Brown and Brenn Method for Gram + and Gram - Bacteria

EMS26105

Components:

Crystal Violet Stain Solution 1% Aqueous

Sodium Biocarbonate 5% Aqueous

Basic Fuchsin Stock Solution 0.25%

Basic Fuchsin Working Solution

Gram's Iodine Solution

Acetone-Alcohol 1:1

Picric Acid Acetone Solution 0.1%

Acetone

Acetone:Xylene

Fixation:

Formalin, 10% Buffered Neutral

Section:

Paraffin, 6µm
 *Recommended technique includes a control slide.

Staining Procedure:

1. Deparaffinise and hydrate to distilled water.
2. Mix 1.0ml (20dr.) Crystal Violet, 1% Aq. with 5 drops Sodium Bicarbonate, 5%, Aq.; pour onto slides held in a staining rack.  Agitate gently to cover section:  stain slides for 1 min.  Rinse in distilled water.
3. Flood with Gram’s Iodine for 1 min, rinse with water and carefully blot with filter paper to dryness.
4. Decolourise with  Acetone-Alcohol, 1:1 by dropping onto the slide until no more colour runs off.
5. Stain in the Basic Fuchsin Working or (dilute one vol. Basic Fuchsin Stock, 0.25%, Aq., with 10 vol. distilled water), 1 minute; wash in water, blot carefully but not to complete dryness as in step #3.
6. Differentiate in Acetone, i.e. one quick dip, then transfer immediately to the Picric Acid – Acetone Solution, 0.1% to complete. Differentiate until sections show yellowish-pink.
7. Rinse quickly in Acetone, then Acetone-Xylene. Clear in 3-4 changes Xylene alone.
8. Mount.

Results:

Note: See also the Taylor modification of the Brown and Brenn+/- technique noted for the varying differentiation available.  Over-differentiation in the B&B step #6 is a problem with some sections; run the control slides at varying rates to determine the amount for the specific organism.

References:

Brown, J.H. and Brenn, L.  Bull. Johns Hopkins Hosp., 48:69 (1931). AFIP Manual of Histologic Staining Techniques: 3rd. ed., ed. G. Luna; New York: McGraw-Hill Publications, c. 1968, p. 222.