BROWN AND BRENN METHOD FOR
GRAM POSITIVE AND
GRAM NEGATIVE BACTERIA.
DRAFT COPY ONLY.
(Procedure 81).
http://www.netautopsy.org/axsop/axsop081.htm
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United States Government Work, uncopyrighted, public-domain,
DRAFT COPY ONLY. This document does not necessarily represent the views
or policies of any United States Government agency.
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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or in connection with the document or the use or other dealings
made with the document.
See also: Tissue Stains: 49,
51, 52,
55, 56,
69, 72.
PRINCIPLE OF THE TEST.
To stain paraffinized tissue sections for Gram-positive bacteria, and
Gram-negative bacteria, 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.
1% Crystal violet solution.
Crystal violet powder.
5% Sodium bicarbonate solution.
Sodium Bicarbonate powder.
Gram's iodine solution.
Iodine crystals.
Potassium iodide.
Ethylether acetone solution.
Ethyl ether.
Acetone.
0.25% Basic fuchsin solution (stock).
Basic fuchsin solution (working).
Basic fuchsin crystals.
Picric acid acetone solution.
Picric acid.
Acetone.
Acetone xylene solution.
Xylene.
Crystal violet solution.
Sodium bicarbonate solution.
Xylene substitute.
Permount or Histoclad.
Control slide: Mouth infection with mixed flora.
STEP-BY-STEP DESCRIPTION.
FIXATION: 10% Neutral buffered formalin.
TECHNIQUE: Cut paraffin sections at 6 microns.
SOLUTIONS: 1% CRYSTAL VIOLET SOLUTION.
Crystal violet .............................. 1.0 gm
Distilled water ............................. 100.0 ml
5% SODIUM BICARBONATE SOLUTION.
Sodium Bicarbonate.......................... 5.0 gm
Distilled water........................... 100.0 ml
GRAM'S IODINE SOLUTION.
Iodine...................................... 1.0 gm
Potassium iodide............................ 2.0 gm
Distilled water........................... 300.0 ml
ETHYLETHER ACETONE SOLUTION.
Ethyl ether................................ 50.0 ml
Acetone.................................... 50.0 ml
0.25% BASIC FUCHSIN SOLUTION (stock).
Basic fuchsin.............................. 0.25 gm
Distilled water........................... 100.0 ml
BASIC FUCHSIN SOLUTION (working).
Basic fuchsin solution (stock)............. 10.0 ml
Distilled water........................... 100 ml
PICRIC ACID ACETONE SOLUTION.
Picric acid................................. 0.1 gm
Acetone................................... 100 ml
ACETONE XYLENE SOLUTION.
Acetone.................................... 50.0 ml
Xylene..................................... 50.0 ml
STAINING PROCEDURE.
1. Deparaffinize and hydrate to distilled water.
2. Place slides on a staining rack and pour on approx.
1 ml (20 drops) of crystal violet solution and add 5 drops of sodium
bicarbonate solution. Allow to remain for 1 minute. Agitate gently.
(If preferred, the two solutions may be mixed just prior to use.)
3. Rinse in tap water.
4. Flood slides with Gram's iodine solution for 1 minute.
5. Rinse in water and blot with filter paper to complete dryness.
6. Decolorize with ethyl ether-acetone solution dropped on slides
until no more color runs off.
7. Working basic fuchsin solution for 1 minute. Wash in water.
8. Blot gently, but do not allow sections to dry completely.
9. Dip in acetone to start differentiation reaction.
10. Differentiate immediately with picric acid-acetone solution,
until sections are yellowish pink.
11. Rinse quickly in acetone, then in acetone xylene substitute
solution.
12. Clear in xylene substitute, several changes.
13. Mount,
CONTROL: Mouth infection with mixed flora
RESULTS:
Gram positive bacteria.............................. blue
Gram negative bacteria.............................. red
Filaments of Nocardia and Actinomyces............... blue
Nuclei.............................................. red
Other tissue elements............................ yellow
REMARKS: At least 2 slides should be run with varying degrees of
differentiation in order to lessen the possibility of over
differentiation at step 8.
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. 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.
3. 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.