RE: Gram Stain problem

From:"Hagerty, Marjorie A." <>

Hi Richard,
Not sure which procedure you are using. Gram positive will stain dark blue
and gram negative will be pink to red in most gram stains. They are very
small and you will need to view them on high power to see rods, etc. 
If you are using the Brown and Brenn or a variation, there are a few things
I have found helpful in staining  properly.
1. After the rinse, after the Gram's Iodine the slides need to be blotted to
complete dryness. I have even dried them in an oven (60 C) for 5 minutes to
be sure. 
2. Make sure no more color is coming off before completing the
decolorization step after the gram's.
3. After the basic fuchsin solution, wash in water, and then blot the slide
gently, but do not allow section to dry completely. 
4. Proceed rapidly through the differentiation steps. Slides should look
yellowish pink. The ideal slide has essentially no blue staining except the
gram pos. bacteria, although this isn't always the case.
Practice with some controls. Once you get it, you will always be able to do
a perfect stain. 
Don't know if this helps or even applies, regardless, good luck.
We no longer use this type of stain and have gone to a much easier and more
consistent gram stain used in our micro department. Some techs just never
seem to have been able to do a good B&B or Tworts. 

-----Original Message-----
From: []
Sent: Wednesday, February 14, 2001 3:49 AM
Subject: Re: Gram Stain problem

Aidan writes: 

We have been having troubles with our gram stain (paraffin) procedure.  The 
results are not giving sufficient distinction between Gram +ve's and -ve's. 

Can you tell us what the bacteria look like? Instead of +ve's being black
-ve's being pink, are they all black, all pink, or all somewhere in between?

Bye - Richard Horobin 
Institute of Biomedical & Life Sciences, University of Glasgow 
T direct 01796-474 480 --- E 
"What should we expect? Everything." 

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