A fast and cost-effective methodology for Fonsecaea pedrosoi ATCC46428 staining using ESIPT fluorescent dyes.

The microscopic morphology of Fonsecaea pedrosoi ATCC46428 was observed using two benzazole derivatives, 2-(2'-hydroxyphenyl)benzoxazole and 2-(5'-amino-2'-hydroxyphenyl)benzoxazole, which emit intense fluorescence by a proton transfer mechanism in the electronically excited state (ESIPT). The cell surface could be successfully stained with fluorescent dye solutions of 10 microM-10 mM using two different fast and cost-effective procedures. At these concentrations, any structure or dye crystallization could be observed. Concerning the external microstructural details, only the amino derivative allowed the differentiation between hyphae and conidia. These dyes presented some advantages comparing to commercial dyes, since the stained cells showed high chemical, thermal and photochemical stability during the experiments and also after several months of storage at room temperature and normal light exposition. Procedure 1 presented the advantage to be used when heating can change the chemical or biochemical cell composition. On the other hand Procedure 2 showed to be useful as a routine methodology for cells staining. The results allowed to propose a simple and highly sensitive assay to study the F. pedrosoi micromorphology by epifluorescence microscopy. This methodology can probably be extended for other fungi of clinical interest.