RE: [Histonet] GRAM Stain Timing Question

From:"Kellar, Eric C"

According to Bartholomew and Mittwer (1950,1951), the mechanism of stainability can be explained by differences in the permeability of the organism membrane. The bacteria stain by linkage between acid groups of the bacteria and alkaline groups of dye. Iodine forms a complex with the dye and this complex is dissociated by alcohol. If alcohol passes easily through the membrane, decolorization is rapid and the reaction is Gram negative. If the membrane is hardly or not at all penetrated by alcohol, the reaction is Gram positive. The condition of the membrane of the organism is the determining factor. 

Horobin and Bancroft say that, if specimen slides are taken into differentiator when still wet, then de-colorization can be many times =66aster, leading to dye loss and thus an apparent lack of Gram positive organisms. Standardize your procedure by always introducing specimens into differentiator either wet or blotted dry and always run a known Gram positive/negative control.

The histologist Gram who introduced the method in 1884 states that if you differentiate in alcohol, "Differentiate until no more stain comes away". I=66 using acetone, "Flood slides for not more than 2-5 seconds".

Still works for me!

Eric C. Kellar
Quest Diagnostics 
Histology Laboratory Supervisor
South Florida

 -----Original Message-----
From: []  On Behalf Of Breeden, Sara
Sent:	Wednesday, February 28, 2007 2:16 PM
Subject:	[Histonet] GRAM Stain Timing Question

I seem to do a lot of GRAM stains and although I've modified some of the
times to suit my pathologists' tastes, I have never been able to pin
down the first differentiation time in 100% alcohol.  Today, I'm using
10 quick dips although it's only because the wind is blowing and the
moon is on the rise.  Can someone give me a more specific guideline for
differentiating after the Gram's Iodine and before the Safranin? I'd
like to leave a more definite legacy when I retire than "differentiate
in 100% alcohol or acetone"...  I'm thankin' you in advance.  And I
think we all know who the Purple Haze Generation are on THIS media,
don't we???


Sally Breeden, HT(ASCP)

NM Dept. of Agriculture

Veterinary Diagnostic Services

PO Box 700

Albuquerque, NM  87106



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