This presentation is about ZN staining.

1. ALL ABOUT
MICROBIOLOGY

2. ZIEHL-NEELSEN
(ZN) STAINING
SABA GHAFOOR

3. ZN staining
■ Ziehl- Neelsen staining is used to
identify acid fast organisms
Mycobacterium species including
Mycobacterium tuberculosis,
Mycobacterium ulcerans, and
Mycobacterium leprae.

4. INTRODUCTION
■ Mycobacterium, unlike other bacteria do not stain well by
gram staining.
■ Mycobacterium tuberculosis and Mycobacterium ulcerans are
strongly acid-fast as compared M. leprae is only weak acid
fast.

5. Principle
■ They stained by Carbol fuchsin combined with phenol. The
stain binds to mycolic acid in the mycobacterial cell wall. The
stain binds to mycolic acid in the cell wall of Mycobacterium.
Decolorizing solution is applied that removes the red color
from the background cells except Mycobacterium which retain
the dye. Therefore referred as acid-fast bacilli AFB.

6. Reagents required in ZN staining
 Carbol fuchsin stain (Primary dye)
 Acid alcohol ( Decolorizer)
 Malachite green/ Methylene blue dye (counterstain)

7. Procedure
1. Heat fix the smear
2. Apply primary stain for 30 seconds on the smear.
3. Heat the stain until vapors just begin to rise about 60℃.
4. Wash off the stain.
5. Cover the stain with alcohol for 15-20 seconds and wash off.
6. Cover the stain with counterstain by applying methylene blue
or malachite green.

9. Cont.…
■ Examine the smear microscopically, using 100X oil immersion
objective.

10. Precautions
■ Do not overheat the stain.
■ Do not touch the smear.
■ Smears that are too thick may not stain properly.

11. Results
■ Acid-fast bacilli  red, straight, or slightly curved rods may
appear beaded
■ Cells  Green
■ Background material  Green

13. Reporting of sputum smear
NUMBER OF BACILLI GRADE
NoAFB seen Negative
1-9 AFB/100 fields Report the exact number
10-100AFB/100 fields +
1-10 AFB / field ++
More than 10 AFB per field +++