- Observation and result
- 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.
- 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:
- Bacterial culture (fresh)
- Carbol fuchsin
- Acid alcohol
- Methylene blue
- Water bath
- Glass slide
- Inoculating loop
- Blotting paper
- 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.
- Observe under the microscope and record the colour test
- Classify the bacteria i.e. acid-fast and non-acid fast.
- Also describe their morphology and arrangement of cells.
- M. smegmatis cells appear red coloured indicate acid fast reaction and S. aureus appear blue colour and show non-acid reaction.
|Acid-fast||Mycobacterium smegmatis, Mycobacterium tuberculosis|