The document describes the Ziehl-Neelsen staining technique developed for the identification of acid-fast microorganisms, particularly Mycobacterium tuberculosis, by distinguishing them from non-acid fast bacteria. It details the procedure, principles, reagents, and applications of this staining method. Additionally, it highlights the significance of the structural composition of acid-fast bacteria and mentions potential modifications and applications in various microbiological examinations.

1. M. J. P. ROHILKAND
UNIVERSITY,
BAREILLY

2. Introduction
Acid fast
microorganism
Cell wall structure
Principle
Reagent
Procedure
Result
Modification
Applications
Content

3. Introduction
 In 1882, Paul Ehrlich developed the Acid Fast Staining technique.
 Later, Ziehl and Neelsen developed it in 1883 and hence the technique is also called as
"Ziehl-Neelsen Staining Technique".
 The main aim of this technique was to distinguish bacteria between acid fast groups and
non-acid fast groups.
 This test is exclusively used for identification of Mycobacterium tuberculosis, a
causative agent of TB.
 It is a member of Mycobacterium genus.
 In Ziehl & Neelsen staining method, the acid-fast bacteria appear to be red bright bacilli
with a light blue background.

4.  Acid-fast organisms are characterized by wax-like nearly impermeable cell wall,
containing mycolic acid along with large amount of fatty acids, waxes, and complex
lipids.
 The acid-fast microorganisms are resistant to decolorization by acid due to the
composition of cell wall.
 Microorganisms that are not easily stained by basic stains such as gram staining.
Acid–fast Microorganism
 ACID-FAST BACTERIA
 Mycobacterium tuberculosis,
 M.phlei,
 M.leapre,
 Nocardia farcinica
 N.nova
 N.brasiliensis
 NON-ACID FAST
BACTERIA –
 E.coli,
 Staphylococcus aureus.
 OTHER ACID-FAST
STRUCTURE
 Bacterial endospore
 Head of sperm

5. Cell Wall Structure of Acid fast Bacteria

7. Principle
Acid-fast mycobacteria contain mycolic acid in their outer membrane, making
the cells waxy and resistant to staining with aqueous based stains such as the
Gram stain.
The primary stain, carbol fuchsin is applied to the cells, and heat and phenol
are used to allow the stain to penetrate into the waxy surface of acid-fast
microorganisms.
The excess stain is removed with treatment by acid alcohol (ethanol and
hydrochloric acid).
A secondary stain, methylene blue, is then applied to the cells.

8. Regents
Primary dye: carbol fuchsin(basic fuchsin with phenol)
Decolorizer : sulfuric acid, alcohol, acid alcohol
Counterstain : methylene blue, malachite green
 CARBOL FUCHSIN
• Basic fuchsin (powder) 1gm
• Phenol (crystalline) 5ml
• Alcohol(95% or100%) 10ml
• Distilled water 100ml
DECOLORIZER
ACID ALCOHOL(3%HCl in 95% ethyl
alcohol)
• Concentrated hydrochloric acid 3ml
• Distilled water 2ml
• Ethyl alcohol 95ml
 COUNTERSTAIN
METHYLENE BLUE(0.25%)
• Methylene blue 0.25gm
• Acetic acid 1ml
• Distilled water 99ml

9. Procedure
 STEP 1.
• Prepare smear.
 STEP 2.
• Add pour 1% Carbol Fuchsin - the primary stain.
• Heat the slides gently until you see vapors, but do not boil. (for3 to 5 min)
• Rinse slides gently with tap water.
 STEP 3.
• Add pour 20% Sulphuric acid- the decolorizing solution. ( for 15 sec)
• Rinse the slides with tap water.
• Tilt to drain excess water.
 STEP 4.
• Pour 0.1% Methylene Blue - the counter stain. (for 30 sec)
• Rinse the slides gently with tap water.
• Tilt the slides to drain excess water.
• Finally, place slides on rack to air-dry.
• STEP 5.
• Examine under microscope.

12. • Acid-fast bacteria retain the primary dye, carbol-fuschin,
and stain pink.
• Non-acid fat bacteria take up the methylene blue dye and
appear blue.
• We can see in given below picture acid fast bacteria
Mycobacterium tuberculosis has rod shape structure with
pink colour.
• And background with blue colour.
Result

13. 1.Mycobacterium leprae
Same method is used with 5% sulphuric acid due to less mycolic acid in cell wall.
2.Nocardia and Legionella
1% sulphuric acid is used
2.Stool Specimen
We use 3% acid alcohol instead of sulphuric acid so as to stain Cryptosporidium parvum.
Modifications

14. Applications
 Used for examination and identification of Mycobacterium species.
 Used to differentiate between acid- fast and non-acid fast bacilli
 Used for the identification of some fungal species such as
Cryptosporidium.

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