemically defined medium was de-scribed which supported the growth of the para-sitic phase (spherule) of the dimorphic fungus Coccidioides immitis. Certain physicochemical factors were shown to influence the growth of spherules in this medium. The work to be de-scribed here is an extension of the investigation of the nutritional and environmental factors which affect the spherulation in vitro of C. tm-mitis. MATERIALS AND METHODS C. immitis strain Mll was used in this investi-gation. Experimental cultures were incubated for 72 hr at 34 C on a reciprocating shaker (operating through a 412 in stroke at 96 excur-sions per min) in a liquid basal medium composed of ammonium acetate, 0.016 M; glucose, 0.022 M; KH2PO4, 0.003 M; K2HPO4, 0.003 M; MgSO4-7H20, 0.0016 M; and ZnSO4-7H20, 1.24 X 10-5 M, with a final pH of 6.3 after autoclaving. Fifty ml of medium in 250-ml Erlenmeyer flasks were inoculated with 1 X 107 arthrospores. The flasks were closed with cotton-packed modified thistle tubes inserted through rubber stoppers. The inoculum was prepared by transferring arthro-spores from a stock agar slant to a liquid syn-thetic medium (Basal no. 2 of Roessler et al., 1946; with 4 ppm Zn+ as ZnSO4) and incubat-ing for 14 days under similar conditions. This culture was stored at 5 C and discarded after 1 month. Spherule yields were determined by micro-scopic examination of wet mounts stained with lacto-phenol-blue. The degree of spherulation and total growth was estimated visually for each culture and assigned an arbitrary number on a scale ranging from 0 to 8. Endospore cultures were filtered by gravity through 6 layers of surgical gauze to remove trace amounts of hyphae. The endospore yields were measured by viable counts, using the pour-plate technique with a plating medium composed of peptone, 1 per cent; glucose, 2 per cent; yeast autolysate, (yeast-75, Vico Products Company) 0.1 per cent; and agar, 2 per cent; adjusted to a final pH of 6.9 to 7.2. Plates were incubated for 48 hr at 34 C, and an additional 16 hr at 25 C,