Comparative Analysis of Thioflavin T and other Fluorescent Dyes for Fluorescent Staining of Bacillus subtilis Vegetative Cell, Sporulating Cell, and Mature Spore.

Thioflavin T, a cationic benzothiazole dye, is typically used to detect the amyloid fibrils. In this study, we analyzed the staining properties of B. subtilis cells using several fluorescent dyes, including Thioflavin T analogues, 2-(4'-Methylaminophenyl) benzothiazole (BTA-1), and 2-(4-aminophenyl) benzothiazole (APBT). Thioflavin T stained vegetative cells in the early log phase and outer layer structures of forespores and mature spores. The inner parts of forespores and heat-killed mature spores were also stained with Thioflavin T. Congo red, auramine O, and rhodamine B stained forespores and mature spores similar to Thioflavin T. In contrast, APBT and BTA-1 fluorescence was detected in the outer layers of vegetative cells, mother cells, forespores, and mature spores, indicating that they bind to the cell membrane and/or cell wall. The combination of the fluorescent dyes used in this study will help analyze morphogenetic processes during sporulation and the damage mechanisms of vegetative cells and spores.