ACID FAST STAINING

Zeihl Neelsen’s (ZN) technique

This method is a modification of Paul Ehrlich's (1882) original method for differential
staining of tubercle bacilli and other acid-fast bacilli.

Certain bacteria are easily stained by carbol fuctsin but decolourize completely when
treated with acid-alcohol. On the other hand it is difficult to stain certain other micro
organisms with carbol fuchsin but once stained they retain the colour even after acid-alcohol
treatment. Such organisms which retain the stain after acid-alcohol decolourization are
referred as acid-fast organisms.

Acid fastness has been ascribed to a lipid peculiar to acid fast bacilli, a high
molecular weight hydroxy acid wax containing carboxyl group (mycolic acid). The carbol
fuchsin combines with the mycolic acid to form a complex that is stable and is not
decolourized.

The ordinary aniline stain solutions do not readily penetrate the substance of the
tubercle bacillus and other acid fast bacilli and are therefore unsuitable for staining it. The
use of powerful staining solutions containing phenol and the application of heat helps the
stain to penetrate the bacillus which withstand the action of powerful decolourizing agents
for considerable time and thus still retain the stain when everything else in the smear has
been decolourized. The stain used consists of basic fuchsin and phenol. When mineral acid
is added, basic fuchsin combines with it to form a compound that is yellowish brown in
colour and readily dissolved out of all the structures except acid-fast bacteria. Any strong
acid can be used as a decolourizing agent, but 20% sulphuric acid usually used. Acid-
alcohol may be used instead and generally gives cleaner films.

In order to show structures and cells including non-acid fast bacteria, that have been
decolourized, and to form contrast with red stained bacilli the preparation to stained with
methylene blue/malachite green.

SOLUTIONS

(1) ZEIHL NEELSEN'S (CONC) CARBOL FUCHSIN

Basic fuchsin 10g

Absolute alcohol 100ml

Solution of phenol

5% in Distilled Water to 1000ml

Dissolve the dye in the alcohol and add to the phenol solution.

(2) ACID - ALCOHOL

Conc. Hydrochloric acid 3ml

95% alcohol 97ml

Department of Veterinary Microbiology, Rajiv Gandhi Institute of Veterinary Education & Research, Pondicherry
(4) COUNTER STAIN - LOEFFLER'S METHYLENE BLUE

Saturated sol. of methylene blue

in alcohol 300m1
KOH 0.01% to 1000m1

PROCEDURE

1. Make a smear with the material of suspected sample. The smear should be thick. Air
dry and fix the smear.

2. Place the smear on the staining rack; flood the smear with ZN carbol fuchsin. Heat
the slide from below with the help of a spirit lamp until steam rises.

Note: Do not allow it to boil. The stain must not be allowed to evaporate and dry on
the slide. If necessary pour more carbol fuchsin to keep the whole slide covered with
stain.

The heating and staining is continued for about 8 minutes.

3. Allow the stain to cool down. Wash with water.

4. Decolorize the slide with Acid-alcohol until the slide appears colourless.

5. Wash the slide with water.

6. Counter stain with Loeffler's alkaline methylene blue of 30 seconds.

7. Wash with water, blot dry and examine under the oil immersion objective.

Acid fast bacilli stain bright red, while the tissue cells and other organisms are stained blue.

OBSERVATION:

Arrangement

EXAMPLES

Mycobacterium tuberculosis
Mycobacterium bovis
Mycobacterium paratuberculosis - Johne's bacillus
Mycobacteria are also called ACID FAST BACILLI or AFB.

Department of Veterinary Microbiology, Rajiv Gandhi Institute of Veterinary Education & Research, Pondicherry