In situ fluorescent staining of yeast biofilms

Studies of microbial biofilms have focused mostly on bacterial attachment, resulting in an underdeveloped knowledge base for other microorganisms, including yeasts. Comprehension of yeast biofilms may be critical for therapeutic, control and exploitation strategies in clinical, industrial and natural environments. Visualization techniques form a generic part of biofilm studies, and recent development of fluorescent probes have enhanced localization, quantification and metabolic aspects of microorganisms. Limited application of these probes in yeast biofilms, however, has been reported. We evaluated a series of cell wall stains, live/dead probes, and yeast mitochondrial probes using both planktonic and biofilm cells of species of Cryptococcus , Saccharomyces and Schizosaccharomyces , including comparison with a filamentous fungus, Penicillium glabrum . Our results confirm the difference in metabolic functioning of planktonic versus attached matrix‐enclosed cells, with different concentrations of fluorescent probes, as well as staining periods, applicable to the two microbial lifestyles. Shortcomings in fluorophore properties, with fast photobleaching, and background fluorescence, are also indicated. Exopolymeric interference in visualization, and recalcitrance of biofilms to penetration of stains, are highlighted. In conclusion, the spectrum of parameter variation, and deviation from supplier information, exemplify the need for empirical evaluation before significant results can be obtained when staining biofilms.