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ACID FAST STAINING

  • The acid-fast stain is a laboratory test that determines if a sample of tissue, blood, or other body substance is infected with the bacteria that causes tuberculosis and other illnesses.

HISTORY

  • In the late 19th century, Koch and Ehrlich simultaneously introduced a method for staining the previously undetectable Mycobacterium tuberculosis. Modifications by Ziehl and Neelsen produced the commonly used carbol-fuchsin solution which requires steaming to drive the stain in. (The melting point for mycolic acid is 56oC). Muller and Chermock modified carbol-fuchsin for use at room temperature by addition of a surfactant (wetting agent). A solution of acid-alcohol removes stain from most cellular and tissue elements. The mycolic acid, however, resists penetration by this differentiating agent, leaving acid-fast bacteria red against a colorless background. Light green or methylene blue is used as a counterstain to aid in the localization of cellular material on the specimen. The acid fast stain is used routinely on sputum samples for preliminary diagnosis of active tuberculosis.

TYPES

  1. ZIEHL-NEELSEN STAIN
    The Ziehl–Neelsen stain, also known as the acid-fast stain, was first described by two German doctors: the bacteriologist Franz Ziehl (1859–1926) and the pathologist Friedrich Neelsen (1854–1898). It is a special bacteriological stain used to identify acid-fast organisms, mainlyMycobacteria.

  2. KINYOUN STAIN
    The Kinyoun stain is a method of staining acid-fast microorganisms, specificallyMycobacterium and Nocardia. The procedure for Kinyoun staining is similar to the Ziehl-Neelsen stain, but does not involve heating the slides being stained.

  3. AURAMINE-RHODAMIN STAIN
    The auramine-rhodamine stain (AR), also known as the Truant auramine-rhodamine stain, is ahistological technique used to visualize acid-fast bacilli using fluorescence microscopy, notably species in the Mycobacterium genus.

  4. AURAMINE PHENOL STAIN
    Auramine phenol stain is a stain used in clinical microbiology and histology to identifytuberculosis mycobacteria.

PROCEDURES

  1. Make a thin bacterial smear on a separate slide for each organism provided. You will especially have to work the M. smeg. with the loop. It will be helpful to add a loopful of water. Make one slide a mixed sample by adding a loop from both.

  2. Flood the slide with carbol-fuchsin (3-4 drops) and place over a steaming water bath for 5 minutes, adding stain as the edges begin to evaporate. Try not to drip stain into the boiling water. A dime sized piece of paper towel placed directly on the smear can help hold the dye on the smear.

  3. Remove from bath and cool. Rinse with distilled water and blot gently with bibulous paper. Allow the slide to cool.

  4. Remove the excess satin by tilting the slide and dripping acid-alcoholover it for 30 seconds.

  5. Rinse with distilled water and blot gently.

  6. Differentiate organisms by flooding the slide with acid-alcohol and allowing to stand for two minutes.

  7. Rinse with distilled water and blot gently.

  8. Flood the slide with methylene blue for at least one minute.

  9. Rinse and blot dry. Observe.

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