This 1963 study examined how water activity, solutes, and temperature affect the viability and heat resistance of freeze-dried bacterial spores from four species: Bacillus megaterium, B. stearothermophilus, Clostridium bifermentans, and C. botulinum type E. Spores stored at water activity values between 0.2 and 0.8 at 25°C maintained excellent viability over 6 years in both air and vacuum. However, storage at extremely low water activity (0.00) or very high moisture (1.00) caused significant viability loss. Notably, aerobic Bacillus species showed viability loss in vacuum at water activity 1.00, while anaerobic Clostridium species showed loss in air at the same conditions. When certain solutes—particularly carbonyl compounds like DL-glyceraldehyde and diacetyl—were added before drying, they induced spore death during storage, with effects more pronounced at higher temperatures and water activities. Heat resistance measurements after storage revealed that spores maintained at intermediate water activities (0.4-0.8) best retained their original heat resistance. The authors recommend storage at water activities between 0.2 and 0.43 in vacuum for optimal preservation of both viability and heat resistance.

Key findings

• Freeze-dried bacterial spores maintained stable viability when stored at water activity values between 0.2-0.8 for over 6 years at 25°C, but showed marked viability loss at extremely dry (aw 0.00) or saturated (aw 1.00) conditions.
• Aerobic Bacillus species lost viability in vacuum at saturated moisture, while anaerobic Clostridium species lost viability in air under the same conditions, suggesting differential sensitivity to oxygen during germination.
• Certain low molecular weight solutes, particularly carbonyl compounds (glyceraldehyde, diacetyl), induced spore death during storage at otherwise favorable water activities, with effects accelerated at higher temperatures.
• Storage in vacuum generally better preserved the original heat resistance of spores compared to storage in air, especially at intermediate water activity values (0.4-0.8).
• For optimal preservation of freeze-dried spores, storage at water activity values between 0.2-0.43 in vacuum is recommended.