Assessment of bacterial endospore viability with fluorescent dyes
Article first published online: 23 FEB 2004
Journal of Applied Microbiology
Volume 96, Issue 4, pages 684–692, April 2004
How to Cite
Laflamme, C., Lavigne, S., Ho, J. and Duchaine, C. (2004), Assessment of bacterial endospore viability with fluorescent dyes. Journal of Applied Microbiology, 96: 684–692. doi: 10.1111/j.1365-2672.2004.02184.x
- Issue published online: 23 FEB 2004
- Article first published online: 23 FEB 2004
- 2003/0172: received 28 February 2003, revised 29 October 2003 and accepted 30 October 2003
- Bacillus spores;
- flow cytometry;
- fluorescence microscopy;
- viability assays
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, DiBAC4(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 < 0·02).
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.