B. Papp szerk.: Studia Botanica Hungarica 29. 1998 (Budapest, 1999)

Vasas, Gizella, Bohus, Gábor; Locsmándi, Csaba: Genetic resource collection of macrofungi in Hungary

caying fungi in buildings. These pure cultures were also used for testing antipathogenic and anticarcinogenic effects of the fungicides produced by the macrofungi. Enzyme production rates were also studied in these traditional pure culture collections. The continuously increasing number of the isolates made it necessary to use the more feasible storing technique in liquid nitrogen, instead of the periodic transfer method. This technique allows a gradual enlargement of the collection and ensures a good prevention against infections and other risk factors. CRYOPRESERVATION OF MACROFUNGI IN LIQUID NITROGEN For pure cultures a small piece of the broken fruiting body of a well identified fungus specimen is isolated by a sterile scalpel from the contact zone of the cap and the stalk. The isolates are placed on agar slants. The fruiting bodies should not to be too old, because isolates from young fruiting bodies show better viability. If the fruiting bodies are wet, the probability of infections will increase significantly. Several isolates can be taken from the same fruiting body collection. However, they should be transferred only, if their morphological properties and growth parameters are similar. After substrate colonization the cultures are transferred to Petri plates. At the required size of the mycelium web agar disks of 8 mm diameter are cut by sterile cork-borer. Then the disks are placed into polypropylen cryotubes (2 ml) and closed by screw cap. The plastic cryotubes, in contrast with the glass tubes, are very advantageous to the vitality of the cultures (SIMIONE et al. 1977), and they are not fragile. However, it might be disadvantageous, that after repeated use they do not fit close, becoming permeable for the nitrogen in the liquid phase. Three disks are placed in each tubes, and then a cryoprotective substance, 1.5 ml 10% glycerine is added. Five tubes should be prepared in this way from each isolates. The next step of the procedure is the pre-freezing. Its crucial point is to find the most suitable timing of cooling and re-heating, i.e. the best cooling speed. Instead of using very expensive freezers equipped by built-in computer, a cheaper and more simple method was elaborated in our department. There is a temperature gradient in the nitrogen vapour above the liquid nitrogen surface. Placing the material to be frozen at different heights above the surface of the liquid nitrogen a freezing curve (Fig. 1) can be prepared. Usually ca. 1 °C/min temperature decrease is the most optimal for the cultures. After the cultures have been cooled down to -40 °C (this takes about 1 hour), they can be sunken in the liquid nitrogen.