Question

List the steps for the Acid Fast Stain Procedure:

List the steps for processing specimens for AFB (sputum, gastric lavage, etc.):

Answer 1

Acid fast bacteria contain special lipid substances in their cellwalls and are cannot be stained by general procedure like Gram staning. Acidfast staining is also known as Ziehl-Neelsen staining. This staining method differentiates Acidfast group from nonacidfast bacteria. The basic dye carbol fuschin used in acid fast staining helps to soluble the lipid substances like mycolic acid present cellwall of Mycobacterium tuberculosis and enbles it to appear stained. The steps in the procedure are as follows:

• Clean glass slide is taken and smear is prepared under sterile conditions
• The smear is air dried and heat fixed
• Carbol fuschin stain is added to the smear.
• Then the smear is heated till vapors are just observed and the smear is allowed to stay for 5 minutes. Overheating should be avoided.
• The stain is washedoff with clean and sterile water.
• The smear is then added with decolorizing agent containing 3% acid/ alcohol v/v mixture till the smear turns pale in color.
• The smear is then washed.
• Then the counter stain like methylene blue or malachite green is added and allowed to stay for 2 to 3 minutes.
• The smear is then washed with water
• The slide is wiped gently and air dried
• Finally, the slide can be observed under 100X oil immersion objective.
• The acidfast bacilli appear bright red or purple in color while nonacidfast bacilli appear blue in color.