Acid Fast Staining

Ziehl Neelsen Staining is used to stain acid fast bacteria esp mycobacteria. The name
is derived from bacteriologist Franz Ziehl (1859–1926) and the pathologist
Friedrich Neelsen (1854–1898).

Soon after Koch’s discovery, Paul Ehrlich developed a stain for Mycobacterium
tuberculosis, called the alum hematoxylin stain. Franz Ziehl then altered Ehrlich’s
staining technique by using carbolic acid as the mordant. Friedrich Neelsen kept
Ziehl’s choice of mordant but changed the primary stain to carbol fuchsin.

Ziehl and Neelsen’s modifications together have developed the Ziehl-Neelsen stain.

Acid-fast mycobacteria contain mycolic acid in their outer membrane, making the cells
waxy and resistant to staining with aqueous based stains such as the Gram stain.

Acid Fast stain is a differential stain that helps to distinguish between acid fast and
non-acid fast cells. The primary stain, carbolfuchsin, is applied to the cells and phenol
is used to allow the stain to penetrate into the waxy surface of acid-fast
microorganisms. The excess stain is removed with treatment by 1% sulfuric acid.

A secondary stain, methylene blue, is then applied to the cells.

Some Acid-Fast Organisms

Bacterial origin

Mycobacterium tuberculosi
s
Mycobacterium leparae
Mycobacterium smegmatis
Mycobacterium fortuitum
Nocardia asteroides
Nocardia brasilnensis
Nocardia caviae

Fungal origin

Fungal spores
Reagents :

Primary Stain : Ziehl Neelsen Stain (is a Carbol Fuchsin based stain) (500 ml)

Preparation:

Weigh 5 gm of basic Fuchsin in approx. 150 ml warm water to dissolve the powder.
Separately, melt 100 % Phenol in warm water.
Add 30 ml of the above melted Phenol and 50 ml Methanol to the dissolved Carbol
Fuchsin.
Make up the volume to 500 ml with distilled water.
Filter the stain and store.
Refilter at the time of staining.

Counterstain : Methylene Blue (0.1%, 500 ml) or Brilliant Green

Weigh 0.5 gm Methylene blue and dissolve in 500 ml distilled water.

Acid Decolorizer: Decolorizing Agent (100 ml)

In 80 ml distilled water, add 20 ml Sulfuric acid.

(Acid Alcohol decolorizer : hydrochloric acid and ethanol can also be used)
Acid Fast Staining - Ziehl Neelsen

Protocol

1. Air dry the smear of Mycobacterium smegmatis on a glass slide to fix the smear.
2. Flood the slide with Carbol fuchsin and allow to stain for 10-15 min with intermittent
heating in between (till you see white fumes).
3. Wash the slide in gentle running water or with a spray bottle.
4. Add Decolorizer and incubate for 5 min to remove the fuchsin stain. Optional: Repeat
decolorizer treatment for 2 min in case purple stain is still visible.
5. Wash the slide in gentle running water or with a spray bottle.
6. Flood the slide with Methylene Blue counterstain for 30 sec.
7. Wash the slide in gentle running water or with a spray bottle.
8. Air dry and observe under the microscope.
Variations of ZN Staining

The Kinyoun method or Kinyoun stain (cold method), developed by Joseph

J. Kinyoun, is a procedure used to stain acid-fast species of the bacterial genera
Mycobacterium and Nocardia and the apicomplexan genus Cryptosporidium.
It is a variation of a method developed by Robert Koch in 1882.

Unlike the Ziehl-Neelsen stain (Z-N stain), the Kinyoun method of staining does not
require heating.

In the Ziehl-Neelsen stain, heat acts as a physical mordant while phenol (carbol of
carbol fuschin) acts as the chemical mordant. Since the Kinyoun stain is a cold method
(no heat applied), the concentration of carbol fuschin used is increased.

Expected
Result
• Mycobacterium tuberculosis ATCC 25177 (H37Ra) Dark pink to red
bacilli

• Escherichia coli ATCC 25922 Blue or green

1. Acid-fast mycobacteria will appear as dark pink to red bacilli against a blue
(methylene blue) or green (brilliant green) background when examined
microscopically.

2. Mycobacteria are typically slender, 1 to 10-µm long rods that may appear curved or
bent.

3. Individual bacilli may display heavily stained areas and area of alternating stain,
producing a beaded appearance.

4. Some nontuberculous mycobacteria may appear pleomorphic, appearing as long

filaments or coccoid forms, with uniform staining.

5. Mycobacterium kansaii are often recognized by their large size and cross-banding
appearance.

6. When a carbol fuchsin smear is read a minimum of 300 fields should be examined
before the smear is reported as negative.

7. To verify the staining procedure and staining intensity of the acid-fast organisms it is
recommended that a positive and negative control slide be included with each run of
stains.

8. Rapidly growing mycobacteria may also stain thus AFB stain cannot distinguish
Precautions :