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InvisibleWheedhead!
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Agar recipe for cloning oysters "UPDATE PICS 16/12"
    #2142356 - 11/28/03 04:43 PM (18 years, 2 months ago)

allright, so i prepared some PDA h202 agar, went to the store and bought some fresh oyster shrooms. Cut a piece off the stem (inside) with a clean scalpel, placed the piece on the agar. Its been 6 days and there is no growth on the agar.............. but the transferred piece is fluffy.

Is there a problem with oysters and h2o2? I gave some plates to a friend, he cloned some cubes (at the same time as i did the oysters) and there is plenty of growth on the cube plates...but the oysters doesnt like the pda agar or what?
Is there a special recipe for oysters??

Need some advice here...


Edited by Wheedhead! (12/16/03 05:44 PM)


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Invisiblevampirism
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Re: Agar recipe for cloning oysters [Re: Wheedhead!]
    #2142368 - 11/28/03 04:48 PM (18 years, 2 months ago)

well,
1) it takes a few days for mycelium to get used to h202, that might be the problem
2) could just be a poor strain to clone, I've heard that alot of store-bought species specially made to be especially hard to clone ( cubies arent like that imo)

personally i haven't used pda so I can't help you in that respect, but i find oysters like DFA.. heh.




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Offlinezeronio
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Re: Agar recipe for cloning oysters [Re: ]
    #2148610 - 12/01/03 07:07 AM (18 years, 1 month ago)

Oysters don't have problems with H2O2 and the standard recipies work fine.
It's possible that you got contamns around the transfered piece, that's why it's getting fluffy but it's not spreading on agar. Try to transfer the "fluffy" part to new dishes.


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InvisibleMilletV
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Re: Agar recipe for cloning oysters [Re: zeronio]
    #2149018 - 12/01/03 12:12 PM (18 years, 1 month ago)

The piece will fluff up before it starts to grow on the agar. Sometimes h2o2 will cause a bit of lagtime before the mycelium starts to colonize the plate.

patience...........


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InvisibleWheedhead!
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Re: Agar recipe for cloning oysters [Re: Millet]
    #2149151 - 12/01/03 12:55 PM (18 years, 1 month ago)


Its been 10 days now with zero growth on the Agar, the cubes are doing just fine though.


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InvisibleG a n j a
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Re: Agar recipe for cloning oysters [Re: Wheedhead!]
    #2149235 - 12/01/03 01:32 PM (18 years, 1 month ago)

Well, maybe the oysters were beyond cloning, due to age.
I have cloned many shop bought oysters on

Potato Dextrose (Yeast-extract) Agar (PD(Y)A)
broth from boiling 150 grams sliced potatoes
in 500ml water for 30 minutes(add water to 500ml)
(You can use 5 g of instant potato flakes instead)
9 g agar agar

with .84 ml of 17% h2o2 per 500ml

and oysters seem to love this, with growth
in days, is there not even any mycelium fluff
on the flesh pieces them selve? As if not, i would say start
again.

Better luck next time mate.


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Offlinecreekfarmer
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Re: Agar recipe for cloning oysters [Re: G a n j a]
    #2151091 - 12/02/03 06:38 AM (18 years, 1 month ago)

I've had better luck with MEA. Was the tissue from the base of the stem? Try to use tissue from the cap instead.


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InvisibleWheedhead!
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Re: Agar recipe for cloning oysters [Re: G a n j a]
    #2151233 - 12/02/03 10:14 AM (18 years, 1 month ago)


The pieces are all nice fluffy, but the myc is not spreading. Guess ill have to wait, meantime i think ill prepare some new plates and buy the oysters from a diferent shop (maybe i should ask the manager if he would call me when they get a fresh delivery of shrooms?) and make some new transfers.

some of the pieces transferred were cut from the stem and others from the cap.

The only mistake i know i made was to use too big chunks of shroom. But that should not prevent growth??? or??



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OfflineSuntzu
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Re: Agar recipe for cloning oysters [Re: Wheedhead!]
    #2151342 - 12/02/03 10:54 AM (18 years, 1 month ago)

IME, even if there was some bacteria hitching a ride, the culture would still grow across the agar. When you look at the plate from underneath, does there appear to be bacterial growth? [odd-looking patches]. You may not see them if the plate was poured thick.
Because it is 'fluffing up', it sounds like it's trying to go back into vegetative growth. It could be poor/old genetics or possibly the agar formulation. . .vigorous oyster cultures will colonize just about any standard media. I'd recommend hanging on to the plates [for observation] and trying again with a new clone and new plates.


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InvisibleG a n j a
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Re: Agar recipe for cloning oysters [Re: Wheedhead!]
    #2157292 - 12/04/03 03:01 AM (18 years, 1 month ago)

Quote:

The pieces are all nice fluffy




sounds like you just need to wait a bit longer, once
theyve fluffed up, its just a matter of time before
it moves onto the agar.


Good luck.


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Offlinekeela
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Re: Agar recipe for cloning oysters [Re: G a n j a]
    #2166716 - 12/07/03 04:24 PM (18 years, 1 month ago)

Just wait a while longer. It took me 3 weeks using store bought oysters (I also made the mistake of using big chunks, there was 50% bacteria contamination) to colonize about half of a dish (PDYA, with peroxide) but after that it seemed to speed up a bit. For about 2 weeks it stayed on the flesh but soon it spread out.

On the other hand, maybe my strain is also not so vigorous - its been 10 days on white wheat in quart jars (having been shaken twice) and the growth looks nothing like what it looks like in pictures.


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InvisibleWheedhead!
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Re: Agar recipe for cloning oysters [Re: keela]
    #2169176 - 12/08/03 04:53 PM (18 years, 1 month ago)

Ladies and gentlemen.............we have growth.

Yesterday i checked the plates, and they were growing  :laugh: Nice pure white mycelium. Today the growth has doubled and i think the plates will be fully colonised within a wekk or two. I will post some pics later (just have to move my lazy ass and get my camera at a friends house)

Thank you people for telling me to keep the plates, I was loosing hope :wink:

Now I have to decide what substrate to use for fruiting, im thinking agar to popcorn (spawn) and then plain straw(substrate)? Any ideas?
Also I ould like to know the exact incubation temp and the fruiting conditions for oysters (I know I need the bible,,TMC,, its on my christmas wish list )

I have been searching the shroomery for pictures of oyster myc on agar to compare my experiment, but i didnt find any. So if anybody has an url or remembers a post on this subject, please share.

I just  :heart: The Shroomery


Edited by Wheedhead! (12/08/03 05:15 PM)


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InvisibleG a n j a
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Re: Agar recipe for cloning oysters [Re: Wheedhead!]
    #2169464 - 12/08/03 07:20 PM (18 years, 1 month ago)

TMC gives Pleurotus ostreatus as
Spawn run :78-84?f

Pinning : 55-60?f

High humiderty through out.

Good luck, I would suggest plan straw as well, make sure
its well chopped up, as it makes a better block, the smaller
the straw is shredded IME.

:thumbup:


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InvisibleG a n j a
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Re: Agar recipe for cloning oysters [Re: G a n j a]
    #2169483 - 12/08/03 07:35 PM (18 years, 1 month ago)



Is a P. pulmonarius



Is a clone from Anno which is a P. ostreatus I think


(I could be wrong, I can't find the paper work lol I'll
double check tomorrow, but there def oysters)


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OfflineIndubitableness
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Registered: 11/30/03
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Re: Agar recipe for cloning oysters [Re: G a n j a]
    #2171348 - 12/11/03 07:28 PM (18 years, 1 month ago)

I used potato dextrose agar i made in an oven to clone oysters recently as well, i only inoculated them four days ago and i've already got growth on the agar, although no fluffing.


Okay guys, this hasn't much to do with it but i need to know something. What is the optimum temperature to add h2o2 to the agar? i'm assuming if you add it while it's too hot it's going to deteriorate rapidly and basically become hydrogen and oxygen with no anti-contam effects. If anyone knows the best temp or at least a proven temp let me know. (i have had success by just guessing based on how hot it feels, but i'd rather be precise)


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Invisiblepuggymalone
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Re: Agar recipe for cloning oysters [Re: Indubitableness]
    #2171369 - 12/11/03 07:48 PM (18 years, 1 month ago)

130 F will be cool, at 140 F the H2O2 will break down and you will loose some of the benifits (if not all) of the addition. Go for 6cc per litre of agar.
hope I was helpful
puggy :rasta:


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InvisibleG a n j a
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Re: Agar recipe for cloning oysters [Re: Indubitableness]
    #2171371 - 12/11/03 07:49 PM (18 years, 1 month ago)

130?f or 55?c is when i add it, it got that from here
http://www.shroomery.org/index/par/23462
and it works well for me.


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InvisibleG a n j a
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Re: Agar recipe for cloning oysters [Re: G a n j a]
    #2171378 - 12/11/03 07:52 PM (18 years, 1 month ago)

be carefull of your h2o2 % before doing the math. mines 17% but then 9% and 3%
are also common.


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Invisiblepuggymalone
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Re: Agar recipe for cloning oysters [Re: G a n j a]
    #2171962 - 12/12/03 02:26 AM (18 years, 1 month ago)

ganja,
shit i totally forgot about that, i was just assuming everyone was buying the same stuff i was. (oops:) what I was using is 3%
puggy


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InvisibleG a n j a
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Re: Agar recipe for cloning oysters [Re: puggymalone]
    #2172016 - 12/12/03 03:15 AM (18 years, 1 month ago)

You should be fine then, as I think most of the
formulars are set for the 3% :thumbup: but its
worth being aware of :grin:


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Offlinepluteus
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Re: Agar recipe for cloning oysters [Re: G a n j a]
    #2172061 - 12/12/03 03:38 AM (18 years, 1 month ago)

I use 30% and fried my cultures to death first time round...
that's 0.6 ml / l for 30% folks

for cloning from wild, I prepare antibiotic media, peroxidated media, and also antibiotic + peroxidated media. I have the most success with the antibiotic stuff, but every so often I find a species that's easier to isolate on combination media. e.g. bird's nest fungi

Antibiotic + peroxidated media also seems to work quite well for cloning from rhizomorphs, such as you find growing in woodchip beds underneath colonies of P. cyanescens. Sometimes I quickly rinse the rhizomorph in dilute ethanol first.


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InvisibleG a n j a
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Re: Agar recipe for cloning oysters [Re: pluteus]
    #2172107 - 12/12/03 04:05 AM (18 years, 1 month ago)

My 17% goes in at .84ml per every 500ml.
Do you know of any antibiotic addatives I
can get hold of in the uk ? As that would
be real handy for strating spores from
wild picked mushrooms :cool:

Do antibiotic's stop bacteria ?


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Offlineragadinks
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Re: Agar recipe for cloning oysters [Re: G a n j a]
    #2173352 - 12/12/03 06:45 PM (18 years, 1 month ago)

I do not know what the situation is like in the UK. I got gentamycin from my local doc ( but he's a relative ... :smirk: ). You might also be able to get it from a vet. I have heard that you can get antibiotics in some pet shops. They sell it in order to cure tropical fish. As far as I know gentamycin is the best since you can pressure cook it. Here is a link that describes some other antibiotics and their effect on bacterias.


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InvisibleG a n j a
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Re: Agar recipe for cloning oysters [Re: ragadinks]
    #2174291 - 12/13/03 08:21 AM (18 years, 1 month ago)

Thank you, good link there :cool:
Now I just need to track some down, luckily theres a large farm shop with a big vet section near me, I'll try there first. Gentamycin seems too be the best alrounder.


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Offline284_27
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Re: Agar recipe for cloning oysters [Re: G a n j a]
    #2174353 - 12/13/03 10:09 AM (18 years, 1 month ago)

am not trying to hijack the thread , but how what ratio of gentamycin is added ?


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Offlineragadinks
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Re: Agar recipe for cloning oysters [Re: 284_27]
    #2174371 - 12/13/03 10:39 AM (18 years, 1 month ago)

I think it is 100 mg per liter. Have a look at this thread.


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Offlinepluteus
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Re: Agar recipe for cloning oysters [Re: ragadinks]
    #2174727 - 12/13/03 03:00 PM (18 years, 1 month ago)

I add at 100mg/litre. I have several sources for gentamycin sulphate here in the UK, but it's quite an expensive substance - around ?140 for 5 grams of 60% purity = 50 litres of antibiotic agar media at ?2.80 each

I find that it never seems to eradicate bacteria at this concentration, just - when it works - reduces them to maneagable levels (little blobs as opposed to big splodges) so that it's generally much easier to isolate clones.

I've got a friend who uses novobiocin with success, but I can't remember if it's autoclavable. It is, however, much cheaper. As regards autoclavability, I don't think antibiotic carefully added to cooling media really constitutes a serious vector of contamination anyway, definitely not in my experience.

Gentamycin works by disabling part of the cell machinery (a ribosome protein) that makes other proteins. It does this in a range of organisms, including - according to my jar of it - Gram-negative and Gram-positive bacteria, and mycoplasma, but - crucially - not basidiomycetes. Resistance is commonplace, so it is really just another tool and not a cure-all.


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InvisibleWheedhead!
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Re: Agar recipe for cloning oysters "UPDATE GROWTH" [Re: Wheedhead!]
    #2183052 - 12/16/03 05:43 PM (18 years, 1 month ago)

Ok, so I finally moved my lazy ass to my friends house and got my camera.  I just thought I ould share some pics of the above mentioned projekt.

These Plates were all made 22-11-03. 













2 plates has some yellow on them, it looks like mycelium piss to me? Anyway ill just use pieces without it.
Tomorrow ill transfer some vedges to corn.

Enjoy the pics  :cool:


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InvisibleG a n j a
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Re: Agar recipe for cloning oysters "UPDATE GROWTH" [Re: Wheedhead!]
    #2183298 - 12/16/03 06:53 PM (18 years, 1 month ago)

nice pics, good job :thumbup:
That is more than likely mycelium piss as i see it
far more in oysters than cubensis.


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