Peroxide agar in kitchen tek- huge success!

[Darklight]

Just thought I'd let you know:

I have had a 100% sterile success rate with the peroxide agar kitchen tek, both with the cooked method ( no autoclaving ) and the autoclaved method. Methods and comparison below

Dispensing and media transfer was done under absolutely not sterile conditions, in order to see if the tek could be replicated anywhere ( those of you who know my kitchen can stop laughing ). I was a tad sloppy with my transfer tek as well to see what would happen

Reporting it here because sometimes the signal/noise ratio can be a little loud elsewhere

Batch 1- autoclaved + 6ml/L 3% H2O2

Chinese sauce containers were autoclaved in a bag for 20 min

100ml liquid MEA autoclaved for 20 min, consisting of the following

• 20g/L light malt extract
• 0.1g/L garden lime
• 0.1g/L potassium phosphate dibasic
• 15g/L gelcarin ( 20g/L agar works just as well )

Media was not subject to a pH test

Post autoclave the media was allowed to cool and just above setting point 6ml/L 3% H2O2 was added.

Media was swirled heavily so that the inner surfaces of the bottle were fully coated

Dispensed immediately into sauce containers *on the kitchen bench* in the open air

Lids were placed lightly over the containers and 1hr later the plates were completely sealed after inoculation with various species

Batch 2- cooked 1hr + 6ml/L 3% H2O2

Chinese sauce containers were autoclaved in a bag for 20 min

100ml liquid MEA cooked for 1hr by placing media container in an open saucepan. Media container lids were left loose. Water came up the the media level- bottles weren't more than 3/4 immersed. Cooked at a slow boil for 1hr

• 20g/L light malt extract
• 0.1g/L garden lime
• 0.1g/L potassium phosphate dibasic
• 15g/L gelcarin ( 20g/L agar works just as well )

Media was not subject to a pH test

Post autoclave the media was allowed to cool and just above setting point 6ml/L 3% H2O2 was added.

Media was swirled heavily so that the inner surfaces of the bottle were fully coated

Dispensed immediately into sauce containers *on the kitchen bench* in the open air

Lids were placed lightly over the containers and 1hr later the plates were completely sealed after inoculation with various species

Sterile ( non-peroxide MEA ) library cultures were opened and haphazardly used ( left open for much of the inoculation process ) to inoculate the plates above using a scalpel blade which was only cleaned and flamed between species

The following species were placed in the centre of the agar of each container

• Reishi ( Ganoderma lucida )
• Blue Oyster ( Pleurotus spp )
• Lion's mane ( Hericium erinaceus )
• Elm Oyster ( Hypsizgus ulmanarius )

Plates were sealed with Austraseal and incubated in the dark at 22C

2 plates from the cooked group and 2 plates from the autoclaved group were left uninoculated as controls to check for contamination during dispensing

By my judgement it was all a bit haphazard and I didn't believe it would work. Even a contam rate of 10% per batch would have been acceptable

At +1 week there is no contamination, anywhere, and growth is good for the Reishi and Elm Oyster. Still waiting on the Blue Oyster and Lion's Mane, but plenty of time yet- those parent cultures could have been a little old- I have storage/ library cultures and can reinoculate from them easily at +3 weeks

If you are thinking about the peroxide tek for agar- give it a go. I've only made it sound complex cos I wanted to write it all up so you know I took all the steps. I now pronounce this part of the tek piss easy https://www.shaman-australis.com/forum/applications/core/interface/imageproxy/imageproxy.php?img=&key=ed93ee4b8a158835e0af19ead9c794b11af03de360911f7858b9da338588c45d

Edited very fast, because I am an idiot and forgot to put the decimal point in

Edited May 29, 2013 by Darklight

[watertrade]

Thanks Darklight - I used the peroxide agar method for years with good success - very few contaminations.

My method is essentially the same of yours but with only 7ml/L of 3% H2O2 into autoclaved media (all kinds of different media used) also the H2O2 was added when the temperature of the media was just below 60 degrees.

My standard media would be

• 20g/L light malt extract
• 2g/L Yeast
• 20g/L agar
• *optionally (and often) I would add a pinch of any range of flours, wheat rice, corn etc. I also liked to add powdered straw/ wood/ manure depending species I was growing.

I actually promised Worowa that I would reinstate his fungal collection since he moved - so I will be doing this all again soon!

Edit: I was going to say 60ml/L H2O2 seams like too much peroxide to me. (?) & apparently some species don't like H2O2. although I have yet to find one though.

Edited May 29, 2013 by watertrade

[Darklight]

Shit mate, you're right! I used 0.6% H2O2- that's 6ml/L of 3% peroxide

Glad you pointed that out. I'll change the original post

Hope no-one copied and pasted it, thanks for bringing it to my attention so fast!

[watertrade]

I figured it was something like that

[obtuse]

I too have used the H2O2 method with great success. certainly thumbs up from me.

i think mine might be a little stronger with between 8 ml/L to 10 ml/L 3% H2O2, but i dont notice any problems.

Thanks Darklight for posting. Its great to have a scientific protocol floating around rather than try this or that which is what i am more inclined to write lol

Cheers, Ob.

Edited May 29, 2013 by obtuse

[Bigred]

I use the hydroponic h2o2 and it has colloidal silver in it . I use it in a lot and it works wonders .

Thanks for the tek, its great for newbies and will give it a try next batch.

[seachangeau]

Just thought I'd let you know:

I have had a 100% sterile success rate with the peroxide agar kitchen tek, both with the cooked method ( no autoclaving ) and the autoclaved method. Methods and comparison below

Dispensing and media transfer was done under absolutely not sterile conditions, in order to see if the tek could be replicated anywhere ( those of you who know my kitchen can stop laughing ). I was a tad sloppy with my transfer tek as well to see what would happen

Reporting it here because sometimes the signal/noise ratio can be a little loud elsewhere

Batch 1- autoclaved + 6ml/L 3% H2O2

Chinese sauce containers were autoclaved in a bag for 20 min

100ml liquid MEA autoclaved for 20 min, consisting of the following

• 20g/L light malt extract
• 0.1g/L garden lime
• 0.1g/L potassium phosphate dibasic
• 15g/L gelcarin ( 20g/L agar works just as well )

Media was not subject to a pH test

Post autoclave the media was allowed to cool and just above setting point 6ml/L 3% H2O2 was added.

Media was swirled heavily so that the inner surfaces of the bottle were fully coated

Dispensed immediately into sauce containers *on the kitchen bench* in the open air

Lids were placed lightly over the containers and 1hr later the plates were completely sealed after inoculation with various species

Batch 2- cooked 1hr + 6ml/L 3% H2O2

Chinese sauce containers were autoclaved in a bag for 20 min

100ml liquid MEA cooked for 1hr by placing media container in an open saucepan. Media container lids were left loose. Water came up the the media level- bottles weren't more than 3/4 immersed. Cooked at a slow boil for 1hr

• 20g/L light malt extract
• 0.1g/L garden lime
• 0.1g/L potassium phosphate dibasic
• 15g/L gelcarin ( 20g/L agar works just as well )

Media was not subject to a pH test

Post autoclave the media was allowed to cool and just above setting point 6ml/L 3% H2O2 was added.

Media was swirled heavily so that the inner surfaces of the bottle were fully coated

Dispensed immediately into sauce containers *on the kitchen bench* in the open air

Lids were placed lightly over the containers and 1hr later the plates were completely sealed after inoculation with various species

Sterile ( non-peroxide MEA ) library cultures were opened and haphazardly used ( left open for much of the inoculation process ) to inoculate the plates above using a scalpel blade which was only cleaned and flamed between species

The following species were placed in the centre of the agar of each container

• Reishi ( Ganoderma lucida )
• Blue Oyster ( Pleurotus spp )
• Lion's mane ( Hericium erinaceus )
• Elm Oyster ( Hypsizgus ulmanarius )

Plates were sealed with Austraseal and incubated in the dark at 22C

2 plates from the cooked group and 2 plates from the autoclaved group were left uninoculated as controls to check for contamination during dispensing

By my judgement it was all a bit haphazard and I didn't believe it would work. Even a contam rate of 10% per batch would have been acceptable

At +1 week there is no contamination, anywhere, and growth is good for the Reishi and Elm Oyster. Still waiting on the Blue Oyster and Lion's Mane, but plenty of time yet- those parent cultures could have been a little old- I have storage/ library cultures and can reinoculate from them easily at +3 weeks

If you are thinking about the peroxide tek for agar- give it a go. I've only made it sound complex cos I wanted to write it all up so you know I took all the steps. I now pronounce this part of the tek piss easy

Edited very fast, because I am an idiot and forgot to put the decimal point in

gee thanks so much - can i put this onto a document?

[Darklight]

gee thanks so much - can i put this onto a document?

Sure, thanks for asking https://www.shaman-australis.com/forum/applications/core/interface/imageproxy/imageproxy.php?img=&key=ed93ee4b8a158835e0af19ead9c794b11af03de360911f7858b9da338588c45d

[worowa]

I've used from 3mL/L to 25mL/L...always works, except for a "stropharia rugoso-annulata" (never got it to fruit, so not sure if it was the real deal). Mycelium moves slower with peroxide, but that's a good thing usually. 1000's of plates, no contams. Still get the occasional grain spawn contam, but I've never used peroxide in grain.

My current recipe, which probably needs updating to avoid Nestle is 1 tablespoon malted milo, 1 tablespoon organic black strap mollasses, 1 cup of freshly brewed black coffee, 25 grams agar (1 packet swan brand), top up with boiled tap water to 2L. The extra agar makes it set thick, which makes it easier to take wedges out. This is nice and dark, easy to see mycelium. Add peroxide between 50 and 60 degrees.

[mesq]

Bump!

I'm glad I found this thread. I used peroxidated agar, pouring in the open with good success many years ago. Recently logged onto the shroomery where i saw Roger Rabbit has largely disregarded peroxidated agar as uneccessary and irrelevant. My experience was good with h2O2 so i think ill use it again soon

[mesq]

Also used it in my dextrose water as shown by this ancient and blurry photo sure it takes a little bit longer for the mycelium to take hold, but I think it saves a lot of mucking about

[lindsay]

h2o2 great stuff for cleaning up wild mushrooms. I put this whole

Trametes in a 3% h202 for 30sec then chopped up and put on

h202 plates.

it took a few days to start growing and a week to get to the edge of

a 90mm plate. yep peroxide does slow the growth.

the next transfer on to a non-h2o2 and this thing unleashed itself.

this is a 90mm plate, 36hrs after the transfer.

actually is scares me, I don't know what I have captured.

[LokStok]

turkeytail is a monster on plates! (and grain, and plugs, and straw, and....)

[drpotato]

I had a 110% success rate adding 2ml during the open air boil (in a flask tho) then 3ml (both instances 6%) right before pouring.

By 110% i mean that it was so effective that a couple cultures didnt even grow, they got killed by the shock, though i later discovered my oyster was actually fast growing mold. My cubensis however grew really slow. but a couple covered the plate which was neat. I tried again without using a SAB for the pour, half contaminated by germinating and expanding from the sides of the lid. it wont germinate off the agar but it can still colonize it