Re: [Histonet] GRAM Stain Timing Question

From:Rene J Buesa

  This is how I used to do it (to everybody's acceptance):
  2-slides horizontal in a rack flooded with crystal violet and 5 drops of bicarbonate-phenol x 5 min
3-rinse one at a time in running water and return to the staining rack to be flooded with Grams's iodine x 5 min
  4- one at a time rinse in tap water and BLOT with paper towels
  5- one at a time immerse in acetone UNTIL NO MORE BLUE comes out of the slide (usually 1-2 dips in acetone)
  6- quick rinse in tap water and place in Coplin jar
  7- add 40 mL of distilled water in the Colplin jar + 3 mL of alcoholic basic fuchsin x 5 min
  8-after that blot (do not rinse) with paper towels one at a time
  9- prepare 5 Coplin jars (3 with acetone + 2 with xylene)
  10-in the first one add 40 mL of acetone + 7 mL of stock picro-acetone sol.
  11-one at a time run the slides in the 5 Coplin jars. In the first immerse the slides until no more red comes out (usually 1-2 dips).
  12-after that move quickly the slides through the other 4 Coplin jars
  13-leave in xylene until coverslip.
  To your specific question: see #5 (until no more blue comes out of the section).
  Hope this will help you!
  René J.
"Breeden, Sara"  wrote:
  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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