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Does H2O2 KILL spores or just inhibit germination?
    #1788870 -

I know I've repeatedly read that H2O2 kills spores, but what's to say it doesn't just inhibit their germination? Has anyone read/done any work to show this?

I searched, but didn't find anything helpful. Thanx!

--
Micro


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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1788904 -

http://www.ncbi.nlm.nih.gov:80/entrez/qu...p;dopt=Abstract

--
Micro


--------------------
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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1788910 -

I have wondered this myself and did some searching just now.
According to this, peroxide kills spores.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: MrMaddHatter]
    #1788922 -

Yeah, but that's bacteria....

--
Micro


--------------------
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(Avatar is Maxxy, a character by Mizzyam, RIP)

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1788989 -

I would think it has the same effect on most spores, be it bacteria or fungus. But I have no idea if theres a difference between bacteria spores or fungus spores.
It states:
hydrogen peroxide that abolish spore
germination by a) causing very severe damage to the
spore?s inner membrane or b) damaging spores such that
initial events in germination can occur, but the germinated
spore cannot swell and grow, respectively.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: MrMaddHatter]
    #1789265 -

peroxide and spores mutually annihilate each other.
as the peroxide destroys the spores, it is itself consumed.
so you can overwhelm a dilute concentration of peroxide in an area by using massive amounts of spores so that some still survive after the peroxide is gone, such as with peroxidated agar.


--------------------
Admin @ mycotopia.net
Mycotopia

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: Hippie3]
    #1789666 -

Oh -- I was under the impression that you can't grow from spores on peroxidated agar -- I guess it just kills a certain precentage, then.  It "stuns" the mycelium at first, too -- is this the same idea?

That would explain what it said in the article from the link I posted above.  Here's another:

Mol Cell Biochem. 1999 May;195(1-2):169-72.  Related Articles, Links 


Effects of chloroperoxidase and hydrogen peroxide on the viabilities of Aspergillus flavus conidiospores.

Jacks TJ, De Lucca AJ, Morris NM.

Southern Regional Research Center, USDA, New Orleans, LA 70179, USA.

The effects of chloroperoxidase [EC 1.1.1.10] and hydrogen peroxide on the viabilities of quiescent and germinating conidiospores of an aflatoxigenic fungus, Aspergillus flavus, were determined. Hydrogen peroxide was found moderately lethal and chloroperoxidase produced a 30-fold increase in the lethality of hydrogen peroxide to germinating conidia, which were 75-fold more susceptible to chloroperoxidase than were quiescent conidia. According to infrared examinations of fungal corpses, mortality occurred by oxidation rather than peroxidative chlorination.

Madd -- I think fungal spore walls are mostly chitin and glucans.  It seems bacterial endospores are different, but also have some resistance to H2O2:

The 65-kDa CotA protein is an abundant component of the outer coat layer. CotA is a highly thermostable laccase, assembly of which into the coat is required for spore resistance against hydrogen peroxide and UV light.

(from: http://www.ncbi.nlm.nih.gov/entrez/query...;dopt=Abstract)

Thanks :wink:

--
Micro 


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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1789841 -

micro said:
Oh -- I was under the impression that you can't grow from spores on peroxidated agar -- I guess it just kills a certain precentage, then.



think of it like an anti-matter/matter collision.
each spore destroyed consumes a bit of the peroxide.
on agar, peroxide cannot move.
so if you streak an esp. thick smear of spores on,
there will be enough to both neutralize the peroxide there and still have enough survive intact to germinate.
but if the peroxide is stronger, then no spores survive.



Quote:
It "stuns" the mycelium at first, too -- is this the same idea?


not exactly.
living mycellia is a more advanced organism and has enzymes it releases to break-down the peroxide without destroying itself.
but that process does take time and slows down any growth until the mycellia has eliminated the peroxide.


--------------------
Admin @ mycotopia.net
Mycotopia

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: Hippie3]
    #1790237 -

Neat! Thanks, I didn't know that.

--
Micro



--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: Hippie3]
    #1790239 -

Very interesting.

Thanks for the link Micro :thumbup:

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: MrMaddHatter]
    #1792068 -

Not to knock hippie, but I just want to point out that unless he's found some studies in the last week or so, he's just guessing at exactly how this works.

The facts are the same, use a heavy spore streak on peroxidated agar and it will germinate.

I think hippies got a pretty good idea of how it works, it makes sense, but it's just conjecture.

I'd also note that some people have guessed that peroxide can cause genetic damage to germinating spores, and several have repeated this as if it were a fact. I've seen no reason, or sound explanation for why this might be the case, or any evidence that it's true, so unless a scientific study shows differently, I will dismiss it as a remote possibility at worst .


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: mycofile]
    #1792122 -

Well, back to the drawing board, I guess, then, unless some information can be cited. Thanks, Myco. It made sense to me at the time, though, because fungi can produce peroxidases....

I would think you're selecting out a substrain resistant to the H2O2 which is why I don't use it. As for the mutagen thing, it's an oxidizer so I would think it could promote mutations, but I don't think it could actually cause them. Sort of along the lines of taking antioxidants to counter oxidative mutations.

--
Micro


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: mycofile]
    #1792994 -

mycofile said:
Not to knock hippie, but I just want to point out that unless he's found some studies in the last week or so, he's just guessing at exactly how this works.



Actually, he's got it down pretty straight, and this is old info.

Living cells contain an enzyme that breaks down hydrogen peroxide into water and oxygen. They need this because all cells produce H2O2 in small amounts during metabolic functions, and need to break down this poison.

If you overwhelm an organism with H2O2, and it can't produce enough enzyme to neutralise the stuff, then it'll die. This is what happens to lone spores on a plate.

On the other hand, aggregate cells have a rather easy time dealing with H2O2 because they can produce more enzyme. The thing is, it's -not- a matter-antimatter kinda thing -- one molecule of enzyme can destroy many, many molecules of peroxide.

If you laid down a large spore streak, then a lot of spores would die, but the release of the enzymes would create a peroxide-less area in the agar directly beneath and around the spores, which the spores that made it through could take advantage of.

I've never heard of peroxide causing genetic damage, but I suppose that it's technically possible. I don't think that any damaged spores would be likely to grow and reproduce, though.


--------------------
"Do we want the stars? We can have them. Can we borrow cups of fire from the sun? We can and must and light the world." --"On the Shoulders of Giants", Ray Bradbury

All of my posts are full of fiction and blatant lies.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1792998 -

If I have only one print of a species, would you recommend heavy streaking on peroxidated agar or light to normal streaking on regular agar? First time agar user here.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: YidakiMan]
    #1793045 -

Sev -- yeah, I guess it makes sense, but mycelium lives fine on peroxidated agar while other species that should also produce peroxidases seem not to, like bacillus for example. It may have to do with cell wall, too, which is made of chitin, but I don't know.

I think Hippie's right up to a point, but myco's point is that there is probably no research to back it up.

--
Micro


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)

Edited by micro (08/08/03 05:07 PM)

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1793164 -

lol
i'm not just making this shit up as i go along, folks.
and my matter/anti-matter analogy applied only to spores and peroxide, so the enzyme factor wasn't the point since that only applied to living tissue, not spores, which is what afterall, i was talking about.


--------------------
Admin @ mycotopia.net
Mycotopia

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: Hippie3]
    #1793243 -

Spores secrete enzymes but not as many as a live cell would. Even bacterial endospores secrete enzymes.

I understand the analogy but it isn't very specific. Nothing really colides and destroys each other like in matter/antimatter; the peroxide gets reduced into something else which requires something in the spore to get oxidized; in this case it would have to attack the protinaceous outer coat, first. This doesn't convince me that this is how all the peroxide gets reduced in the case of spores and that it's not an effect of enzymes. Bacterial endospores even make peroxidases.

I think this is where the term "conjecture" comes into play. I'd also need to see data on how much peroxide is actually reduced and how much is still in the media after the spores germinate and survive.

I think we're all stuck here, so I think I'll email R. R. Wayne, Ph.D. and see if I get a reply. I'll post a copy of the email if I do.

--
Micro


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)

Edited by micro (08/08/03 06:14 PM)

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1793352 -

lol
any analogy breaks down when you get specific,
i was merely trying to give an easy-to-understand
illustration of what's it's like,
not trying to be technically accurate on all science.
but for the record,
no, it's not exactly like matter/anti-matter.
the only real similarity, the point i was going for, is that the spores and the peroxide mutually annihilate.
that's all.


--------------------
Admin @ mycotopia.net
Mycotopia

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1795050 -

Yep. Old news. It is possible to germinate (a large amount of) spores on peroxidated agar. I reported the phenomenon to Rush Wayne in 1999 and a year later it was published in part 2 of his manual at page 5.

The reason that this technique is not really promoted is that there is an older technique for 'spore germination without HEPA/Glovebox' which works without peroxide and with only tiny specs of spores.

Let's call it the test tube tek. It is the technique which Paul Stamets and Andrew Weil used to clone a couple of highly desired medicinal mushrooms in China and take those to the US. All you need for it is a screwcap slant of agar, a metal pin or neelde and a lighter.

And no I am not going to describe it in detail here. The technique is also in the 2nd manual of Rush Wayne. See WWW MYCOMASTERS COM (page 6&7 of manual II). And of course there need to be dots . between the www and the mycomasters as well as between the mycomasters and the com but or some weird reason it is impossible to link to the original source here?!

Yachaj

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: mycophreak]
    #1795063 -

Myco -- Dr. Wayne explained to me why in an email he sent me this morning:

Quote:
Hello Micro,

Thanks for your note.

The selectivity of peroxide comes entirely from the fact that a greater amount of peroxide-decomposing enzymes can be mustered by multicellular or clumped organisms than can be mustered by individual spores or cells. All organisms produce the enzymes, but the isolated ones are easily overwhelmed by peroxide, whereas the multicellular ones can defend themselves against it.

--Rush Wayne




"Clumped" would be talking about the spores. I know Sev said this above, but it clarifies the fact that spores are clumped and produce the enzymes, too, in addition to the mycelium. The mycelium should produce more enzymes than the spores, too. I never thought the fact that they are clumped would be a factor.

--
Micro


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)

Edited by micro (08/09/03 11:15 AM)

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1796147 -

clumped is just another word for concentrated,
thus the principle remains the same.
a concentration [clump] of spores can locally over-whelm the peroxide and germinate.
like i said way back a few posts ago..


--------------------
Admin @ mycotopia.net
Mycotopia

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: Hippie3]
    #1796168 -

a friend recently drew up a cc or 2 into a spore
syringe, mistaking it for a culture syringe.

(lesson: no smokey while workey)

afoaf told him, well, good luck, I think you've
killed her.

he came back a few weeks later and said that
he had knocked up some brf jars just for shits
and giggles and that they had germinated and
were showing PHENOMENAL growth.

true story.


--------------------
All I know is The Growery is a place where losers who get banned here go.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: afoaf]
    #1799327 -

Yaeh, Hippie -- thanks for your vague post. You'd get 1/2 cerdit if I were grading you on a test....

--
Micro


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1800002 -

Hippie3 has a good analogy, and this statement in Dr Rush?s e-mail to Micro backs it:

?All organisms produce the enzymes, but the isolated ones are easily overwhelmed by peroxide, whereas the multicellular ones can defend themselves against it.?

Appaerently there are peroxidase enzymes inside the spores, which will decompose the peroxide, but the spore is destroyed in the process. The spores are not fully alive like the mycelium, which can produce its own peroxidase enzymes as needed.

The peroxidase enzyme can only decompose a certain amount of peroxide before it gets burned out and stops working. Therefore, using a large clump of spores will eventually create an area of non-peroxidated agar media for the unaffected spores (those protected inside spore clumps) to germinate.

Assume each spore had one peroxidase, and that peroxidase could catalyze one molecule of peroxide. You take a spore clump containing 150 spores; 100 spores exposed on the outside of the clump and 50 contained within. You place the spore clump on peroxidated agar. The spore clump occupies a surface area containing 100 molecules of peroxide. The 100 spores on the outside of the clump are destroyed while decomposing the 100 molecules of peroxide present in that area. This leaves that area of agar media without any peroxide, yet there are still 50 spores left that can germinate and mate with each other. Therefore, the spore clump has OVERWHELMED the peroxide in the agar.

The peroxidase enzyme and peroxide are similar to Hippie3?s matter and anti-matter. They both will eventually cancel each other out.


--------------------
If it's not broken, then the government will try to fix it until it is.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: mah2b1]
    #1800019 -

As for AFOAF, the spores syringes made with peroxide probably underwent the same process, leaving behind only a few viable spores that were protected in clumps. Those clumps went on to germinate.

There could be many explanations for the aggressiveness of the mycelium produced. One may be that there were less sub-strains produced (from the viable spores mating) that would compete amongst themselves for available substrate. Or, the use of the peroxide (antibiotic chemical) weeded out the bad spores leaving behind the stronger more viable spores for mating after the peroxide was decomposed. These are only two reasons for your friend?s success, after all your friend botched up the syringes to begin with, but it is an interesting bit of information.

What was the concentration/formula of the peroxide solution used to make these accidental spore syringes (may come in useful when working with contaminated prints)?


--------------------
If it's not broken, then the government will try to fix it until it is.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1800937 -

suit yerself,
you can count on that being the very last time i waste my time trying to educate you.
i teach you something you didn't know,
Quote:
Neat! Thanks, I didn't know that.


and you then repay me with this shit ?
Quote:
Yaeh, Hippie -- thanks for your vague post. You'd get 1/2 cerdit if I were grading you on a test....





--------------------
Admin @ mycotopia.net
Mycotopia

Edited by Hippie3 (08/11/03 01:01 PM)

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: Hippie3]
    #1801473 -

This has gotten silly. The answer to the original question/post, as hippie just pointed out, is that spores can germinate in the presence of peroxide, and we have some practical guidlines for how to make that work.

I really didn't mean to sow the seeds of dissent against hippie, I just like to be pretty careful in describing how I think something works vs the way it's been proven to work. I think hip's original anologies of overwhelming clumps and matter/anti-matter are good anologies for a general understanding of the practical applications. But I've not seen scientific proof that it's exactly right. It very well could be a matter of strains whose spores are more peroxide resistant being selected, like with bacteria and antibiotics. I don't really think so, I think hippie and Wayne are on the right path, but I just haven't seen proof of it, and I only leave the possibility of Wayne being wrong because he stated in his first book that this was impossible.

And yes, this is old news. The method was first brought to my attention back when drooldonkey first opened, so that was years ago.

And yes, it's not necessary, mycophreak talks about using slants, and simply flaming the lip before opening. Not to mention, I know plenty of people who have decent success doing open air agar. Especially when you use peroxidated agar for the 2nd and subsequent plates, the first one doesn't NEED to be peroxidated.

So, unless somebody does have a truly scientific breakdown of the processes involve, then there is no need arguing. It can be done. We know how to make it more likely to be successful (high spore concentration). Why argue?

Oh and to
Quote:
If I have only one print of a species, would you recommend heavy streaking on peroxidated agar or light to normal streaking on regular agar? First time agar user here.


Since a print has plenty of spores to streak hundreds of plates, peroxidated or not, I wouldn't recomend one over the other. It's just a matter of your personal situation. I mean if you've got a flow hood, you wouldn't use the peroxide. Either way you should be succesful if you follow the proper guidlines and principles.


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: mah2b1]
    #1810838 -

it wasn't my intention to imply that growth
was phenomenal because of the peroxide.
moreso, it was phenomenal in spite of the
peroxide in the syringe.

it was a 10cc syringe, he prolly drew up an
additional 2 cc's of 7% peroxide.

it was definitely a dark syringe and the strain
tends to be quite voracious during colonization.

maybe just enough surivived to keep hope alive.


--------------------
All I know is The Growery is a place where losers who get banned here go.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: afoaf]
    #1813012 -

Yeah, good clarification, in spite of rather than because, I was a little confused about exactly what you meant too.


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: afoaf]
    #1814910 -

Thanks for the feedback AFOAF.

I have some thick prints that I know are contaminated. I am hoping that this spore clump protection idea will work with mild peroxide treatment.

I am figuring that the contams are only on the outsides of the spore clumps, and that if I hydrate them with water and then treat them with peroxide that it will kill off most of the contams. This should give me a better chance to obtain some clean mycelium for a tissue transfer.


--------------------
If it's not broken, then the government will try to fix it until it is.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: mah2b1]
    #1816790 -

Yeah, but you could just germinate them on agar, then isolate the mycelium for the transfer. If it's just a matter of not wanting to mess with agar, BRF paste is a good alternative. The flat surface of these substrates just makes the isolation and transfer a lot easier.


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: mycofile]
    #2129389 -

The peroxidase enzymes came from the gills of the mushroom and are just stuck to the spores.




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Re: Does H2O2 KILL spores or just inhibit germination? [Re: mickywilliams2]
    #2129867 -

Fact or guess?


--------------------
Grow Mushrooms from Spores - PF-Tek for Simple Minds

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: Anno]
    #2130320 -

guess


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Re: Does H2O2 KILL spores or just inhibit germination? [Re: mickywilliams2]
    #2130480 -

Spores produce enzymes -- they clump together and can produce more enzymes for a given volume than something like bacteria (or isolated spores) could because of the fact that they clump together. When it's in a liquid, for example, it obviously can't exhaust all the peroxide in the area around it -- it just neutralizes it.

This confuzed me for a while.... It's the clumping of spores or the organization of the hyphae that enables it to exhaust the peroxide -- it doesn't work for bacteria, because of this.

Oh, and I hate to beat a dead horse, but I forgot about this post. I didn't mean to start an argument, but Hippie -- what you said:

Quote:
and my matter/anti-matter analogy applied only to spores and peroxide, so the enzyme factor wasn't the point since that only applied to living tissue, not spores, which is what afterall, i was talking about.



is implicitly wrong -- that's why I meant your post was vague. The spores do produce peroxidases, and they clump, so they're able to knock out the peroxides in a given area because they produce more enzymes per given area than a bacteria, for example, since they do not clump.

Oh, yeah, and:

Quote:
"clumped is just another word for concentrated,
thus the principle remains the same."



No, it is not. Spores clump -- bacteria does not.

--
Micro


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #2131419 -

depends on the concentration :smile:.....

high percentages of H2O2 are known to cause organic matter to explode....(we're talking 60+%)





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Re: Does H2O2 KILL spores or just inhibit germination? [Re: Elemicin]
    #2134321 -

I would assume we're talking about the concentration used in agar, since that's what this thread is about....

--
Micro


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #2137195 -

micro said:
Quote:
hippi3 said:"clumped is just another word for concentrated,
thus the principle remains the same."


No, it is not. Spores clump -- bacteria does not.

--
Micro


While I have no wish to enter this argument I do have to agree with micro on that one. Bacteria may be concentrated but are in no way "connected" to one another except during mitosis(?). The spores, OTOH, actually web/knit themselves together with mycelium, as is their nature. That is all.


--------------------
To be honest, I fear the OTD. ---TheMadConductor

Now I'm gonna have to start getting down on my knees and blowing mushrooms... ---RogerRabbit

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Mushrooms, Mycology and Psychedelics > Advanced Mycology
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