- Top of page
- Material and methods
- Bacterial strains and growth conditions
- Sporulation and spore purification
- Spore counts, purity evaluation and DNA staining
- Spore irradiation
- Culturability evaluation by plate counts
- Respiratory activity
- Membrane active pumping assessment
- Membrane integrity testing
- Levan digestion
- Fluorescence microscopy, image analysis and flow cytometry
- Statistical analyses
- Spore characteristics and culturability
- Respiratory activity assay
- Membrane energization
- Membrane permeabilization
- Levan degradation
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.