FLOW CYTOMETRIC QUANTITATION OF YEAST A NOVEL TECHNIQUE FOR USE IN ANIMAL-MODEL WORK AND IN-VITRO IMMUNOLOGICAL ASSAYS

Animal models of fungal and other infectious diseases often require th at the number of organisms in tissue be quantified, traditionally by g rinding organs, plating them on agar and counting colony forming units (CFU). This method is labor intensive, slow as some fungi require two weeks of culture and limited in reliability by poor plating efficienc y. To circumvent these problems, we developed a flow cytometric method to quantify yeast. In vitro cultured Blastomyces dermatitidis, Crypto coccus neoformans, Candida albicans and Histoplasma capsulatum yeast w ere labelled with specific monoclonal or polyclonal antibodies to stai n surface determinants or with Calcofluor to stain cell-wall chitin, A defined number of fluorescently labelled beads were added prior to ac quisition by flow cytometry as a reference standard for quantitation. Beads were readily distinguished from yeast by forward scatter, side s catter and intensity of fluorescence. Cultured yeast were enumerated b y both standard CFU determination and flow cytometry in a range of 10( 2) to 10(7) cells. Only flow cytometry enabled discrimination of live and dead yeast by using appropriate fluorescent dyes. The flow cytomet ric method was applied to murine models of histoplasmosis and blastomy cosis to quantify the burden of fungi in the lungs of infected mice. L abelling yeast with Calcofluor alone resulted in unacceptably high lev els of nonspecific binding to mouse cell debris. In contrast, labellin g H. capsulatum with a rabbit polyclonal antiserum and B. dermatitidis with a monoclonal antibody to the surface protein WI-1 permitted accu rate quantitation. We conclude that this flow cytometry technique is r apid, efficient and reliable for quantifying the burden of infection i n animal models of fungal disease. The technique also should lend itse lf to performing cytotoxicity assays that require discrimination of li ve and dead fungi, or phagocytosis assays that require discrimination of intracellular and extracellular organisms. (C) 1998 Elsevier Scienc e B.V.