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Amazon Shop for: Agar, Laminar Flow Hood, Potato Dextrose

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OfflineFr0ggy
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Registered: 12/13/02
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Agar help please
    #1534794 - 05/09/03 03:44 PM (13 years, 6 months ago)

I'm trying to clone some shrooms but the tissue just rots on the agar. What could I be doing wrong?

Here's the recipe I'm using:

1 litre water
300 grams potatoes
20 grams agar agar
10 grams dextrose
6 ml hydrogen peroxide


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Ribbit ?


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Invisiblemicro
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Re: Agar help please [Re: Fr0ggy]
    #1534816 - 05/09/03 03:55 PM (13 years, 6 months ago)

What do you mean rots? Is there a bacterial infection?

The recipie sounds ok -- by 300 g potatoes, you do mean the infusion from (the water after boiling them,) right?

--
Micro


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OfflineFr0ggy
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Re: Agar help please [Re: micro]
    #1534826 - 05/09/03 03:58 PM (13 years, 6 months ago)

There is no sign of bacterial infection and from what I've read bacteria can't survive on peroxidated agar anyway!


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Ribbit ?


Edited by Fr0ggy (05/09/03 04:04 PM)


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Invisiblemicro
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Re: Agar help please [Re: Fr0ggy]
    #1534838 - 05/09/03 04:06 PM (13 years, 6 months ago)

Yes, the peroxide will inhibit it. It could still live on the fruitbody itself, though.

You want to rub the whole outside of the fruitbody with rubbing alcohol under a flow hood/glovebox and then with 2 sterile scalples cut it in half the long way and then take a piece of meat to culture from the inside of the fruitbody. It should be sterile if you do this right.

I know it may sound silly, but I'll ask again -- you did use potatoe water from boiling, right? Also -- how long did you sterilize it for? You should only need 15 min @ 121C/250F

--
Micro


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Edited by micro (05/09/03 04:08 PM)


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OfflineFr0ggy
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Re: Agar help please [Re: micro]
    #1534865 - 05/09/03 04:15 PM (13 years, 6 months ago)

I just use the broth from the boiled potatoes, not the actual potatoes themselves.

The tissue was taken from inside the cap itself with a sterile scalpel, I didn't think rubbing alcohol was essential in tissue cloning and yes, I did sterilize.

I've read that it takes a while for the myc to adjust to the peroxide but this is getting ridiculous !  :crazy:



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Ribbit ?


Edited by Fr0ggy (05/09/03 04:22 PM)


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Invisiblemicro
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Re: Agar help please [Re: Fr0ggy]
    #1534898 - 05/09/03 04:26 PM (13 years, 6 months ago)

Only 2 things I can think of is bacteria or the dextrose carmelized from over sterilizing the media.

I always use rubbing alcohol just because the outside isn't sterile and the scalple touches it, too, but the way you described sounds like it should work.

--
Micro


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OfflineFr0ggy
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Re: Agar help please [Re: micro]
    #1534914 - 05/09/03 04:30 PM (13 years, 6 months ago)

Hmmm.. I'm going to make up another batch of agar and just try again I think.

Thanx for the help anyway, I appreciate it.

5 shrooms for you !  :laugh: 


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Invisiblemicro
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Re: Agar help please [Re: Fr0ggy]
    #1534974 - 05/09/03 04:45 PM (13 years, 6 months ago)

n/p, thanks and good luck!

--
Micro


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OfflineCultyVader
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Re: Agar help please [Re: micro]
    #1535065 - 05/09/03 05:18 PM (13 years, 6 months ago)

I find that dogfood agar works great and its very easy to make. I use Kibbles n' bits and the clones and spores have no problem growing contaminates grow very well too :wink:


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Invisiblecalicyco
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Re: Agar help please [Re: CultyVader]
    #1535078 - 05/09/03 05:23 PM (13 years, 6 months ago)


Try not putting any peroxide in it this time.



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InvisibleRoadkill
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Re: Agar help please [Re: Fr0ggy]
    #1536365 - 05/10/03 04:37 AM (13 years, 6 months ago)

Potato Dextrose Yeast Agar (PDYA)

500 ml water
9 to 10 grams agar
7 grams of honey
5 grams of instant potato flakes (Potato Buds brand)
1 gram nutritional yeast

Cut out a wedge from the stem.
Cut away any outside part of the stem.

Dip it in a 10 to 1 mix of Water and Hydrogen Peroxide.

Then put wedge onto agar.

This is the results.
South African clone


1st transfer of the South African


The South African is a Mad Man!



--------------------
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Invisiblemicro
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Re: Agar help please [Re: calicyco]
    #1536390 - 05/10/03 04:58 AM (13 years, 6 months ago)

"Try not putting any peroxide in it this time."

6 ml peroxide/L shouldn't be too much -- mycomasters.com recommends 6-8.
A great way to inhibit bacteria.

--
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Edited by micro (05/10/03 04:59 AM)


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Invisiblewoodrow
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Re: Agar help please [Re: micro]
    #1536436 - 05/10/03 05:38 AM (13 years, 6 months ago)

The agar recipe you are using is not the easiest or the best. Try one of the others suggested. If you are making peroxide agar, the peroxide must be added after the agar has been sterilized and before it gets hard otherwise the H2O2 will be destroyed by the heat. I find an easy way to make peroxide agar is to take regular agar and flood it for a few seconds with peroxide straight from the bottle and then let the agar drain.
Some bacteria can grow on peroxide agar. If you spread a bit of the rotted tissue across the agar, you should see colonies forming from whatever it is that that is causing the rot.
It might help to dip the tissue in straight peroxide before placing it on the agar.


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OfflineBaby_Hitler
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Re: Agar help please [Re: Fr0ggy]
    #1536477 - 05/10/03 06:56 AM (13 years, 6 months ago)

When I'm cloning I usually just tear the mushroom to expose it's innards. That way I don't get contamination from the outside surface inside the mushroom.


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OfflineFr0ggy
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Re: Agar help please [Re: Roadkill]
    #1536676 - 05/10/03 11:07 AM (13 years, 6 months ago)

Thanks for the recipe Road. I can't get the Potato Buds brand in my part of the world but will look for a suitable alternative.
Quote:

the peroxide must be added after the agar has been sterilized and before it gets hard otherwise the H2O2 will be destroyed by the heat.



I add H202 when the temperature falls to 130F so as not to break down the peroxide to it's trace elements.
Quote:

When I'm cloning I usually just tear the mushroom to expose it's innards. That way I don't get contamination from the outside surface inside the mushroom.



Yes, this is what I do as well. Slicing the mushroom with a scalpel tends to drag outer contams into the inner tissue.

What I failed to mention is I'm trying to clone store bought shrooms (agaricus bisporus, portobello, pleurotus pulmonarius, pleurotus cornucopiae, pleurotus djamor and shiitake) which may not be the freshest of specimens although their well within the sell by date on the packs..

Could this possibly be my problem?


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Invisiblemicro
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Re: Agar help please [Re: Fr0ggy]
    #1536860 - 05/10/03 01:32 PM (13 years, 6 months ago)

Yes, that's your problem. They really have to be 24-48 hrs old at the most.

--
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OfflineFr0ggy
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Re: Agar help please [Re: micro]
    #1536875 - 05/10/03 01:40 PM (13 years, 6 months ago)

Damn and blast !

Back to the drawing board !


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InvisibleMagashM
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Re: Agar help please [Re: Fr0ggy]
    #1537578 - 05/10/03 08:19 PM (13 years, 6 months ago)

Your on the right track. Experaments are the best way to learn.



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Amazon Shop for: Agar, Laminar Flow Hood, Potato Dextrose

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