Z n staining

MBBS Students

1. ZN Staining (Ziehl Neelsen ) Arun Kumar Parthasarathy

2. 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

3. 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

4. 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.

5. Materials and methods Microscope with 100x objective Cedar wood oil or liquid paraffin oil Bacterial culture clean glass slide Spirit lamp ZN Staining stain :- Carbol fuchsin – Primary stain 20% H2S04 or acid alcohol – Decolourizer 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 )

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

7. 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.

8. 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

9. 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

10. Thank You