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United States Government Work, uncopyrighted, public-domain,
DRAFT COPY ONLY. This document does not necessarily represent the views
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This document is provided "as is", without warranty of any kind,
express or implied, including but not limited to the warranties
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PRINCIPLE OF THE TEST.
To stain paraffinized tissue sections for Treponemal organisms,
required in the diagnosis of certain diseases encountered
in anatomic pathology, especially syphilis.
SPECIMEN REQUIRED.
All human tissue excised at surgery, outpatient clinics, and postmortems,
fresh or in fixative, along with a filled-out Tissue Examination Form
(U. S. Standard Form 515, USSF515).
REAGENTS, INSTRUMENTATION.
Distilled water.
Triple Distilled water.
Acidulated water.
10% Neutral buffered formalin.
Paraffin sections at 6 microns.
1% Aqueous citric acid.
1% Silver nitrate solution (for impregnation).
2% Silver nitrate solution.
Silver nitrate, ACS crystals.
5% Gelatin solution.
Fisher G-7 Gelatin, high grade (Type B).
0.15% Hydroquinone solution.
Hydroquinone crystals, photographic grade.
Control slides: Melanotic skin.
STEP-BY-STEP DESCRIPTION.
FIXATION: 10% buffered neutral formalin. Do not use chromate fixative.
TECHNIQUE: Cut paraffin sections at 6 microns.
SOLUTIONS: Use chemically clean glassware.
ACIDULATED WATER.
Triple Distilled water................................... 1000.0 ml
Add enough 1% aqueous citric acid to bring water to pH 4.0
1% SILVER NITRATE SOLUTION (FOR IMPREGNATION).
Silver nitrate, ACS crystals................................. 1.0 gm
Acidulated water............................................. 100 ml
2% SILVER NITRATE SOLUTION.
Silver Nitrate, ACS crystals........................... 2.0 gm
Acidulated water ...................................... 100 ml
5% GELATIN SOLUTION.
Fisher G-7 Gelatin, high grade (Type B)
Isoelectric Point......................................... 10.0 gm
Acidulated water..............,............................ 100.0 ml
0.15% HYDROQUINONE SOLUTION.
Hydroquinone crystals, photographic grade.................... 0.5 gm
Acidulated water........................................... 100.0 ml
Place the 2% silver nitrate, 5% gelatin, and the 0.15% hydroquinone
in separate flasks, in a flotation bath at 54o C during slide
impregnation in 1% silver nitrate.
REMARKS.
It may be necessary to prolong development
of sections for the demonstration of Donovan bodies.
Spirochetes are usually found in connective tissue.
NOTE:
Use paraffin coated forceps, particularly at steps 2 and 3.
Use acid clean glassware.
CONTROL:
Melanotic skin.
REFERENCES.
1. Swisher BL.
Microorganisms. Chapter 16, pp. 325-344.
In: Bancroft JD, Gamble M.
Theory and Practice of Histological Techniques. Fifth Edition.
Edinburgh: Churchill Livingstone. 2002;:325-344.
ISBN 0-443-06435-0, 796 pages.
2. Warthin AS, Starry AC.
A more rapid and improved method of demonstrating spirochetes.
Am J Syphil, Gonorrh, Venereal Dis. 1920;4:97.
3. Luna LG.
Manual of Histologic Staining Methods
of the Armed Forces Institute of Pathology. Third Edition.
New York: McGraw-Hill Co. 1968;:.
4. Prophet EB, Mills B, Arrington JB, Sobin LH, eds.
Laboratory Methods in Histotechnology.
Washington, DC: Armed Forces Institute of Pathology. 1992;:.
ISBN 1-881041-00-X, 278 pages.
5. Mikel UV, ed.
Advanced Laboratory Methods in Histology and Pathology.
Washington, DC: Armed Forces Institute of Pathology. 1994;:.
ISBN 1-881041-13-1, 254 pages.