Subject: clean clones

From: [email-masked] (Paul Stewart)
Date: 25 Mar 1995 11:50:11 -0400

I have been establishing cultures of shiitake and oysters from grocery 
stores to practice my clean techniques, establish my home lab, and 
prepare for the real thing from wild specimens when things thaw out and 
start sprouting. After exhausting all other sources of contamination, I 
am left with the contaminants brought home from the store. (If all the 
other produce I buy is this dirty it's no wonder my fridge looks like a 
cool composter half the time...why do they call it a "crisper" :-) )

I have tried wiping the outside of the mushrooms with ethanol (70%), but 
still get bacteria in my cultures. I keep cultures at 25-26 C. I have 
done lots of plant tissue culture and so tried rinsing the shrooms in 70% 
isopropyl alcohol for 30 sec, followed by 2% bleach + 0.2% dish detergent 
for 10 min, with shaking, then three rinses in sterile water.  The 
oysters were glassy and translucent throughout. I squeezed out most of 
the water, but they still looked pretty wimpy. The shiitake, being more 
dense, held up better. Two days later, the cultures (on PDA + peptone and 
wood) already show bacterial colonies.

The question is, is there a standard disinfection procedure that will 
clean into the gills, while still leaving viable tissue. I always take 
tissue from the inside, just above where the stem meets the cap, 
especially for oysters, whose cap is too thin to excise from. I'd like to 
solve this problem before I need to clone a valuable specimen. Never had 
this problem with chanterelle or boletus...the fungi carried into culture 
with these species seem to be beneficial if not symbiotic.

Is contamination from cloned storebought a problem others have 
encountered and solved? (frustration mounts with each passing week!)

BTW...while I'm going on and on...I posted a question recently regarding 
using a horizontal oil drum with a hatch cut in the side as a rolling 
spawn generator. Any comments yet on this?

			Thanx...Paul Stewart
			<[email-masked]>

---

From: [email-masked] (Jim King)
Date: 25 Mar 1995 17:51:26 -0400

>                        <[email-masked]>

Try sandwiching the contaminated culture between two layers of agar 
medium. Place the sterilized agar out of a non-innoculated petrie 
dish directly on top of your  contaminated culture. The mycelium 
should grow up thru the second layer cleaning itself by friction.
Do this as soon as you see the contaminant. I learned this from Paul
Stamets at one of his workshops. His books " Growing Gourmet and 
Medicinal Mushrooms " and " The Mushroom Cultivator " are a must for
any sincere amateur or professional mushroom grower. They cover all
aspects from scratch to finish. His company is FUNGI PERFECTI ...
e-mail:[email-masked] Tel:360-426-9292/800-780-9126 Fax:360-426-9377

Enjoy, Somananda < [email-masked] >

---

From: [email-masked] (Jim King)
Date: 25 Mar 1995 17:58:41 -0400

>Enjoy, Somananda < [email-masked] >

Adding to that ... why wood in the agar ?! that may be your source of 
contamination as it may not sterilize properly in the short time that 
you sterilize agar medium.

---

From: [email-masked] (RandBio)
Date: 26 Mar 1995 10:41:18 -0500

Acquire a medium called Mycocel or Micobiotic agar.  There are two
inhibitors. 
Chloramphenicol for bacteria and Cycloheximide for saprophitic fungi. 
Using the aformentioned layering technique - make your medium sandwich
with one layer of PDA and the other C&C medium.

--------------------------------------------------------------------------
-------------------------------------------
Finishing in second place, makes one the first loser.
--------------------------------------------------------------------------
------------------------------------------
Robert Randolph
Randolph Biomedical
401-826-1407
[email-masked]