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Preservation of Cultures
Cultures of fungi frequently need to be stored for long periods, either to serve as reference strains or for repeated use in long-term experiments. The biggest danger is the possibility of cultural degeneration, leading to the loss of important characteristics such as pathogenicity or the ability to sporulate. In many laboratories the usual way to maintain cultures is frequent transfer onto agar media. If this is to be done, it is essential that a weak medium is used to reduce the risk of degeneration and to reduce the frequency of subculturing. Once cultures are established on fresh medium, they should be stored at low temperature (refrigerator or cold room). Several tubes or plates of each culture should be stored: these should be checked for signs of degeneration and contamination at every transfer and only those which retain wild type characteristics used to start the next generation of cultures.
If cultures are to be maintained for more than a few months, more specialised storage techniques should be used.
Lyophilisation, or freeze-drying, is the method of choice for long-term preservation of most fungi and is used routinely in most major culture collections. Its major drawback is the requirement for specialised equipment. There are also many fungi which cannot be freeze-dried successfully, especially Oomycetes. It is best suited to fungi which sporulate well in culture.
More than 12,000 isolates have been lyophilised at the Fusarium Research Laboratory since 1971 by drying colonised carnation leaf-pieces in small glass ampoules under high vacuum (10-1 - 10-2 Torr). The ampoules are prepared by inserting a small cotton wool plug and then autoclaving in a loosely covered beaker. Four leaf-pieces from a culture two to three weeks old and initiated from a single conidium are aseptically transferred to the ampoule which is replugged, labelled then heated and drawn out to an hour-glass shape using a gas torch. The lyophilising unit is equipped with a refrigerated vacuum chamber to enhance drying. After 18 hr the ampoules are sealed under high vacuum and stored at 5°C. All common species of Fusarium have been successfully lyophilised using this technique and have retained viability after 20 years storage.
Cultures can be revived by aseptically plating the dried leaf pieces onto CLA. The ampoule is first surface sterilised before it is shattered to release the leaf pieces.
Silica gel is the preferred method of storage in some laboratories because it does not require expensive apparatus and it is easy to use. Cultures can be repeatedly taken from a single storage tube, although contamination must be avoided. The major disadvantage is a gradual decline in viability but this can be overcome by replacement with fresh material. Survival of fungi preserved by this method depends upon an abundant production of conidia.
Silica gel (non-indicating) is dry heat sterilised at 180°C for 1.5 hr in screw cap culture tubes then pre-cooled in an ice bath before use. A concentrated conidial suspension is prepared by adding several colonised carnation leaf pieces to tubes containing 2 mL sterile skim milk. An aliquot, 0.3ml, of the suspension of conidia is distributed over 3cm3 of crystals to moisten them. Silica gel will fuse if too much moisture is added. A vortex mixer is used to redistribute the conidia and then the culture tubes are placed in an ice bath until they cool as heat is released in the reaction between moisture and silica gel. Both conidia and milk solids are absorbed onto the silica gel surface but the fungus does not colonise the substrate and so the chance of degeneration is restricted.
This is similar in many respects to silica gel storage. A loam soil with 20% moisture content is placed in small (approx 30 mL) glass universal bottles to about 1/3 full, and the bottle autoclaved twice 24 hours apart. One mL of spore suspension in sterile water is added, and the cultures left to grow for 2 days (fast growing species) to 10 days (slow growing species) at room temperature. The bottles are then stored in a refrigerator. Cultures are revived by sprinkling a few grains of soil onto fresh medium. Many fungi can survive for several years in soil storage. However, the method is less reliable than for silica gel storage because the fungus colonises the soil both before and during storage, increasing the potential for degeneration and the consequent loss of morphological characters.
This is a cheap, simple method that is particularly suitable for Pythium and Phytophthora. Agar blocks 5 mm square are cut from the margin of a young, actively growing fungal colony. These are placed in sterile water in a McCartney bottle and the cap screwed down. The bottles are stored under cool conditions (15° preferred). Cultures can be stored for 1-2 years, with some species surviving up to 5 years. Cultures are revived by removing a block of agar from the bottle and placing mycelium side down on fresh medium.
The dehydration of colonised leaf pieces over silica gel for 48 hr can be used as a temporary method of storage or for culture transfer through the postal system. The colonised leaf-pieces are taken from cultures approximately 2 weeks old and placed in small sterile (gamma irradiated) envelopes. During dehydration over silica-gel the envelopes are left partly open.
Cultures can be revived by placing the leaf pieces on CLA. Alternatively conidia in sporodochia on the dry leaf pieces can be used to initiate new cultures using the germinated single conidium transfer technique.
Holotype specimens grown on PDA are required when lodging a formal description of a species or subspecies.
Cultures are initiated from single germinated conidia and grown under standard conditions of temperature and light for 2 to 3 weeks. Cultures are then killed by exposing the plates to formalin in a closed container for 3 days. Preservation of the culture is achieved using agar and glycerine. Agar, 3 g is dissolved in 147 mL water, which is then dispensed as 6ml aliquots into test tubes before autoclaving. The lid of the culture dish is inverted, 1.5-1.75 mL glycerine is added and then the 6 mL aliquot of hot agar is poured over the glycerine. The culture is aseptically lifted from the Petri dish and floated on the mixture in the lid. Cultures are then allowed to dry in a drawer for 3-5 days covered with a sheet of white paper. When dried, the culture is rubbery and can be removed from the Petri dish for storage.
This procedure was refined for use with Fusarium species at the Fusarium Research Center, Pennsylvania State University.