Bacteria Preservation: Refrigeration, Freezing and Freeze Drying
Between stock cultures, mutant strains, and genetically engineered variants, the number of individual bacterial cultures which any one lab can accumulate can be numerous. Indeed, the number of variations created in the process of engineering one plasmid can be astounding. And most labs will hold on to all those and other variations as you’ll never know what you might need tomorrow. Consequently, preserving all those bacterial cultures and genetic variants is something to be approached with thought.
A bacterial culture in a capped tube is in a closed environment. Though the culture may start healthy, given time the number of viable cells will decrease to zero. The goal of preserving the cultures is to slow that death rate so that when the culture is revisited, some of the cells are still viable and available for culturing. The reasons the cells die can be numerous, but in every instance are based on the inherent chemistry of the cells and their environment. If the deleterious chemical reactions can be slowed or halted, then the overall culture will remain viable for a longer period of time.
There are two basic approaches to slowing the rate of deleterious reactions in a culture of bacteria. The first is to lower the temperature which decreases the rate of all chemical reactions. This can be done using refrigerators, mechanical freezers, and liquid nitrogen freezers. The second option is to remove water from the culture, a process which can be tricky and involves sublimation of water using a lyophilizer.
Following is a brief discussion of the major options for preserving bacteria. The strengths and weakness of each option is reported.
Bacteria can survive for a short period of time at 4°C. For strains that are used daily or weekly, cultures grown on agar slants or plates can be stored in a refrigerator assuming that precaution has been taken to avoid contamination. Cultures should be prepared using standard techniques and then sealed before storing. For slants, we recommend using screw capped tubes. For cultures on Petri dishes, the plates need to be sealed with Parafilm. Sealing the plates not only helps to prevent molds from sneaking into the plates, but it slows the agar from drying. For anything over a week or two, cultures can be stored as stabs in small, flat-bottomed screw capped vials. In this technique, vials are filled with a small amount of agar medium (e.g., 1 ml) and sterilized. Bacteria are then introduced into the solidified agar with a sterile needle. The culture is incubated overnight with loose caps and then stored at 4°C with tight caps. Cultures stored in stabs are more resistant to drying and contamination, but they will lose viability more quickly than frozen stocks. The length of time a stab can remain viable is dependent upon the strain. Some manuals claim that stabs are good for a year however it is unwise to make that assumption unless it is tested.