Scribd
Upload
210 views10 pages

Acid Fast Staining: Dr.T.V.Rao MD 1

The document discusses acid-fast staining and the Ziehl-Neelsen staining technique used to identify Mycobacterium tuberculosis and other acid-fast bacteria. Acid-fast bacteria contain high amounts of lipids and waxes in their cell walls, allowing them to retain stains like carbol fuchsin even after being treated with acid alcohol. The Ziehl-Neelsen staining method involves staining samples with carbol fuchsin, then decolorizing with acid alcohol before counterstaining with methylene blue. This leaves acid-fast bacteria stained red against a blue background under a microscope.

Uploaded by

RoBin Bacani
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
210 views10 pages

Acid Fast Staining: Dr.T.V.Rao MD 1

The document discusses acid-fast staining and the Ziehl-Neelsen staining technique used to identify Mycobacterium tuberculosis and other acid-fast bacteria. Acid-fast bacteria contain high amounts of lipids and waxes in their cell walls, allowing them to retain stains like carbol fuchsin even after being treated with acid alcohol. The Ziehl-Neelsen staining method involves staining samples with carbol fuchsin, then decolorizing with acid alcohol before counterstaining with methylene blue. This leaves acid-fast bacteria stained red against a blue background under a microscope.

Uploaded by

RoBin Bacani
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
You are on page 1/ 10

ACID FAST STAINING

Dr.T.V.Rao MD 1
Mycobacterium are Acid Fast
Bacilli
• Mycobacterium are Gram-resistant (waxy cell
walls), non-motile, pleomorphic rods, related to
the Actinomyces. Most Mycobacteria are found
in habitats such as water or soil. However, a few
are intracellular pathogens of animals and
humans. Mycobacterium tuberculosis, along with
M. bovis, M. africanum, and M. microti all cause
the disease known as tuberculosis (TB) and are
members of the tuberculosis species complex.
Each member of the TB complex is pathogenic,
but M. tuberculosis is pathogenic for humans
while M. bovis is usually pathogenic for animals.
Dr.T.V.Rao MD 2
•
Acid-Fast
Acid-fast cells contain a
Stain
large amount of lipids and
waxes in their cell walls
– primarily mycolic acid
• Acid fast bacteria are
usually members of the
genus Mycobacterium or
Nocardia
– Therefore, this stain is
important to identify
Mycobacterium or Nocardia

Dr.T.V.Rao MD 3
Ziehl-Neelsen stain
• Ziehl-Neelsen staining is used to stain species
of Mycobacterium tuberculosis that do not
stain with the standard laboratory staining
procedures like Gram staining.
• The stains used are the red colored Carbol
fuchsin that stains the bacteria and a counter
stain like Methylene blue or Malachite green.

Dr.T.V.Rao MD 4
Acid-Fast Organisms
• Primary stain binds cell wall mycolic acids
• Intense decolorization does not release
primary stain from the cell wall of AFB
• Color of AFB-based on primary stain
• Counterstain provides contrasting
background

Dr.T.V.Rao MD 5
AFB Staining Methods
• Zeihl Neelsen’s-
hot stain
• Kinyoun’s-cold
stain
• Modifications

Dr.T.V.Rao MD 6
Ziehl- Neelsen Procedure
Make a smear. Air Dry. Heat Fix.
2. Flood smear with Carbol Fuchsin stain
– Carbol Fuchsin is a lipid soluble, phenolic compound, which is able to
penetrate the cell wall
3. Cover flooded smear with filter paper
4. Steam for 10 minutes. Add more Carbol Fuchsin stain as
needed
5. Cool slide
6. Rinse with DI water
7. Flood slide with acid alcohol (leave 15 seconds). The acid
alcohol contains 3% HCl and 95% ethanol,
or you can declorase with 20% H2 S04
– The waxy cell wall then prevents the stain from being removed by the
acid alcohol (decolorizer) once it has penetrated the cell wall. The acid
alcohol decolorizer will remove the stain from all other cells.
Dr.T.V.Rao MD 7
Ziehl- Neelsen Procedure
(continued)
8. Tilt slide 45 degrees over the sink and add acid
alcohol drop wise (drop by drop) until the red color
stops streaming from the smear
9. Rinse with DI water
10. Add Loeffler’s Methylene Blue stain (counter stain).
This stain adds blue color to non-acid fast cells!!
Leave Loeffler’s Blue stain on smear for 1 minute
11. Rinse slide. Blot dry.
12. Use oil immersion objective to view.

Dr.T.V.Rao MD 8
1 2 3

4 5 6

7
Ziehl-Neelsen
stain
Dr.T.V.Rao MD 9
How the Acid fast bacteria appear

Dr.T.V.Rao MD 10

You might also like