FLOW CYTOMETRIC ANALYSIS OF GERM TUBE FORMATION IN CANDIDA-ALBICANS

The transition from the blastospore to the hyphal phase in Candida albicans is an important step in the pathogenesis of candidiasis. We present the application of flow cytometry (FCM) to the analysis of germ tube (GT) formation in large numbers of yeast cells. FCM parameters of 90 degrees vs. forward angle light scatter, GT-specific fibrinogen (Fbn) binding fluorescence, and relative DNA content were used to provide a means of monitoring GT formation over time. FCM measurements of 90 degrees vs. forward angle light scatter distinguished blastospores from GTs, whereas detection of Fbn binding to GTs by indirect immunofluorescence was used to quantitate GT formation. Under conditions favoring germination, a five-fold average increase in Fbn binding was observed over a 120 min period in clinical and type culture strains of C. albicans, but not in a GT-deficient mutant of C. albicans. Cell sorting was used to confirm that Fbn binding correlated with percent GT formation and increased GT length. DNA analysis by FCM showed increasing DNA content in cells undergoing germination. Combined FCM measurements of light scatter, Fbn binding and DNA content provide a method of monitoring germination in entire yeast cell populations quickly and easily and provide a basis for correlation with other biological changes associated with germination.