ZN STAIN / ACID FAST STAIN

INTRODUCTION

·Introduced by Paul Ehrlich & Carl Weigert & further modified by Ziehl & Neelsen (ZN).

·ZN stain also known as Acid fast stain.

·Some acid fast bacteria include Mycobacterium tuberculosis which is responsible for tuberculosis & M. laprae cause Leprosy.

·Commonly used staining technique for acid fast bacteria.

PRINCIPLE OF GRAM STAIN

• Acid fast bacteria contain high amount of lipids, fatty acids & alcohol found in mycobacterium. Mycolic acid (a wax) which is found in every acid fast bacteria due to lipid contents, acid-fastness these bacteria take only ZN stain.

REQUIREMENT FOR GRAM STAIN

# Material

A.Specimen:

• Sputum

• Urine

• Stool

• Pleural fluid

• CSF

B.Slides:

• Clean and sterile slides for preparing smears.

C.Bunsen Burner:

• For heat-fixing bacterial smears to the slides.

D.Reagent:

• It include:

a)Carbol fuchsin

b)20% H2so4 (sulphuric acid)

c)Methylene blue

E.Water:

• Distilled water for rinsing during the staining process.

F.Microscope:

#Method

·Take clean glass slide.

·Now place one drop saline/water into the slide.

·Transfer small amount of specimen onto the slide.

·Spread the sample in thin film.

·Heat fix the prepared smear.

PROCEDURE

·Step1- Apply carbol fuchsin onto the prepared smear for 5 to 8 minute, Gentle heat is given for deep penetration of the stain.

·Step2- Wash with rinsing water

·Step3- The stained smear is decolourised with 20% sulphuric acid & washed with water. This step should be repeated till the pink/red colour stops coming out.

·NOTE- In case of lepra bacilli 5% sulphuric acid & for nocardia 1% sulphuric acid is used for stain.

·Step6- Wash with water.

·Step7- Counterstain with methylene blue for 2 minute.

·Step8- Wash the smear with water for removal extra stain.

·Step9- Air dry the smear for microscopy.

INTERPRETATION OF RESULT

• The results of Ziehl-Neelsen (ZN) staining are based on the microscopic examination of stained slides.

• Positive Result: Acid-fast bacteria (AFB) retain the red or pink color of the carbol fuchsin stain. They are easily identified under the microscope by their characteristic appearance, which includes rod-shaped cells with a red/pink colour.

• Negative Result: The absence of red/pink stained cells suggests the absence of acid-fast bacteria in the specimen.

CONCLUSION

• ZN stain is most common staining technique for stain acid fast bacteria.

• Introduce by Ziehl & Neelsen

• ZN staining plays a pivotal role in the early diagnosis of infectious diseases, especially tuberculosis

• Cost effective method

• A positive ZN staining result indicates the presence of acid-fast bacilli, marked by red or pink color bacilli.

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