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Resurrecting a Better Method for Long-Term Storage of Mushroom Cultures
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.
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.
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.
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.
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.
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.
(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)
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Below is a compilation of texts about the water storage method:
Oei, Peter. 1991. MANUAL ON MUSHROOM CULTIVATION - TECHNIQUES, SPECIES AND
OPPORTUNITIES FOR COMMERCIAL APPLICATIONS IN DEVELOPING COUNTRIES. Transfer
of Technology for Development (Amsterdam) and Technical Center for Agricultural
and Rural co-operation (Wageningen), ISBN 90 70857 22 7, p 256:
"A simple technique is to grow the culture on an agar medium and to keep small pieces of colonizad agar floating in demineralized water. If 100 ml bottles are used, then these should be fille with 75 ml demineralized water. Sterilize the bottles for two hours, let them cool, then transfer aseptically small pieces from the agar culture. Put about three to four pieces of 0.5 x 0.5 square centimeters in each bottle. Always inoculate at least three bottles per strain, so some contamination occurs then there is still a back-up. Strains can easily be recovered by taking a piece of the agar out of the water and transferring it to a new slant. This operation is not as messy as the mineral oil technique. Strains can be kept for at least one year without losing vigour (Exept for VOLVARIELLA VOLVACEA, which cannot stand prolonged storage at temperatures below 12 centigrade)."
The following quote is taken from Smith, D. and Onions, A.H.S.: THE PRESERVATION AND MAINTENANCE OF LIVING FUNGI, SECOND EDITION (1994), CAB International, Wallingford, Oxon. ISBN: 0 85198 902 0 (italics in the original text are replaced by CAPITALS)
"WATER STORAGE The method used at the International Mycological Institute (IMI) is as follows: 1. 6mm cubed agar blocks are cut from the growing edge of a fungal colony. 2. The blocks are placed in sterile distilled water in McCartney bottles and the lids are tightly screwed down, they are stored at 20-25 centigrade. 3. Retrieval is by removal of a block and placing, mycelium down, on a suitable medium. Storage periods of 2-3 years have been obtained with species of PHYTOPHTHORA and PYTHIUM at IMI before any loss of viability was noted (Onions&Smith, 1984). These cultures showed some deterioration in pathogenicity but the majority were able to infect their host. Viability deteriorated rapidly after 2 years storage and 42% (21/50) of the isolates were dead at 5 years. Growth may sometimes occur during storage in water. This will be reduced if the spores or hyphae are removed from the surface of agar media and no medium is transferred. This method of storage was originally described by Castellani (1939, 1967) who stored fungi pathogenic to man. Figueiredo (1967) was able to keep 22 plant pathogens without any loss in pathogenicity. Figueiredo & Pimentel (1975) subsequently reported 10 years of successful storage by this means. Boeswinkel (1976) stored 650 plant pathogens including representatives of the OOMYCOTA, ASCOMYCOTA, BASIDIOMYCOTA and mitotic fungi. They all remained viable and pathogenic for 7 years. Clark & Dick (1974) reported successful results with OOMYCOTA, Ellis (1979) with ENTOMOPHTHORALES, PYRENOMYCETES, HYMENOMYCETES, GASTEROMYCETES and HYPHOMYCETES, and Marx and Daniel (1976) with ectomycorrhizal plant pathogens.
Boeswinkel, H.J. (1976) Storage of fungal cultures in water. TRANSACTIONS OF THE BRITISH MYCOLOGICAL SOCIETY 66, 183-185.
Castellani, A. (1939) Viability of some pathogenic fungi in sterile distilled water. JOURNAL OF TROPICAL MEDICINE AND HYGIENE 42, 225-226.
Castellani, A. (1967) Maintenance and cultivation of common pathogenic fungi of man in sterile distilled water. Further researches. JOURNAL OF TROPICAL MEDICINE AND HYGIENE 70, 181- 184
Ellis, J.J. (1979) Preserving fungus strains in sterile water. MYCOLOGIA 71, 1072-1075
Figueiredo, M.B. (1967) Estudes sobre a aplicacao de Castellani para conservacao de fungos patogenos en plantas. BIOLOGICO 33, 9-15.
Figueiredo, M.B. & Pimentel C.P.V. (1975) Metodos utilizados para conservacao de fungos na micoteca de Secao de Micologia Fitopatologica de Instituto Biologico. SUMMA PHYTOPATHOLOGICA 1, 299-302 Marx,
D.H. & Daniel, W.J. (1976) Maintaining cultures of ectomycorrhizal and plant pathogenic fungi in sterile water cold storage. CANADIAN JOURNAL OF MICROBIOLOGY 22, 338-341
Onions, A.H.S.&Smith, D. (1984) Current status of culture preservation
and technology. In: CRITICAL PROBLEMS OF CULTURE COLLECTIONS (edited by L.R.
Batra&T. Iigima). Osaka: Institute of Fermentation Osaka"