Preservation of Freeze-Dried Cultures

Microorganisms for quality control work should be purchased from an international culture collection registered with ECCO or the WDCM. Collections interested in joining the European Culture Collection's Organization (ECCO) have to apply for membership, and have to provide documentation, while the World Data Center on Microorganisms is a worldwide register of Institutions holding microorganisms, without criteria for acceptance. We therefore recommend only the ECCO- or WDCM- Collections distributing a recent (max. 3 year-old) Catalogue of Cultures. The CCUG would be pleased to provide coordinates to the ECCO collections.

End-user's preservation of subcultures for quality control
Most microbiological laboratories have to maintain microorganisms to be able to fullfill quality control criteria set up by different official bodies. Some of the microorganisms in common use are robust, other are fragile. Since the quality controls are aiming at checking quite different parameters, from simple growth controls to much more sophisticated analysis, the storage conditions must be adequate for the preservation of cell structures, genes and plasmids.

Preservation in liquid nitrogen (at very low temperature) is for most tissues and microorganisms the optimal method. Freeze-drying is the preferred method for long-term preservation of microorganisms bound to be distributed through ordinary mail. Most lyophilized cultures will remain viable for decades or centuries if kept in the dark at a temperature of +4°C to +8°C. (Some fragile organisms like Helicobacter constitute an important exception, and storage of freeze-dried Helicobacter pylori should not exceed 3 years). However, some ampoules may not have been sealed perfectly and other ampoules may crack during storage. We therefore recommend to revive the cultures distributed within a few weeks.

The most widespread method for local preservation is probably the 'bead' technic. The CCUG gives the following general recommendation for the maximum storage time for microorganisms frozen in small aliquots or on beads kept in freezers under optimal conditions. In case of malfunction of the freezer the time limits must be cut considerably down, and in case of thawing, the 'microbank' should be reconstituted from original lyophilized cultures distributed by international culture collections. Customers are encouraged to purchase 2 or more ampoules of the same organism at the same time at substantially reduce prices. Duplicate ampoules may be kept for decades and will be immediately available to the customer in case of freezer breakdown.

Principle of the 'bead technic': ".... Individual coloured beads are packaged approximately 25 beads in a cryovial containing cryopreservative. The beads are acid washed and are of a porous nature allowing microorganisms to readily adhere onto the bead surface. After inoculation (and removal of excess fluid) the cryovials are kept at -70°C for extended storage. When a fresh culture is required, a single bead is easily removed from the vial and used to directly inoculate a suitable bacteriological medium." (Description of the Pro-Lab Diagnostics Microbank (Product Code PL 160))

Alternative method:
The CCUG also recommends freezing in small aliquots. The suspending fluid used for freeze-drying supplied by our Medium Department (Phone: 031.342 4651 or Substrat) is excellent:
Horse serum 300 ml
Glucose, pro analysi 30 g
'Lab Lemco' Oxoid L29 0.5 g
Bacto peptone Difco 0018 0.8 g
Water, high purity 100 ml

Sterile-filtered 'Mist. desiccans' Fry and Greaves, 1951 (NCTC formulation)