The Acid fast staining procedure

A couple of months ago I had to learn the acid fast method of staining and write about it. So this blog contains what I learned about that topic. “The acid fast stain… is an important diagnostic stain,” (Talaro, 2008) performed so that one may be able to differentiate between those bacteria containing mycolic acid in their cell walls (which therefore may be classified as acid fast staining bacteria) and those which are nonacid-fast staining. This unique structural characteristic of acid fast bacteria renders its cells impervious to most staining agents; however this type of staining procedure allows for adhesion to this specific type of waxy cell wall (using a combination of dyes) and thus, differential identification. The procedure is done by first smearing and then heat fixing a culture of bacteria to a clean slide. The smear is then covered with adsorbent paper and saturated with Carbolfuchsin. The slide is then placed over a beaker filled one quarter of the way with boiling, distilled water (Ziehl-Neelsen Method) for a period of five minutes. This method is used so that the dye may adhere more readily to the acid fast bacterial cells and prevent drying out. The paper is then removed and the smear is then washed carefully by allowing distilled water to run through it. Next, acid-alcohol is made to cover the smear for one minute in order to decolorize those bacteria which are nonacid –fast staining. Then again the smear is washed gently with water. After this, the counterstain, Methylene blue is applied to the stain for one minute so that the non-acid fast bacteria may be dyed and identified during viewing. This is followed by one final washing of the smear with distilled water. Lastly the specimen is blotted dry using Kim Wipes and viewed under oil immersion magnification. It is expected that those bacteria which contain mycolic acid within their cell walls will retain the fuchsia hue of the Carbolfuchsin dye, while the other cells within the smear will stain purple with the counterstain, Methylene blue.