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United States Government Work, uncopyrighted, public-domain,
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PRINCIPLE OF THE TEST.
To stain paraffinized tissue sections for fungi,
required in the diagnosis of certain diseases
encountered in anatomic pathology.
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.
10% Neutral buffered formalin.
Paraffin sections at 6 microns.
4% Chromic acid solution.
5% Silver nitrate solution.
3% Methenamine solution (hexamethylenetetramine).
5% Borax solution.
Silver nitrate, 5% solution.
Methenamine, 3% solution.
1% Sodium bisulfite solution.
0.1% Gold chloride solution.
2% Sodium thiosulfate solution.
0.2% Light green solution.
Permount or Histoclad.
Control slides: Lung with Pneumocystis carinii infection,
or lung with aspergillosis.
STEP-BY-STEP DESCRIPTION.
FIXATION: 10% buffered neutral formalin
TECHNIQUE: Cut paraffin sections at 6 microns.
SOLUTIONS:
4% CHROMIC ACID SOLUTION.
5% SILVER NITRATE SOLUTION.
3% METHENAMINE SOLUTION (Hexamethylenetetramine).
5% BORAX SOLUTION.
METHENAMINE-SILVER NITRATE SOLUTION (stock).
Silver nitrate, 5% solution..................... 5.0 ml
Methenamine, 3% solution...................... 100.0 ml
A white precipitate forms but immediately dissolves on
shaking. The clear solution remains usable for months.
Store in refrigerator.
METHENAMINE-SILVER NITRATE SOLUTION (working).
Methenamine-silver nitrate solution (stock).... 25.0 ml
Distilled water................................ 25.0 ml
Borax, 5% solution.............................. 2.0 ml
Make fresh prior to use.
1% SODIUM BISULFITE SOLUTION.
0.1% GOLD CHLORIDE SOLUTION.
Gold chloride, 1% solution..................... 10.0 ml
Distilled water................................ 90.0 ml
This solution may be reused repeatedly.
2% SODIUM THIOSULFATE SOLUTION.
0.2% LIGHT GREEN SOLUTION.
CONTROL: Lung with P carinii infection or lung with aspergillosis
STAINING PROCEDURE.
1. Deparaffinize and hydrate to distilled water.
2. Oxidize in 4% chromic acid solution for 1 hour.
3. Wash in tap water for a few seconds.
4. Sodium bisulfite solution for 1 minute to remove any residual chromic acid.
5. Wash in running water for 5 - 10 minutes.
6. Rinse with three or four changes of distilled water.
7. Place in freshly mixed working
methenamine-silver nitrate solution
in oven at at 58-60o C
for 60 minutes or until sections turn yellowish-brown.
8. Rinse in six changes of distilled water.
9. Tone in gold chloride solution for 2 - 5 minutes.
10. Rinse in distilled water.
11. Remove unreduced silver with sodium thiosulfate (hypo) solution for 2 - 5 minutes.
12. Wash thoroughly in tap water.
13. Counterstain with working light green solution for 30 - 45 seconds.
14. Dehydrate in 95% alcohol, absolute alcohol and clear,
two changes each.
15. Mount.
RESULTS:
Fungi...................................... sharply delineated in black
Mucin.................................... Light to dark grey
Inner parts of mycelia & hyphae............ old rose
Background................................. pale green
If Nocardia is suspected, two s1ides should be processed, one for 60
minutes and one for 90 minutes in the methenamine-silver nitrate solution,
to assure a positive demonstration of any filaments of Nocardia that may
exist. Slides previously stained with most other stains may be used by
removing cover glass in xylene substitute and running through Flex to water.
Subsequent chromic acid treatment removes any remaining stain.
REFERENCE.
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. Lopez JF, Grocott RG.
Demonstration of Histoplasma capsulatum in peripheral blood
by the use of methenamine-silver nitrate stain (Grocott's).
Am J Clin Pathol. 1968 Dec;50(6):692-694.
PMID: 4177859.
PubMed Entry
3. Grocott RG.
A stain for fungi in tissue sections and smears
using Gomori's methenamine-silver nitrate technic.
Am J Clin Pathol. 1955 Aug;25(8):975-979.
PMID: 14398663.
PubMed Entry
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.