ZIEHL NEELSEN ACID FAST BACILLI STAINING (DIRECT SMEAR ) - PURPOSE, PRINCIPLE, PROCEDURE AND RESULT

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Purpose:   To demonstrate acid-fastbacilli in the given specimen.

Principle: Mycobacterium possesses cell walls that contain Mycolic acid (long-chain fatty acids).  The presence of Mycolic acid and lipids in the Mycobacterial cell wall accounts for the property of resisting decolorization with strong acids. Once stained with primary stains, they resist decolourisation with strong acids. This feature is called Acid fastness and it is associated with the intact cell wall and the presence of large quantities of lipidecolorisationds and mycolic acid.

Performance characteristics: 

This test cannot find out the exact species of Mycobacteria. It can only differentiate whether the Acid Fast Bacilli are present or not. Further tests must be performed to confirm the species of Mycobacteria spp.

Type of samples:  

Sputum, transtracheal aspiration, laryngeal swab, bronchoscopic specimens, bronchoalveolar lavage.  Extra-Pulmonary Specimens: Urine, Gastric lavage, tissue and body fluids.

Recommended sample collection: Early morning sample after goggling. Avoid saliva-mixed samples.  

Type of sample container: Sterile containers 

Required equipment and reagents:

          Equipment: Light microscope.

         Biological safety cabinet.

 

         Reagents: Strong Carbol Fuchsin.

         25% Sulfuric acid.

          Methylene blue.

Procedure: 

1.    Prepare a thin smear of the sputum on a clean glass slide and heat fix the smear allowing it to dry.

2.    Flood the smear with Strong Carbol Fuchsin and heat the slide for one minute such that steam comes off from the stain. Do not allow it to boil.

3.    Allow the slide to cool and wait for 5 minutes.

4.    Wash with running water.

5.    Decolourize with 25% Sulfuric acid over the smear and keep for 1-2 minutes.

6.    Wash with water.

7.    Counterstain with Methylene blue for 1 minute.

8.    Gently wash it with water and allow it to dry.

9.    View under oil immersion objective.

Quality control procedures: Check the reliability of stains with known sample bacteria, whenever new batches of stain are used

Positive control: Known positive sample (Mycobacterium sp.)

Negative control: Known negative sample

Results and clinical interpretation:  If any definite bacilli (pink /red rods) are seen, report the smear as AFB positive and give an indication of the number of bacteria present as follows:

RNTCP guidelines for AFB quantification

Number of AFB seen (1000X magnification)

         Report                                               

     0 AFB/100 fields                                     

                 No AFB seen

1-9 AFB/100 fields

                 Scanty

10-99 AFB/Minimum of 100 fields

                     1+

1-10 AFB/field in minimum of 50 fields

                     2+

>10 AFB/field in minimum of 20 fields

                     3+

   

Acid Fast Bacilli in Ziehl Neelsen

References:

1.    Practical Medical Microbiology by Mackie & McCartney 14th Edition, Page No-796 – 798.

2.    Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Sixth edition by Washington et al., 2006Page no; 1064-1124.

3.    Bailey and Scott’s Diagnostic Microbiology, 12th edition by Betty et al., 2007, Page no: 478-509.

 

 


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