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Fr0ggy
AmphibianExperimenter
Registered: 12/13/02
Posts: 85
Loc: Amazonian Lily Pad
Last seen: 19 years, 6 days
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Agar help please
#1534794 - 05/09/03 01:44 PM (20 years, 10 months ago) |
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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
-------------------- Ribbit ?
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micro
bunbun has a gungun
Registered: 05/09/03
Posts: 7,532
Loc: Brick City
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Re: Agar help please [Re: Fr0ggy]
#1534816 - 05/09/03 01:55 PM (20 years, 10 months ago) |
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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
-------------------- Any research paper or book for free (Avatar is Maxxy, a character by Mizzyam, RIP)
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Fr0ggy
AmphibianExperimenter
Registered: 12/13/02
Posts: 85
Loc: Amazonian Lily Pad
Last seen: 19 years, 6 days
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Re: Agar help please [Re: micro]
#1534826 - 05/09/03 01:58 PM (20 years, 10 months ago) |
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There is no sign of bacterial infection and from what I've read bacteria can't survive on peroxidated agar anyway!
-------------------- Ribbit ?
Edited by Fr0ggy (05/09/03 02:04 PM)
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micro
bunbun has a gungun
Registered: 05/09/03
Posts: 7,532
Loc: Brick City
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Re: Agar help please [Re: Fr0ggy]
#1534838 - 05/09/03 02:06 PM (20 years, 10 months ago) |
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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
-------------------- Any research paper or book for free (Avatar is Maxxy, a character by Mizzyam, RIP)
Edited by micro (05/09/03 02:08 PM)
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Fr0ggy
AmphibianExperimenter
Registered: 12/13/02
Posts: 85
Loc: Amazonian Lily Pad
Last seen: 19 years, 6 days
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Re: Agar help please [Re: micro]
#1534865 - 05/09/03 02:15 PM (20 years, 10 months ago) |
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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 !
-------------------- Ribbit ?
Edited by Fr0ggy (05/09/03 02:22 PM)
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micro
bunbun has a gungun
Registered: 05/09/03
Posts: 7,532
Loc: Brick City
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Re: Agar help please [Re: Fr0ggy]
#1534898 - 05/09/03 02:26 PM (20 years, 10 months ago) |
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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
-------------------- Any research paper or book for free (Avatar is Maxxy, a character by Mizzyam, RIP)
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Fr0ggy
AmphibianExperimenter
Registered: 12/13/02
Posts: 85
Loc: Amazonian Lily Pad
Last seen: 19 years, 6 days
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Re: Agar help please [Re: micro]
#1534914 - 05/09/03 02:30 PM (20 years, 10 months ago) |
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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 !
-------------------- Ribbit ?
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micro
bunbun has a gungun
Registered: 05/09/03
Posts: 7,532
Loc: Brick City
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Re: Agar help please [Re: Fr0ggy]
#1534974 - 05/09/03 02:45 PM (20 years, 10 months ago) |
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n/p, thanks and good luck!
-- Micro
-------------------- Any research paper or book for free (Avatar is Maxxy, a character by Mizzyam, RIP)
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CultyVader
Dark SporeApprentice
Registered: 03/13/03
Posts: 595
Last seen: 11 years, 6 months
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Re: Agar help please [Re: micro]
#1535065 - 05/09/03 03:18 PM (20 years, 10 months ago) |
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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
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calicyco
member
Registered: 05/03/03
Posts: 355
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Re: Agar help please [Re: CultyVader]
#1535078 - 05/09/03 03:23 PM (20 years, 10 months ago) |
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Try not putting any peroxide in it this time.
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Roadkill
Retired Shroomery Mod
Registered: 12/11/01
Posts: 22,674
Loc: Montana
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Re: Agar help please [Re: Fr0ggy]
#1536365 - 05/10/03 02:37 AM (20 years, 10 months ago) |
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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!
-------------------- Laterz, Road Who the hell you callin crazy? You wouldn't know what crazy was if Charles Manson was eating froot loops on your front porch! Brainiac said: PM the names with on there names, that means they have mushrooms for sale.
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micro
bunbun has a gungun
Registered: 05/09/03
Posts: 7,532
Loc: Brick City
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Re: Agar help please [Re: calicyco]
#1536390 - 05/10/03 02:58 AM (20 years, 10 months ago) |
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"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. -- Micro
-------------------- Any research paper or book for free (Avatar is Maxxy, a character by Mizzyam, RIP)
Edited by micro (05/10/03 02:59 AM)
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woodrow
journeyman
Registered: 03/17/03
Posts: 142
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Re: Agar help please [Re: micro]
#1536436 - 05/10/03 03:38 AM (20 years, 10 months ago) |
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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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Baby_Hitler
Errorist
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Re: Agar help please [Re: Fr0ggy]
#1536477 - 05/10/03 04:56 AM (20 years, 10 months ago) |
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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.
-------------------- "America: Fuck yeah!" -- Alexthegreat “Nothing can now be believed which is seen in a newspaper. Truth itself becomes suspicious by being put into that polluted vehicle. The real extent of this state of misinformation is known only to those who are in situations to confront facts within their knowledge with the lies of the day.” -- Thomas Jefferson The greatest sin of mankind is ignorance. The press takes [Trump] literally, but not seriously; his supporters take him seriously, but not literally. --Salena Zeto (9/23/16)
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Fr0ggy
AmphibianExperimenter
Registered: 12/13/02
Posts: 85
Loc: Amazonian Lily Pad
Last seen: 19 years, 6 days
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Re: Agar help please [Re: Roadkill]
#1536676 - 05/10/03 09:07 AM (20 years, 10 months ago) |
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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?
-------------------- Ribbit ?
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micro
bunbun has a gungun
Registered: 05/09/03
Posts: 7,532
Loc: Brick City
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Re: Agar help please [Re: Fr0ggy]
#1536860 - 05/10/03 11:32 AM (20 years, 10 months ago) |
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Yes, that's your problem. They really have to be 24-48 hrs old at the most.
-- Micro
-------------------- Any research paper or book for free (Avatar is Maxxy, a character by Mizzyam, RIP)
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Fr0ggy
AmphibianExperimenter
Registered: 12/13/02
Posts: 85
Loc: Amazonian Lily Pad
Last seen: 19 years, 6 days
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Re: Agar help please [Re: micro]
#1536875 - 05/10/03 11:40 AM (20 years, 10 months ago) |
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Damn and blast !
Back to the drawing board !
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Magash
Da Bud Guru
Registered: 07/25/02
Posts: 5,876
Loc: Near Hilo
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Re: Agar help please [Re: Fr0ggy]
#1537578 - 05/10/03 06:19 PM (20 years, 10 months ago) |
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Your on the right track. Experaments are the best way to learn.
-------------------- All creatures tremble when faced with violence. All creatures fear death, all love life. If we can only see ourselves in others, then how could we possibly hurt another creature? Join us at the Growery!
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