ACID-FAST STAINING

Acid-Fast Staining

Content:

About:

It was developed by Paul Ehrlich in 1882.
Acid fast staining is a kind of differential staining.
This method is used for the identification of mycobacterium and other bacteria which retain carbol fuchsin (primary stain) after the treatment of strong acid and methylene blue.

Principle:

Mycobacteraium contain a waxy substance composed of mycolic acid in its cell wall.
These mycolic acid are carboxylic acid with up to 90 carbon atoms chain.
Mycolic acid in addition with other lipids serves as barrier and prevents the entry of dye inside the bacterial cell.
The dye used in this method is carbol fuchsin (lipophilic dye) which binds with the acid and lipid in the cell wall and gives red colour.
Binding property of the dye is related to the carbon chain length of the mycolic acid.

Requirements and reagents:

Steps:

On a clean slide prepare a smear of Mycobacterium smegmatis and Staphylococcus aureus on another slide.
Air dry and heat fix.
Pour some drops of carbol fuchsin on both the smears.
Place the slides in steam for 3-5 min, to avoid smear from drying add more stain time to time.
Cool the slide for some time and wash with distilled water.
Pour acid alcohol for 20-30 second to decolorize the smear or until the smear gives pink colour.
Wash slide with distilled water.
Add few drops of counter stain i.e. methylene blue to the smears for 1-2 minutes.
Wash and blot dry with a blotting paper.

Observation:

Results:

M. smegmatis cells appear red coloured indicate acid fast reaction and S. aureus appear blue colour and show non-acid reaction.

Example:

Types	Example
Acid-fast	Mycobacterium smegmatis, Mycobacterium tuberculosis
Non-Mycobacterial bacteria	Nocardia