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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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PRINCIPLE OF THE TEST.
To stain paraffinized tissue sections for Acid Fast Bacilli, required in the
diagnosis of certain diseases encountered in anatomic pathology, especially
tuberculosis and Mycobacterium avium-intracellulare.
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.
1% Sulfuric acid solution.
Methylene blue solution (working).
Xylene-peanut oil solution.
Zeihl-Neelsen carbol fuchsin solution.
Sulfuric acid solution.
Working methylene blue solution.
Xylene substitute.
Permount or Histoclad.
Control slide: Lymph node with atypical mycobacterial infection.
STEP-BY-STEP DESCRIPTION.
1% SULFURIC ACID SOLUTION
METHYLENE BLUE SOLUTION (working).
STAINING PROCEDURE.
1. Deparaffinize through two changes of xylene-peanut
oil solution for 12 minutes.
2. Drain, wipe off excess oil and blot to opacity.
The residual oil helps prevent shrinkage and injury of section.
3. Zeihl-Neelsen carbol fuchsin solution for 30 minutes.
4. Wash in tap water for 3 minutes.
5. Differentiate slides individually with sulfuric acid solution,
until sections are faint pink, about 1 minute.
6. Wash in running water for 3 minutes.
7. Counterstain lightly with working methylene blue solution.
8. Rinse off excess methylene blue in tap water.
9. Blot and let stand for a few minutes to air dry thoroughly.
10. Dip slides in xylene substitute before mounting.
11. Mount.
CONTROL: Lymph node with atypical mycobacterial infection
RESULTS:
Acid fast bacilli..................... red.
Nocardia filaments.................... red
Lepra bacilli......................... red
Background............................ pale blue
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. Neelsen F.
Ein Casuistischer Beitrag zur Lehre von Tuberkulose.
[German: A casuistic contribution in the doctrine of tuberculosis].
Zentralbl f d medizin Wissensch. 1883;21:497.
3. Ziehl F.
Zur Färbung des Tuberkelbacillus.
[German: Regarding staining of the tubercle bacillus].
Dtsch med Wochenschr.1882;8:451.
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.