Ziehl-Neelsen/
Acid Fast Staining
by
SAIFULLAH
LECTURER
Department of MLT Pathology
Northwest Institute of Health Science
Ziehl-Neelsen
Introduction
The Ziehl–Neelsen stain, also known as the acid-fast stain,
was first described by two German doctors: the bacteriologist Franz
Ziehl 1859–1926) and the pathologist Friedrich Neelsen (1854–1898).
It is a special bacteriological stain used to identify acid-fast organisms,
mainly Mycobacterium.
Cont……
Mycobacterium tuberculosis is the most important of this group because it
is responsible for tuberculosis (TB).
Acid-fast organisms like Mycobacterium contain large amounts of lipid
substances (60%) within their cell walls called mycolic acids.
Cont……
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Step:1
Prepared smear slide, When dried, fix the slides using the blue flame of a
Bunsen burner. With forceps, pass the slide briefly through the flame 3
times, smear side up.
Heat fixation ensures that the sputum will stick to the glass slide.
Excessive heating could damage the bacilli.
Standard reagents
o Carbol Fuchsin _ Primiary Stain
o 20 % Sulfuric Acid/3% HCL_ Decoloriser
o 95% Acid/Alcohol _ Decoloriser
o Methylene Blue /Malachite Green_Counterstain
Note:
5% Sulfuric Acid for _ [Link].
1% Sulfuric Acid for _Nocardia Species.
Cont…….
Carbol fuchsin is used as a dye to detect acid fast bacteria because it is
more soluble in the cells wall lipids than in the acid alcohol. It is primary
stain.
Acid Alcohol is used in histology and cytology to remove excess stain. It
is also used as decolorizing agent.
Methylene blue is then applied to counterstain any cells which have been
decolorized. It is a special bacteriological stain used to identify acid-
fast organisms, mainly Mycobacteria.
Cont……
Step: 2
flood the entire slide with carbol fuchsin.
Step : 3
Using a Bunsen burner, heat the slide slowly until they are streamlining.
Step:4
Rinse the slide with water.
Gently wash the stain from each slide with a stream of cold
water until all free stain has washed away. Always rinse gently
so the smear is not washed off of the slide.
Cont…..
Step: 5
Cover each slide with decolorizing solution such as acid alcohol.
Leave this on the slides for 3 minutes. If under-decolorized, sputum
contents other than the TB bacilli may remain stained. This could lead to a
false positive result.
Cont…..
Rinse slides again carefully with water and till each slide to
remove excess water.
If the slide is still pink, an additional amount of decolorizing
solution can be reapplied for 1 to 3 minutes.
Cont….
Step:6
Next, counter-stain with methylene blue for l minute
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Step: 7
Rinse slides again carefully with water and tilt each slide to remove
excess water.
Finally, tilt each slide and place in a slide block to air dry.
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Cont…..
Examine the smear microscopically, using the 100x oil
immersion objective.
Cont……
• Negative Report: “Negative for
• acid-fast bacilli where no organisms have been observed in 100
fields
.
• Positive Report:. Positive for acid-fast bacilli : Provide AFB
quantitation.
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