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Offlineanevsky
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Registered: 07/30/08
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Basic agar germination questions - search is doing me wrong..
    #10085944 - 04/01/09 08:48 AM (15 years, 9 months ago)

I know for a fact that these questions have been answered millions upon millions of times, but I'm not seeing crap in the search, and I've done about every permutation on the words.. so here goes, post links if you know of them, otherwise, please contribute.  Thanks.

It has been a little over a week that I have placed spores on MEA in petri dishes.  Sterile procedure was followed to a T.  The plates themselves are very clean, no signs of contams, but also no signs of mycellium.  The spores are Reishi and come from a print easily a year old.  In, now, eight days, there are NO signs of any growth good or bad.  I can still see the dark clumps of spores that I placed on the agar with my loop.

Here are some places where I could have gone wrong:

1. I mixed the MEA per instructions.  This MEA comes from Fungi Perfecti and calls for 50g/1l.  I used it at that level.  It is VERY thick, and looking through the agar side, undissolved granules are CLEARLY visible.  I did my best to completely dissolve the mea in distilled water, and shook like crazy before pouring.  I guess it was just too concentrated though the instructions said that was the right ratio.  My question is - some threads talk about overly rich agar plates (in terms of this thick, granulated pour) as being a contributing factor to spores germinating poorly.  Can some trusted cultivators (not in the literal sense) comment on this?  True or not?

2. The agar plates were poured and then wrapped in parafilm.  Up until today they were sitting in their bag that was tied up.  I realized this was not ideal for FAE, so I pulled them out.  What is one to do?  Keep them bagged while germinating, or let them breath as much as they want to?  Something tells me that just for germination there is PLENTY of fresh air inside the dish itself since no growth has happened, but I am often wrong.

3. The plates are "incubating" at a temp somewhere in the mid 60s.  Quite cold I know.  This will absolutely influece the amount of time growth will take - but beyond this slowness, is this a bad idea? 

So, agar density, fae for dishes, and temps..

Thanks all,
A

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InvisibleStarrider
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Registered: 11/09/08
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Re: Basic agar germination questions - search is doing me wrong.. [Re: anevsky]
    #10086301 - 04/01/09 10:27 AM (15 years, 9 months ago)

You should have the temp between 72-78. 60 is to low. I incubate mine at 76 and have good results every time.


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OfflineRogerRabbitM
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Re: Basic agar germination questions - search is doing me wrong.. [Re: Starrider]
    #10088992 - 04/01/09 06:07 PM (15 years, 9 months ago)

Since the spores were a year old, they probably dried out and need to re-hydrate before germination.

Stamets MEA is good stuff, but I always mix it a bit weak, especially when germinating spores.  I've used it for so many years, I simply put 3 1/2 tablespoons into .7 liter.  If I'm not mistaken, a tablespoon of his agar powder weighs 7 grams, so the recommended mix is 7 tablespoons per liter.  By mixing it a tad weak, the agar isn't quite so solid, thus better for spores.

I'd say you're fine.  If another week goes by without germination, add a couple of drops of sterilized water over the top of the spores.  If possible, get the temp up to 75F.  Parafilm is good, and I also usually stick the freshly poured dishes back into the sleeve they came in, and then after inoculation, I put three or four dishes into ziplock sandwich size bags, which I inflate with sterile air from the flowhood before sealing.
RR


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Offlineanevsky
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Re: Basic agar germination questions - search is doing me wrong.. [Re: RogerRabbit]
    #10089049 - 04/01/09 06:13 PM (15 years, 9 months ago)

Much thanks RR.  I'll post back to this thread when I see growth.

Best,
A

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