Spore washing experiment [ dilute bleach ]
Posted 17 December 2006 - 07:45 AM
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.
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.)
Posted 17 December 2006 - 07:54 AM
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 ?
Posted 17 December 2006 - 08:44 AM
Posted 17 December 2006 - 10:20 AM
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.)
Posted 17 December 2006 - 10:39 AM
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.
Posted 17 December 2006 - 12:28 PM
bleach has shown remarkable results before
this needs more attention and investigation.
very nice work/discovery!
Posted 17 December 2006 - 02:48 PM
Nice work! :bow:
Posted 17 December 2006 - 04:46 PM
Posted 18 December 2006 - 09:14 AM
Posted 09 January 2007 - 06:22 PM
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.
Posted 09 January 2007 - 06:53 PM
can't hurt, might help.