TB Stain (Ziehl-Neelsen)

(Heating Method)

 

Intended use:

For identification of acid-fast bacteria of the genus Mycobacterium, which includes the tuberculosis-causing Mycobacterium tuberculosis.

 

Principle:

This staining kit is based on the Ziehl-Neelsen stain (a carbofulfuchsin solution) recommended by the WHO. Acid-fast bacteria such as Mycobacterium tuberculosis and Mycobacterium leprae are difficult to stain because of their lipoid capsule in the cell wall. However, once the lipoid capsule is stained with an enhanced dye such as carbolfuchsin, the newly formed compound will resist decolorization from acid-alcohol and retain the original color stained (red), which can be easily differentiated from other microorganisms that stilladsorb the counter blue stain.

 

Methods:

1. Fix smear with flame and then add Carbolfuchsin. Heat slowly and keep the stain steaming for 5 minutes.

    Avoid vaporizing to dry or boiling.

2. Wait  minutes to cool, rinse with water gently (Blot up the slide gently with bibulous paper)

3. Decolorize for 2 minutes with Acid Alcohol.

4. Rinse with water gently (if red color is still visible on the surface of specimen, add Acid Alcohol again until red

    is completely decolorized).

5. Apply Methylene blue for 30 seconds. Rinse with water gently and air dry. Examine the finished slide under a

    microscope.

 

Specifications:

 

Contents	3vialsx100ml/kit	3Btlsx250ml/kit	3x(6Btlsx500ml)/kit	Components
Carbolfuchsin	1x100ml	1x250ml	6x500ml	Carbolfuchsin, Phenol
Acid Alcohol	1x100ml	1x250ml	6x500ml	Ethanol, Hydrochloric acid
Methylene blue	1x100ml	1x250ml	6x500ml	Methylene blue

 

Precaution:

1. After each use, cap the reagent bottle immediately to avoid vaporization.

2. For sputum specimen, the smear should be properly thickened to increase detection rate. For thicker smear,

    the counterstaintime must be controlled. The microscopic examination will be affected if background is too

    dark.

3. Occasionally the intensity of red stain may be different for the same acid-fast bacterium. Note the difference

    in red stain intensity when observed.

4. For kit storage, avoid exposure to extreme high or low temperature and sunlight.

5. When acid-fast staining pathologic slides, heating stain method is recommended. For better results, the

    staining time of Carbolfuchsin should be longer than 5 minutes.

 

Expected Results:

Acid-fast Mycobacterium species will appear bright red, while other bacteria and cells will be blue.
Mycobacterium tuberculosis is a rod-shaped (straight or curved rod) Gram-positive bacterium, about 1.3 to 3.5 µm long and 0.3 to 0.5µm wide. It does not form flagella or spores. Mycobacterium can be readily stained by acid-fast stain. Under the microscope, acid-fast bacteria may appear rods in clump, or as particles when metamorphosing. At lower cell, certain special shapes, such as “V”,”Y” and ”T”, can be formed via the linkage of 2~3 cells.