[Histonet] RE: Gram Control
Susan.Walzer <@t> HCAHealthcare.com
Mon Jun 15 02:14:11 CDT 2009
We take some fresh tissue (we use umbilical cord) and have micro incubate half with gram positive and the other with negative. After a few day we fix the tissues and put a piece of each in blocks. We then do a modified gram stain using acetone and the stain always looks great. See below:
A. 1% Crystal Violet Solution
B. 1% Basic fuchsin Solution
C. Gram's Iodine Solution ( 1 gm iodine, 2 gm. potassium iodide in 300 ml. distilled water.)
D. Gallego's Differentiating Solution:
1. distilled water................100ml
2. Formalin, 37%....................2 ml
3. acetic acid......................1 ml
E. Picric Acid-Acetone ( available from Polyscientific #S1867)
A. Deparaffinize and dehydrate to water.
B. Place in 1% crystal violet for 1 min.
C. Rinse in tap water.
D. Place in Gram's Iodine for 1 min.
E. Rinse in tap water.
SUBJECT: BROWN AND HOPPS GRAM STAIN PAGE 2 OF 2
F. Decolorize in acetone until background is clear.
G. Wash immediately in tap water.
H. Place in 1% basic fuchsin 1 min.
I. Rinse in tap water.
J. Place in Gallego's differentiating solution, 2 changes, 1 minute each.
K. Rinse in tap water.
L. Place in acetone for 30 seconds.
M. Place in picric acid-acetone for 2 minutes.
N. Place in acetone-xylene, 2 changes.
O. Clear in xylene and coverslip.
A. Gram-positive bacteria.....................blue
B. Gram-negative bacteria......................red
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gomez, Milton
Sent: Saturday, June 13, 2009 4:33 PM
Subject: [Histonet] Gram Control
Good afternoon Histonetters:
May someone share a gram control block for special stains? May you also share how can I prepare one in my lab?
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