Acid fast staining method is one of the differential staining techniques. This technique was first developed by Ziehl and later on, modified by Neelsen, hence this method is also known as Ziehl-Neelsen staining technique. In the year 1883, Neelsen used Ziehl’s carbol fuchsin and heated then decolourized with an acid alcohol followed by counter-stained with methylene blue. Thus Ziehl-Neelsen staining technique was developed as acid fast staining (Table).
Objectives:
• To differentiate bacteria into acid fast groups and
non-acid-fast groups.
• It is used for those microorganisms which are not stained
by simple or Gram staining methods, particularly the member of genus
Mycobacterium which are resistant to other stains.
Principle:
The smear is prepared and stained with carbol fuchsin which
solubilizes the lipoidal material present in the Mycobacterial cell wall and by
heating, carbol fuchsin further penetrating through the lipoidal wall and entering
into the cytoplasm, resulting in all cells appearing red. Then the smear is
decolourized with 3% HCl in 95% alcohol (decolourizing agent) but the acid fast
cells are resistant due to the presence of a large amount of lipoidal material
in their cell wall that prevents the penetration of decolourizing solution.
Then the smear is stained with a counterstain, methylene blue and only
decolourized cells are absorbed and appear blue while acid-fast cells retain
the red colour (Table)
Table: Cell colours with various dyes
|
Application
of |
Reagent |
Cell
colour |
|
|
Acid-fast |
Non-acid
fast |
||
|
Primary dye |
Carbol
fuchsin |
Red |
Red |
|
Decolourizer |
Acid alcohol |
Red |
Colourless |
|
Counterstain |
Methylene
blue |
Red |
Blue |
Acid Fast Staining Procedure:
1. Prepare bacterial smear on a clean and grease-free slide,
using a sterile technique.
2. Allow the smear to air dry and then heat fix.
3. Cover the smear with carbol fuchsin stain.
4. Heat the stain until the vapour just begins to rise about
60°C. Allow the heated stain to remain on the slide for 5 minutes.
5. Wash off the stain with clean water.
6. Cover the smear with 3% v/v acid alcohol for 5 minutes or
until the smear is sufficiently decolourized, i.e. pale pink.
7. Wash well with clean water.
8. Cover the smear with a malachite green stain for 1–2
minutes, using the longer time when the smear is thin.
9. Wash off the stain with clean water.
10. Wipe the back of the slide clean, and place it in a
draining rack for the smear to air dry.
11. Examine the smear microscopically, using the 100 X oil
immersion objective.
Acid-fast: Mycobacterium tuberculosis, Mycobacterium
smegmatis.
Non-Mycobacterial bacteria: Nocardia; Coccidian
Parasites: Cryptosporidium.
Fig: A. Non acid fast: Blue colour; B. Bright red to
intensive purple, Red, straight or slightly curved rods