A novel method for the whole-cell detection of environmental microorganisms using hemin and tyramide signal amplification (Hemin-TSA) with a desired fluorescent dye

A method called hemin-tyramide signal amplification (Hemin-TSA) was developed for visualization of environmental microorganisms using hemin and tyramide signal amplification. In Hemin-TSA, hemin, which has peroxidase activity, is bound to microbial cells, and a desired fluorescent dye is deposited on the microbial cells by a hemin-catalyzed TSA reaction. The protocol was initially optimized in terms of hemin concentration, hemin binding time and repeated reaction times of TSA. Hemin-TSA showed a comparative or improved signal-to-noise ratio compared to DAPI staining. The shapes of fluorescent signals obtained from microbial cells were almost morphologically identical to those observed in phase contrast microscopy. Hemin-TSA staining provided more accurate cell counts than DAPI staining, especially for actively growing cells, for which two or three spotty DAPI signals were obtained from a single cell. In addition, microbial cells that were not detected by DAPI staining were detected by Hemin-TSA with fluorescein, which enabled us to avoid high non-specific fluorescence under UV excitation. The method developed in this study allows us to visually detect microbial cells in various environments with the characteristics of better cell morphological identification, improved enumeration accuracy and selectivity of fluorescent dyes.