Lousie, here is our procedure. It is a microwave - not sure that is what you
wanted. When it says 10 to 20 minutes in reducer, 10 is better. Needs to
have a pretty light yellow background so spirochetes will show well.

Marg

EISENHOWER MEMORIAL HOSPITAL 		 Rancho Mirage, California

DEPARTMENT:  ANATOMIC PATHOLOGY          			Orig 4-87,
Rev 7-94
 
Reformatted 6-96
	
Rev. 10/99
TITLE:  SPIROCHETE STAIN:  MICROWAVE STEINER

____________________________________________________________________________
____________________________

PURPOSE:  To demonstrate argyrophilic organisms.

PRINCIPLE:  The organisms demonstrated by this method are argyrophilic; that
is, they will adsorb silver from a silver solution but the adsorbed silver
must be chemically reduced to the visible metallic form.  Hydroquinone is
the reducing agent or "developer."

SPECIMEN REQUIRED:  Cut paraffin sections at 4-5 microns.

REAGENTS: 

 1% Silver Nitrate
 Silver Nitrate    			.5 gm
 DI Water         			50 mL

REACTIVE! Poison, may be fatal if swallowed.Causes burns. Dust inhalation
may cause lung damage. Strong oxidizer. Do not get into eyes, on skin, or on
clothing.

1% Uranyl Nitrate
Uranyl Nitrate     			.5 gm       
	DI Water         				50 mL   

This solution may be re-used but discard after 2 months.
 
DANGER! Strong oxidizer, contact with other matierial may cause fire.
Harmful if swallowed or inhaled. Causes kidney and liver damage. Caution
radioactive material. 

     	0.04% Silver Nitrate
	Silver Nitrate 				.04 gm     
	DI Water         				100 mL    

Make fresh each time and filter before use.

     		2.5% Gum Mastic
	    	Gum mastic      			 2.5 gm 
	     	Absolute Alcohol 			100 mL

Allow Gum Mastic to dissolve in the alcohol for 24 hours, then filter the
solution until it is clear yellow.      Refrigerate at 4C.  This solution
may be reused, but do not pour used solution back  into the stock bottle.
Discard if solution is not clear or if it separates.

     		
		 
		2% Hydroquinone
	    	 Hydroquinone      			 1 gm
	     	 DI Water         			 50 mL 

MAKE FRESH EACH TIME.  Fresh Hydroquinone should always be used, and
anhydrous hydroquinone should be discarded and replaced after 1 to 2 years. 

POISON! DANGER, vapor and dust hazardous. May be fatal if swallowed. Causes
eye and skin irritation. Target organs affected: Eyes, skin, Throat,
Respiratory and Gastrointestinal Tract, Liver and Kidneys.

    		 Reducing Solution
	   	 2.5% Gum Mastic  			 20 mL
	    	 Hydroquinone   			 50 mL
	     	Absolute alcohol  			 10 mL

Make just before use & filter with #4 filter paper & add:

	0.04% Silver Nitrate  5 mL

	Mix gently & do not filter after addition of silver nitrate.

THE SOLUTION WILL HAVE A MILKY APPEARANCE WHEN GUM MASTIC IS ADDED.   

QUALITY CONTROL:  Tissue containing spirochetes, C pylori, Legionella
organisms, or Cat Scratch positive bacteria must be used depending on the
organism to be demonstrated.  Use chemically clean glassware, and take care
that no metal comes in contact with the staining solutions.

PROCEDURE:

Before staining, place a plastic Coplin jar in a 45 to 50 C water bath to
heat.  Prepare the reducing solution and place in the preheated Coplin jar.
(Use only vented plastic Coplin jars in the microwave, be sure that they are
loosely capped, and place them inside a loosely closed plastic bag.)

1. Deparaffinize and rehydrate tissue to distilled water.

1. Sensitize sections in preheated 1% aqueous uranyl nitrate at 60 C in
waterbath for 15 minutes.

1. Rinse in DI water until cross contamination possibility is eliminated.

1. Place slides in 1% Silver Nitrate and put into microwave oven for 30-45
seconds or until solution reaches boiling point.  Remove from oven and allow
slides to stand in hot Silver Nitrate 5 minutes 
     (SEE "PROCEDURE NOTES")  or 1% Silver Nitrate preheated to 60 C in a
waterbath for 1 to 1/2       
      hours.

1. Rinse in DI water - 3 changes.

1. Dehydrate in 2 changes of 95% alcohol.

1. Dehydrate in 2 changes of 100% alcohol.

1. Treat with 2.5% Gum Mastic for 5 minutes.

1. Allow to air dry for 1 minute.

1. Rinse in DI water - 2 changes.

1. Reducing solution at 45 C in waterbath for 10-20 minutes or until
sections have developed satisfactorily with spirochetes at desired
intensity, and background is light yellow.  Avoid an intensely stained
background as it may interfere with spirochete identification.

1. Rinse in distilled water to stop reduction.

1. Dehydrate, clear and mount.
	 
RESULTS:

Spirochetes & other nonfilamentous bacteria   	 Dark brown to black

Background    				                          Light
yellow

PROCEDURE NOTES:

1. At EMC we have been using the microwave procedure and have found, in
general, that the 2-3 minutes in the hot Silver Nitrate has not been
sufficient.  Therefore after step 11, it has been necessary to repeat steps
4 through 11 again.  It is therefore recommended that longer time be given
in the silver nitrate to begin with.  Perhaps begin with 5 minutes.  

2. The color of the background is sometimes related to the chemicals used in
processing the tissue.  The preferred background color is a bright but soft
yellow.  If too much background color is achieved, the spirochetes may be
obscured as they may be few in number.  The desired color of spirochetes is
black.  To check for proper development, remove slide when it is yellow to
light brown macroscopically, put into DI water, dehydrate quickly in 95%
alcohol, absolute alcohol and xylene, drop a coverslip on section and
examine under the microscope.  If spirochetes are not yet black in control
section, rehydrate to distilled water and return to reducer.  A bacteria
control may be stained, as bacteria stain much more rapidly and can be used
to control the progress of the spirochete control.

3. This procedure is completely satisfactory for the demonstration of Cat
Scratch bacteria.

REFERENCES:

1. Garvey, W., Fathi, A., and Bigelow, F.:  Modified Steiner for the
Demonstration of Spirochetes.  Journal of Histotechnology 8:15-17, 1985.
Further modified slightly by Billie Swisher, Experimental Pathology Branch,
Division of Host Factors, Center for Disease Control.

2. Winter issue, 1994-95, of the newsletter for the California Society of
Histotechnologists.

-----Original Message-----
From: Renton, Lousie, Mrs [mailto:177louie@chiron.wits.ac.za]
Sent: Thursday, March 29, 2001 2:06 AM
To: histonet@pathology.swmed.edu
Subject: Steiner method


hi everybody,

PLease would someone be kind enough to share with me a method 
for Steiner's stain (for spirochaetes). We do not have access to 
American texts which wouuld have this methodology.

Best regards
Louise