Acid-Fast Procedure

1. Prepare smears of organisms to be stained.

2. Heat fix the smears.

3. Cut or tear absorbent paper (bibulous paper) to fit the slide leaving one end for handling.  Do not allow the paper to protrude beyond the slide, but the smears must be covered.

4. Place the slide on wire gauze on a ring stand.

5. Saturate the paper with carbolfuschin.

6. Heat the slides with a hand-held bunsen burner until steam can be seen rising from the surface.  Alternately remove the burner and reheat the slide to maintain steaming for 3-5 minutes.  As the paper begins to dry during the staining process add a drop or two of carbolfuschin to keep the slide moist.  Adding too much stain will cool the slide (and drip on the bench).  Overheating the slide or letting it dry will distort the cells.  Under heating the slide will fail to stain acid-fast cells.

7. At the end of staining remove the paper with tweezers and wash the slide thoroughly.

8. Drain the slide.

9. Decolorize with acid-alcohol for 30 seconds.

10. Rinse, drain, and counterstain with methylene blue for 45 seconds.

11. Rinse, blot, and examine.  First observe each organism on its separate smear.  Then examine the mixed smear.

12. Acid-fast organisms will appear red and non-acid-fast organisms will be blue.

Results of an Acid-fast Stain
Lab Procedures