INTRODUCTION TO ACID FAST STAINING
Acid fast staining technique was developed in 1882 by Paul Ehrlich and modified by Ziehl & Nielsen in 1890 hence called Z.N. Staining or Ziehl-Neelsen staining.
Certain bacteria and Actinomycetes have many components in the cell wall and their cell wall has little permeability. Majority of bacteria can be stained with simple stain but the number of Mycobacteria and Nocardia can only be stained by the special technique known as Acid-fast staining technique.
It was first used for Mycobacterium tuberculosis. Also, this staining technique can be used to stain all the species of Mycobacterium.
PRINCIPLE / MECHANISM OF ACID FAST STAINING
In the cell wall of Acid-fast Bacteria, a lipid called Mycolic acid is present that is a waxy substance. Mycolic acid resists the staining of the bacterial cell by simple stains or using simple techniques but when Carbol fuchsin & heat is applied, the Mycolic acid layer gets weaken and becomes porous, due to which the carbol fuchsin enters the cell wall.
During decolorization, a special decolorizer containing sulfuric acid or Hydrochloric acid is used. Bacterial cells that get decolorized and appear colorless were said to be Non Acid- fast bacteria and those which do not get decolorized even with this powerful decolorizer were sad to be Acid-Fast Bacteria.
After decolorization, the Acid fast bacteria appear Red in color whereas the Non-acid fast bacteria appear as colorless bodies. To distinguish them easily a counterstain i.e. Methylene blue is applied which stains the Non-acid fast bacteria and they appear as blue colored bodies under the microscope.
REQUIREMENTS FOR ACID FAST STAINING
- Bacterial culture/ Sputum sample
- Glass Slide
- Wash bottle
- Distilled water
- Acid-Fast Decolorizer
- Inoculating loop
- Spirit lamp / Bunsen burner
- Carbol Fuchsin
- Methylene blue
PROCEDURE OF ACID FAST STAINING
1.) Prepare a thin smear of the specimen on a clean grease free glass slide.
2.) Fix the smear onto the slide by passing over the flame of Spirit lamp or Bunsen burner.
3.) Gently flood the smear with Carbol Fuchsin dye for 5 minutes.
4.) Gently heats the slide till fumes appear & keep the smear moisten with dye.
5.) Wash the smear with Distilled water using wash bottle.
6.) Decolorize the smear using Acid-fast decolorizer.
7.) Wash the smear using wash bottle.
8.) Cover the smear with Methylene blue dye for 1 minute and then wash with distilled water using wash bottle.
9.) Air dry the smear and observe under the microscope at 100X.
INTERPRETATION OF ACID FAST STAINING
⇒ Acid-fast Bacteria appear Red in color.
⇒ Non- Acid fast Bacteria appear blue in color.