ZN Staining

(Ziehl Neelsen)

Arun Kumar Parthasarathy

 Introduction
Ziehl-Neelsen staining (Acid Fast) technique is a
differential staining technique
It was initially developed by Ziehl and modified later
by Neelsen hence the name Ziehl-Neelsen stain
Neelsen used carbol fuchsin with heat and added a
decolorizing agent acid-alcohol and counter stain
methylene blue.
The use of acid-alcohol in the technique so it is called as
acid-fast stain
 Where it is used?
Microorganisms that are not easily stained by basic
stains such as gram staining, negative staining
Examples:- Mycobacterium, Actinomycetes., Nocardia,
Cryptosporidium etc.
These microorganisms have thick cell wall made up of
lipodial complexes known as mycolic acid
 Principle
The ziehl Neelsen stain uses basic fuchsin and phenol
compounds to stain the cell wall of mycobacterium spp.
Mycobacterium does not bind readily to simple stains
Use of heat along with carbol-fuchsin and phenol allows
the penetration through he bacterial cell.

Mycolic acid:- on its cell wall making it waxy,

hydrophobic and impermeable.
Mycolic acids are β-hydroxycarboxylic acids made up of
90 carbon atoms – defines the acid fastness of the
bacteria.
 Materials and methods
Microscope with 100x objective
Cedar wood oil or liquid paraffin oil
Bacterial culture
clean glass slide
Spirit lamp
ZN Staining stain :-
1. Carbol fuchsin – Primary stain
2. 20% H2S04 or acid alcohol – Decolourizer
3. Methylene blue – Counter stain

Carbol fuchsin contains:-

Distilled water, Basic fuchsin, ethyl alcohol and phenol crystals

Preparation of acid alcohol (3% HCl in 95% of ethyl alcohol)

 Procedure
1. Keep slide in staining rack
2. Flood smear with carbol fuchsin and heat gently until it
produces fumes by spirit lamp
3. Allow it to stand for 5 minutes and wash it off with gently
flowing tap water.
4. Add 20% H2S04 and leave it for 2-3 minutes and wash it off
with gently flowing tap water.
5. Flood the smear with Methylene blue dye and wait for 1
minute and wash it off with gently flowing tap water.
6. Air dry the slide and observe under microscope with oil
immersion objective lens
 Interpretation

Acid fast bacteria retain primary dye (Carbol fuchsin) –

Pink in colour E.x Mycobacterium, Cryptosporidium,
Nocardia, Isospora etc.
Non acid fast bacteria does not retain primary dye- Blue
in colour.
 RNTCP grading

What is RNTCP?
Revised National Tuberculosis Control Program
Recently changed name, NTEP (National Tuberculosis
Elimination program)
RNTCP Grading : The number of bacilli seen in a smear reflects
disease severity and patient infectivity
It is given by WHO
It is mostly used in Pulmonary tuberculosis
 Modified acid fast stain
Cold Method with Gabbet’s methylene blue stain:-
The smear is flooded with basic fuschin-phenol
stain and allow to stand at RT for 10 mins and counter
stain Gabbet’s methylene blue stain for 2 mins.
Kinyoun’s Method:-
Nocardia and legionella resist decolourization by
1% cold sulphuric acid
Acid fast stain using by 5% sulphuric acid:-
M. leprae resist decolourization by 5% sulphuric
acid
Thank You