Re: Bob Schoonhoven, dig out your methyl green counterstain again

<< Previous Message | Next Message >> (Gayle Callis),
Date:Tue, 14 Sep 1999 13:58:54 -0400 (Eastern Daylight Time)

Don't recall seeing the question posted but here it is:

Please note that this is not MY procedure but I do not remember where I got it.

Methyl Green Counterstain

a)	Acetic Acid, Glacial (aldehyde-free) 
b)	Sodium Acetate, Anhydrous 
c)	Methyl Green


1 )    0.1 N Acetic Acid: Add 6ml of glacial acetic acid to 1000 ml of filtered
distilled water.  

2)	0.1 N sodium Acetate: Add 4.102 g of sodium acetate to 500 ml of  distilled water.

  3)      Buffer:
   a) Add 755 ml of 0.1 N acetic acid to 265 ml of 0.1 N sodium acetate.
b)	Adjust pH to 4.2 with NAOH.

4)	Methyl Green 
a)	Add 20.0 g of methyl green to the buffer solution.
b)	Filter the methyl green solution.

STORAGE: room temperature
SHELF LIFE: Indefinite while in storage; when in use, the shelf life is 2 weeks or
until staining becomes too weak.

1)	After color development of slides, rinse in distilled water briefly.

2)	Place slides in methyl green solution for 3 to 5 minutes, or as long as necessary
to get the
         staining desired. (slides which have been denatured, i.e. as for
         typically need far less Counterstain 30 seconds or so.)

3)	Do not rinse in water.  The methyl green washes out too easily.  Go directly into
          alcohol for a brief time (about 10 dips) .

4)	Continue dehydrating through one more 9S% alcohol, then 3 changes of 100% alcohol,
         allowing the slides to stay in the last alcohol for 2-3 minutes.

  5)	     Clear through 3-4 changes of xylene, and coverslip.

best regards,
Robert Schoonhoven
Laboratory of Molecular Carcinogenesis and Mutagenesis
Dept. of Environmental Sciences and Engineering
University of North Carolina
Chapel Hill, NC 27599
office 919-966-6343
   Lab 919-966-6140
   Fax 919-966-6123 

Don't go around saying the world owes you a living; the world owes you
nothing; it was here first. 
Mark Twain [Samuel Langhornne Clemens] (1835-1910) 

-- Begin original message --

> From: Gayle Callis <>
> Date: Mon, 13 Sep 1999 22:37:15 -0600
> Subject: Bob Schoonhoven, dig out your methyl green counterstain again
> To:
> Bob Schoonhoven has a methyl green counterstain that works very nicely, of course
> I have volunteered his expertise to come forth with the recipe/method.
> Sorry, I am at home without my manual here, can't recite recipe
> Gayle Callis

-- End original message --

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