Acid-Fast stains: are particularly helpful when the organism’s cell wall is thick and waxy (lipoidal)—resisting the other (more conventional) staining methods. Acid-Fast staining requires 3 reagents: the primary stain; the decolorizing agent; and the counterstain.
The primary stain is carbol fuchsin— dark red, 5% phenol. Carbol fuchsin is lipid-soluble which allows it to penetrate the thick lipoidal cell wall. Heat (e.g. steam from water bath) helps to facilitate penetration (Ziehl-Neelson method). Instead of heat, one may use a wetting agent (Tergitol) in the Kinyoun method.
The decolorizing agent is an acid-alcohol of 3% HCl and 95% ethanol. Before using the decolorizing agent, the slide must cool in order for the waxy lipoidal wall to harden and retain the primary stain better. When the decolorizing agent is applied, the color is removed from those non-lipoidal structures or those structures that did not retain the primary stain well.
The counterstain is methylene blue which stains all of the decolorized structures (non acid-fast) blue.
Clinical significance. Helpful in detecting members of the Mycobacterium which are especially pathogenic such as M. tuberculosis and M. leprae.
Summary of procedure.
- Prepare a smear and heatfix.
- Warm a beaker of water on a hotplate.
- Flood the slide with carbol fuchsin and place slide over the beaker. Alternatively, flood smear with Tergitol and let it stand for 5-10 minutes (then skip to step 6).
- Steam for 5 minutes. Do not let stain evaporate—you may add more carbol fuchsin during this process. Do not let water boil or get too hot (you will blow your slide).
- Remove slide from the steam, and let slide cool.
- Gently flush slide with deionized water.
- Decolorize by sparingly adding the decolorizing agent drop by drop until the run-off is a pale tinge of red (almost clear looking). Do not overdo this!
- Gently flush slide with deionized water.
- Counterstain using methylene blue for 2 minutes.
- Gently flush slide with deionized water.
- Blot with bibulous paper.
Reference
Cappuccino, J. G., & Welsh, C. (2018). Microbiology: A laboratory manual.
