Generally, freezing a suspension of living cells may result in a decrease in viability.16, 17 As the bacterial cell suspension is cooled below the freezing point, ice crystals begin to form and the concentration of solutes in the suspension increases. The formation of intracellular ice crystals can result in the damage of cellular structures. This effect can be minimized if water within the cells is allowed to escape by osmosis during the cooling process. A slow cooling rate, generally -1°C to -10°C per minute, facilitates this progression. However, as the cells lose water, they shrink in size and will quickly lose viability if they surpass a minimum threshold volume. The addition of cryoprotectant agents, such as glycerol or dimethylsulfoxide (DMSO), will mitigate these effects.18, 19 For the preservation of bacterial cultures, ATCC recommends using glycerol as a cryoprotectant.
The standard procedure for cryopreservation is to freeze cells slowly until they reach a temperature below -70°C in medium that contains a cryoprotectant. Vials are then transferred to a liquid-nitrogen freezer to maintain cultures at temperatures below -130°C.
The recovery of cryopreserved cells requires the rapid thawing of the bacterial suspension in a 37°C water bath. The entire contents of the vial are then transferred to an appropriate growth medium. There are numerous factors that can affect the viability of recovered bacterial strains. These critical parameters can include the composition of the freeze material, the growth phase of the bacterial strain, or the concentration of bacterial cells within the solution. To obtain optimal cell viability upon recovery, modify the cryopreservation procedure for each bacterial strain, being sure to harvest cultures during the late logarithmic phase of growth.
Contact ATCC for more information on the cryopreservation of bacterial strains. ATCC bacterial strains that are frozen are commonly cryopreserved with a medium consisting of a final concentration of 10% sterile glycerol.
Glycerol and DMSO are the most common cryoprotectant agents. ATCC predominantly recommends using a 20% glycerol stock at a final concentration of 10%. However, if the bacterial strain is sensitive to glycerol, a 50% DMSO stock can be used at a final concentration of 5%. In contrast, glycerol can be sterilized via autoclavation whereas DMSO must be sterilized by filtration. Care should be used when handling any DMSO solution as it will rapidly penetrate intact skin and may carry toxic contaminants along with it.
Use only reagent-grade DMSO or glycerol. Store both in aliquots protected from light. ATCC offers DMSO (ATCC® 4-X™) that has been thoroughly tested for use, as well as TSB with 10% glycerol (ATCC® 20-2200™) for the cryopreservation of non-fastidious bacteria. Overall, the optimum formulations for individual bacterial strains must be determined empirically.
- Cryopreservation vials
There are two materials to choose from for cryopreservation vials: glass or plastic. Glass vials are more difficult to work with; they need to be sterilized before use, they need to be sealed with a hot flame, and they can be difficult to open. However, they are preferred for long-term storage (many years) of valuable cultures and are considered fail-safe once properly sealed.
If cryopreservation in glass ampoules is not possible, plastic vials can be used. Plastic vials come in two varieties: those with an internal thread and silicone gasket, and those with an external thread.
Vials with an internal-thread were the first commercially available, but have some disadvantages over the external-thread version. For example, while the silicone gasket provides an excellent seal, it needs to be tightened just right; the vial will leak if the seal is too tight or too loose. For the storage of cryopreserved stocks, ATCC uses plastic vials.
- Controlled-rate freezing chambers
There are several means to achieve a cooling rate of -1°C per minute. The best method involves the use of a computer controlled, programmable electronic freezing unit (such as Thermo Scientific* CryoMed Freezers), which rigorously maintains this rate of cooling. This is the method used exclusively at ATCC. Such equipment is relatively expensive and absolutely necessary for only the most sensitive strains.
A less costly approach is to place the cryopreservation vials into an insulated chamber and cool for 24 hours in a mechanical freezer at -70°C or colder. There are several commercially available freezing chambers which achieve a cooling rate very close to the ideal -1°C per minute (CoolCell® LX; ATCC® ACS-6000™). Alternatively, the vials can be placed into a polystyrene box, with 15 mm (3/4 inch) thick walls and 1L capacity that is packed with paper, cotton wool, or foam peanuts for insulation.
Liquid Nitrogen Freezing Storage
The ultra-low temperatures (below -130°C) required for long-term storage can be maintained by specialized electric freezers, or more commonly, by liquid nitrogen freezers. There are two basic types of liquid nitrogen storage systems: immersing vials in the liquid or holding vials in the vapor phase above the liquid. The liquid-phase system holds more nitrogen and thus requires less maintenance. However, there is always a chance that some liquid will enter improperly sealed vials, which may then explode when retrieved. For this reason, ATCC strongly recommends storage in vapor-phase.
Vapor-phase storage systems create a vertical temperature gradient within the liquid nitrogen container. The temperature at the bottom of the container will be -196°C, whereas the temperature at the top will vary depending upon the amount of liquid nitrogen at the bottom and the length of time the container is open. To ensure the safe storage of cells, maintain sufficient levels of liquid nitrogen in the container so that the temperature at the top is -130°C or colder. All storage systems should be equipped with temperature alarms.
The procedure below will work for most non-fastidious bacterial strains and should be modified as needed. Additional information on the preservation of fastidious bacterial strains can be found in the latter segment of this chapter. Freeze medium formulations for ATCC bacterial strains can be found on the ATCC website.
- In preparation for freezing, grow the bacterial strain under
optimal conditions in an appropriate medium as to retain the
salient features of the strain. Bacterial strains should be grown
to late log phase.
- When freezing bacteria, add 5 to 10% glycerol or DMSO in culture medium. Glycerol is usually prepared
in aqueous solution at double the desired final concentration for freezing. It is then mixed with an
equal amount of cell suspension.
- Label the appropriate number of vials with the name of the bacterial strain and the date. Aliquot 1 to
1.8 mL of the bacterial suspension to each vial and seal. Seal plastic ampoules tightly with the screw
cap. Seal glass ampoules with a gas-oxygen torch, pulling the neck of the ampoule as it is rotated in
- Allow the cells to equilibrate in the freeze medium at room temperature for a minimum of 15 minutes
but no longer than 40. After 40 minutes, cell viability may decline if DMSO is used as the cryoprotectant.
- Place the vials into a pre-cooled (4°C), controlled-rate freeze chamber and place the chamber
in a mechanical freezer at -70°C (or colder) for at least 24 hours. Alternately, use a pre-cooled (4°C)
programmable freezer unit set to cool the vials at -1°C per minute until a temperature below -40°C is
achieved and then set the temperature to abruptly drop to -130°C.
- Quickly transfer the vials to liquid nitrogen or a -130°C freezer.
- Record the location and details of the freeze.
- After 24 hours at -130°C, remove one vial, restore the bacterial strain in culture medium, and determine
the viability and sterility.
Recovery of Cryopreserved Cells
The frozen bacterial solution needs to be warmed as rapidly as possible and then immediately transferred to an appropriate growth medium. Some bacterial strains may take more time than normal to fully recover from cryopreservation.
- Prepare a culture vessel that contains at least 10 mL of the appropriate growth medium equilibrated
for both temperature and pH.
- Remove the vial from the liquid nitrogen freezer and thaw by gentile agitation in a 37°C water bath (or
a bath set at the normal growth temperature for that bacterial strain). Thaw the strain rapidly until all
ice crystals have melted (approximately 2 minutes).
- Remove the vial from the water bath and decontaminate it by dipping in or spraying with 70% ethanol.
Follow strict aseptic conditions in a laminar flow hood for all further manipulations.
- Unscrew the top of the vial and transfer the entire contents to the prepared growth medium.
- Examine the cultures after an appropriate length of time.