Lyophilizing bacteria involves a series of steps to remove water from the bacterial culture while preserving the integrity and viability of the cells. Generally, a bacterial culture is first prepared in a suitable growth medium and allowed to reach the desired growth phase. Next, the culture is harvested, typically by centrifugation or filtration, to separate the bacterial cells from the growth medium. The cells are then resuspended in a cryoprotective solution containing substances like glycerol or sucrose to protect them during freezing and drying.
After resuspension, the bacterial suspension is rapidly frozen, often by immersion in liquid nitrogen or placement in a freeze-dryer with pre-cooled shelves. Rapid freezing is crucial to prevent the formation of large ice crystals that could damage the bacterial cells. Once frozen, the lyophilization chamber’s pressure is reduced, and heat is applied to induce sublimation, removing water directly from ice crystals in the bacterial suspension. This primary drying step is essential for removing the majority of the water content from the bacterial cells while maintaining their viability.
Following primary drying, residual bound water molecules are removed during secondary drying at slightly elevated temperatures. This ensures complete dehydration of the bacterial cells and enhances their stability during storage. Once lyophilization is complete, the dried bacterial cells are typically sealed in vials or containers under inert gas to prevent moisture uptake and contamination.