ZIEHL –NEELSEN

STAINING
.
It was first discovered by Ehrlich Paul in 1882.Later more
developed by Ranz Ziehl and Frederich Neelson
• Principle:Certain organisms,such as members of Genus
Mycobacterium and some other organisms do not take
Gram staining as their cell wall contain mycolic acid ,that
is long chain fatty acid.It is difficult to stain those
organisms but once is stained they resist decolorization
by acid and alchohol as they retain the color of primary
staining.
• Procedure:
• Prepared and fixed smear is supplied as Tuberculosis is a
highly contagious disease.
• Primary staining:A small amount of carbol fuchsin is
taken in test tube and heated till fumes appear.Then the
• Decolorization:done by 20% sulphuric acid until the smear is sufficiently decolorized(pale
pink) for 1min.Background cells,tissue fibres and other organisms stain pale pink.thwn
washed with gently tap water.
Aim: To differentiate between acid fast and non acid fast
Counter staining:The smear is covered with methylene blue for 1min.
The slide is washed by distilled water and pat dry.
Aim:to color the background for better visualization
After completion of the procedure ,it is examined under microscope.
At first,the smear is examined with 40x high power field to see the staining color and
distributionof material.
Then with 100x oil immersion to report the bacterial cells.
• Interpretetion: Mycobactrium tuberculosis:Bright red,straight or slightly curved ,singly
arranged ,rod like structure with beaded apperance against blue background.
• Mycobacterium leprae:Thick ,stout ,short in cigar bundle appearance.
• Actinomyces: filamentous.
• Comment:Acid fast bacilli:present /not present.
• NB:should not write “no AFB seen” as it can be mistaken as asingle name is NoAFB” is
an organism.
• Modified Ziehl Neelson staining:If any other concentrations than 20 % H2SO4 is
used,then it is called modified Ziehl Neelson staining..
• For example,in case of M.leprae the concentraion of sulphuric acid is 5% and in case of
Actinomyces and Nocardia it 1%.