Flow cytometric detection of yeast by in situ hybridization with a fluorescent ribosomal RNA probe

Abstract

An in situ hybridization procedure with a fluorescent RNA probe applied to yeast detection by flow cytometry is described. Saccharomyces ceerevisiae cell were permeabilized to allow the use of a biotinylated rRNA probe of about ≈ 100 nucleotides, by formaldehyde treatment, followed by a limited enzymatic hydrolysis of the cell wall. Hybridization was performed with cells in suspension and hybrids were detected with streptavidin-FITC (fluorescein isothiocyanate) by microscopy and flow cytometry. The signal obtained with yeast antisense rRNA probe was 30 times higher than the nonspecific signal obtained with a bacterial sense rRNA probe. The yeast-specific signal was not eliminated after posthybridization RNase treatment althoug it completely disappeared when cells were treated with RNase before hybirdization, confirming the rRNA was the target. This study is the first demonstration of the use of in situ hybridization for yeast detection by flow cytomertry. With appropriate probes, the procedure could be used also for detection of specific yeast populations.