Laboratory Exercise 9

ACID FAST STAINING

Majority of bacterial organisms are stainable by either simple or Gram staining

procedures, but there are few genera, particularly by members of the genus Mycobacterium that
are resistant and can only be visualized by the acid-fast method. Since M. tuberculosis and M.
leprae represent bacteria that are pathological to humans, the stain is of diagnostic value in
identifying these organisms. The characteristic difference between Mycobacteria and other
microorganisms is the presence of a thick, waxy (lipoidal) wall that makes penetration by stain
extremely difficult. Once the stain has penetrated, however it cannot be readily removed even
with the vigorous use of acid alcohol as decolorizing agent. Because of this property, these
organisms are called acid-fast, while other microorganisms which are easily decolorized by acid
alcohol are non-acid fast.

Objectives: At the end of the experiment, the student must be able to:
1. Understand the biochemical basis of the acid-fast stain
2. Perform an acid-fast stain
3. Differentiate bacteria into acid-fast and non-acid fast groups

Procedure:
A. Preparation of smear

Materials: microscope slides, inoculating loops, alcohol lamp, TSB broth cultures of E. coli and
Mycobacterium phlei, sputum

Procedure:
1. Prepare a smear from broth culture of E. coli and Mycobacterium phlei.
2. Sputum smear preparation. Using sterile technique, prepare sputum smear of moderate
thickness. Use whitish or yellowish granular structures in the specimen. (Note: For your
protection, use mask. You are dealing with airborne bacteria. Dry the smears and heat fix.
3. Allow the smear to air dry and then heat-fix.
4. Place the slide on a hot plate that is within a chemical hood, and cover the smear with a piece
of paper towel that has been cut to the same size as the microscope slide.
5. Saturate the paper with Ziehls carbolfuchsin. Heat for 3 to 5 minutes. Do not allow the slide
to dry out, and avoid excess flooding! Also, prevent boiling by adjusting the hot plate to a
proper temperature. A boiling water bath with a staining rack or loop held 1 to 2 inches above
the water surface also works well.
6. Remove the slide, let it cool, and rinse with water for 30 seconds.
7. Decolorize by adding acid-alcohol drop by drop until the slide remains only slightly pink. This
requires 10 to 30 seconds and must be done carefully.
8. Rinse with water for 5 seconds.
9. Counterstain with alkaline methylene blue for about 2 minutes.
10. Rinse with water for 30 seconds.
11. Blot dry with bibulous paper.
12. Examine the slide under oil immersion and record your results. Acidfast organisms stain red;
the background and other organisms stain blue or brown.
13. Examine the prepared slide of Mycobacterium tuberculosis under oil immersion and record
your results.
Study Questions:

What is the purpose of heat fixation? What happens when too much heat is applied?

1. What is the purpose of the heat during the acid-fast staining procedure?
2. What is the function of the counterstain in the acid-fast staining procedure?
3. Are acid-fast bacteria gram positive or gram negative? Explain your answer.
4. For what diseases would you use an acid-fast stain?
5. What makes a microorganism non-acid-fast?
6. What chemical is responsible for the acid-fast property of mycobacteria?
7. Is a Gram stain an adequate substitute for an acid-fast stain? Why or why not?