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Invisiblemicro
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Does H2O2 KILL spores or just inhibit germination?
    #1788870 - 08/07/03 04:25 PM (13 years, 3 months ago)

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!

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Invisiblemicro
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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1788904 - 08/07/03 04:34 PM (13 years, 3 months ago)



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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1788910 - 08/07/03 04:36 PM (13 years, 3 months ago)

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


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Invisiblemicro
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Re: Does H2O2 KILL spores or just inhibit germination? [Re: MrMaddHatter]
    #1788922 - 08/07/03 04:40 PM (13 years, 3 months ago)

Yeah, but that's bacteria....

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1788989 - 08/07/03 04:56 PM (13 years, 3 months ago)

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 - 08/07/03 06:26 PM (13 years, 3 months ago)

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.


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Re: Does H2O2 KILL spores or just inhibit germination? [Re: Hippie3]
    #1789666 - 08/07/03 08:22 PM (13 years, 3 months ago)

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:

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1789841 - 08/07/03 09:24 PM (13 years, 3 months ago)

Quote:

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.


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Re: Does H2O2 KILL spores or just inhibit germination? [Re: Hippie3]
    #1790237 - 08/07/03 11:04 PM (13 years, 3 months ago)

Neat! Thanks, I didn't know that.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: Hippie3]
    #1790239 - 08/07/03 11:04 PM (13 years, 3 months ago)

Very interesting.

Thanks for the link Micro :thumbup:


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Re: Does H2O2 KILL spores or just inhibit germination? [Re: MrMaddHatter]
    #1792068 - 08/08/03 02:22 PM (13 years, 3 months ago)

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 .


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Re: Does H2O2 KILL spores or just inhibit germination? [Re: mycofile]
    #1792122 - 08/08/03 02:40 PM (13 years, 3 months ago)

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.

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Re: Does H2O2 KILL spores or just inhibit germination? [Re: mycofile]
    #1792994 - 08/08/03 06:50 PM (13 years, 3 months ago)

Quote:

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.


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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1792998 - 08/08/03 06:51 PM (13 years, 3 months ago)

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 - 08/08/03 07:04 PM (13 years, 3 months ago)

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.

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


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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1793164 - 08/08/03 07:35 PM (13 years, 3 months ago)

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.


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Re: Does H2O2 KILL spores or just inhibit germination? [Re: Hippie3]
    #1793243 - 08/08/03 07:54 PM (13 years, 3 months ago)

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.

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


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InvisibleHippie3
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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1793352 - 08/08/03 08:27 PM (13 years, 3 months ago)

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.


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Re: Does H2O2 KILL spores or just inhibit germination? [Re: micro]
    #1795050 - 08/09/03 12:40 PM (13 years, 3 months ago)

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 - 08/09/03 12:52 PM (13 years, 3 months ago)

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.

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Edited by micro (08/09/03 01:15 PM)


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