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Offlinegray1
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Registered: 04/30/01
Posts: 430
Loc: brooklyn
Last seen: 14 years, 7 months
Re: a few questions for an agar guru...
    #304761 - 05/03/01 10:08 AM (15 years, 7 months ago)

parafilm: i assume you wish to seal a closed plate, if so, cut a thin strip and begin to stretch it by holding one end down with your finger. gently stretch it as thin as it will go. the strip should wrap around the plate a little more than two times. with your finger nail, scratch the loose end up into the crevice of where the bottom and top lid come together.
yes, theoretically you could perpetuate a culture indefinately on agar, however, not very well with the same exact material. when it's time to change the agar, say ~ every 2months, it would be best innoculate the new slant/p[etri dish with the old sample, and allow it to grow for a short period. as long as you are storing fresh growth, you can perpetuate a culture.
bacteria can be stored for years at -80 degrees celsius (your home freezer atop the fridge is ~-20) in a solution of media that is 15% glycerol. however, freeze/thaw cycles to take away from the viability. i have yet to try this for fungi.
hope this helps

c12h16n24ohdmt


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OfflineElektrolurch
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Registered: 05/02/00
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Re: a few questions for an agar guru...
    #308402 - 05/03/01 12:07 PM (15 years, 7 months ago)


It looks like I wasn't direct enough. Yes I store cultures. In a fridge (4 C to 6 C) they have to be transfered the latest every 6 month (sooner is better, e.g. 3 or 4 month)

>So could you preserve a strain forever by storing the >culture in the fridge, reviving it every few monthes by re->isolating it, and changing the agar medium ever so often?

I thought I answered this one right at the beginning!

>Does anyone have trouble with the para film not sticking to >the petri?

I don't use parafilm with Petri's. But parafilm sticks on itself very well, actually it should only be used this way, so overlay it with itself.

If you are going to store cultures, then DON'T use Petri dishes, use slant-tubes, like I extendly wrote in the last post!

>Does anyone here pre pour??? I am thinking of trying it.

Yeah, but don't store prepoured dishes longer than 1 or 2 month, because:

a) Water evaporates from the media and it dries.
b) the nutrients are transformed due to chemical processes, especially if you added some kind of base or acid to adjust pH

>You didn't answer my question...

I expected that you at least thanked me for the extended explanation of a storage method I posted. If you ask if storage is even possible, then I assume that the best answer is to give you a method for doing so.

Elektrolurch



--------------------
"For all the time spent in that room
The doll's house, darkness, old perfume
And fairy stories held me high on
Clouds of sunlight floating by.", Pink Floyd '67


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OfflineTriple_3
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Registered: 03/21/01
Posts: 99
Last seen: 15 years, 5 months
Re: a few questions for an agar guru... [Re: Elektrolurch]
    #308482 - 05/03/01 02:18 PM (15 years, 7 months ago)

senescence ocurs when the origional mycelia has expanded and split so many times that it changes, more aborts , forms mutations

It matters how much it has grown (how many generations of petri dishes)
not necessarily the time

Paul Stamets has a scale to determine the viability of a strain based upon it's generation.... ?

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OfflineTriple_3
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Registered: 03/21/01
Posts: 99
Last seen: 15 years, 5 months
Re: a few questions for an agar guru... [Re: Triple_3]
    #308484 - 05/03/01 02:20 PM (15 years, 7 months ago)

prepouring sucks.... but it works,more carmelization,uneven growth...

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Offlinepsilocybes
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Registered: 01/09/01
Posts: 4
Last seen: 15 years, 6 months
Re: a few questions for an agar guru... [Re: gray1]
    #309090 - 05/04/01 05:36 AM (15 years, 7 months ago)

Alright. I got another question. After inoculating a petri how long should I wait before transfering mycelium to new petris? When the petri is 100% colonized or should I wait till the mycelium shows strong growth?

Also what should I use for a sterile air environment? I am interested in what you guys use?



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Offlinegratefulredhead
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Registered: 04/27/01
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Re: a few questions for an agar guru... [Re: psilocybes]
    #309151 - 05/04/01 09:39 AM (15 years, 7 months ago)

hey man--be cool to the folks who are trying to help you out... here's some info that might be of use:


(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. =





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.
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.
References:
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"




--------------------


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Offlinegratefulredhead
member

Registered: 04/27/01
Posts: 487
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Last seen: 1 year, 8 months
Re: a few questions for an agar guru...
    #309934 - 05/05/01 02:38 PM (15 years, 7 months ago)

elektrolurch--love the info. and the drawings!!!



--------------------


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OfflineElektrolurch
enthusiast
Registered: 05/02/00
Posts: 307
Loc: Germany
Last seen: 10 years, 8 months
Re: a few questions for an agar guru... [Re: gratefulredhead]
    #310505 - 05/06/01 02:01 PM (15 years, 7 months ago)

Thanx,

Your info is almost complete though ;)

Elektrolurch



--------------------
"For all the time spent in that room
The doll's house, darkness, old perfume
And fairy stories held me high on
Clouds of sunlight floating by.", Pink Floyd '67


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Offlinegratefulredhead
member

Registered: 04/27/01
Posts: 487
Loc: Off the map....
Last seen: 1 year, 8 months
Re: a few questions for an agar guru... [Re: Elektrolurch]
    #310643 - 05/06/01 05:18 PM (15 years, 7 months ago)

never too much info! ;-)

this hobby gets to be a bit obsessive... :-))



--------------------


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