Assessment of bacterial endospore viability with fluorescent dyes

Abstract

Aim: To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. Methods and Results: Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC 4 (3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P < 0AE02). Conclusion: It is possible to use CTC and Live/Dead BacLight to rapidly test endospore viability and evaluate the proportion of spores in a preparation that could not be recovered with plate count. Significance and Impact of the Study: This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.

DNA unmasked in the red rain cells of Kerala

“…5c, d). This is consistent with DAPI-DNA complex specific fluorescence (Banerjee & Pal, 2008;Laflamme et al, 2004;Tanious et al, 1992;Setlow et al, 1991;Hamada & Fujita, 1983), indicating the presence of DNA in the core of red rain cells. Certain nanoparticles fluorescing in blue are also apparent near the periphery of these cells (Fig.…”

Flow cytometry analysis of germinating Bacillus spores, using membrane potential dye

“…Moreover, no germinating spores were observed (phase-dark spores), i.e. all spores were phase-bright spores (Laflamme et al 2004). Obtaining a homogenous preparation is important, since this study emphasizes on the early detection of Bacillus spore germination.…”

“…Some of these assays have been validated directly on Bacillus spores (Comas-Riu and Vives-Rego 2002; Laflamme et al 2004). Although those tests gave new information about spores, they did not provide rapid information on the germination potential of the organism.…”

Low temperature atmospheric pressure plasma sources for microbial decontamination

“…The bacteria with intact membrane are able to exclude the dye, while bacteria with damaged membrane take up the coloring agent. This allows a differentiation by fluorescent signals between healthy and non living microorganisms (LaFlamme et al, 2004). Proof for demolished membranes or other morphological disorders can also be given by electron microscopy (Laroussi et al, 2003) or atomic force microscopy (Pompl et al, 2009;Hähnel et al, 2010a;Kuo et al, 2006 Boucher, 1985;ISO 17665, International Standard, 2006).…”