Acid-Fast Stain(Ziehl-Neelsen staining) Technique: Principle,Procedure, and result
Introduction:
Acid Fast staining is an important differential
staining procedure or technique. It is most commonly used to identify Mycobacterium spp. techniques which
was first developed by Ziehl and later on modified by Neelsen. So this method
is also called Ziehl-Neelsen staining techniques. Neelsen in 1883 used Ziehl’s carbol-fuchsin
and heat then decolorized with an acid alcohol, and counter stained with
methylene blue. Thus Ziehl-Neelsen staining techniques was developed.
Principle:
Majority of the bacteria are
stained with simple stain and Gram-stain but certain bacteria do not do so
because they have waxy components of the cell wall, hence their cell wall has
limited permeability. Such bacteria belong to genera
like Mycobacterium and Nocardia; and are stained by acid-fast stain; the latter
is used to identify Mycobacterium tuberculosis and M. leprae, the pathogens
responsible for tuberculosis and laprosy, respectively.
Acid-fast bacteria may be defined as those cells which keep the
color of the primary dye (carbol fuchsin) even after the process of
decolorization by the acid-alcohol solution.Acid-fast
bacteria are coated with a thick waxy material, mycolic acid,
which makes the bacterial cells highly resistant to the penetration of dyes.
The penetration of dye is promoted by using heat as mordant. The heat invades
the dye through the waxy coat and into the cytoplasm.
Requirements:
ØPrimary stain : 0.3% Carbol Fuchsin
– Dissolve 50g phenol in 100ml ethanol (90%) or methanol(95%). Dissolve 3g
basic fuchsin in the mixture and add distilled water to bring the volume to 1
L
Ø Decolorization solution : 25% Sulphuric acid
Ø Counter
stain : 0.3% methylene blue or malachite green
- Make a thin smear of the material for study and heat fix by passing the slide 3-4 times through the flame of a Bunsen burner or use a slide warmer at 65-75 C. Do not overheat.
- Place the slide on staining rack and pour carbol fuschin over smear and heat gently underside of the slide by passing a flame under the rack until fumes appear (without boiling!). Do not overheat and allow it to stand for 5 minutes.
- Rinse smears with water until no color appears in the effluent.
- Pour 20% sulphuric acid, wait for one minute and keep on repeating this step until the slide appears light pink in color (15-20 sec).
- Wash well with clean water.
- Cover the smear with methylene blue or malachite green stain for 1–2 minutes.
- Wash off the stain with clean water.
- Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not blot dry).
- Examine the smear microscopically, using the 100x oil immersion objective.
Result and Interpretation:
- Acid Fast Bacilli : Red, straight or slightly curved rods, occurring singly or in small groups, may appear beaded
- Cells :
Green (malachite green) or Blue (methylene blue)
- Background material : Green (malachite green) or Blue (methylene blue).
Reference
1. heory and Practice of
Histological Techniques, John D Bancroft, 6th ed, p314
2. Dorner, W. 1926. Un procédé
simple pour la colouration des spores. Le Lait 6:8-12.
3. Schaeffer AB, Fulton M
(1933). "A simplified method of staining
endospores". Science. 77: 194.
4. Endospore Stain Protocol from American Society for Microbiology website Archived 2012-06-01 at the Wayback Machine
Tags
Microbiology