[Histonet] GRAM Stain Timing Question
Rene J Buesa
rjbuesa <@t> yahoo.com
Wed Feb 28 14:40:16 CST 2007
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!
"Breeden, Sara" <sbreeden <@t> nmda.nmsu.edu> 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,
Sally Breeden, HT(ASCP)
NM Dept. of Agriculture
Veterinary Diagnostic Services
PO Box 700
Albuquerque, NM 87106
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