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

Authors

Ephrem Debebe Zegeye, Marina Aspholm

Published in

Current protocols. Volume 6. Issue 10. Pages e70480.

Abstract

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.

PMID:
42832334
Bibliographic data and abstract were imported from PubMed on 06 Oct 2026.

Read full publication at:
Please sign in to see all details.

Stats

1-terrible, 9-excellent. How would you rate this publication? Sign in in to submit your rating.

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.