Amyloid Histology Stain for Rapid Bacterial Endospore Imaging

Abstract

Bacterial endospores are some of the most resilient forms of life known to us, with their persistent survival capability resulting from a complex and effective structural organization. The outer membrane of endospores is surrounded by the densely packed endospore coat and exosporium, containing amyloid or amyloid-like proteins. In fact, it is the impenetrable composition of the endospore coat and the exosporium that makes staining methodologies for endospore detection complex and challenging. Therefore, a plausible strategy for facile and expedient staining would be to target components of the protective surface layers of the endospores. Instead of targeting endogenous markers encapsulated in the spores, here we demonstrated staining of these dormant life entities that targets the amyloid domains, i.e., the very surface components that make the coats of these species impenetrable. Using an amyloid staining dye, thioflavin T (ThT), we examined this strategy.

Dynamic staining of Bacillus endospores with Thioflavin T

“…Following the methods we previously reported [10,12], we inoculated the Bacilli, sporulated the cells, and purified the endospores. Bright field and epifluorescence microscopy allowed us to examine the samples (Fig.…”

“…Bright field and epifluorescence microscopy allowed us to examine the samples (Fig. 1a, b) [10,12]. We recorded the kinetics of fluorescence staining with ThT using protocols developed in our lab [11].…”

“…Furthermore, we previously reported no observable change on the endospores' ability to germinate after exposure to ThT [10].…”

“…post-treatment steps for the visualization of endospores [10]. Furthermore, we previously reported no observable change on the endospores' ability to germinate after exposure to ThT [10].…”

Detection and differentiation of foodborne pathogenic bacteria in mung bean sprouts using field deployable label-free SERS devices

“…For example, PCR is based on nucleic acid amplication and consequently cannot discriminate nucleic acid amplied from viable and nonviable bacteria. 22 As one of the feasible way to realize "real-time" detection, Raman spectroscopy has the potential for rapid detection of a wide range of chemical and biological substances. Immunological detection, such as enzymelinked immunosorbent assay (ELISA), has the advantage of being specic to bacterial type and strain, but it also requires multiple steps, varied chemical reagents, and incubation time making this method impractical for "real-time" detection in the eld.…”