As mentioned above, the temperature at which frozen bacteria are stored affects how long they can be stored while remaining viable. Freezing and thawing cells at an appropriate rate and maintaining the frozen stocks at the proper storage temperature help to minimize damage from the freezing process. Also, the greater the cell density, the better the recovery is after thawing the cells. For most bacteria, a density of 107 cells/mL will result in adequate recovery if all conditions are properly maintained.1-2
Cryoprotectants: As water in cells is converted to ice, solutes accumulate in the residual free water. This localized increase in salt concentration can denature biomolecules.3 Furthermore, ice crystal formation can damage cell membranes. Additives that are mixed with the bacterial suspension before freezing lower the freezing point and protect cells during freezing to minimize the detrimental effects of increased solute concentration and ice crystal formation. The most commonly used cryoprotectants are dimethylsulfoxide (DMSO) and glycerol, which are typically used at 5-15% (v/v). Non-permeable additives used as cryopreservants, such as polysaccharides, proteins and dextrans, adsorb to the surface of microorganisms and form a viscous layer that protects membranes, making these agents particularly useful for cryopreservation.4 Other commonly used additives include blood serum, ethylene glycol, methanol, skim milk, yeast extracts and tripticase soy.4
Freezing samples: To prepare glycerol stocks, the glycerol is first autoclaved and allowed to cool. The appropriate volume of glycerol is added to a suspension of log-phase bacteria and vortexed to dissociate the cells and ensure even mixing of the bacteria with the glycerol. After aliquoting the suspension into cryogenic screw-cap vials, the cells are snap-frozen by immersing the tubes in either ethanol-dry ice or liquid nitrogen and then stored in freezers (‑20 to -80°C) or liquid nitrogen (-150°C).5 Repeated thawing and refreezing of the bacterial stocks will reduce cell viability and should be avoided. When recovering strains with antibiotic selection markers, culturing them on selective media will ensure that the bacterial stocks were not contaminated.
Freeze drying: Bacteria can be freeze-dried by suspending log-phase cells in a lyophilization medium and then freeze drying the suspension. Not all bacteria can be successfully freeze-dried.6-8 Certain strains might not survive the process or die rapidly once freeze dried. The best way to determine if a strain is amenable to freeze drying is to empirically evaluate its stability post-freeze drying while maintaining a live culture as a backup. Once freeze dried, it is best to store the bacteria at or below 4°C.
Storing bacterial samples requires careful consideration about how they will be used, availability of the storage unit and space within the unit. The time it takes to prepare a sample for storage, how precious the sample is and whether the strain is compatible with the desired storage condition must also be considered. Furthermore, the storage unit must be carefully monitored, as inconsistent temperature fluxuations can often occur in institutional freezers commonly used in research facilities.9 Aseptic technique must be strictly observed during sample handling and stock preparation to avoid cross-contamination between strains and contamination from air movement and handling. Finally, good record keeping is critical for maintaining bacterial samples. Downloadable forms available online can help keep track of all events surrounding the handling of a specific sample. With careful planning and handling, you can ensure that your bacterial sample remains viable for the entire study period and for future studies.
1. Simione, F.P. and Brown, E.M. (1991). ATCC Preservation Methods: Freezing and Freeze Drying. American Type Culture Collection, Rockville, Maryland.
2. Simione, F.P. (1992). Key issues relating to the genetic stability and preservation of cells and cell banks. J Parenter Sci Technol 46:226-32.
3. De Paoli, P. (2005). Biobanking in microbiology: From sample collection to epidemiology, diagnosis and research. FEMS Microbiology Reviews 29:897-910
4. Huba'lek, Z. (2003). Protectants used in the cryopreservation of microorganisms. Cryobiology 46: 205-29.
5. Moore, L.W. and Rene, V. (1975). Liquid nitrogen storage of phytopathogenic bacteria. Phytophathology 65:246-50.
6. Miyamoto-Shinohara, Y., et al. (2008). Survival of freeze-dried bacteria. J Gen Appl Microbiol 54(1):9-24.
7. Miyamoto-Shinohara, Y., et al. (2006). Survival curves for microbial species stored by freeze-drying. Cryobiology. 52(1):27-32.
8. Miyamoto-Shinohara, Y., et al. (2000). Survival rate of microbes after freeze-drying and long-term storage. Cryobiology. 41(3):251-5.
9. Su, S.C., et al. (1996). Temperature variations in upright mechanical freezers. Cancer Epidemiol Biomarkers 5(2):139-40.