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Cloning Method - 1
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Nan (Nanook)
Posted on Tuesday, October 23, 2001 - 06:31 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

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 a private email = distribution service sponsored by members of the Cultivation Interest =
= Group of the Oregon Mycological Society. =


From the mycoculture.org archives, additional info on water storage, including a quote from PF on cubensis storage, where he answers the age-old question, "How long will my spore syringe last?":

From: "Perf. Fungi E."
Date: Thu, 2 Apr 1998 22:22:33 +1
Subject: Quotes about water storage
Below is a compilation of texts about the water storage
method, which I found today.
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.

*******************************************************

Preserve your collection on agar slants. Seal tubes with parafilm and refrigerate (some fungi will not survive refrigeration, however, and must be stored at room temperature). Cultures should be checked semi-annually or annually. If very long storage is desired, pour sterile mineral oil over the mycelium in the culture tube. Cultures may remain viable for over 20 years using this technique. Culture collections at major institutions are stored under liquid nitrogen.

Spore prints stored in sealed bags can be stored for years. To rehydrate spores, touch the spores with a sterilized implement and transfer to sterile water. Let stand 6-12 hours and plate a drop or two on suitable agar. [Liquid Culture]

In starting a culture from a spore print, factors controlling sexual reproduction must be recognized. In a tetrapolar fungus, recall that only one fourth of the spores from any one mushroom are fully compatible with any random spore from that same strain. Two of the four pairings will form illegitimate pairings and make clamp connections, but cannot fruit. Thus, two thirds of the random dikaryons formed will be of the illegitimate type. These illegitimate pairings cannot be recognized in culture. One way around this problem is to
inoculate with a large number of spores and take a tissue culture of the best mushroom that appears. This is the simplest method but the strains produced in this manner still must be evaluated thoroughly.

Another method is to start a culture from multispore germinations. As the colony grows from these spores, sectors will be produced. Sectors are wedge-shaped
areas of differing physical or growth characteristics of the mycelium. Each sector is a unique strain of the mushroom. The different strains formed from the multispore germinations will eventually sort themselves out. As the colony grows and the cultivator repeatedly transfers from one sector, a colony is eventually obtained that no longer produces new sectors. This is one way of isolating a pure strain. Several strains may be isolated from the same original petri plate in this way.

Shroom Glossary
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Veteran Cosmic Rocker (Silverpen)
Posted on Saturday, December 01, 2001 - 02:17 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Ok, it's been almost two years since I got my first pf syring from Fanaticus. I was able to make a print off the first batch and have continued with the same strain, using them over and over by taking print after print. I don't know if it's my imagination or not, but my last batch does not seem to be as potent as previous batches, even using the same tek. To boost the viability of the spores, as well as possibly improving the potency, would it help to mix newly ordered pf spores with my old ones? Does Fanaticus basically continue with the same strain too? Any suggestions or insight on this subject would be appreciated.
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Lichen (Lichen)
Posted on Saturday, December 01, 2001 - 03:04 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

it must be your imagination, because spores, no matter what, are 'new life', a brand-new and vigorous 'seed' that will produce a strong, viable source of the species that can and will continue the mushrooms to time indefinite...it's the nature of reproduction.
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ralph (Ralphster44)
Posted on Saturday, December 01, 2001 - 04:25 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

You should keep changing your substrate.
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ion ewe (Ion)
Posted on Saturday, December 01, 2001 - 06:13 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Tolerance, perhaps?

-that guy
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hippie3 (Hippie)
Posted on Saturday, December 01, 2001 - 12:55 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

no, ralph hit on it.
over long term, it's important to maintain the full genetic variety of the strain.
if you continually grow on the same substrate, the strain loses some of it's strength.
so you must switch it around from time to time.
in this case, it's prolly too late for that so i'd recommend getting some fresh samples from pf.
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Veteran Cosmic Rocker (Silverpen)
Posted on Sunday, December 02, 2001 - 12:18 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Hummm...ok, change the substrate? Does that mean I should use something besides brown rice flour?
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hippie3 (Hippie)
Posted on Sunday, December 02, 2001 - 11:45 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

correct, you should grow out the occasional batch on rye or birdseed, dung, straw, etc.
mix it up some, so the strain is forced to keep adapting.
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Paranoid Android234 (Jinks420)
Posted on Tuesday, December 11, 2001 - 04:16 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Does anyone know why mixing two different strains doesn't result in a new strain? has anyone tried this? If they are from the same species why not? what defines a species is the ability to interbreed. I could have sex with a different "so called race" and yes that would be genetically different then the parents (the shading of skin color for example)(phenotypically speaking) Also why would continiously using the same substrate result in weaker strains (I mean a weaker trip) - mutation in the chromosome? would interbreeding the strains allow more genetic viability?
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Cragith Kilbonith (Kilborn)
Posted on Tuesday, December 11, 2001 - 06:45 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

you cant mix 2 strains and get 1 strain using pf tek, least i dont believe???(check out pf's site he has something on it) i heard that you just get 2 diffrent strains growing on the cake, if it even makes it that far. the only time i mixed strains was the albino x indian. i got it to pin like a beast but none became fully mature, anyone else had luck? ya if you even get fruits i bet you would just get alot of mutents aye? i just set the cake in the 'maid and didnt mark it after being birthed but im pretty damn sure it was the cake that produced about 2 albinos and the rest aborts, and they grew very small.
hope this helped