AFB (Ziehl-Neelsen Stain), Tissue Test Cost & Procedure

Ziehl-Neelsen acid-fast dyeing procedure is utilized to smudge Mycobacterium species. Identification of AFB in dyed and acid-washed smudges investigated microscopically may furnish the initial bacteriologic proof of the existence of mycobacteria in a clinical sample. Stain microscopy is a rapid and comfortable strategy that can be accomplished.

There are two strategies generally manipulated for acid-fast staining:

• Carbolfuchsin strategies which comprise the Ziehl-Neelsen and Kinyoun methods
• Fluorochrome strategy utilizing auramine-O or auramine-rhodamine stains

The precept of the Ziehl-Neelsen Method of Acid-Fast Staining

Mycobacteria, which do not smudge generously by Gram dye, are smudged with carbol fuchsin integrated with phenol.

In the ‘hot’ ZN procedure, the phenol-carbol fuchsin pigment is heated to facilitate the pigment to infiltrate the waxy mycobacterial compartment wall.

In the ‘cold’ procedure known as Kinyoun Method, smudges are not heated but the penetration is accomplished by boosting the amount of essential fuchsin and phenol and incorporating a ‘wetting mechanism’ chemical.

Exhibition and Preparation

Due to the overnight proliferation of discharges, first morning samples are better likely to generate reasonable healing of AFB.

Immediate Smear : Smear set instantly from a patient sample before processing.
Indirect Smear : Smear set from a processed sample after centrifugation

Reagents needed:

• Carbol fuchsin stain
• Acid alcohol 3% v/v
• Malachite green 5 g/l

Ziehl-Neelsen taining Procedure

>>The period required: is 45 minutes.

Acid-fast bacilli staining strategy

• Formulate the sputum smudge
• Scatter the sputum evenly over the prominent area of the slip using a constant rotational activity. The instructed extent of the smudge is about twenty mm by ten mm.
• Spot glides on the dryer with smeared texture upwards, and air dry for approximately half an hour.
• Warmth bribe parch smear.
• Warmth fascination with untreated samples may NOT kill M. tuberculosis whereas liquor fascination is bactericidal.
• Application of a preliminary pigment
• Coat the smudge with carbol fuchsin pigment
• Warm the smudge until the moisture just starts to grow.
• Do not overheat. Add different stains if crucial. Allow the heated pigment to stay on the slide for five minutes.
• Wash off the pigment with sterile water.
• Application of decolorizer
• Coat the smudge with three % v/v acid alcohol for two-five minutes or until the smudge is adequately decolorized, i.e. pale pink. Inspection to notice that no additional red pigment runs off the surface when the slip is pitched. Adding a bit of additional decolorizer for very dense glides or those that resume to “bleed” red stain.
• Wash nicely with sanitary moisture
• Counterstaining
• Coat the pigment with a malachite green dye for one-two minute.
• Wash off the pigment with sterile water
• Brush the rear of the slide sterile, and spot it in a draining shelf for a a smudge to air dry .
• Investigate the smudge microscopically.