(First published in in _Mushroom,the Journal_, Winter 1998 edition, with clarifications added by the author, April 1998.) Copyright(c)1998 by Joseph C. Kish, All Rights Reserved. _______________________________________________________________________ Resurrecting a Better Method for Long-Term Storage of Mushroom Cultures _______________________________________________________________________ by Joseph C. Kish *** clarifications Sooner or later every mushroomer with an interest in edible fungi ends up with a collection of cultures, and then there is the problem of storage. I began searching for better methods for storing my cultures a long time ago. Agar Slants: The usual method of storage is in slants on nutrient agar. This works fine short term, but many strains start losing viability when kept on a single nutrient for an extended period. Every couple of months they need to be moved to an agar with a different nutrient. Some strains of Agaricus appear to start the dying process anyway, as though agar is not the media they prefer. Rating: 2 bells. Mineral Oil: Overlaying the slants with sterile mineral oil keeps the sample from drying out, and acts as an oxygen barrier. The oil increases The time between transfers to about six onths, however, the cultures must be refrigerated, and the oil is messy. Rating: 1 bell. Deep Freeze: It would really be nice to have access to liquid nitrogen, that stores cultures at -384F (-196 C), that essentially stops all of the metabolic activity. If I had that resource, the cultures would store indefinitely. I tried using a chest freezer at -5 F (-18C). The culture is mixed with glycerol (glycerin from the drugstore) to prevent ice crystals from damaging the culture. It is stored as a frozen mush in slants. This method stores quite well, but many mushroomers do not have access to a chest freezer. Refrigerator freezers kill the cultures, as they cycle high and low. Rating: 2 bells. Sawdust / Spawn: Most cultures will colonize in sawdust. Small baby-food jars 2/3 full of hardwood sawdust (80%), wheat bran (15%), gypsum (5%) is moistened to perfection and sterilized. The jars are inoculated. When fully colonized, they are refrigerated. A single grain of spawn is transferred to an agar plate to start a new copy. This method stores cultures for more than a year. The best yet. Rating: 4 bells. Distilled Water: I came upon an article written by Michael D.Graham,a microbiologist at ATCC (1) that described the storage of yeast in sterile distilled water. What a brilliant idea! If that method stores yeast, It should work well on gourmet mushroom cultures, too. It's easy to do, very space efficient, allows the cultures to be stored at room temperature, and maintains thier viability for years. I contacted the author, he indicated that edible fungi stores even better than yeast, and you can store the spores as well as the hyphae often for decades! Rating: 10 bells. Sterile water was first used to preserve cultures by Castellani in 1939 (2). Since then, many scientists have used this method; McGinnis et al. in 1974 (3) and Odds in 1991 (4) reported that they were able to maintain viable cultures for more than three years, without degredation. This technique satisfies many different interests: Castellani's pathogenic fungi, Odds interest was in pathogenic yeast, and McGinnis' interest was in a wide range of fungi, yeast, and bacteria. The distilled water preserved all of them. I suggest you obtain copies of these scientific papers from your University library, and you'll be impressed, too. Storage Method: Obtain dram vials from your laboratory supplier and fill them about half full (about 3mL) with distilled water, loosly cap the vials and sterilize them in a pressure cooker for 30 minutes @250 F. Half dram vials or test tubes with screw caps would also work well. *** About six milliliters of sterile distilled water is pipetted aseptically into a freshly growing culture. The fragments of hyphae are dislodged by lightly scraping the aerial growth with the same pipette, and the resulting suspension is withdrawn and transferred to a sterile glass vial. Put plenty of inoculum into each vial to insure success. Screw the lid on tight and wrap Parafilm around the top of the vial to make sure it is airtight. When you come back in a few years,you would not want to find that the water had evaporated. Store the vials at room temperature away from direct sunlight. A bookshelf or wall cabinet is an excellent place. If conditions deteriorate, and the room should become unbearably hot, the vials can be refrigerated, but that is not normally necessary. In the distilled water envirnment, the mushroom culture enters a dormant state, and it is held in stasis. The Rude Awakening: Under aseptic conditions, simply dip a sterile loop into the vial, *** and streak the mycellia-rich water onto an agar plate. It will start to reanimate on being in a nutrient source and oxygen. The first four methods keep cultures alive with three items: food, water, and oxygen. If they lack any of these, It's goodbye. Instead of trying to keep them alive,there is a better way: In sterile distilled water, with no food, oxygen, or minerals. This method was in use almost 60 years ago, but was apparently lost due to lack of communication. References (1) M.D.Graham, "A Simple,Practical Method for Long Term Storage of Yeast", Brewing Techniques 5, March/April (1997), pp 58-62 (2) S.Castellani,"Viability of Some Pathogenic Fungi in Distilled Water", Journal of Tropical Medicine and Hygiene 42, pp 225-226 (1939) (3) M.R.McGinnis,A.A.Padhye,and L. Ajello,"Storage of Stock Cultures of Filamentous Fungi,Yeasts, and Some Aerobic Actinomycetes in Sterile Distilled Water", Applied Microbiology 28, pp 218-222 (1974) (4) F.C.Odds, "Long Term Laboratory Preservation of Pathogenic Yeast in Water",Journal of Medical and Veterinary Mycology 29, pp 413-415(1991) ========================================================================= = Article distributed by The+mycoculture@teleport.com, a private email = = distribution service sponsored by members of the Cultivation Interest = = Group of the Oregon Mycological Society. = =========================================================================