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Spore washing experiment [ dilute bleach ]


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#1 rip

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Posted 17 December 2006 - 07:45 AM

I hope someone finds this useful.

I received a fresh outdoors Azurescens print with good vibes from FSRE. (Thanks!) First I tried to germinate the spores on agar. Unfortunately the print was heavily contaminated with a mold which produced spores before any mushroom mycelia could be detected.

So I decided to try washing the spores before putting them on agar. I could not find any other useful info exept some mold spores are quite resistant to H2O2 or moderate heat, but suspectible to sodium hypochlorite. I tried one hour treatment of 3%, 1%, 0.3% and 0.1%, (30, 10, 3 and 1 ml/l,) dilutions of cheap household bleach with 5% or less of sodium hypochlorite, roughly 20, 6, 2, 0.6 mMol/l or less of NaOCl.

After a few week incubation, looks like one hour of 3% and 1% bleach kills all azurescens spores and everything else and 0.3% and 0.1% appear to not affect germination. I was expecting less steep treshold and and used way too much spores to make a colony count. I've detected no mold or other contaminants. Washing spores with 0.3% bleach might make possible to even skip the agar step with outdoors azurescens spores. Bleach tolerance of spores from different mushroom species may be different.

The Tek:

Hydrate the spores with small amount of water and mix a drop of the spore suspension with 0.3% diluted bleach in a small test tube. Let settle for 1 hour in room temperature. Remove most of the bleach with a syringe and discard, suspend the spores from the bottom of the tube in small amount of sterile water and spread on agar. If you have access to a centrifuge or NaOCl of known concentration, use em.

(Disclaimer: I have no prior experience with azurences or other woodlovers.)

Attached Thumbnails

  • azu_washed_1.jpg
  • contaminated_spores1.jpg


#2 spacecake

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Posted 17 December 2006 - 07:54 AM

How do you discard the bleach from the spores ?
Do you let the spores sink to the botttom and the suck up the water/bleach ?

..Maybe you can put the (contaminated)spores in a kind of thea bag first,..and then put the theabag in the water/bleach solution ?

#3 golly

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Posted 17 December 2006 - 08:44 AM

That's pretty interesting Rip...If your experiment can be repeated ,then that could be a really helpful Tec...thanx for posting...

#4 rip

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Posted 17 December 2006 - 10:20 AM

I just let the spores to sink to bottom of the tube and then sucked out as much of the bleach solution I could. Pros of course would use a centrifuge. Please replicate this
and try it with other species.

Next I'll have to work with my the azu mycelium. I live in zone 4, so I have not much hope for outdoors furting. (I'll try it anyway.)

#5 cyberd0c

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Posted 17 December 2006 - 10:39 AM

Hi rip, and very good thinking. I tried successfully in the past a spore filtration tek which yielded sterile spores from wild specimens. you can read about it here
http://mycotopia.net...ead.php?t=11788
the only disadvantage is that it is a little bit expensive.
using dilute bleach simplifies the process and makes it a lot cheaper, but if you want to separate the spores short of a centrifuge you can use one of the micron filters with pore size of 5-8 micron.
I will test your tek with a wild cube and tex that i received recently. If one has to duplicate your experiment one need to use the same initial bleach concentration, i.e. 5% and then dilute. some commercial brands can reach up to 7%, so be careful.
GL

#6 Hippie3

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Posted 17 December 2006 - 12:28 PM

very interesting
bleach has shown remarkable results before
this needs more attention and investigation.
very nice work/discovery!
:bow:

#7 dial8

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Posted 17 December 2006 - 02:48 PM

I recall several members trying something similar a while back. Nothing conclusive was ever established. Thanks for sharing. I hope your results can be duplicated.

Nice work! :bow:

#8 rip

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Posted 17 December 2006 - 04:46 PM

I'd suggest trying few different dilutions to find the optimum. I've taken mycelium germinated from the 0.3% treated spores to be safe but the 0.1% too still appears at least visually clean of contaminants. Household bleach certainly is not reagent grade stuff. And I have no idea of bleach tolerance of other species. (I had no info about azurescens, I made a guess and tried it.) Good luck!

#9 StroFun

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Posted 18 December 2006 - 09:14 AM

Thank you for posting your experiment up here i really hope your results can be reliably repeated :teeth:

#10 zeegos

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Posted 09 January 2007 - 06:22 PM

im about to try this out justnow. i have my 0.3% bleach solution accurately measured out, a psilocybe cyanescens print and some plastic tust tubes.
how sterile should my procedure be? should i sterilize any equipment?
i know i'll have to have a syringe full of sterile water so i can suck some of the spores out of the bleach solution.

#11 Hippie3

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Posted 09 January 2007 - 06:53 PM

i'd be as sterile as possible,
can't hurt, might help.




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