Cryoprotection

Cryopreservation refers to the use of a very low temperature (below approximately –130° C [–202° F]) to store a living organism. Organisms (including many types of bacteria , yeast , fungi , and algae) can be frozen for long periods of time and then recovered for subsequent use.

This form of long-term storage minimizes the chances of change to the microorganism during storage. Even at refrigeration temperature, many microorganisms can grow slowly and so might become altered during storage. This behavior has been described for strains of Pseudomonas aeruginosa that produce an external slime layer. When grown on a solid agar surface, the colonies of such strains appear like mucous drops. However, when recovered from refrigeration storage, the mucoid appearance can be lost. Cryopreservation of mucoid strains maintains the mucoid characteristic.

Cryostorage of bacteria must be done at or below the temperature of –130° C [–202° F], as it is at this temperature that frozen water can form crystals. Because much of the interior of a bacterium and much of the surrounding membrane(s) are made of water, crystal formation would be disastrous to the cell. The formation of crystals would destroy structure, which would in turn destroy function.

Ultralow temperature freezers have been developed that achieve a temperature of –130° C . Another popular option for cryopreservation is to immerse the sample in a compound called liquid nitrogen. Using liquid nitrogen, a temperature of –196° C [–320.8° F] can be achieved.

Another feature of bacteria that must be taken into account during cryopreservation is called osmotic pressure. This refers to the balance of ions on the outside versus the inside of the cell. An imbalance in osmotic pressure can cause water to flow out of or into a bacterium. The resulting shrinkage or ballooning of the bacterium can be lethal.

To protect against crystal formation and osmotic pressure shock to the bacteria, bacterial suspensions are typically prepared in a so-called cryoprotectant solution. Glycerol is an effective cryoprotective agent for many bacteria. For other bacteria, such as cyanobacteria, methanol and dimethyl sulfoxide are more suitable.

The microorganisms used in the cryoprotection process should be in robust health. Bacteria, for example, should be obtained from the point in their growth cycle where they are actively growing and divided. In conventional liquid growth media, this is described as the mid-logarithmic phase of growth. In older cultures, where nutrients are becoming depleted and waste products are accumulating, the cells can deteriorate and change their characteristics.

For bacteria, the cryoprotectant solution is added directly to an agar culture of the bacteria of interest and bacteria are gently dislodged into the solution. Alternately, bacteria in a liquid culture can be centrifuged and the "pellet" of bacteria resuspended in the cryoprotectant solution. The resulting bacterial suspension is then added to several specially designed cryovials. These are made of plastic that can withstand the ultralow temperature.

The freezing process is done as quickly as possible to minimize crystal formation. This is also referred to as "snap freezing." Bacterial suspensions in t cryoprotectant are initially at room temperature. Each suspension is deep-frozen in a step-wise manner. First, the suspensions are chilled to refrigerator temperature. Next, they are stored for a few hours at –70° C [–94° F]. Finally, racks of cryovials are either put into the ultralow temperature freezer or plunged into liquid nitrogen. The liquid nitrogen almost instantaneously brings the samples to –196° C [–320.8° F]. Once at this point, the samples can be stored indefinitely.

Recovery from cryostorage must also be rapid to avoid crystal formation. Each suspension is warmed rapidly to room temperature. The bacteria are immediately recovered by centrifugation and the pellet of bacteria is resuspended in fresh growth medium. The suspension is allowed to adapt to the new temperature for a few days before being used.

Cryoprotection can be used for other purposes than the long-term storage of samples. For example, cryoelectron microscopy involves the rapid freezing of a sample and examination of portions of the sample in an electro microscope under conditions where the ultralow temperature is maintained. If done correctly, cryoelectron microscopy will revel features of microorganisms that are not otherwise evident in conventional electron microscopy. For example, the watery glycocalyx , which is made of chains of sugar, collapses onto the surface of a bacterium as the sample is dried out during preparation for conventional electron microscopy. But glycocalyx structure can be cryopreserved. In another example, cryoelectron microscopy has also maintained external structural order on virus particles, allowing researchers to deduce how these structures function in the viral infection of tissue.

See also Bacterial ultrastructure; Donnan equilibrium; Quality control in microbiology