TB Stain (for pathologic Acid-Fast Stain)

 

Intended use:

This kit is based on the Ziehl-Neelsen method recommended by the WHO and it is a chemistry stain intended for the detection of acid-fast bacteria such as Mycobacterium tuberculosis.

 

Principle:

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 still adsorb the counter blue stain. Phenol is mordant and is able to improve the staining ability.

 

Methods:

1. Deparaffin tissue, add Carbolfuchsin, heat with small flame and keep the stain steaming for 5~10 minutes. Avoid vaporizing to dry or boiling.

2. After cooling, rinse with water gently. Blot up the slide gently with bibulous paper.

3. Decolorize for 1~2 minutes with Acid Alcohol.

4. Rinse with water gently (if red is still visible on the surface of specimen, add Acid Alcohol again until red is completely decolorized. Then continue rinsing with water).

5. Apply Methylene Blue for 20~30 seconds. Rinse with water gently.

6. Differentiate in 95% ethanol for 5~10 seconds.

7. Dehydrate in absolute ethanol and clear with xylene. Mount with mounting media.

8. Alternatively, the dehydration step shown above can be replaced by air drying.

9. Examine the finished slide under an oil immersion lens.

 

Specifications:

 

Contents	4vialsx20ml/kit	Components
Carbolfuchsin	1x20ml	Carbolfuchsin, Phenol
Acid Alcohol	2x20ml	Ethanol, Hydrochloric acid
Methylene Blue	1x20ml	Methylene Blue

 

 

Precaution:

1. When deparaffin tissue, xylene can be substituted by a mixture of gasoline and turpentine (1:1). Subsequently, without ethanol, rinse with running water for an extended time and stain. This xylene-free procedure reduces the destruction of bacteria capsules, increasing the detection rate of acid-fast bacteria. 

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

3. For sputum specimen, the smear should be properly thickened to increase detection rate. For thicker smear, the counterstain time must be controlled. The microscopic examination will be affected if background is too dark.

4. Occasionally the intensity of red stain may be different for the same acid-fast bacterium. Note the difference in red stain intensity when observed.

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

 

Expected Results:

The acid-fast bacteria (Mycobacterium tuberculosis) appear red; other bacteria and appear 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 0.5 µm 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 density, certain special shapes, such as “V”, ”Y” and ”T”, can be formed via the linkage of 2~3 cells.