Topic ACID-FAST STAINING

Topic: Acid fast staining

Content: Aim, principle, reagents used, procedure, observation, grading of AFB & report.

Introduction: It is a method to stain acid fast bacteria like Mycobacterium slender rod bacilli.
They do not stain readily but once stained, resist decolourisation due to presence of lipid rich
waxy materials i.e, mycolic acid on the cell wall. So they are called acid fast bacilli.

There are 2 methods of staining acid fast bacteria

1. Hot staining method. Eg:- Zielh-Neelsen staining.

2. Cold staining method. Eg:- Kinyoin staining.

ZIEHL-NEELSEN STAIN

Aim: To stain the given sputum smear for the detection of Acid-fast bacilli.

Principle

When the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the
Mycobacterial cell wall but by the application of heat, carbol fuchsin further penetrates through
lipoidal wall and enters into cytoplasm. Then after all cell appears red. Then the smear is
decolorized with decolorizing agent but the acid fast cells are resistant due to the presence of
large amount of lipoidal material in their cell wall which prevents the penetration of decolorizing
solution. The non-acid fast organism lack the lipoidal material in their cell wall due to which
they are easily decolorized, leaving the cells colorless. Then the smear is stained with
counterstain, methylene blue. Only decolorised cells absorb the counter stain and take its color
and appears blue while acid-fast cells retain the red color

Reagents used:

a) Strong carbol fuchsin – Primary stain

b) 20% H2SO4 – Decolouriser
c) Methylene blue – Counter stain

Procedure

1. Place the sputum smear slide on staining rack.

2. Cover the smear with strong carbol fuchsin.
 3. Heat the slide with spirit lamp & remove it when fumes arise.
4. Stop heating when fumes are observed & never heat to boil & the stain must not be
allowed to dry.
5. Keep it for 5-8 minutes.
6. Decolourise the smear with 20% H2SO4 for 1-2 minutes.
7. Wash the slide under running tap water.
8. Counter stain with methylene blue for 2 minutes.
9. Wash the slide under tap water.
10. Air dry the smear & examine under oil immersion objective lens (100X) microscpope.

Observations

1. Acid -fast bacteria retains primary stain that is carbol fuschin & appear pink in colour.
2. Non-acid fast bacteria gets decolorised & retains methylene blue stain & appearblue in
colour.
3. Inflammatory cells or pus cells appears blue in colour.
Acid-fast bacilli

Pus cells &

inflammatory
cells

Fig:- Acid-fast stain smear

Grading of AFB smear

No. Of AFB Fields Report

0 300 fields AFB not seen
1-2 300fields Doubtful, repeat smear
1-9 100 fields 1+
1-9 10 fields 2+
1-9 1 fields 3+
>10 1 fields 4+

Report: 0 acid-fast bacilli in 300 fields. Hence the given sputum smear is negative for Acid-fast
bacilli.

1. Textbook of Medical Lab Technology - Praful B. Godkar, Darshan P. Godkar

2. Textbook of microbiology by - Ananthanarayan and Paniker
3. Microbiology, an introduction – Tortora, Funke case