Acid Fast staining
A K Gupta
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Acid Fast Stain
(Ziehl-Neelsen method)
Theory :
Many bacterial cells are easily stained with simple stains or
using the Gram stain. A few types of bacteria, such as the
mycobacteria and Nocardia species, do not stain using
these techniques because their walls are not permeable to
the dyes in common staining regimens.
• There are more than 70 species of mycobacteria
• Two are major pathogens:
1. Mycobacterium tuberculosis
2. Mycobacterium leprae
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The cell walls of the
mycobacteria contain
large amount of fatty
waxes (mycolic acid)
within their cell wall giving
the cell walls a high lipid
content that resist
staining by ordinary
methods.
This characteristic is
thought to be the reason
these bacteria are difficult
to stain. It require a
special stain Acid Fast
stain. 3
To view these cells in samples staining
requires higher concentrations of
the dye solution and/or a heating
period penetrate the lipid cell wall
reach the cytoplasm. Cytoplasm once
stained resist decolorization because
acid-alcohol can not dissolve the cell
wall and penetrate beneath it
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• The expression “acid fast” is derived from the
observation that even with the addition of
hydrochloric acid to the alcohol decolorizer,
some of the stained cells retain the primary
stain (carbolfuchsin). Cells that release the
primary stain (carbolfuchsin) with decolorizing
will be visible after the counterstaining step is
complete. Bacteria described as acid fast will
appear red when examining specimens using
bright-field microscopy. Non-acid-fast cells
and field debris will appear blue.
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History:
In 1882 Robert Koch reported the discovery of the tubercle
bacillus and described the appearance of the bacilli resulting
from a complex staining procedure. During the same time
period other researchers, intending to improve on Koch’s
method, introduced modifications to the reagents and the
procedure. Franz Ziehl and Neelsen used the primary stain
basic fuchsin. This method became known as the Ziehl-
Neelsen(Z-N staining) method in 1890s. In this method heat
is used to help drive the primary stain into the waxy cell
walls of the cells. The use of heat in this method has been
the reason that this technique is called the “hot staining”
method.
The Ziehl-Neelsen method has endured as a reliable and
effective way to demonstrate the acid-fast bacteria.
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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 backgroun
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Smear Preparation
1. Clean a glass slide (be sure to remove any dust or
crushed glass debris) .
2. Prepare the sample.
3. Using a microbiological loop, apply a small sample of
the specimen to the slide by slowly spreading the liquid to
make a thin film; If you are using solid matter from a
colony, be sure to choose a very minute sample and spread
it into a very thin film. Applying the cells before adding
water (or other mixing fluid) will help the cells adhere to
the slide. The size of the film should be about 1 cm in
diameter. Avoid any actions that would splatter droplets of
the sample in the surrounding area.
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Acid-Fast Procedure
1.Prepare smears of organisms to be stained.
2.Allow the smear to dry completely.
3.Heat fix the smears.
3. Cut or tear absorbent paper (bibulous paper) to fit
the slide leaving one end for handling. Do not allow
the paper to protrude beyond the slide, but the
smears must be covered.
4. Place the slide on the stand.
5. Saturate the paper with carbolfuschin.
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• 6. Heat the slides with a hand-held bunsen
burner until steam can be seen rising from the
surface. Alternately remove the burner and
reheat the slide to maintain steaming for 3-5
minutes. As the paper begins to dry during
the staining process add a drop or two of
carbolfuschin to keep the slide moist. Adding
too much stain will cool the slide (and drip on
the bench). Overheating the slide or letting it
dry will distort the cells. Under heating the
slide will fail to stain acid-fast cells.
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7. At the end of staining remove the paper and
wash the slide thoroughly.
8. Drain the slide.
9. Decolorize with acid-alcohol for 15-30 seconds.
( 1% HCl in 7o % alcohol or 20 % sulphuric acid)
10. Rinse, drain, and counterstain with 2%
methylene blue for 30 sec to 1minute.
11. Rinse, blot, dry and examine under oil
immersion microscope.
12. Acid-fast organisms will appear red and non-
acid-fast organisms will be blue.
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Precautions
• During steaming the smear, the stain should
not be allowed to evaporate. If needed, some
additional stain should be added.
• Prior to decolourization, the smear should be
cooled, which allows the waxy cell substances
to harden.
• During decolourization, the reagent should be
added drop by drop until carbol fuchsin fails
to wash from smear.
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An example of an acid-fast stain Mycobacterium tuberculosis
An example of an fluorescence stain
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• Thank You
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