Leprosy Bacillus Stain

Assay-kits

Product code: ACMBS-08
Specifications: 4x100mL, 4x250mL (Box)
Intended use: Mainly used for Mycobacterium staining (e.g. Leprosy bacillus) etc. acid-fast stain
Store temperature: 5℃～30℃
Expiry date: 24 Months

More Details

PrincipleMain componentsSample requirementTest procedureResult interpretationMethod limitationPrecautions

Principle

Mycobacterium 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. Because the acid resistance of leprosy bacilli is relatively weak, it cannot decolorization with traditional 3% hydrochloric alcohol solution. It can only decolorization with low concentration of hydrochloric acid or dilute sulfuric acid, otherwise the red will easily be removed. This product uses low concentration of hydrochloric acid as a decolorization solution.

Main components

Reagent composition	Main ingredient
1. Carbolfuchsin solution	Basic fuchsin, Phenol
2. Hydrochloric acid alcohol solution	Ethanol, Hydrochloric acid
3. Methylene blue solution	Methylene blue

Sample requirement

Take the nasal mucous swab or skin lesion scraping smear, and the diameter of the membrane was 5～7mm, the thickness was uniform and containing blood was not qualified. If the specimen is taken with blood, red blood cells should be removed. The commonly used method is to add 1% hydrochloric acid or 5% glacial acetic acid solution before fixing the smear, and then gently rinse it with water after 1～2 minutes and then stain it. If the smear contains less blood, it can be treated with hemolysis.

Test procedure

1. After natural drying, the smear is placed on the staining rack and the distance between the slides is more than 10mm. Heating and fixing (the slide is heated by flame for 4 times in 5 seconds).
2. Fill the slide with the carbolfuchsin solution and heat it until steam appears. Stop heating and keep staining for 10 minutes. (Do not increase the temperature too high to avoid boiling or evaporating to the dry). Or at room temperature, it does not need to be heated, staining 30 minutes by adding carbolfuchsin solution. (The conditions and results of staining without heating are more stable and worthy of promotion).
3. The running water slowly washed away the stain solution and drained off the remaining water on the specimen.
4. Add acid alcohol solution and covered slide, decolorizing 1～2 minutes. (Shake the slide from time to time until there is no red shedding).
5. Rinse slowly for 10～20 seconds with running water, and drain off the remaining water on the slide. If there is still red in the specimen, then decolorizing again until the specimen is colorless or pale pink.
6. Add methylene blue solution, staining for 30～60 seconds.
7. Rinse with running water gently and air dry. Examine the finished slide under a microscope.

Result interpretation

In light blue background, acid-fast bacteria such as leprosy are red, and other bacteria and cells are blue. The length of the Leprosy bacilli was 1～8 um, and the wide between 0.2～0.5 um, slightly curved elongated bacilli, morphologically like tuberculous bacilli, but relatively thick and short; No flagellum and no spores. Arranged in parallel or bundled or stacked into a cluster. Sometimes it is called "leprosy cells" in the foamy cells of the lesion.

Method limitation

Only for the staining of acid-fast bacteria.

Precautions

1. Do not let the staining solution on the slides dry out during staining.
2. The specimen after smearing, should not be too thick and the specimen is too thick to be difficult decolorizing. It is necessary to prolong the decolorization time and easily cause the background to be too deep to affect the microscopic examination.
3. Due to cedarwood oil can dissolve fuchsin dye, discoloring the dyed material; And it is easy to dry and coagulate, causing damage to the oil lens, therefore, the prohibition of the use of cedarwood oil, must use special immersion oil.
4. If you want to save the after observation smear, use the filter paper or absorbent paper to dry the immersion oil, then use the swab to dip the wet alcohol, gently wipe off the immersion oil on the top of the specimen, wipe the intensity less, and guard against the specimen falling off.
5. When the room temperature is too low in winter, the staining time should be lengthened properly.
6. Each reagent after use, please quickly cover, to avoid evaporation.
7. For kit storage, avoid exposure to extreme high or low temperature and sunlight.
8. This kit must be used by professionals and the interpretation of results.
9. Should carefully read the instruction manual before using. Can only be used before expiration date, and good personal hygiene protection.
10. After use, disposal of waste should comply with the hospital or the environmental protection department requirements.
11. The production date, production batch number and expiration date on the package.

References

1. Li Wenzhong. Modern leprosy [M] Shanghai science and Technology Press, 2006.

2. People's Republic of China national health and Family Planning Commission of medical affairs authority. The clinical laboratory operating procedures M. Fourth edition. Beijing: People's Medical Publishing House, 2014.

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