To find out the Mycobacterium sp., Using this staining technique.

1. Ziehl-Neelsen staining
By
K.Sanjay
BD230907

2. Principle
• Acid fast staining is the Differential staining
technique which was first developed by Ziehl and later on
modified by Neelsen. So, this method is also called Ziehl -
Neelsen staining techniques.
• Neelsen in 1883 used Ziehl’s Carbol -fuchsin and heat
then decolorized with an acid alcohol, and counter stained
with Methylene blue.
• Thus, a Ziehl-Neelsen staining technique was
developed.

3. • Ziehl & Neelsen staining method is used for those
microorganisms which are not staining by Simple or
Gram staining method, particularly the member of
genus Mycobacterium, Nocardia and
Cryptosporidium.
• The primary stain is Carbol fuschin, the second
stain is Acid alcohol. In Acid alcohol (3 %
hydrochloric acid and 95 % ethanol) and is used as
decoloring agent. The final reagent is Methylene blue
that act as Counter stain.

4. Materials Required
• Glass slide
• Inoculation loop
• Bunsen burner
• Carbol fuschin
• Acid alcohol (3 % hydrochloric acid and 95 % ethanol)
• Methylene blue
• 72 hours culture of Mycobacterium sp.
• Microscope.

5. Procedure
Clean and dry glass slide.(Using 75%ethanol)
Prepare thin smear of given bacteria.(Inoculation loop)
Heat fixation(Direct flame)
Added few drops of Carbol fuschin.
Heat fixation upto 3 to 5 minutes(Heat act as a Mordant)

6. Washed with low flow tap water.
Added the acid alcohol(Wash it, immediately).
After, added the methylene blue for 2 minutes.
Wash and dry it, properly.
Observe the slide under microscope(100X) with oil
immersion.

7. Result
Acid fast: Bright red to intensive purple.
Non-acid fast: Blue color.

8. Acid fast bacilli(Red) and Non-acid fast bacilli(blue)