This presentation mainly focuses on explanation about acid fast staining, their principle, reagents and procedure. And also about acid fast organisms. And a detailed explanation about the importance of Ziehl-neelson stain and errors occured during the procedure.

1. Z I E H L N E E L S E N S TA I N I N G
O V YA P U G A L E N T H I A R U N A

2. C O N T E N T S
• Introduction
• Acid fast staining
• Ziehl-neelsen staining
• Principle
• Reagents
• Procedure
• Acid fast organisms
• Modified zn staining
• Importance of Ziehl-Neelsen stain
• Errors
• Bibliography

3. I N T R O D U C T I O N
• It was first discovered by Earlich in 1881 and modified by Zeihl
& Neelsen.
• Acid fast staining is an important differential staining
procedure. It can be used to identify mycobacterium tuberculosis
and M. leprae these bacteria as well as other mycobacteria have
cell wall containing lipids constructed from mycolic acid. Which
prevents dye from binding to cells. ZN method uses high
concentration of phenol and carbol fuchsin, as well as a wetting
agent, to drive carbol fuchsin into mycobacterial cells. once this
dye has penetrated, the cells are not easily decolonized by
acidified alcohol and thus are said to be acid fast.

4. A C I D FA S T S TA I N I N G
• The acid-fast stain is a differential stain used to identify
acid-fast organisms such as members of the genus
Mycobacterium .
• Acid-fast organisms are characterised by wax-like, nearly
impermeable cell walls; they contain mycolic acid and
large amounts of fatty acids, waxes, and complex lipids.
• Acid-fast organisms are highly resistant to disinfectants
and dry conditions. Because the cell wall is so resistant
to most compounds.

5. Z I E H L - N E E L S E N
S TA I N I N G
• Z N stain is a modification
of Ehrlich’s original
method for differential
staining of acid fast bacilli
by use of aniline gentian
violet followed by strong
nitric acid.

6. P R I N C I P L E
• Acid fastness has been ascribed to the high content and variety of lipid,
fatty acid and higher alcohols found in tubercle bacilli.
• A lipid particular to acid fast bacilli is a long chain fatty acid (mycolic acid)
• Acid fast is not a property of lipid alone but depends also on integrity of
the cell wall.
• The ordinary aniline dye solution do not readily penetrate the acid fast
bacilli.
• So by use of powerful staining solution that contain phenol and
application of heat , the dye can be made to penetrate the bacillus.

7. • Phenol will solubilise the cell wall and heat will
increase the stain penetration.
• Once stained the tubercle bacilli will withstand the
action of powerful de colourising agents for
considerable period of time , retains the primary stain
when every thing else has been decolonized.

8. Z I E H L N E E L S E N A C I D - FA S T S TA I N I N G
1. Add one lapful of sterile water to a microscope slide.
2. Make a heavy smear of Mycobacterium smegmatis. Mix
throughly with a loop. Then transfer a small amount
of Staphylococcus epidermidis to the same drop of
water.
3. Air dry and heat fix.
S M E A R P R E PA R AT I O N

9. • Cover the smear with
carbolfuchsin dye.
Carbolfuchsin a potential
carcinogen. Hence
wearing a gloves is
mandatory.
• Place a piece of paper
towel on top of the dye.
Be sure the paper towel is
saturated with the dye.

10. • Dry heat for 2 minutes.

11. Cool and rinse with water
De-colourise with acid-
alcohol for 15-20 seconds.

12. Wash the top and bottom of slide with water and clean
the slide bottom well.

13. Counterstain with
Methylene Blue for 30
seconds to 1 minute.
Wash and blot the slide with
bibulous paper.

14. C O N C L U S I O N
Acid-Fast
Staining
Cell Colour Cell Colour
Procedure Reagent
Acid Fast
Bacteria
Non Acid Fast
Bacteria
Primary Dye Carbolfuchsin Red Red
Decolourizer Acid-Alcohol Red Colorless
Counter Stain Methylene Blue Red Blue

15. A C I D FA S T O R G A N I S M S
• All Mycobacteria - M. tuberculosis, M. leprae and atypical
Mycobacterium.
• Actinomyces – Nocardia ,Rhodococus
• Bacterial spores
• Cysts of some coccidian parasites: Cryptosporidium
parvum, Isospora belli.
• A few other parasites: Taenia saginata eggs, Hydatid cysts.

16. Cyclospora
Isospora

17. M O D I F I E D Z N S TA I N I N G
• A modified acid-fast staining method was developed
for rapid detection of Mycobacterium tuberculosis and
its forms, where in carbol fuchsin and dioxogen were
mixed into the sputum smear. With this method, the
dyeing time is shortened and heating is not required.

18. I M P O R TA N C E O F Z I E H L - N E E L S E N S TA I N
• Acid fast staining reaction of mycobacteria, along with
their characteristic size and shape, is a valuable aid in
the early detection of infection and in the monitoring
of therapy for mycobacterium disease.
• The presence of acid fast bacilli in the sputum,
combine with a history of cough, weight loss and chest
radiographic evidence of pulmonary infiltrate, is the
presumptive evidence of active tuberculosis.

19. E R R O R S
• Causes of false positive smears: Food particles,
Precipitated stains, Saprophytic AFB , SporesFibers
and pollens.
• Causes of false negative smears: Inadequate
specimen, inadequate storage of specimens,Failure to
select suitable material to prepare smear,Inadequate
preparation of smears,Inadequate examination,
Mistakes in labelling, false recording.

20. B I B L I O G R A P H Y
• https://www.uwyo.edu/virtual_edge/units/
acidfast_stain.html
• PRESCOTT’S MICROBIOLOGY (tenth edition) 2017
pg:33, JOANNE M. WILLEY, LINDA M. SHERWOOD,
CHRISTOPHER J. WOOLVERTON.
• Textbook of Microbiology,C. K. Jayaram Paniker
and R. Ananthanarayan (tenth edition) 2017. Pg :136 -
137

21. T H A N K Y O U