Ziehl-Neelsen (Acid Fast) staining technique

Introduction
The cell wall of Mycobacterium sps typically contain waxy substance (mycolic acid) that makes it
impervious to staining by aqueous staining solutions. These bacteria can not be stained by simple
stains or even by Gram staining. These bacteria can however be stained by drastic measures, and
once stained can not be readily decolourized by weak mineral acids. Hence, these bacteria are
called acid fast bacilli and the staining method is called acid fast staining. It is also a type of
differential staining method.
Acid fast staining was introduced by Ehrlich in 1882. It was subsequently modified by Ziehl and
Neelsen. There are two types of acid fast staining: hot method and cold method. Ziehl-Neelsen is
a hot method of acid fast staining. The components of Ziehl-Neelsen stain include primary stain
(strong/concentrated carbol fuchsin), decolourizer (20% H2SO4) and counterstain (Loeffler's
methylene blue).
Strong carbol fuchsin solution is basic fuchsin dissolved in phenol (carbolic acid). Heating the slide
helps to soften the waxy material on the bacterial cell wall. The waxy material is hydrophobic to
aqueous solution but not to phenolic solution of basic fuchsin. Hence strong carbol fuchsin is able
to stain the cell. Upon staining, they tend to resist decolourization by 20% H2SO4 (sulphuric acid).
The background is then stained by Loeffler's methylene blue.
One of the main applications of acid fast staining is the diagnosis of pulmonary tuberculosis.
Sputum sample obtained from the patient is used to make thin and uniform smear on glass slide.
The readymade smear is provided to you.
Requirements: Smear on glass slide, staining rack, spirit lamp, Strong carbol fuchsin, 20%
H2SO4, Loeffler's methylene blue, blotting paper, immersion oil, microscope.
Procedure:
Keep the slide with the smear facing upwards on the staining rack. Flood the entire slide with strong
carbol fuchsin solution. Heat the slide with gentle waving from below using lighted spirit lamp until
fumes arise from the stain. Do not let the stain solution boil or dry out. Pour more stain if required.
Allow the fumes to disappear and repeat the process of heating the slide two more times. Allow
the slide to cool down. Pour off the stain and decolorize the smear using 20% H2SO4 for at least
one minute. Wash the slide under gentle stream of running tap water. Examine the slide for signs
of proper decolourization. The smear must be almost colourless or faintly pink. Repeat the process
of decolourization until the smear get properly decolourized. Wash the smear and place the slide
back on the rack. Clean the other side of the slide with 20% H2SO4, if it is stained as well. Cover
the smear (NOT the entire slide) with few drops of Loeffler's methylene blue and allow it to act for
a minute. Was the slide in tap water and dry it using blotting paper. Place a drop of immersion oil
on the smear and observe it under 100x objective.

Observation:
Pink coloured, slightly curved bacilli in singles or in small clumps seen against a blue background
of epithelial cells and pus cells.
Inference:
The given smear contains one of the acid fast bacilli (May be Mycobacterium tuberculosis).
 Gram Staining:
Principle, Procedure, Interpretation, Examples ram
Staining is
G the common, important, and most used differential staining techniques in microbiology, which was
introduced by Danish Bacteriologist Hans Christian Gram in 1884. This test differentiate the bacteria
into Gram Positive and Gram Negative Bacteria, which helps in the classification and differentiations
of microorganisms. Principle of Gram Staining When the bacteria is stained with primary stain Crystal
Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some
are decolorized by alcohol.

The cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called
peptidoglycan and lipid content is low. Decolorizing the cell causes this thick cell wall to dehydrate
and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell. So the
ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the thick layer of
peptidoglycan of gram positive bacteria and appears blue or purple in color.

In case of gram negative bacteria, cell wall also takes up the CVIodine complex but due to the thin
layer of peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed
off. When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows
the crystal violet-iodine complex to leach out of the cells. Then when again stained with safranin,
they take the stain and appears red in color.

Reagents Used in Gram Staining • Crystal Violet, the primary stain • Iodine, the mordant • A
decolorizer made of acetone and alcohol (95%) • Safranin, the counterstain

Procedure of Gram Staining 1. Take a clean, grease free slide. 2. Prepare the smear of suspension on
the clean slide with a loopful of sample. 3. Air dry and heat fix 4. Crystal Violet was poured and kept
for about 30 seconds to 1 minutes and rinse with water. 5. Flood the gram’s iodine for 1 minute and
wash with water. 6. Then ,wash with 95% alcohol or acetone for about 10-20 seconds and rinse with
water. 7. Add safranin for about 1 minute and wash with water. 8. Air dry, Blot dry and Observe
under Microscope.

Interpretation Gram Positive: Purple Color Gram Negative: Red Color