[Histonet] RE: Gram Control

[Walzer Susan]

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:

III.  SOLUTIONS:

      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)
            Acetone- Xylene

IV.   PROCEDURE:

      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.

V.    RESULTS:

      A. Gram-positive bacteria.....................blue

      B. Gram-negative bacteria......................red

      C. Background...............................yellow

-----Original Message-----
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
To: Histonet
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?
 
Thanks,
Milton

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