Subject: Re: mycorrhizae practical

From: Peter Herman x5495 <[email-masked]>
Date: Fri, 24 Mar 1995 09:22:22 -0700 (MST)

Dear Paul,

Here is the lab I use, I got it from a past MS student who now works 
with VAM as part of her job.   	 just used it last week and it worked 
like a charm.  I would be interested in your lab as I have a GC (I do 
both ARA assays and FAMES now and again)

VA Mycorrhiza Lab
Kormanic and McGraw Procedure

Samples:  Each student should bring in 2 plants, 1 monocot and 1 dicot,
preferably that they can identify.  The samples should include most or all
of the roots.  Bag samples in plastic or foil to keep tissue fresh. 

Rinse:  Wash roots thoroughly but gently under running tap water to remove
adhering soil.  Then rinse in distilled water. Staining Procedure:  Read
and be familiar with these steps BEFORE you come to lab! 

1. Place roots in a beaker with 10% KOH, autoclave for 10 min (15 psi)

2. Pour off KOH and rinse with tap water until rinse water runs clear (no
trace of brown). 

3. Cover with alkaline H2O2 solution for 10-20 min, until roots are
bleached. 

4. Rinse at least 3 times in water to remove H2O2. 

5. Cover with 1% HCl and soak for 3-4 min, pour off solution. Do Not
Rinse! 

6. Cover with acid fuchsin stain solution and autoclave for 10 min (15
psi). 

7. Place in destain solution. Transfer to fresh destain to clear specimen. 
Do not rinse.  Make mount in destain solution.  May be held * 2 years in
destain. 

Reagents: Alkaline H2O2: 3 ml NH4OH, 30 ml 10% H2O2, 967 ml water.  Must
be made fresh with each use! 

Acid Fuchsin:  874 ml lab grade lactic acid (glacial), 63 ml glycerin
(=glycerol) 63 ml water, 0.1 g acid fuchsin. 

Destain: 874 ml lab grade lactic acid (glacial), 63 ml glycerin 
(=glycerol) 63 ml  water.

*   Peter Herman, Dept. of Biology	Phone: +1 505 646 4532    *
*   New Mexico State University		Fax:   +1 505 646 5665    *
*   Box 30001, Dept. 3AF					  *
*   Las Cruces, NM 88003 USA		e-mail [email-masked]    *