Veterinary Epidemiology and Zoonosis

EXERCISE-13: DIAGNOSIS OF TUBERCULOSIS

Acid fast staining or Ziehl-Neelsen staining

• The high mycolic acid content of certain bacterial cell walls, like those of Mycobacteria , is responsible for the staining pattern of poor absorption followed by high retention.
• The most common staining technique used to identify the mycobacterium bacilli is the Ziehl-Neelsen staining method, in which the acid fast bacilli are stained bright red and stand out clearly against a blue background.
• Examples: All Mycobacteria - M.tuberculosis, M.leprae, M.smegmatis and Nocardia.

Materials required

Materials required	Quantity and purpose
Microscopic slid	2 numbers
Spirit lamp	One
Ziehl-Neelsen staining kit	To stain the smear
Light microscope	For examination of stained slides
Imersion oil	For examination at 100X

Procedure

• Prepare a smear on a clean glass slide and air dry.
• Fix the smear with gentle heat (should not be over heated).
• Flood the smear with Carbol Fuchsin stain solution (primary stain) and heat under the slide with spirit lamp to make the Carbol Fuchsin stain to pierce the cell wall mycolic acid components (heat to steaming for 5 minutes with a low flame, do not boil the stain and do not permit drying of the smear).
• Allow the slide to stand in hot solution for 5 minutes.
• Wash with running tap water.
• Decolourize with acid-fast decolourizer for 2 minutes or until no more stain comes off in the washing (if washinf is not thorough, may get false positive result).
• Wash with running tap water.
• Flood with methylene blue stain (counter stain) for 30 seconds.
• Wash with running tap water and air dry/blot to dry.
• Examine under oil immersion.

Interpretation of result

• Acid fast positive bacilli stain brilliant red colour against blue background.