Metabolism, Macromolecular Synthesis, and Nuclear Behavior of Cryptococcus albidus at 37 C

Abstract

The temperature-sensitive events which prevent Cryptococcus albidus from growing at 37 C were investigated. Cultures incubated at 37 C immediately after inoculation did not increase in optical density nor in cell numbers, and by 24 h 90% of cells in such cultures were deformed and dead. When cultures in log phase were shifted from 23 to 37 C the optical density increased but the cell numbers did not. Morphological observations revealed that the increase in turbidity at 37 C represented enlargement and distortion of cells without appreciable replication. Uptake and incorporation of '4C-leucine were similar at 23 and 37 C. There was no difference in 14CO. evolution from cells at either temperature. Uptake and incorporation of adenine-8-14C into RNA was slightly lower in cells incubated at 37 C. There was, however, a 60% reduction in incorporation of adenine-8-14C into DNA after 3 hr at 37 C. Nuclear staining revealed that nuclear migration did not occur in cells incubated at 37 C. Thus the data indicate that both adenine incorporation into DNA and nuclear migration prior to nuclear division by C. albidus are temperature sensitive.The ability to grow at 37 C is one of the criteria used to distinguish various species in the genus Cryptococcus. Strains of C. albidus generally do not grow at 37 C, but many remain viable at that temperature for some time. Metabolism, macromolecular synthesis, and nuclear behavior of this yeast at 23 and 37 C were studied in an attempt to identify the temperature-sensitive step(s) which precludes growth at the higher temperature.MATERIALS AND METHODS Organism. The strain of C. albidus used in this study (no. 378, stock culture collection of this Department) was grown on slants of glucose-peptone-agar (2) at room temperature for 3 days and subsequently refrigerated. Subcultures were made at monthly intervals. The cells were grown and prepared for the experiments as described previously (12).Growth of C. albidus. The growth of the fungus was measured by 3 methods: (i) nephelometry, (ii) direct hemocytometer counts, and (iii) quantitative plate counts.Nephelo triple-baffled culture flasks (Bellco, Vine-land, N.J.), each containing 20 ml of glucose-peptonebroth, were used in those experiments in which growth was measured by increases in turbidity. The flasks were inoculated with 0.4 ml of a 24-h broth culture and incubated at 23 or 37 C on a rotary-action shaker (model "V", New Brunswick Scientific Co., New Brunswick, N.J.). At determined intervals, the turbidity of the broth was measured with a Klett-Summerson photoelectric colorimeter (Klett Mfg. Co., Inc., New York, N.Y.). Viability of the cells was determined by eosin-y dye exclusion (13).Direct counts were made on cultures prepared and inoculated as in the preceding paragraph. A 1-ml sample was withdrawn at determined intervals and appropriately diluted. The cell numbers were then counted in a hemocytometer.The inoculum for quantitative plate counts was grown in 30 ml of glucose-peptone-broth for 24-h at 23 C. The cells were harvested,...