Fluorescent Brighteners with Nitrocellulose, Nylon, and Polyvinyl Chloride Membrane Filters for In Situ Staining of Fungi in Cultures

Abstract

Objective: To test the fluorescent brightening (FB) staining method, in which fungi are grown on cellulose nitrate filter membranes, dried, and rendered transparent with oil after staining with lactophenol cotton blue and to encourage use of FBs to eliminate the need for drying and clearing the membranes, therefore simplifying the procedure and reducing the time to perform it. Methods: Uvitex 2B (U2B) and 9 calcofluor white (CFW) reagents were used with 25 genera or species of fungi grown on nitrocellulose (NC), nylon (N), and polyvinyl chloride (PVC) filter membranes placed on a variety of growth media. For microscopic examination, membranes were removed from the surface of the growth medium and placed on microscope slides, with a drop of FB added to each; the preparations were then coverslipped and examined via violet light epi-illumination. Results: With many of the fungi tested, if growth was not completely inhibited, it was restricted on the PVC compared to that on the NC and N membranes and directly on a medium surface. The fluorescence of fungal structures and their contrast with the background were favorable with all 3 types of membranes and all the FBs tested. For staining yeasts on membranes from media containing polysorbate 80 (TWEEN 80), U2B is preferable to CFW because TWEEN 80 interferes with CFW staining. Conclusion: FB staining of fungi grown on NC or N membrane filters provides a rapid and simple method to examine fungal growth, especially young cultures, with minimal disturbance. * LPCB : lactophenol cotton blue; FBs : fluorescent brighteners; CFW : calcofluor white; U2B : uvitex 2B; NC : nitrocellulose; N : nylon; PVC : polyvinyl chloride; PBS : phosphate-buffered saline; cSt : centistokes