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MODIFIED BROWN AND BRENN METHOD FOR GRAM
POSITIVE AND GRAM NEGATIVE BACTERIA
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Brenn Method for gram positive and gram negative bacteria |
FIXATION: 10% buffered neutral formalin.
TECHNIQUE: Paraffin sections cut at 5 μm.
SOLUTIONS:
Hucker-Conn Solution, Modified
Crystal violet, C.I. 42555 ------------------------------------------------------- 0.8 gm
Ethyl alcohol, absolute ---------------------------------------------------------- 8.0 ml
Ammonium oxalate ------------------------------------------------------------- 4.0 gm
Distilled water -------------------------------------------------------------------- 392.0 ml
Dissolve the crystal violet in the ethyl alcohol, and the ammonium
oxalate in the distilled water. Add the ammonium oxalate to the
crystal violet and mix. Filter through Whatman #1 filter paper before
use.
Modified Grams Iodine Solution
Iodine ------------------------------------------------------------------------------ 2.0 gm
Potassium iodide ----------------------------------------------------------------- 4.0 gm
Distilled water -------------------------------------------------------------------- 400.0 ml
Dissolve the potassium iodide in a little of the distilled water, add the iodine and dissolve. Add the remainder of the distilled water.
Ethyl Alcohol-Acetone Solution
Ethyl alcohol, absolute ---------------------------------------------------------- --- 50.0 ml
Acetone ---------------------------------------------------------------------------- 50.0 ml
0.5% Basic Fuchsin Solution (Stock)
Basic fuchsin or pararosanilin, (C.I. 42500) -------------------------------------- 0.5 gm
Distilled water -------------------------------------------------------------------- 100.0 ml
Dissolve with the aid of heat and a magnetic stirrer.
Basic Fuchsin Solution (Working)
Basic fuchsin solution (stock) -------------------------------------------------- --- 5.0 ml
Distilled water -------------------------------------------------------------------- 35.0 ml
Picric Acid-Acetone Solution
Picric acid -------------------------------------------------------------------------- 0.05 gm
Acetone ---------------------------------------------------------------------------- 100.0 ml
Acetone- Xylene solution
Acetone ---------------------------------------------------------------------------- 50.0 ml
Xylene ----------------------------------------------------------------------------- 50.0 ml
Use both positive and negative control slides.
1. Deparaffinize and hydrate to distilled water.
2. Hucker-Conn solution for 2 minutes.
3. Rinse quickly with two changes of distilled water.
4. Gram's iodine solution for 1 minute.
5. Rinse quickly with two changes of distilled water. Blot the slide but not the tissue section.
6. Decolorize by dipping in the ethyl alcohol-acetone solution until the blue color stops coming off.
7. Rinse in two changes of distilled water, several dips in each.
8. Working basic fuchsin solution for 30 seconds.
9. Rinse in two changes of distilled water, three dips in each. Blot the slide but not the tissue section.
10. Place in acetone for 5 seconds.
11. Differentiate and decolorize by placing in picric acid-acetone for 10 seconds.
12. Dip several times in acetone-xylene.
13. Clear in xylene, four changes.
14. Mount with synthetic resin.
Gram positive bacteria --------------------------------------------- blue
Gram negative bacteria -------------------------------------------- red
Filaments of Nocardia and Actinomyces ---------------------------- blue or red
Nuclei -------------------------------------------------------------- red
Other tissue elements ---------------------------------------------- yellow
This staining method is a modification of Taylors modification of the Brown-Brenn stain for bacteria. Not only does it reduce the staining time, but it also eliminates the use of ether; a laboratory safety hazard.
The tissue section should not be allowed to dry out at any point during the staining procedure. If the section is allowed to dry out after treatment with the modified Grams iodine, decolorization with alcohol-acetone will be difficult and inadequate. If the section is allowed to dry out after staining with basic fuchsin, differentiation with picric acid-acetone will be inadequate. Nothing can be done to remedy these problems.
Brown, J.H., and Brenn, L.: A method for the differential staining of Gram-positive and Gram-negative bacteria in tissue sections. Bull. Johns Hopkins Hosp. 48:69-73, 1931.
Taylor, R.D.: Modification of the Brown-Brenn Gram stain. Amer. J. Clin. Path. 46: 472-474, 1966.
Churukian, C.J., and Schenk, E.A.: A method of demonstrating Gram-positive and Gram-negative bacteria. J. Histotechnology 5:127-128, 1982.
Image Examples:
Diagnostic Application: