ACID FAST STAINING

 

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

1. Add one loopful of sterile water to a microscope slide.
2. 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.