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Electron Microscopy Sciences

Technical Data Sheets

Brown and Brenn Method for Gram + and Gram - Bacteria

EMS Catalog # 26105-series

Fixation:

Formalin, 10% Buffered Neutral   (#15740)

Section:

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

Staining Procedure :

  1. Deparaffinize and hydrate to distilled water.
  2. Mix 1.0ml (20dr.) Crystal Violet, 1% Aq., (#26105-01) with 5 drops Sodium Bicarbonate, 5%, Aq., (#26105-02); 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, (#26105-04), 1 min., rinse with water and carefully blot with filter paper to dryness.
  4. Decolorize with  Acetone-Alcohol, 1:1, (#26105-05) by dropping onto the slide until no more color runs off.
  5. Stain in the Basic Fuchsin Working, (#26105-3A) or (dilute one vol. Basic Fuchsin Stock, 0.25%, Aq., (#26105-03) 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, (#26105-07), i.e. one quick dip, then transfer immediately to the Picric Acid – Acetone Solution, 0.1%, (#26105-06) to complete. Differentiate until sections show yellowish-pink.
  7. Rinse quickly in Acetone, then Acetone-Xylene, (#26105-08). Clear in 3-4 changes Xylene, (#23400) alone.
  8. Mount.

Results:

Gram+ Bacteria, Nocardia and Actinomyces Filaments Blue
Gram- Bacteria, Nuclei Red
Additional tissue elements Yellow

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.

Online Ordering

Brown and Brenn Method for Gram+ and Gram- Bacteria is available online from the EMS Catalog. For ordering or product information, click here.