Evaluation of endospore purification methods applied to Bacillus cereus

Research on spore inactivation techniques requires highly pure spore suspensions, but the efficacy of commonly used purification methods has not been systematically compared. By providing this comparison, the present study will help researchers improve the consistency and validity of their experiments. The current study evaluated alternative purification methodologies applied to Bacillus cereus endospores on the basis of criteria of purity, yield, time investment and effect on integrity of spores. The ASTM E2111-00, ethanol, heat shock, daily water washes and lysozyme treatment methods were applied to B. cereus cultures incubated for 3 and 10 days. Purities achieved ranged from 67 to 99% spores as a proportion of total colony forming units in the purified suspension, whereas yields ranged from less than 1 to 39% spores in proportion to total colony forming units of B. cereus culture. TEM images showed that only lysozyme treatment substantially altered spore morphology. Ethanol treatment is optimum for 3-day B. cereus cultures, whereas the ASTM method is most favorable for 10-day-old cultures. Lysozyme treatment is the least attractive purification method because of its large time investment and damage to spores.