LABORATORY EXERCISE 4: ACID FAST-BACILLI STAINING AUGUST 18, 2017

BMLS-3A

Acid Fast Stain (Ziehl–Neelsen Staining of Acid Fast Bacilli)

Acid fast stain was discovered by Ehrlich (1882), who found that after staining with aniline dyes, tubercle
bacilli resist decolorization with acids. The method, as modified by Ziehl and Neelsen, is in common use now.

Principle
Some bacteria, such as mycobacteria are resistant to aniline dyes and do not readily penetrate the substance of the
tubercle bacillus and are therefore unsuitable for staining it. The dye can be made to penetrate the bacillus by the use
of a powerful staining solution that contains phenol, and the application of heat. Once stained the tubercle bacillus
cannot be decolorized even with powerful decolorizing agents for a considerable time and thus still retains the stain
when everything else in the microscopic preparation has been decolorized. Hence, they are called Acid–fast bacilli
(AFB). The stain used consists of basic fuchsin, with phenol (acts as a-mordant) added. The dye is basic and its
combination with a mineral acid used as decolorizer produces a compound that is yellowish brown in color which is
readily dissolved out of all structures except acid-fast bacteria. Any strong acid can be used as a decolorizing agent,
but 20% sulfuric acid (by volume) is usually employed. In order to show structures and cells, including nonacid- fast
bacteria, that have been decolorized, and to form a contrast with the red-stained bacilli, the preparation is
counterstained with methylene blue or malachite green. Acid fastness has been ascribed to the high content and
variety of lipids, fatty acids and higher alcohols found in tubercle bacilli. A lipid peculiar to acid fast bacilli, a high
molecular weight hydroxyl acid wax containing carboxyl groups (mycolic acid) is acid fast in the free state. Acid-
fastness depends also on the integrity of the cell wall besides lipid contents.

Ziehl–Neelsen (Z–N) Staining or Acid Fast Staining

A fixed smear is provided for Ziehl–Neelsen (Z–N) staining. If the smear is unfixed, it is fixed by passing the dried
slide, film downwards, three times slowly through the flame, or by heating through the glass slide (the slide is held,
film upwards) in the top of the Bunsen flame for a few seconds so that the slide becomes hot.

Procedure

1. The slide containing fixed smear is covered with carbol fuchsin. The carbol fuchsin is left on the slide for 5–10
minutes with intermittent heating during that period. Heat the slide until the steam rises, but without boiling. (Do
not allow the stain to dry, to counteract drying more solution of stain is added to the slide and the slide reheated).
2. Wash in tap water.
3. The stained smear is decolorized with ACID ALCOHOL. The red color of the preparation is changed to
COLORLESS. After about 1 min in the acid, wash the slide with water, and pour on fresh acid. Repeat this
procedure several times. When it is complete, the film, after washing, is only very faintly pink. (3 mL HCI and 97
mL ethanol).
4. The smear is counterstained with a contrasting dye such as methylene blue for 1–2 minutes. Malachite green can
also be used as counterstain instead of methylene blue.
5. Wash with water, blot with clean paper, dry and mount.
6. Examine under oil immersion (× 100) objective. Acid-fast bacilli appear red (color of carbol fuchsin) while other
organisms, tissue cells and debris are stained blue or green according to the counterstain used
Important Points in Observation
1. Acid-fast bacilli such as Mycobacterium tuberculosis appear red while other organisms, tissue cells and debris are
stained blue or green according to the counterstain used
2. Show your observations to the examiner by focusing a good stained field of your smear as such and by drawing a
well labeled diagram using colored pencils.
Acid-fast Organisms
1. All mycobacteria are acid-fast e.g. Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae,
atypical mycobacteria.
2. Nocardia asteroides, Nocardia braziliensis.
3. Cryptosporidium-a protozoan coccidian parasite, which causes opportunistic infections in AIDS, is acid-fast.
4. Bacterial spores are weakly acid-fast.