(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.
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
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
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
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
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
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,
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,
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
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"