This is our procedure:

Marg Hagerty
Supervisor, Anatomic Pathology
Eisenhower Medical Center
39-000 Bob Hope Drive
Rancho Mirage, CA 92270


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.  The final major step is counterstaining; Neutral Red and
Fast Green are used for this purpose. Gram negative bacteria stain red and
the background is green.


APPLICATIONS: This procedure will stain gram positive and negative bacteria
without the toxic and dangerous chemicals picric acid and ether. 


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


REAGENTS:

1.	Crystal Violet Oxalate, working solution
	Newcomer Supply (cat#10422)

2.	Gram's Iodine (Made at EMC - use same as used in de-B5 set up)

	PLEASE NOTE HMIG SAFETY INFORMATION FOR THIS CHEMICAL!
	THIS SOLUTION IS POISON AND IS VERY CORROSIVE.  
          THE VAPORS ARE HARMFUL.  USE ADEQUATE VENTILATION.



3.	Acetone (Baxter)

	PLEASE NOTE HMIG SAFETY INFORMATION FOR THIS CHEMICAL!
	AVOID CONTACT WITH SKIN AND EYES; XTREMELY FLAMMABLE, 	USE NEAR
FIRE EXTINGUISHER WITH ADEQUATE VENTILATION.

4.	Twort Stain Set, Newcomer Supply, (cat# 14034)
	
	a.	Solution A, Neutral Red

	b.	Solution B, Fast Green
		Newcomer Supply, (cat# 14034)
	
	STORAGE AND HANDLING: KEEP WELL CLOSED. STORE IN A DRY, 	WELL
VENTILATED AREA AT ROOM TEMPERATURE. STORE 	AWAY FROM HYPOCHLORITES AND
STRONG OXIDIZERS. KEEP 	AWAY FROM HEAT, FLAME AND IGNITION SOURCES.
	

QUALITY CONTROL:   Tissue containing both gram-positive and gram-negative
bacteria.


PROCEDURE:

1.	Deparaffinize and hydrate to distilled water.

2.  	Using dropper technique, place Crystal Violet-Oxalate on slides for
30 seconds.   

3.	Rinse quickly in distilled water.

4.	Treat with Gram's Iodine for 20 seconds.

5.	Rinse quickly is distilled water.

6.	Decolorize by gently dipping in acetone in a coplin jar until no
more purple color comes off. (2 quick 	dips)

7.	Rinse quickly in distilled water.

8.	Twort Stain for 2 minutes. Prepare immediately before use. Combined
stain must be used within 	10 minutes of preparation. Combine in a  50
mL coplin jar:

	a.	9 mL of solution A, Neutral Red
	b.	1 mL of Solution B, 1.0% Fast Green
	c.	30 mL distilled water
9.	Rinse quickly in distilled water, shake excess off and carefully
blot dry on paper towels.

10.	Agitate quickly in coplin jar of clean acetone to dehydrate.

11.	Two changes of xylene and mount.


RESULTS: 

Gram positive bacteria 	                    dark blue              
Gram negative bacteria            		        red
Filaments of Norcardia and Actinomyces    blue
Nuclei                                     		        red
Background                                	        light green
RBCs                                                             green to
greenish blue


PROCEDURAL NOTES:

1.	Crystal Violet Oxalate solution is stable and may last up to two
years, requring only occasional 	filtering.	

2.	Gram positive organisms seem to stain exceptionally dark, making it
harder to overdifferentiate with 	acetone.

3.	Do not reuse Twort stain once it is mixed. The green loses its
effectiveness within a half hour.

4.	Do not place slides in alcohol after Twort stain. The neutral red
will be removed.	


REFERENCE:

1)	Bancroft and Stevens, "Theory and Practice of Histologic Technique",
Third Ed., St. Louis, 	980, The 	C.V. Mosby & Company.
	
	With modifications developed at VAMC, Madison WI.






             

-----Original Message-----
From: gary powers [mailto:elephantsneedus@erols.com]
Sent: Sunday, September 12, 1999 3:43 PM
To: histonet@pathology.swmed.edu
Subject: Twort's gram stain


Would anyone have a procedure for Twort's gram stain?

Thanks,

Marian