ACID FAST STAINING
AIM:
To
perform Acid Fast staining for the given bacterial culture
PRINCIPLE:
When the smear is stained with
carbol fuchsin, it solubilizes the lipoidal material present in the
Mycobacterial cell wall but by the application of heat, carbol fuchsin further
penetrates through lipoidal wall and enters into cytoplasm. Then after all cell
appears red. Then the smear is decolorized with decolorizing agent (3% HCL in
95% alcohol) but the acid fast cells are resistant due to the presence of large
amount of lipoidal material in their cell wall which prevents the penetration
of decolorizing solution. The non-acid fast organism lack the lipoidal material
in their cell wall due to which they are easily decolorized, leaving the cells
colorless. Then the smear is stained with counterstain, methylene blue. Only
decolorized cells absorb the counter stain and take its color and appears blue
while acid-fast cells retain the red color.
MATERIALS REQUIRED:
Bacterial culture, Glass slides,
Inoculation loop, Bunsen burner, Microscope
PROCEDURE: Smear
Preparation
- Add one loopful of sterile
water to a microscope slide.
- Make a heavy smear of given
bacterial culture. Mix thoroughly with your loop.
3. Air dry and heat fix.
4. Cover the smear with carbolfuchsin dye. Place
a piece of paper towel on top of the dye. Be sure the paper towel is saturated
with the dye.
5. Dry heat for 2
minutes.
6 Cool and
rinse with water. Decolorize with acid-alcohol
for 15-20 seconds.
7. Wash the
top and bottom of slide with water and clean the slide bottom well.
8. Counterstain
with Methylene Blue for 30 seconds to 1 minute.
9.
Wash and blot the slide with bibulous paper. Focus 10X - then use oil
immersion.
RESULT: Acid fast: Bright red to intensive
purple, Red, straight or slightly curved rods, occurring singly or in
small groups, may appear beaded Non-acid fast: Blue color.
Mycobacterium tuberculosis visualization using the Ziehl–Neelsen stain.
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