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Offlineeve.kalika

Registered: 09/11/14
Posts: 26
Last seen: 10 years, 30 days
Agar Technique
    #20569363 - 09/15/14 02:19 AM (10 years, 4 months ago)

Hi everyone. I'm totally new here and to mushroom cultivation. I decided to jump straight into the more "advanced" techniques, though, because I work as a lab technician and have a science degree, so the materials, equipment, and aseptic technique are all familiar to me.

In fact, that's where my question comes in. At work I plate samples for yeast and mould on a regular basis, using PDA in petri dishes. The technique I've been taught is different from what I see described on this forum and in the videos I've watched. If someone just handed me a spore syringe and told me to cultivate mycelium in a petri dish, I would add the sample first, then only pour in a small amount of agar and swirl the dish around until the bottom of the dish was covered. In any plating, the only time I let the agar solidify before adding the sample is when I'm air sampling or using a filter.

So I'm wondering if there's a reason why people use so much agar (looks like people are using 1/4" or so), and why they let the agar solidify before adding the spores. Clearly, it works, I'm just wondering if it's better than what I would have done or if it's just the popular way to do it around here.


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OfflinecronicrMFacebook
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Registered: 08/07/11
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Re: Agar Technique [Re: eve.kalika]
    #20569390 - 09/15/14 02:50 AM (10 years, 4 months ago)

we add the spores after it has cooled because spores are not thermotolerant and we want the growth on top only to grow acoss the agar so we can see and isolate sectors we like and or clean growth


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OfflineKizzle
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Registered: 08/30/11
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Re: Agar Technique [Re: cronicr]
    #20569488 - 09/15/14 04:29 AM (10 years, 4 months ago)

For one spores from prints aren't necessarily sterile. One of uses of agar is to isolate clean mycelium from any contaminants. If you spread the spores evenly like that any contaminants would get spread evenly too.


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Offlineeve.kalika

Registered: 09/11/14
Posts: 26
Last seen: 10 years, 30 days
Re: Agar Technique [Re: Kizzle]
    #20572961 - 09/15/14 09:35 PM (10 years, 4 months ago)

Quote:

Kizzle said:
For one spores from prints aren't necessarily sterile. One of uses of agar is to isolate clean mycelium from any contaminants. If you spread the spores evenly like that any contaminants would get spread evenly too.



What temperatures can they survive in?


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Offlinepawnzy
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Registered: 03/04/14
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Re: Agar Technique [Re: eve.kalika]
    #20572988 - 09/15/14 09:39 PM (10 years, 4 months ago)

Quote:

eve.kalika said:
Quote:

Kizzle said:
For one spores from prints aren't necessarily sterile. One of uses of agar is to isolate clean mycelium from any contaminants. If you spread the spores evenly like that any contaminants would get spread evenly too.



What temperatures can they survive in?




probably the same ones humans comfortably can, maybe colder though.

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OfflineKizzle
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Re: Agar Technique [Re: pawnzy]
    #20573085 - 09/15/14 09:54 PM (10 years, 4 months ago)

The spores cans survive the lowers temperatures agar can drop to before gelling. I think the thermal death temperature for the spores is around 120-130F while the mycelium is lower, around 106F. Of course it depends on how they're exposed to those temperatures.


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Offlineeve.kalika

Registered: 09/11/14
Posts: 26
Last seen: 10 years, 30 days
Re: Agar Technique [Re: Kizzle]
    #20573565 - 09/16/14 12:05 AM (10 years, 4 months ago)

That's about the temperature we store the agar in, actually. We keep it in a bath after it's made, so it's not fresh out of the autoclave hot when we use it.

It makes sense about having the mycelium on the surface, and about the agar not being too hot, although I don't quite understand the bit about contaminants. Wouldn't it be easier to separate contaminants if they weren't all in the exact same spot as the mycelium?

Also, in my lab, we add antibiotics to the PDA to keep bacteria from killing the fungus. I understand that the most common contaminants in mycelium cultivation would be competing fungus, such as mould, but I wonder if bacteria would also kill mycelium the way it kills yeast and mould. I haven't seen any mention of using antibiotics. Although, if everything's sterile it shouldn't make much difference.


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Demented Knife-wielding Escaped Lunatic Libertarian Zombie Mutant

Edited by eve.kalika (09/16/14 12:07 AM)

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InvisiblePastywhyteMDiscord
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Re: Agar Technique [Re: eve.kalika]
    #20573994 - 09/16/14 05:18 AM (10 years, 4 months ago)

Bacteria won't kill the myc on the plate but it will ride along. I have used antibiotics which do work well but there are other non antibiotic techniques that work very well. Most will center on the myc ability to grow through agar which will the strip bacteria riding along off.

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Offlinepawnzy
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Re: Agar Technique [Re: Pastywhyte]
    #20574006 - 09/16/14 05:32 AM (10 years, 4 months ago)

mushrooms are amazing

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Offlineeve.kalika

Registered: 09/11/14
Posts: 26
Last seen: 10 years, 30 days
Re: Agar Technique [Re: eve.kalika]
    #20579845 - 09/17/14 02:41 PM (10 years, 3 months ago)

Are spores harmed by UV light? Our laminar flow hood has a UV light.


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Offlinepawnzy
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Re: Agar Technique [Re: eve.kalika]
    #20579850 - 09/17/14 02:42 PM (10 years, 3 months ago)

+5 for asking

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OfflineKizzle
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Re: Agar Technique [Re: pawnzy]
    #20580360 - 09/17/14 04:43 PM (10 years, 3 months ago)

Quote:

It makes sense about having the mycelium on the surface, and about the agar not being too hot, although I don't quite understand the bit about contaminants. Wouldn't it be easier to separate contaminants if they weren't all in the exact same spot as the mycelium?



Yes, but generally the spores are streaked across the surface to some degree. It's enough to separate them some but not enough to redistribute contaminants back into the clean areas. Some people do use antibiotic agar but it's also possible to isolate clean mycelium without it.

Strong UV radiation can potentially kill about anything on the surface that's directly exposed to it. You wouldn't want use an UV sterilizer on something you're trying to grow.


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Offlineeve.kalika

Registered: 09/11/14
Posts: 26
Last seen: 10 years, 30 days
Re: Agar Technique [Re: Pastywhyte]
    #20582273 - 09/18/14 01:27 AM (10 years, 3 months ago)

Quote:

Pastywhyte said:
Bacteria won't kill the myc on the plate but it will ride along. I have used antibiotics which do work well but there are other non antibiotic techniques that work very well. Most will center on the myc ability to grow through agar which will the strip bacteria riding along off.



What antibiotic do you use? What are the other techniques?


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Offlineeve.kalika

Registered: 09/11/14
Posts: 26
Last seen: 10 years, 30 days
Re: Agar Technique [Re: Kizzle]
    #20582277 - 09/18/14 01:28 AM (10 years, 3 months ago)

Quote:

Kizzle said:
Quote:

It makes sense about having the mycelium on the surface, and about the agar not being too hot, although I don't quite understand the bit about contaminants. Wouldn't it be easier to separate contaminants if they weren't all in the exact same spot as the mycelium?



Yes, but generally the spores are streaked across the surface to some degree. It's enough to separate them some but not enough to redistribute contaminants back into the clean areas. Some people do use antibiotic agar but it's also possible to isolate clean mycelium without it.

Strong UV radiation can potentially kill about anything on the surface that's directly exposed to it. You wouldn't want use an UV sterilizer on something you're trying to grow.



Thanks for the info. :smile:


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