Skill Lab – 4
Ziehl Neelsen Staining
Introduction
The Ziehl –Neelsen staining, also known as Acid Fast staining. It was first described by
two German doctors, Franz Ziehl (1859–1926) and Friedrich Neelsen (1854–1898). It is a special
bacteriological stain used to identify acid-fast organisms, mainly Mycobacterium species. Acid
fast bacilli AFB are called so because these retain the basic Carbol Fuchsin dye, after
decolorization with acid solution. Mycobacteria are extremely difficult to stain by ordinary
methods like Gram Stain. These have thick cell wall containing several complex lipids such as
long chain fatty acids called Mycolic acids, which contribute to organism’s acid fastness.
Acid Fast organisms
Mycobacterium (M.tuberculosis, Atypical Mycobacteria - M. ulcerans, M. leprae.)
Actinomyces (A. israelii)
Bacterial endospores
Nocardia (N. asteroides)
Principle:
Mycobacteria have thick cell wall containing several complex lipids such as long chain
fatty acids called Mycolic acids. The phenolic compound Carbol Fuchsin used as primary stain is
lipid soluble and penetrates the waxy cell wall. The smear is heated to melt the wax, allow the
stain to move into the cell and enhance staining. Acid fast cells resist decolorization by acid. The
background material is decolorized and stained green or blue by counterstains Malachite
green or Methylene blue which provide a
contrast color against which the red acid-fast
bacilli (AFB) can be seen.
Reagents Used
Primary Stain: Carbol Fuchsin stain
Decolorizer: Acid alcohol 3% v/v (3-part
conc. HCl in absolute alcohol) or 20% H2SO4
(Sulphuric acid)
Counter Stain: Methylene blue; 0.5 % w/v or
Malachite green
Ziehl Neelsen Staining
Introduction
The Ziehl –Neelsen staining, also known as Acid Fast staining. It was first described by
two German doctors, Franz Ziehl (1859–1926) and Friedrich Neelsen (1854–1898). It is a special
bacteriological stain used to identify acid-fast organisms, mainly Mycobacterium species. Acid
fast bacilli AFB are called so because these retain the basic Carbol Fuchsin dye, after
decolorization with acid solution. Mycobacteria are extremely difficult to stain by ordinary
methods like Gram Stain. These have thick cell wall containing several complex lipids such as
long chain fatty acids called Mycolic acids, which contribute to organism’s acid fastness.
Acid Fast organisms
Mycobacterium (M.tuberculosis, Atypical Mycobacteria - M. ulcerans, M. leprae.)
Actinomyces (A. israelii)
Bacterial endospores
Nocardia (N. asteroides)
Principle:
Mycobacteria have thick cell wall containing several complex lipids such as long chain
fatty acids called Mycolic acids. The phenolic compound Carbol Fuchsin used as primary stain is
lipid soluble and penetrates the waxy cell wall. The smear is heated to melt the wax, allow the
stain to move into the cell and enhance staining. Acid fast cells resist decolorization by acid. The
background material is decolorized and stained green or blue by counterstains Malachite
green or Methylene blue which provide a
contrast color against which the red acid-fast
bacilli (AFB) can be seen.
Reagents Used
Primary Stain: Carbol Fuchsin stain
Decolorizer: Acid alcohol 3% v/v (3-part
conc. HCl in absolute alcohol) or 20% H2SO4
(Sulphuric acid)
Counter Stain: Methylene blue; 0.5 % w/v or
Malachite green
