FGSC culture preservation methods

(FGN 33:47-48, 1986)
C.H. Wilson

FGSC has collected and stored strains of Neurospora and Aspergillus since 1961. The storage methods described here were developed before then, but the experience of preserving several thousand strains has demonstrated the effectiveness and convenience they provide in preserving conidial ascomycetes.

Most strains of Neurospora and Aspergillus produce large numbers of conidia, and can be routinely stored in silica gel and in lyophil. Both methods are employed for such strains. Upon receipt of a new culture, a transfer is made to a fresh slant. An adequate amount of conidiating material is generally produced within 4 days, the strain is then processed. In the case of sparsely conidiating cultures, the amount of fresh material can be increased by growing more than one slant. The aim is to maximize the number of viable conidia in the suspension fluid used for preservation. Some colonial strains require a longer period before finally producing abundant conidia. Recognition of this character comes through trial and error - with such strains the culture tube can be sealed with a permeable membrane cap to retard drying.

When an adequate amount of material has grown, preservation on silica gel and in lyophil can be accomplished at the same time. The suspension fluid used is nonfat dry milk (7% w/v), autoclaved in 13 X 100 mm tubes for 20 minutes. 2.0 ml is an adequate volume. The mycelial-conidial mass is loaded into the milk with a sterile loop or needle. Normal concentrations of conidia can usually be suspended by agitation with the tip of a Pasteur pipette, but cultures that are largely aconidial will require grinding with a stouter implement. I use a small aluminum rod (4 mm diam) which can be flamed with alcohol. When the vegetative matter is dispersed, the suspension is drawn into a pasteur pipette and dispensed onto the silica gel and into ampules for lyophilization. Silica gel (12-28 mesh) is dispensed into 13 X 100 mm screw cap vials, half filling them. These are plugged with cotton and kept in a 70 C oven. Immediately prior to use, the required number is removed and placed in an ice bath. When cooled, the tube is held horizontally to distribute the silica gel along its length, and approx. 0.25 ml of the suspension described above is dispensed drop-wise from one end to the other. The tube is them capped, shaken briefly but vigorously on a vortex mixer and returned to the ice bath. This dissipates the heat generated as the silica gel absorbs moisture. Once cooled, the silica gel tubes are refrigerated with a desiccant in sealed plastic boxes.

Using the same pipette, several ampules are loaded for lyophilization. Each receives approx. 0.15 ml of the milk-conidial suspension. The ampules are made of 6 mm OD glass tubing, 13-15 cm long, with one end flame sealed, the other plugged with cotton. Groups of ampules are quickly frozen in liquid nitrogen. They are placed in a mesh-bottomed metal bucket and suspended in LN vapor for approx. 2 minutes. After two minutes, the bucket is lowered into the liquid nitrogen, which freezes the ampules and contents. Ampules are then quickly transferred to a desiccator kept in a -25 C freezer. The desiccator, which contains an open dish of Phosphorus pentoxide (phosphoric anhydride), is evacuated and the moisture in the frozen samples sublimes. Within a few days, all the water has left the samples to be absorbed by the desiccant, and only a dried pellet of milk and spores remains in each ampule. They are individually flame sealed while attached to a vacuum sleeve.

Strains that are absolutely aconidial may be impossible to lyophilize, though most can be preserved on silica gel. For these stocks, preservation over liquid nitrogen is the backup storage form. Strains to be stored over liquid nitrogen are suspended in 10% DMSO. Mycelium is ground into small pieces as described and the suspension poured into a 2 ml polypropylene screw-cap vial for freezing and storage at -75 C. These stocks can be sampled by scraping small pieces off the top with a flamed loop or needle.

After being processed by any method, each strain is checked for its genetic characteristics. Silica gels are tested after a week, lyophils when the samples have dried. Nutritional markers are scored, mating type is determined, unusual morphology is noted. Wild collected strains are crossed to testers for the respective species to verify both species and mating type. Aberration stocks are crossed to standard wild types to observe expected ascospore patterns. Such tests are also conducted when anomalous behavior is reported in any stock mailed by FGSC.

The need to make such tests points out the need for persons depositing strains to be complete in describing traits of each strain.