Acid Fast Stain (Ziehl-Neelsen)

Acid Fast Stain (Ziehl-Neelsen)
Acid Fast Stain (Ziehl-Neelsen)


Acid Fast Stain (Ziehl-Neelsen)

  • Product code: ACMBS-04
  • Specifications:  4x100ml, 4x250ml (Box)
  • Intended use: Intended for staining of acid-fast stain of Mycobacteria and other bacteria
  • Store temperature: 5℃~30℃
  • Expiry date: 24 Months

More Details

PrincipleMain componentsSample requirementTest procedureResult interpretationMethod limitationPrecautions


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 still adsorb the counter blue stain.

Main components

Reagent compositionMain ingredient
1. Carbolfuchsin solutionCarbolfuchsin, Phenol
2. Acid alcohol solutionEthanol, Hydrochloric acid
3. Methylene blue solutionMethylene blue

Sample requirement

  • Pick the sputum specimens of cheese-like, pus-like or suspicious part of about 0.05ml, evenly coated on the front of the slide into a 10mmx20mm oval sputum, natural dry. To Stain after heating fixed.

Test procedure

  • 1. The smear after natural drying, placed on the stained rack, slide spacing to maintain a distance of 10mm or more, heat fixed (5 seconds the flame back and forth flame 4 times).
  • 2. Add carbolfuchsin solution covered with slides, heated to steam, stop heating, to maintain staining for 5 minutes. Staining should always be kept sputum membrane is covered with staining solution, if necessary, can add more staining solution. Do not make the dyeing liquid boil. High altitude areas should be appropriate to increase the number of heating and dyeing time.
  • 3. Water from the slide gently washing one end, washed dye, drain.
  • 4. Add acid alcohol solution covered with slides, decolorization 1 to 2 minutes, if necessary, need to wash away the acidic alcohol solution, once again bleaching to the sputum membrane no visible red.
  • 5. Running water from the slide gently rinse 10 to 20 seconds, rinse acid alcohol solution, drain.
  • 6. Dropping methylene blue solution dyed for 30 to 60 seconds.
  • 7. Running water from the slide gently washing one end, washed dye, drain, microscopic examination.

Result interpretation

  • In the light blue background, acid-fast bacteria (such as: Mycobacterium tuberculosis) was red, the other bacteria and cells were blue.Mycobacterium tuberculosis most of the rod-shaped, slightly curved, cell width (0.3~0.6) μm, cell length varies between (0.5~8) μm, most in the (1.5~3.5) μm, oder people can be spiral, well-dyed Mycobacterium tuberculosis, can be found in darker colored particles. Fresh specimens containing more Mycobacterium tuberculosis by acid staining, 100×oil lens observation, both to find a single bacteria, but also to see clusters clustered or branched arrangement of bacteria.

Method limitation

  • For use only with acid-fast bacteria.


  • 1. Do not make the dye dry on the slide when staining.
  • 2. After smearing the sputum membrane can not be too thick or too thin.
  • 3. Due to cedarwood oil can dissolve the complex red dye, so that the dyed material has faded, and easy to dry condensation, causing damage to the oil lens, it is forbidden to use cedar oil, you must use a microscope dedicated mirror oil (immersion oil).
  • 4. In the same acid-fast bacilli, sometimes there will be red shades of the situation, observation should be noted.
  • 5. For the preservation of the observed smear, filter paper or absorbent paper blot oil mirror oil, cotton swab and then gently wipe the alcohol oil above the sputum film, wipe the intensity to be small, wipe off Sputum membrane.
  • 6. Winter temperature is too low, the dyeing time should be extended.
  • 7. After each reagent runs out, please quickly cover, so as to avoid volatilization.
  • 8. Reagent storage, try to avoid high and low temperature environment and direct sunlight.
  • 9. This product should be used by professionals and interpretation of the results.
  • 10. Before use should read the instructions for use, in the period of use, and personal hygiene protection.
  • 11. After use should be hospital or environmental protection department requires the disposal of waste.
  • 12. The production date, production lot number and expiry date, see the packaging.

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