Kathy
you are using a high concentration of methyl green. Normally it would be used as
0.2 to 0.4% solution in 0.1M acetate buffer ph 4.2 as a nuclear counterstain.
Two possibilities are suggested,
One is that your concentration is too high - if my calculation is correct the
final concentration is 2 % and the buffer may not be of sufficient molarity to
maintain the pH. - this in unlikely but possible
There is also the possibility that you have either a higher concentration of
crystal violet (a common contaminant of methyl green) in your powder than normal
or that the bottle is mislabeled and is really crystal violet or another dye.
Check to see whether the Biologic Stain Commission certified this dye.
Barry

kwalters wrote:

> Everytime I make methyl green it turns a gorgeous shade of blue.  I have a
> brand new powder that I am using.
>
> Here is my protocol:  please tell me where I went wrong!!
>
> Thanks for any help!!
>
> Methyl Green
> a ) 0.1 N Acetic Acid: Add 6 ml of glacial acetic acid to 1000 ml of
>          filtered distilled water.
>
> b) 0.1 N sodium Acetate: Add 4.102 g of sodium acetate to 500 ml of distilled
> water.
>
>          c) Buffer:
> 1)Add 755 ml of 0.1 N acetic acid to 265 ml of 0.1 N sodium
> acetate.
> 2) Adjust pH to 4.2 with NAOH.
>
>          d) Methyl Green
> 1) Add 20.0 g of methyl green to the buffer solution.
>                         2) 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.
>
> Kathy Walters                                //
> Central Microscopy Research Facility        / /
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> University of Iowa                        / /\ \
> Iowa City, Iowa 52242                    / /  \ \
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> email: Katherine-Walters@uiowa.edu     |__|    / /
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