Flow cytometric analysis of the DNA synthetic cycle of Candida species

Abstract

The total DNA per cell and DNA synthetic cycle phases were determined by flow cytometry in five Candida isolates including three species: Candida albicans 208R1, Candida tropicalis ATCC 750, and Candida parapsilosis 970, 3138, and ATCC 22019. The cells were prepared for flow cytometry by fixation in Carnoy fixative followed by staining with mithramycin. Marked but stable and reproducible inter- and intraspecific differences in total DNA per cell of stationary-phase cultures were found which did not correlate directly to diphenylamine estimates of the same parameter. This discrepancy was resolved by mathematically converting flow cytometry data into diphenylamine data. The reason for the discrepancy was found in studies of the DNA synthetic cycle of these yeasts: a large but isolate-specific variable proportion of the population is arrested in the S and G2-M phases after the culture passes from exponential to stationary phase. Histograms of exponential-growth-phase Candida isolates demonstrate that the majority of the population is in the G2-M phase of the DNA synthetic cycle. The DNA content of the C. tropicalis and C. parapsilosis isolates studied is as high as or higher than that of C. albicans. Extranuclear fluorescent particles were observed in the C. tropicalis isolate. No equivalent particles could be detected in the other four Candida isolates. The nature of the particles is unknown.