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ranonar
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Hydrogen peroxide agar upside down TEK, interesting little technique 1
#18569849 - 07/17/13 02:43 AM (10 years, 6 months ago) |
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It is a long time ago that I visited the Shroomery. But I still come here every now and then. And this time I found a bunch of comments of RR against the use of hydrogen peroxide in cultures. I feel he is overlooking something.
I use hydrogen peroxide to prevent the germination of contaminants. I have not been successful in killing established (visible) contaminants (molds, bacteria).
I do peroxide in agars (transferring mycelium) but I suppose I do it in a kind of unusual way. I do not use petri dishes. I use 250ml glass jars with screw caps. And especially I incubate them UPSIDE DOWN.
I fill these jars with 25ml peroxidated agar, then I swirl the agar around while it cools, so a thin (as thin as possible) agar film sticks to the inner side of the jar. Then I wait until the agar is solid.
Whatever mycelium I want to grow I put on this inner side of the jar (it sticks to the peroxidated agar film). And then I loosely screw the lid on the jar and I put it upside down on a shelf.
What happens is that the desired mycelium grows upwards over the peroxidated agar film (in the direction of the peroxidated agar layer) while contaminants, if present do not only need to fight the peroxide but if they are bacteria they tend to drop downwards (in the direction of the lid).
In the mean time, most of the desired mycelium (still on the wedge) is not in direct contact with the peroxidated agar film. It is free to actively support the part of the mycelium which is in contact (by producing peroxidase's, i.e. anti-peroxide compounds). The mycelium which has to colonize the peroxidated agar film, and later the agar layer, is slowly adjusted to the peroxide since at first it is only a film. And gravity ensures that the anti-peroxide compounds do not make the agar layer vulnerable for contaminant attack.
You can observe the mycelium growth by using a simple magnifier glass (way easier than on an agar layer on the bottom). The mycelium grows much faster on a film too compared to an agar layer.
The result is a kind of inbetween act, the mycelium is protected by peroxide in Rush Wayne's manner but not poisoned by it (RRs critique). Only the growing part (frontier) of the mycelium is in immediate contact with peroxide, a tiny bit of peroxide at first because it is on an agar film not an agar layer. The peroxide contact is also very short since the mycelium produces peroxidase's. And after use the peroxidase's drip to the lid of the jar.
I do not know if this technique works as well with chlorine or iodine. Peroxide at least is broken down in water and oxygen very fast, by enzymes which every form of life produces. Hydrogen peroxide is a very 'natural' way of combating unicellular contaminants. White blood cells make use of peroxide to kill bacteria. Bees make use of peroxide to keep their nectar uncontaminated until the honey is concentrated enough to do it, etc. So I would not be surprised if a mycelium can be selected for peroxidase production, i.e. peroxide fighting power. Anyway, I have not experienced any slowing down in mycelium growth in this way on agar. But I do like the fact that I do not need a flowhood or glovebox for cloning in this way!
I do not know yet about 'natural' applications of iodine or chlorine and I kind of dislike the residues they leave behind
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PussyFart
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: ranonar]
#18569896 - 07/17/13 03:25 AM (10 years, 6 months ago) |
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You are only using peroxide to "prevent the germination of contaminants", but if you practiced good sterile technique, you would realize that using peroxide just a waste of time, and peroxide, as there would be no contaminants to germinate.
If you make your dishes/whatever in a SAB or in front of a flow hood, there should be very little chance of contams.
Just seems like unneeded overkill to me....not using peroxide works just fine for everyone else.
--------------------
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ranonar
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: PussyFart]
#18569914 - 07/17/13 03:45 AM (10 years, 6 months ago) |
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But I do not need a flowhood!
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ranonar
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: ranonar]
#18569918 - 07/17/13 03:49 AM (10 years, 6 months ago) |
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Of course I do not need petri dishes or pressure canners either. I do not even need guns to make my point
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PussyFart
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: ranonar]
#18569973 - 07/17/13 04:52 AM (10 years, 6 months ago) |
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Quote:
ranonar said: But I do not need a flowhood!
Neither do I, because I have a SAB, good sterile technique, and a 99-100% success rate.

But if a $20 still air box is out of your budget, then you have bigger issues...
Quote:
ranonar said: Of course I do not need petri dishes or pressure canners either. I do not even need guns to make my point 
What point?
That you can take a completely unneeded substance like peroxide and use it with agar to get clean cultures that would have been clean regardless had you just used a little common sense and good sterile technique?
Not trying to be rude here, I see what you are saying about it not negatively affecting the mycellium, but it is just not needed.
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THIS HOBBY IS NOT FOR THE IMPATIENT! PLEASE BE PATIENT, DON'T BE A PATIENT! A Tale of 10 Isolates, GT Cluster Clone Monotubs, RR's Let's Grow Mushrooms DVD, SGFC(Shotgun Fruiting Chamber), Monotub Tek, Damion5050's Coir Tek, TL's Tek List, Frank's Tek List, EvilMushroom666's Pasteurization Tek, How It Should & Shouldn't Look - NEW CULTIVATORS GUIDE *** *** AFGHAN KUSH GROW LOG *** ***
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RogerRabbit
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: ranonar]
#18570105 - 07/17/13 06:19 AM (10 years, 6 months ago) |
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The thing most people don't realize is that peroxide breaks down very fast into water and oxygen in the presence of organic materials such as agar and malt. If one thinks peroxide put on agar more than an hour or two ago is still 'working' they'd be mistaken.
I mostly speak against dipping cultures in peroxide before placing on agar because it stunts them, leading to slower growth which makes it easier for competitor fungi to get the upper hand. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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ranonar
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: ranonar]
#18570178 - 07/17/13 07:03 AM (10 years, 6 months ago) |
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My point is that equipment like PC, SAB or flowhood is completely unneeded when you use hydrogen peroxide in agar, my point is not to get clean cultures from dirty ones but to keep cultures clean.
SABs and flow hoods prevent contaminant spores from landing on agar. Peroxide kills foreign spores after they land. The result is exactly the same: clean cultures.
But you are right in that when you use one thing you do not need another. I like to keep it simple and avoid equipment.
RR wrote: >If one thinks peroxide put on agar more than an hour or two ago is still 'working' they'd be mistaken.
I disagree. Here is a test: prepare a peroxidated agar plate. Do not inoculate. After a week, open it for 5 minutes in a normal still air environment (not a box, just close windows&doors). Agar will not contaminate because of the peroxide. Do the same thing with normal, not peroxidated agar and it contaminates.
But peroxidated agar plates quickly loose their peroxide protection when a peroxidase producing piece of tissue is put on it and it is wet. The water transports the peroxidase's over the agar surface and the peroxide is gone. Water which runs off the mycelium should not be allowed to collect on the agar. That is how I stumbled upon the upside down idea.
>I mostly speak against dipping cultures in peroxide before placing on agar
Agreed. People who dip tissue in peroxide attempt to kill contaminants on the surface of the tissue. This is difficult to do because the peroxidase's produced by the living tissue protect both the tissue and the contaminants (piggybacking). You need a much higher peroxide concentration to pull this off. PF used this method to clone primordia from a cake in an onsterile still air environment. It worked because those primordia are very clean to begin with (grown in a sterile invitro environment) and -because they are so young- in optimum condition to fight high peroxide concentrations.
If you dip a piece of un-sterile mushroom tissue in peroxide and then put it on agar it will usually contaminate. Especially when it is dripping wet and put on top of a flat agar surface (both peroxidated and 'normal').
But if you take some tissue from inside a mushroom stem (i.e. clean tissue) and put it on a peroxidated agar film in the upside down jar method then it will grow out clean, even if you do not use a hepa or still air box.
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noobs1988
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: ranonar]
#18587839 - 07/21/13 12:35 AM (10 years, 6 months ago) |
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Very interesting. I will be experimenting...
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Sillyputty67

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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: noobs1988]
#18598545 - 07/23/13 06:54 AM (10 years, 6 months ago) |
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Im not going down this rabbit hole. Hacker gave you perfect advice. Feel free to flirt with failure. We all do at some point, and afterwards feel free to come around to reason.
-------------------- 1) Everything I ever posted or say is a lie.
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noobs1988
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: Sillyputty67]
#18600447 - 07/23/13 03:37 PM (10 years, 6 months ago) |
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Quote:
Notahacker420 said:
Quote:
ranonar said: But I do not need a flowhood!
Neither do I, because I have a SAB, good sterile technique, and a 99-100% success rate.

But if a $20 still air box is out of your budget, then you have bigger issues...
Quote:
ranonar said: Of course I do not need petri dishes or pressure canners either. I do not even need guns to make my point 
What point?
That you can take a completely unneeded substance like peroxide and use it with agar to get clean cultures that would have been clean regardless had you just used a little common sense and good sterile technique?
Not trying to be rude here, I see what you are saying about it not negatively affecting the mycellium, but it is just not needed.
Quote:
malicom said: Im not going down this rabbit hole. Hacker gave you perfect advice. Feel free to flirt with failure. We all do at some point, and afterwards feel free to come around to reason.
I feel like yall aren't seeing his point. Why go threw all the trouble and money building SAB or buying flow hoods ( which are proven methods)to achieve cleanliness when you can add a little peroxide to your agar and achieve the same effect. Sounds a lot easier to me. And plus, if it works it works. Don't dis it man.,
I think that's all ranonar is trying to say.
Although, I can say that swirling the agar around till it produces a thin film does sound like a pain in the ass when it comes to doing 5+ jars. How long does it take for a thin film to dry using the swirling method?
I wouldn't mind seeing some pictures of what the agar looks like after it dries. So if you wouldn't mind posting a couple pictures, thatd be cool.
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anne halonium
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: noobs1988]
#18600490 - 07/23/13 03:48 PM (10 years, 6 months ago) |
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ive played with h202 agar. not against it, not for it.
i put my reliance on air filters, bleach, and SAB. i didnt see where h202 agar, gave me any advantage.
i did note, that after adding worthwhile amounts, that spores dont do so well on h202 agar.
roger has a strong point , about it degrading quickly. it does.
a really clean grow IMO, doesnt need h202. and, it wont do miracles, for a dirty grow either.
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Edited by anne halonium (07/23/13 03:53 PM)
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RogerRabbit
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: ranonar]
#18600631 - 07/23/13 04:26 PM (10 years, 6 months ago) |
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Another thing I didn't mention above is that peroxide doesn't necessarily kill spores if it's diluted into agar, but simply keeps them from germinating. On agar, we want any contaminants to germinate so we can transfer our healthy mycelium to a new dish. Peroxidated agar tends to bite one later when the cultures are spawned to grains and those dormant spores begin to germinate.
Storing cultures upside down also causes the moisture to leave the agar by gravity over time. This will cause your culture to dry out, and then get soaked when you turn it over again to work. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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ranonar
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: RogerRabbit]
#18618337 - 07/27/13 03:50 AM (10 years, 6 months ago) |
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>peroxide doesn't necessarily kill spores if it's diluted into agar, but simply keeps them from germinating.
While it is correct that peroxide does not necessarily kill spores (if that were true it would be totally impossible to germinate spore prints on peroxidated agar, which is not true), peroxide does not "simply keep them from germinating".
Peroxide does not work that way. It kills cells (spores) by rupturing their walls and then their DNA. Cells (spores) can only defend themselves by producing anti-peroxide enzymes (called peroxidases).
All living cells are capable to do that because hydrogen peroxide is a natural compound. All cells, with the exception of anaerobic bacteria,
produce hydrogen peroxide as a normal part of cellular metabolism (cells produce water as by product of their metabolism. In a small percentage of cases, however, oxygen yields a superoxide radical that may then be converted to hydrogen, for the complete explanation see https://en.wikipedia.org/wiki/Reactive_oxygen_species )
And while spores are, for that reason, equipped with a tiny amount of protective peroxidases from the start they need to produce more of those in order to continue their fight against the peroxide in the agar, which means they need to induce mycelial growth or they die. They can not just wait it out. In tiny amounts peroxide stimulates growth for that reason (and also another reason: the decomposition down of peroxide increases the local availability of oxygen).
Anyway there is a test for that. Mix spores in dilute agar with peroxide, wait a couple of minutes and put them on a suitable medium without peroxide. My bet: they will not gem inate. They never will because they are dead.
>Storing cultures upside down also causes the moisture to leave the agar by gravity over time. This will cause your culture to dry out, and then get soaked when you turn it over again to work.
No, that is not how evaporation works. Water molecules leave the agar layer as vapor and then fill the jar or petri, and eventually leave the container. Then the agar dries out. The molecules are very tiny so the gravity is not very important to them, they just float away. There even is a test for that. Just make two jars with a layer of agar, put one upside down and the other straight up. Do not inoculate. Close the lids and watch how the agar dries out (it may take many months). There is no free water which collects anywhere.
It is a different matter when mycelium is growing on the agar. All oxygen breathers produce lots of water. They have to because they produce carbon dioxide as a waste product. They need to get rid of it, and since carbon dioxide is a smaller molecule than a water molecule it is impossible to evolve or design a pore based membrane which allows carbon dioxide to get out and at the same time keep all the water in. Therefore, all breathable filters and membranes leak a bit of water. Including the mycelial 'skin'. Mycelium may sometimes produce water droplets. Fortunately it is easy to get rid of those droplets: they collect on the lid of the jar (because it is upside down). So all you have to do to keep the mycelium dry when you want to work with it is to lift the (upside down) jar from its lid and put it straight up. Then you can work.
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ranonar
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: anne halonium]
#18618355 - 07/27/13 04:00 AM (10 years, 6 months ago) |
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Swirling the agar around till it produces a thin film does sound like a pain in the ass when it comes to doing 5+ jars. How long does it take for a thin film to dry using the swirling method?
A quick way to do it is to place a couple of jars in a larger box or crate. Then swirl the box or crate around when the agars begins to thicken but is not solid yet. You do not need to keep swirling until the agar is totally solid, just a couple of good swirls is enough.
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fastfred
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: ranonar]
#18671830 - 08/07/13 04:17 AM (10 years, 5 months ago) |
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Quote:
>Storing cultures upside down also causes the moisture to leave the agar by gravity over time. This will cause your culture to dry out, and then get soaked when you turn it over again to work.
No, that is not how evaporation works. Water molecules leave the agar layer as vapor and then fill the jar or petri, and eventually leave the container. Then the agar dries out. The molecules are very tiny so the gravity is not very important to them, they just float away.
Wives tales can't be defeated with simple logic and evidence!
Peroxide has it's place, but it can't replace proper sterile technique. The only thing I use it for is dipping wild specimens. Flooding a dish with it might work well for knocking down spores and some bacteria, but it's certainly not a cure-all.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
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krypto2000
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: fastfred]
#18681388 - 08/09/13 02:16 AM (10 years, 5 months ago) |
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It sounds like introducing hydrogen peroxide, and thus the encouragement for peroxidase production, would essentially allow for a stronger set of mycilium in so far as they would the be capable of utilising more oxygen, and being aerobic, and many of its contaminants being aneroboic, this should not only help reduce contaminants in the short term but have the potential to make the mycilium itself stronger in selecting for this specific chemical change early on. As ranonar said, you just need to look for where the droplets form and cut sections from where they're most concentrated. You could theoretically measure the strength of the peroxidase production by using a controlled agar solution and standard procedures to measure how large the water droplet is from a single monoculture. If it's been selected for it should be reproducable with every petri and even show to react and increase in size with the introduction of hydrogen peroxide.
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ranonar
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: fastfred]
#18681484 - 08/09/13 03:16 AM (10 years, 5 months ago) |
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Dipping with peroxide is -IMHO- not very useful. Peroxide does not kill that fast. Even soaking for a prolonged time in peroxide is not enough to clean contaminated tissue.
I only use peroxide to keep clean substrates (especially agars) clean while they are exposed to an unclean environment (i.e. tabletop in a near still air environment, i.e. close windows&doors). The tissue which I work with do also need to be clean beforehand.
Come to think of it, I do not know a single example of an effective cleaning liquid which is able to instantly free living tissues from piggybacking contaminants (i.e. by dipping). Only mechanical measures (knives, tearing, gravity/putting agar layers upside-down etc.) work for me.
antibiotics may work but they require a couple of days (since bacteria do have to eat them first before they get poisoned). But antibiotics do not work on molds and yeasts so their application range is limited.
All in all, I agree with the statement that you can not reliably use peroxide to get rid of contaminants. You can not do without cloning material (tissue) and a suitable substrate which is clean to begin with. But you can do without a still-air box or flowhood easily. That is to say, I can.
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fastfred
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: ranonar]
#18690016 - 08/11/13 03:40 AM (10 years, 5 months ago) |
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Quote:
Peroxide does not kill that fast. Even soaking for a prolonged time in peroxide is not enough to clean contaminated tissue.
Peroxide does what it does. You can dip something and watch the dirt and bacteria foam away, visibly oxidizing right before your eyes.
A peroxide dip and a bleach dip does quite well at cleaning things. The difference between 1/6 and 0/12 makes or breaks you isolating from the wild. Inside tissue isn't always clean or easy to come by.
Low dose peroxide and peroxidated agar does nothing. If you're even thinking about peroxidase, then you've got the wrong idea from the start.
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nitrous
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: anne halonium]
#27974010 - 09/29/22 01:53 PM (1 year, 3 months ago) |
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I guess it's understood that the H2O2 being used is food grade ie stabilizer free.
One question about hydrogen peroxide in mycology - is it better than/equal to or inferior to isopropyl alcohol?
Again, I'm new to this, but is the standard 'sterilizer solution' 70% IPA?
Thanks Doug
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Baba Yaga
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Re: Hydrogen peroxide agar upside down TEK, interesting little technique [Re: nitrous] 1
#27974335 - 09/29/22 05:53 PM (1 year, 3 months ago) |
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Isopropyl or ethyl alcohol is the sanitizer to use. H2O2 can cause irritation or even burns of eyes, skin, mucosa etc. Depending on the strength this can be really bad.
ISO @ 60-70% is ideal for our hobby.
Please avoid resurrecting old threads and ask questions in a new post. This old crap about H2O2 agar doesn't need to be bumped.
Anyhow, welcome to the shroomery. Hope you are growing some boomers soon and having some fun along the way.
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