Ziehl-neelsen (ZN) staining / Acid Fast Bacillus (AFB)

Acid Fast Bacillus (AFB), e.g. Mycobacterium tuberculosis is stained by ZN staining. Once stained with fuming carbol fuchsin they resist decolourization with 20% H₂SO₄ or acid alcohol.AFB stains red                                                                 

 Acid-fast Stain

Procedure	Reagent	Cell Color Acid-fast Bacteria	Nonacid-fast Bacteria
Primary dye	Carbolfuchsin	RED	RED
Decolorizer	Acid-alcohol	RED	COLORLESS
Counterstain	Methylene blue	RED	BLUE

Method-steps

    1.     Clean the slide and make it greeze free.

    2.     Make a thin Uniform film a sterilized loop.

a.     Solid material (say, a colony).place a loop of clean water on the slide & emulsify the material in it.

b.     Fluid material is used as it is. 

    3.     Dry the film in air.

    4.     Fix the film by slowly passing the slid 3-4 times through a flame.

    5.     Heat Crbol fuchsin in a test tube till fumes appear.

    6.     Cover the slide with fuming carbol fuchsin and keep for 5 minutes.

    7.     Wash with water.

    8.     Decolourization with 2o% H₂SO₄ or acid alcohol until only faint pink color remains.

    9.     Wash with water.

   10.    Counterstain with methylene blue for20-30 seconds.

    11.   Wash with water and dry in air.

*** Focus 10X - then use oil immersion..

Note: The acid-fast Mycobacterium retains carbolfuchsin and stains hot pink.  The Staphylococcus epidermidis is decolorized and the counterstain colors them blue. 

RESULTS:

Acid Fast Bacilli        RED                (Leprae bacilli are short rods)

Nocardia                     RED                (Nocardia organisms are long, thin, and filamentous)

Erythrocytes             PALEPINK Background             BLUE

Note

Reporting of sputum smears

When  any definite red  bacilli are  seen,  report  the smear as ‘AFB positive’, and give an indication of the number  of bacteria present as follows:

More than 10 AFB/field . . . . . . . . . . .  report    

1–10 AFB/field   . . . . . . . .  Report   

10–100 AFB/100 fields. .  Report  

1–9 AFB/100 fields . . . . .  report the exact number

When very few AFB are seen: e.g. when only one or two AFB are  seen,  request  a further  specimen  to examine. Tap water and deionized water (using ‘old’ resin) sometimes contain AFB that resemble tubercle bacilli, and occasionally stained scratches on a slide can  be  mistaken  for AFB although  these  tend  to be in a different focal plane from the smear. Occasionally AFB can be   transferred   from  one smear to another  when the same piece of blotting paper is used to dry several smears.

When no AFB are seen after examining 100 fields: Report the smear as ‘No AFB seen’. Do not report

‘Negative’ because organisms may be present but not seen in those fields examined. Up to three specimens (one collected as an early morning specimen) may need to be examined to detect M. tuberculosis in sputum.