ACID FAST STAINING - ZN STAINING PRINCIPLE , PROCEDURE,OBSERVATION
ACID FAST STAINING - ZN STAINING
INTRODUCTION :
Ziehl-Neelsen staining is also known as acid-fast stain, first introduced by Paul Ehrlich. Ziehl–Neelsen staining is a bacteriological stain used to identify acid-fast organisms, mainly mycobacteria. It is named for two German doctors who modified the stain: the bacteriologist Franz Ziehl (1859–1926) and the pathologist Friedrich Neelsen (1854–1898).
PRINCIPLE :
Acid fastness property of the intact cell depends on the trapping of intracellular mycolate-fuchsin complexes formed in the cell wall during staining. the dye fuchsin is more soluble in phenol than in water or acid alcohol. phenol in turn, is more soluble in lipids present in the cell wall of acid-fast bacteria.
During staining, fuchsin enter the cell. On decolorizer, acid-fast cells resist decolorization, since the primary stain is more soluble in cellular waxes than in decolorizing reagent. non acid-fast bacteria get easily decolorized and are counter stained with dye of different colors like methylene blue or malachite green.
REQUIREMENT :
- Carbol fuchsin stain (filtered)
- Acid alcohol 3% v/v (or 20% sulfuric acid)
- Malachite green 5 g/l (0.5% w/v) or Methylene blue, 5g/l
PROCEDURE :
- Spread the sputum evenly over the central area of the slide using a continuous rotational movement.
- air dry smear.
- Heat fix dried smear.
- Cover the smear will carbol fuchsin stain
- Heat the smear until vapor just begins to rise (i.e. about 60 degree Celsius). Do not overheat (boil or dry). Add additional stain if necessary. Allow the heated stain to remain on the slide for 5 minutes.
- Wash off the stain with clean water.
- Cover the smear with 3% v/v acid alcohol for 2-5 minutes (or 20% sulfuric acid) o
- Wash well with clean water
- Cover the stain with malachite green stain for 1-2 minutes
- Wash off stain with clean water
- air dry smear.
- Examine the smear microscopically
IN SHORT
- Smear
- Heat fix
- add carbol fuchsin
- heat until vapor begin to rise
- wash
- add acid alcohol or sulfuric acid( 2-5 min)
- wash
- counter stain ( malachite green 1-2 min )
-wash
OBSERVATION :
Acid-fast bacteria will be red, while nonacid fast bacteria will stain blue/green
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