FIXATION:        10% buffered neutral formalin.  Do not use fixatives which contain

chromium or mercury.

TECHNIQUE:        Paraffin sections cut at 4-5 um.

SOLUTIONS:

1.0% Uranyl Nitrate-1% Copper Nitrate

Uranyl nitrate --------------------------------------------------------------------        0.4 gm

Copper nitrate -------------------------------------------------------------------        0.4 gm
Distilled water -------------------------------------------------------------------        40.0 ml

0.5% Silver Nitrate

Silver nitrate ---------------------------------------------------------------------        0.205 gm
Distilled water -------------------------------------------------------------------        41.000 ml

2.5% Gum Mastic

Gum mastic ----------------------------------------------------------------------        2.5 gm
Absolute alcohol        ---------------------------------------------------------------        100.0 ml

Allow the gum mastic to dissolve for 24 hours. Filter through

Whatman #4 filter paper and store in a refrigerator at 3-6° C.

2% Hydroquinone

Hydroquinone -------------------------------------------------------------------        0.5 gm
Distilled water -------------------------------------------------------------------        25.0 ml   

Prepare fresh each time.

Reducing Solution

2.5% Gum mastic        ---------------------------------------------------------------            15.0 ml
2% Hydroquinone ---------------------------------------------------------------        25.0 ml
0.5% Silver nitrate --------------------------------------------------------------                0.7 ml

The 0.7 ml of 0.5% silver nitrate is obtained from the 41 ml of

0.5% silver nitrate.

Prepare just before use. The solution will have a milky appearance

but does not need to be filtered.

• Use control slide.

l .        Deparaffinize and hydrate to distilled water.

2.        Place in 40 ml of l % uranyl nitrate and 1% copper nitrate in a plastic Coplin jar, with the cap loosely applied, and microwave at power level 6 (360W) for 40 seconds. Gently agitate the Coplin jar for about 15 seconds. Microwave again at power level 6 for 25 seconds. Gently agitate the Coplin jar for about 15 seconds. Allow the slides to remain in the hot solution for 2 minutes. 

3.        Rinse in five changes of distilled water.

4.        Place in 40 ml of 0.5% silver nitrate in a plastic Coplin jar, with the cap loosely applied, and microwave at power level 6 (360W) for 40 seconds. Gently agitate the Coplin jar for about l 5 seconds. Microwave again at power level 6 for 20 seconds. Allow the slides to remain in the hot solution (80° C) for 2 minutes.

5.        Rinse in three changes of distilled water.
6.        Dehydrate in two changes of 95% ethanol and two changes of absolute ethanol.
7.        Place in 2.5% gum mastic for 1 minute.

8.        Place slides in freshly prepared reducing solution in a plastic Coplin jar with the cap loosely applied, and microwave at power level 4 (240W) for 30 seconds. Gently agitate the Coplin jar for about 15 seconds. Microwave again at power level 4 for 30 seconds. Gently agitate the Coplin jar again for about 15 seconds. Allow the slides to remain in the hot solution (75° C) for 3 minutes. The sections should appear yellow-brown at the end of the impregnation time. If the sections still appear too light, allow them to remain in the reducing solution for 3 more minutes.  If they are still too light, microwave again at power level 4 for 30 seconds.

9.        Rinse in three changes of distilled water.
10.        Dehydrate in graded alcohols.
l1.        Clear in three or four changes of xylene.
12.        Mount with synthetic resin.
       

Spirochetes, Legionella pneumophila, Helicobacter (formerly Campylobacter pyloridis), Alipia felis (cat-scratch bacillus) and other bacteria stain dark brown to black.

The most commonly used staining methods for demonstrating spirochetes and some Gram negative bacteria which stain poorly with tissue Gram stains, are the Deterle, WarthinStarry and Steiner methods. All of these methods require the use of heat to impregnate tissue sections with silver nitrate followed by a reducing solution which converts the silver nitrate to deposits of black metallic silver on the spirochetes and bacteria. The staining time required to perform these methods has been greatly reduced by the use of the microwave oven. We have tried all of these methods and found that they all usually give satisfactory staining results. However, for demonstrating spirochetes the Steiner method produces the most consistent and reliable staining results.

Garvey modified Steiners method and was able to reduce the staining time required to perform the procedure. She used a new developing solution which is easier to prepare than Steiners original formula. The staining time was reduced more by Swisher who used a microwave oven in performing the method. We have been able to further reduce the staining time by performing the reducing step in a microwave oven rather than a hot water bath.

In Swishers method the uranyl and silver nitrate solutions are heated to the boiling point. We have found that when this is done there is the danger of the solutions boiling over.  In the described method the uranyl nitrate is not heated in order to reduce non-specific background staining.  The silver nitrate is heated to only 80˚ C which eliminates the possibility of the solution boiling over.

It is necessary to add a very small amount of silver nitrate to the reducing solution in order to achieve satisfactory staining results. If no silver nitrate is added none of the spirochetes will stain. If the amount of silver nitrate is increased there will be an increase in background staining which may obscure the organisms.

When the solutions are heated in a microwave oven there can be up to a 15° C difference in temperature between the top portion of the solution as compared to that in the lower part of the Coplin jar. Therefore, in order to equalize the temperature of the solutions, the slides are gently agitated in steps 2, 4 and 8. This assures uniformity of staining throughout the tissue sections.

Garvey, W. and Fathi, A.: Modified Steiner for the demonstration of spirochetes. J. Histotechnol. 8:15-17,1985.

Swisher, B.L.: Modified Steiner procedure for microwave staining of spirochetes and nonfilamentous bacteria. J. Histotechnol. 10:241-243, 1987.

Churukian, C.J. and Garvey, W.: Microwave Steiner method for spirochetes and bacteria. J. Histotechnol. 13:45-47, 1990.

Garvey W., Fathi A., Bigelow F. and Wynnchuk M.: Revised modified Steiner to enhance visability of spirochetes. J. Histotechnol. 18:57-60, 1995.