A Guide to 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.
Freezing is a good way to store bacteria. Generally, the colder the storage temperature, the longer the culture will retain viable cells. Freezers can be split into three categories: laboratory, ultralow, and cryogenic. The problem faced by bacteria (and other cells) stored in freezers is ice crystals. Ice can damage cells by dehydration caused by localized increases in salt concentration. As water is converted to ice, solutes accumulate in the residual free water and this high concentration of solutes can denature biomolecules. Ice can also rupture membranes, though this problem is more often associated with cells lacking walls, such as cultured animal cells. To lessen the negative effects of freezing, glycerol is often used as a cryoprotectant. Glycerol is produced by many fish and insects to defend against cold temperatures by depressing the freezing point of the cells, enhancing supercooling, and by protection from ice. With bacteria, adding glycerol to final concentration of 15% will help to keep cells viable under all freezing conditions (see this link for a protocol or this link for a ready to use freezing tube). The following are some specifics for each freezer category.
Laboratory freezers are those that can pull temperatures down to -20 to -40°C. These are single stage systems (one compressor) and often called general purpose freezers. Bacteria can be stored for moderate periods of time, e.g., 1 year, in general purpose freezers. It is best to use freezers without frost-free temperature cycling as this can wreak havoc on cells and other temperature sensitive biomolecules. General purpose freezers are inexpensive and found in most labs, thus they are readily available for storing cultures. The downside is that they are not sufficiently cold for long-term storage.
Ultralow freezers are two stage systems (two compressors each having a different refrigerant) which pull down to around -86°C. Ultralow freezers are very prevalent, but space in them can sometimes be limited and competitive. Ultralow freezers also are much more expensive to purchase, run and maintain. The upside is that cells stored at -80°C tend to remain viable for several years. The lower temperature generated by ultralow freezers substantially reduces chemical reactions within the culture. However, molecular motion still occurs in frozen cells and thus the viability of the culture will decline. It is important to regularly monitor cultures to assess their level of viability.
Cryogenic freezers are very cold and rely on liquid nitrogen or specialized mechanical systems to operate. For biological samples, cryogenic storage should be below -130°C. At this temperature, the molecular motion of water is halted and cells are trapped in a glass-like matrix. Bacteria stored in cryogenic freezers retain their viability for many years. In our laboratory bacterial and yeast cultures have been maintained at -140°C for 15 years without significant loss of viability. Storing cells in cryogenic freezers is the most effective and, as compared to freeze drying, the easiest method for long-term storage. The downside is cost and potential vulnerability of stocks to power outages, mechanical failures, and failed deliveries of liquid nitrogen. Additionally, tubes should never be stored in tanks submersed in liquid nitrogen. Screw cap tubes leak and will pull the nitrogen into the tube along with contaminants ( see link for more information). Liquid nitrogen vapor phase freezers will effectively avoid this problem, but these freezers are very expensive (upwards of $10K) and require large volumes of liquid nitrogen. An alternative is mechanical cryogenic freezers that can go as low as -150°C, but these are also very expensive to purchase (about $20K). Both cryogenic freezers will cost several hundred dollars a month to operate.
In an aqueous system, such as a living cell, water not only serves as the medium for enzymatic reactions, but also spontaneous negative reactions such as free radical formation. Removing water halts both enzymatic and non-enzymatic reactions. Freeze drying is one method of removing this water. Many bacteria can be preserved very effectively by freeze drying. By freezing the cells in a medium that contains a lyoprotectant (usually sucrose) and then pulling the water out using a vacuum (sublimation), cells can be effectively preserved. This method is laborious and requires specialized equipment, but it has the advantage of generating stock cultures that are unaffected by power outages and empty liquid nitrogen tanks. Furthermore, if cultures are routinely shipped to other labs, freeze dried cultures do not require special handling. The downside on freeze drying is that not all cultures react the same way thus some experimentation is required to optimize the process for each strain. For any lab which is serious about producing and maintaining a culture collection, then freeze drying should be included as a major method for preservation.
Details on freeze drying bacteria can be found on the webpage Bacteria Freeze Drying Protocol.