An improved method for the selective detection of fungi in hospital waters by solid phase cytometry

Yeast cells and mould spores can be fluorescently labelled with the viability stain carboxyfluorescein diacetate (CFDA) and detected on a membrane filter by laser scanning (solid phase cytometry, SPC). Although the selectivity of an existing commercial SPC procedure for fungi is ensured by using a 2 microm pore size membrane filter and a pre-incubation on a proprietary spore swelling/activation medium, some bacteria are still co-detected. In the present study, the selectivity for fungi has been enhanced by combining the green fluorescent CFDA with a second red fluorescent label, i.e. TRITC-concanavalin A, targetting fungal but not commonly bacterial cells. Additional improvements resulted from the prolongation of the pre-incubation and from the extra-rinsing of the membrane filter. The improved method was applied to detect fungi in hospital waters, dialysis fluids and endoscopic rinse waters. In general, SPC detected more fungi in water than plate methods. The occurrence of fungi in dialysis fluid and endoscopic rinse water was rare. Evidence for the presence of fungal viable but non-culturable (VBNC) cells in water was weak.