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MICROWAVE MODIFICATION OF THE WARTHIN-STARRY
METHOD FOR BACTERIA
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Warthin-Starry Method for Spirochetes. |
FIXATION: 10% buffered neutral formalin. Do not use dichromate fixatives.
TECHNIQUE: Paraffin sections cut at 5 μm.
SOLUTIONS:
Glycine-Acetic Acid (Stock)
Glycine ------------------------------------------------------------------------- 1.2 gm
Acetic acid, glacial ------------------------------------------------ ------------- 0.3 ml
Distilled water ------------------------------------------------------------------- 100.0 ml
The pH of this solution is approximately 3.5. Store in a refrigerator at 3-6o C.
Glycine-Acetic Acid (Working)
Glycine-acetic acid (stock) ----------------------------------------------------- 3.0 ml
Distilled water ------------------------------------------------------------------- 300.0 ml
The pH of this solution is approximately 3.5.
0.25% Silver Nitrate Solution
Silver nitrate --------------------------------------------------------------------- 0.1 gm
Glycine-acetic acid, working solution ---------------------------------------- ----- 40.0 ml
Store in a refrigerator at 3-6° C.
2% Silver Nitrate Solution
Silver nitrate------------------------------------------------------------------------ 0.2 ml
Glycine-acetic acid, working solution ---------------------------------------- ---- 10.0 ml
Prepare fresh.
0.2 % Hydroquinone Solution
Hydroquinone -------------------------------------------------------------------- 0.2 gm
Glycine-acetic acid, working solution ---------------------------------------- ----- 10.0 ml
4% Gelatin Solution
Gelatin ---------------------------------------------------------------------------- 1.0 gm
Glycine-acetic acid, working solution ---------------------------------------- ------ 25.0 ml
Dissolve by placing on a magnetic stirrer and applying gentle heat.
0.2% Hydroquinone Solution
Hydroquinone -------------------------------------------------------------------- 0.02 gm
Glycine-acetic acid, working solution --------------------------------------------- 10.00 ml
Place the 2% silver nitrate, 4% gelatin, and the 0.2% hydroquinone solutions in separate flasks in a 55°-58° C
oven. These solutions and the 0.25% silver nitrate are prepared immediately before use.
Silver Nitrate-Gelatin-Hydroquinone Developer
Silver nitrate, 2% ---------------------------------------------------------------- 10.0 ml
Gelatin, 4% --------------------------------------------------------------------- 25.0 ml
Hydroquinone, 0.2% ------------------------------------------------------------ 10.0 ml
Combine in order given making certain the solutions are mixed well after each addition. Prepare immediately
before use.
0.1% Tartarazine Solution
Tartarazine, C.I. 19140 --------------------------------------------------------- 0.1 gm
Distilled water ------------------------------------------------------------------- 100.0 ml
Acetic acid ----------------------------------------------------------------------- 0.2 ml
1. Deparaffinize and hydrate to working glycine-acetic acid solution.
2. Place in 40 ml of 0.25% 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 15 seconds. Microwave again at power level 6 for 20 seconds. Gently agitate the Coplin jar again for about 15 seconds. Allow the slides to remain in the hot solution (75°-80°C) for 2 minutes.
3. Place in freshly prepared silver nitrate-gelatin-hydroquinone developer in a plastic Coplin jar, with the cap loosely applied. Place in a microwave oven and microwave at power level 1 for one minute. Gently agitate the Coplin jar for about 15 seconds. Allow the slides to remain in the solution until the sections appear gray. This usually takes about 1 minute but may take longer.
4. Wash quickly and thoroughly in hot running water.
5. Rinse in two changes of distilled water.
6. Counterstain with tartarazine solution for 15 seconds.
7. Rinse in two changes of distilled water.
8. Dehydrate in graded alcohols.
9. Clear in three or four changes of xylene.
10. Mount with synthetic resin.
Alipia felis (cat-scratch bacillus)------------------------------------- black
Legionnaires bacteria---------------------------------------------- black
Klebsiella------------------------------------------------------------ brown to black
Helicobacter (pylori) -------------------------------------------------------- black
Nuclei ----------------------------------------------------------------------- brown-black
Background--------------------------------------------------------- yellow
We prefer this microwave oven method over the regular Warthin-Starry, Deterle and Steiner methods for demonstrating bacteria, namely, because it is easier to do, less time consuming, and gives equally good if not superior results. Remember that all bacteria are stained by these silver methods, they are not specific for Legionella.
The original Warthin-Starry method required the use of 1% silver nitrate to impregnate the tissues prior to placing them in the developer. We found that a 0.1% silver nitrate solution works equally well with less background staining.
The type of gelatin used in this method is important in obtaining satisfactory results. We tried samples obtained from different suppliers including types I-IV from the Sigma Chemical Co., types A and B from Fisher Scientific Products. The results were satisfactory with al1 of these samples except Sigma type IV and Fisher type B. However, we prefer using Fisher type A gelatin because when this gelatin is used there is less background staining.
The use of xylene substitutes in this method is not recommended. We tried Hemo-De (obtained from Fisher Scientific) as the clearing agent and found that the stain faded within 24 hours to the extent that some bacteria and spirochetes were no longer visible.
Kerr, D.A.: Improved Warthin-Starry method of staining spirochetes in tissue sections. Amer. J. Clin. Pathol. 8:63-67, 1938.
Luna, L.G.: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed., New York, McGraw-Hill, 1968, pp. 238-240.
Churukian, C.J. and Schenk, E.A.: A Warthin-Starry method for spirochetes and bacteria using a microwave oven. J. Histotechnol. 11:149-151, 1988.
Image Examples:
Diagnostic Application: