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The Kinyoun stain is a fundamental technique in microbiology, used to identify acid-fast bacteria like Mycobacterium tuberculosis and Mycobacterium leprae. This staining method is based on the Ziehl-Neelsen stain recommended by the WHO.
Principles of Kinyoun Stain
The Kinyoun stain’s principle lies in its use of carbolfuchsin solution, which contains carbolfuchsin and phenol. Acid-fast bacteria possess a unique cell wall with a lipoid capsule, making them challenging to stain. Carbolfuchsin stains this capsule, rendering it resistant to decolorization by acid-alcohol. Consequently, acid-fast bacteria retain their red color, distinguishing them from other microorganisms that absorb the counter-blue stain.
Main Components of the Staining Kit
- Carbolfuchsin Solution: This contains carbolfuchsin and phenol, essential for staining acid-fast bacteria.
- Acid Alcohol Solution: Comprising ethanol and hydrochloric acid, this solution is crucial for decolorization.
- Methylene Blue Solution: With methylene blue as its core component, this solution is used for the final staining step.
To prepare a sample for Kinyoun staining, select sputum specimens with cheese-like, pus-like, or suspicious characteristics. Apply approximately 0.05ml of the specimen evenly onto a 10mmx20mm oval sputum on a glass slide. Air dry the specimen, then heat-fix the slide by passing it over an open flame four times, each lasting 5 seconds.
- After fixation, place the smear on a rack, ensuring a 10mm slide spacing.
- Apply carbolfuchsin solution at room temperature for 10 minutes or more.
- Gently wash one end of the slide to remove excess dye.
- Cover the slide with acid alcohol solution for 1-2 minutes for decolorization. Rinse with water if needed.
- Rinse the slide with running water for 10-20 seconds.
- Apply methylene blue solution for 30-60 seconds.
- Rinse the slide gently with running water before microscopic examination.
Under a microscope against a light blue background, acid-fast bacteria, like Mycobacterium tuberculosis, appear red, while other bacteria and cells appear blue. Mycobacterium tuberculosis typically exhibits rod-shaped or slightly curved cells with varying dimensions. In well-stained specimens, darker-colored particles may be observed. Fresh specimens may show single bacteria or clusters with a branched arrangement.
The Kinyoun stain is specifically designed for use with acid-fast bacteria.
- Prevent dye from drying on the slide during staining.
- Ensure the sputum membrane’s thickness is suitable.
- Avoid cedarwood oil, as it may damage the oil lens.
- Be cautious of variations in red shades within acid-fast bacteria.
- Preserve smears with absorbent paper and gentle wiping.
- Adjust staining time in colder temperatures.
- Promptly cover reagents after use to prevent volatilization.
- Store reagents away from extreme temperatures and direct sunlight.
- Use the Kinyoun stain under professional supervision.
- Adhere to instructions and practice personal hygiene.
- Dispose of waste per guidelines.
- Check the packaging for production and expiry dates.
Kinyoun stain: Crucial in microbiology for detecting acid-fast bacteria, including Mycobacterium tuberculosis and Mycobacterium leprae.
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