Purification and Thioflavin T Staining of Bacillus spp. Endospores for Fluorescence-Based Assays.

We present a simple, low-cost, and efficient protocol for fluorescent staining of Bacillus spp. endospores using the thioflavin T (ThT) dye. The article provides step-by-step procedures for culturing Bacillus paranthracis, Bacillus thuringiensis (2 strains), and Bacillus subtilis, purifying endospores from pure cultures, and staining them with ThT. ThT-stained endospores retain fluorescence for at least 2 years when stored at 4°C in the dark and remain fluorescent following formaldehyde fixation. Importantly, ThT staining does not impair spore viability, and stained endospores germinate normally. In contrast, vegetative Bacillus cells exhibit minimal to no ThT fluorescence. Overall, this robust and straightforward staining protocol is well suited for applications requiring fluorescently labeled endospores, including fluorescence microscopy, and provides a convenient plate-based readout for germinant screening and related assays. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Endospore purification Basic Protocol 2: Fluorescent staining of endospores Support Protocol: Fluorescence microscopy of ThT-stained spores.