Here is the complete procedure. We use more of the decolorizer than the
other kit components so we end up making more up. The only problems I have
seen with this procedure was caused from inadequate decolorization and
overstaining of gram neg. 


EISENHOWER MEMORIAL HOSPITAL          			  Rancho Mirage,
California

DEPARTMENT:  ANATOMIC PATHOLOGY
 
Orig. 3/4/00
 
Page 1 of 3
TITLE:  GRAM POSITIVE & GRAM NEGATIVE BACTERIA:
	      GRAM STAIN
____________________________________________________________________________
_______

PURPOSE

To differentiate as well as demonstrate gram positive and gram negative
bacteria in tissue and in smears.

PRINCIPLE

Variants of the Gram Stain are useful in the determination of whether an
abscess or necrosis is bacterial in origin.  Gram positive fungal filaments
of Nocardia and Actinomyces may also be shown.  This procedure involves the
application of a crystal violet solution, followed by an iodine mordant to
form a dye lake.  Both gram-positive and gram-negative organisms are colored
blue-black after these 2 steps. Decolorization is the third major step, and
its purpose is to render the gram-negative ones colorless while leaving the
blue-black dye lake in the positive ones.  The decolorizer penetrates the
entire cell, and the step is a relative rather than an absolute one.  If
sections are exposed too long to the action of the decolorizing agent, even
gram-positive cells will lose the dye lake and become colorless. Gram
negative bacteria are stained red with seafaring.

SPECIMEN REQUIRED

Formalin-fixed paraffin sections cut at 5 to 6 microns or smears.

REAGENTS

Difco Gram Stain Kit, Becton Dickinson, cat. # 212539, which includes:

1. Gram Cystal Violet
Warning: Harmful by inhalation and if swallowed. Irritating to the eyes,
respiratory and skin. Possible risk of irreversible effects. 

2. Stabilized Gram Iodine
Warning: Harmful in contact with skin. Causes burns. Irritating to eyes,
respiratory tract and skin. 

3. Gram Decolorizer
Warning: Irritation to eyes, respiratory system and skin.

4. Gram Safranin
Warning: Harmful by inhalation or if swallowed. Irritating to eyes,
respiratory system and skin. 
QUALITY CONTROL

Tissue containing both gram-positive and gram-negative bacteria.

PROCEDURE

1. Deparaffinize and hydrate to distilled water.

2. Flood the slide with primary stain (Gram Crystal Violet) and stain for 1
minute. 

3. Remove the primary stain by gently washing with cold tap water. 

4. Flood the slide with Stabilized Gram Iodine and retain on the slide for 1
minute.

5. Remove the mordant by gently washing in tap water.

6. Decolorize by squeezing  the Gram Decolorizer onto the slanted slide
until it runs clear or no more color comes off.

7. Wash the slide gently in cold tap water.

8. Flood the slide with Gram Safranin and stain for 30 -60 seconds.

9. Wash the slide in cold tap water.

10. Quickly dehydrate, clear, and put on coverslipper.

RESULTS

Gram positive bacteria 	                    dark blue              
Gram negative bacteria            		        red
Filaments of Norcardia and Actinomyces    blue
Nuclei                                     		        red



REFERENCES:

1. Bancroft and Stevens, "Theory and Practice of Histologic Technique",
Third Ed., St. Louis, 980, The C.V. Mosby & Company.

2. Gram Stain Product Insert, cat. # 212539, Becton Dickinson, Sparks,
Maryland, 1999.
	








































             

-----Original Message-----
From: marjorie lehman [mailto:Marjorie.Lehman@unilever.com]
Sent: Friday, February 16, 2001 4:21 AM
To: Hagerty, Marjorie A.; RichardWHorobin@aol.com; Histonet
Subject: RE: Gram Stain problem


Would you share the Gram stain you use with us please?  I'm one of those
"never
get it right" ones. Thanks
Marge Lehman

-----Original Message-----
From:	Hagerty, Marjorie A. [SMTP:mhagerty@emc.org]
Sent:	Wednesday, February 14, 2001 8:11 PM
To:	'RichardWHorobin@aol.com'; 'Histonet'
Subject:	RE: Gram Stain problem

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. 
 
Marg

-----Original Message-----
From: RichardWHorobin@aol.com [mailto:RichardWHorobin@aol.com]
Sent: Wednesday, February 14, 2001 3:49 AM
To: Histonet@pathology.swmed.edu
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
and 
-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  RichardWHorobin@aol.com 
"What should we expect? Everything."