Modified Kinyoun’s Staining Procedure

Purpose:
This standard operating procedure (SOP) establishes the standard for staining acid fast bacteria using the Modified Kinyoun’s staining procedure.

Reagents and Materials:

Reagents

Accustain Carbol-Fuchsin Solution
Commercially prepared by Sigma Diagnostics Co. (Catalog # HT80-1-16).
Expiration date provided by manufacturer.
Store at room temperature.

Acid Alcohol
Hydrochloric Acid 5ml
Distilled water 125ml
95% ETOH 350ml
Store in safety cabinet at room temperature.

Methylene Blue Solution

Stock Solution
Methylene Blue 1.4gm
95% Ethyl Alcohol 100ml

Working Solution
Stock solution 25ml
Tap water 225ml

Materials
Tissue slides and control slides for special stain requested.
Microwave to bake slides.
Staining rack, 20 slide capacity.
Coverslips and synthetic mounting media.

Hazards and Precautions:
See Material Safety Data Sheets.

Sample Preparations:
Paraffin tissue section slides cut at 4 microns.

Calibration and Standardization:
A tissue section slide containing acid fast bacteria is stained with each procedure.

Procedure:

1. Deparaffinize and hydrate to distilled water.

2. Carbo-Fuchsin - 5 minutes.

3. Tap water rinse.

4. 1% Acid Alcohol - dip until the red no longer runs off.

5. Tap water rinse.

6. Methylene Blue - 4-6 dips.

Make fresh: Methylene Blue, (Stock Solution) 5ml
Tap water 45ml

7. Distilled water rinse.

8. Dehydrate, clear and coverslip.

Results:
Acid Fast Bacteria - red

Background, other tissue elements - blue

Figure 1. Johne’s disease macrophages with acid-fast bacteria, 1000 X.

Effective: July 1, 2004
Cherie Chapman
James R. Turk

References:

Sheehan DC, Hrapchak BR: Theory and Practice of Histotechnology, 2nd Ed, CV Mosby, St. Louis, 1980, pp. 236-237.