The document summarizes the Ziehl-Neelsen staining technique used to identify acid-fast bacteria like Mycobacterium tuberculosis. The technique involves staining a smear with carbol fuchsin, heating to allow the stain to penetrate the waxy cell walls, washing with acid to decolorize non-acid fast cells, and counterstaining with methylene blue. Acid fast bacteria will appear bright pink against a blue background under the microscope. The document also describes the Kinyoun cold staining method and provides grading scales used to report acid fast bacilli observations.

1. ZIEHL-NEELSEN
STAINING
Dr. Dinesh Kr Jain, MD., Assistant professor,
Department of Microbiology,SMS Medical college, Jaipur

2. ZIEHL-NEELSEN STAINING
PRINCIPLE
• Acid-fast mycobacteria contain mycolic acid in their outer
membrane, making the cells waxy and resistant to staining with
aqueous based stains such as the Gram stain.
• The primary stain, carbol fuchsin is applied to the cells, and heat
and phenol are used to allow the stain to penetrate into the waxy
surface of acid-fast microorganisms.
• The excess stain is removed with treatment by acid alcohol (ethanol
and hydrochloric acid).
• A secondary stain, methylene blue, is then applied to the cells.
• NOTE: Colour Blind workers are advised to use picric acid
solution (7g/l in water) which yields a yellow background.

3. METHOD OF MAKING SMEAR
1. Vortex concentrated sediment, un-concentrated sputum, other purulent
material, or stool. Place 2 to 3 drops on the slide from the end of the
broom-stick parallel to the slide and slowly spread the liquid uniformly to
make a thin smear.
2. For cerebrospinal fluid (CSF) sediment, vortex thoroughly and apply to
the slide in heaped drops. A heaped drop is allowed to air dry, and a
second application of sediment is placed on the same spot and allowed to
dry. A minimum of three layers, applied to the same 1-cm diameter circle,
should facilitate detection of small numbers of bacilli. (Note: Some
laboratories have stopped performing acid-fast stains on CSF because
positive stains are extremely rare.)
3. Fix the smear at 80° C for 15 minutes or for 2 hours at 65° to 70° C on an
electric hot plate. (Note: Survival of mycobacteria at this temperature has
been reported; handle all specimens with proper precautions.)

4. Ziehl-Neelsen staining
Procedure
4. Rinse with Tap Water & Decolorize
with 25% sulphuric acid
1. Pour 1 % Carbol Fuchsin
5. Let it stand for 10-15 seconds &
rinse gently with tap water
6. Pour 0.1% methylene blue &
leave for 1 minute
3. Carbol fuchsin 3-5 mins2. Gently heat until vapour rises

5. Acid fast bacilli will appear stained pink,
straight curved rods with blue background due
to methylene blue

6. Reporting
• Read at least for 5 minutes & 100 high power field before reporting a
negative result.

7. REVISED RNTCP GUIDELINES
AFB observation Grade
More than 10 AFB per field in atleast 20 fields 3+
1-10 AFB per field in atleast 50 fields 2+
10-99 AFB per 100 fields 1+
1-9 AFB per 100 fields Scanty
(Actual no.)
No AFB seen in atleast 100 field Negative

8. Acid-fast organisms/structures Sulfuric acid (%) needed
for Decolorlzation
Mycobacterium tubercuJosis 25%
Mycobacterium leprae 5%
Nocardia 1%
Acid·fast parasites such as Cryptosporidium,
Cyclospora, Isosopra, Microsporidia, Taenia
saginata (segments and eggs), hooklets of hydatid
cyst and eggs of Schistosoma mansoni
1%
Bacterial spore 0.25-0.5%
Sperm head 0.5-1%
Legionella micdadei 0.5-1%

9. Kinyoun modification (Cold Method)
Identification of acid-fast Mycobacterium spp. and parasites such as
Cryptosporidium and Isopora spp.
PRINCIPLE
• Acid-fast mycobacteria contain mycolic acid in their outer membrane,
making the cells waxy and resistant to staining with aqueous based stains
such as the Gram stain.
• The primary stain, carbol fuchsin, is applied to the cells and phenol is used
to allow the stain to penetrate into the waxy surface of acid-fast
microorganisms.
• The excess stain is removed with treatment by 1% sulfuric acid.
• A secondary stain, methylene blue, is then applied to the cells.

10. RESULTS
• Acid-fast organisms, Mycobacterium spp., will appear pink.
• Nonacid-fast organisms will appear blue.
• In addition, background material should stain blue.
LIMITATIONS
• May be less sensitive than the Ziehl-Neelsen method.
• Smears that are too thick may not properly stain.

11. • Smear prepared from material
obtained from a necrotic
tuberculoma of the lung stained
with the kinyoun acid-fast stain.
Note the relatively short, thin,
beaded, slightly curved, red-
staining acid-fast bacilli

12. REFRENCES
• Bailey & Scott’s Diagnostic Microbiology
• Koneman’s color atlas and textbook 7th Edition
• Mackie & McCartney Practical Medical Microbiology 14th Edition

13. THANK YOU
13th September, 1853
166th Birth-Anniversary of Christian Gram