6.2 By freezing

Freezing damages bacterial cells due to the localised increase in salt concentration and the damage to cell membranes caused by the forming of ice crystals. Despite this, most bacteria survive well.

Key factors to successful bacterial freezing are:

• a large initial cell density of at least 10⁷ CFU per mL
• using cryoprotectants for freezing (e.g. glycerol; DMSO)
• ‘snap freezing’.

Reductions in bacterial viability occur during the initial freezing and each time the bacteria are thawed. Thus, the greater the initial cell density and the more protected the cells, the better the isolate’s survivability. Efficient bacterial freezing kits are also available on the market.

It is a good idea to preserve each isolate in several screw-cap cryogenic vials so that the same sample is not taken out of the freezer each time the isolate is required. It is not necessary to thaw the whole vial each time the isolate is needed – the frozen surface can be scraped to obtain a small amount of bacteria.

Snap freezing in dry ice or liquid nitrogen limits cell damage. The frozen bacteria can then be stored between ‑20°C and -80°C, or in liquid nitrogen at -150°C. If snap freezing is not possible, it is acceptable to put unfrozen bacteria directly into -70°C or -80°C or into liquid nitrogen. Direct storage at -20°C is not recommended as it is likely to drastically reduce viability.