Acid-Fast Staining of Sputum Sample

The video begins by explaining that the sputum slides are already prepared and heat-fixed. Ordinarily, one would create a smear and heat fix it before starting the staining process.

The first step in the staining procedure is to cover the smear with carbol fuchsin and leave it on for 3 minutes. After 3 minutes, rinse the carbol fuchsin off the slide and shake off the excess.

Next, use acid alcohol for the decolorizing step. Add it drop by drop until the pink or purple color stops running from the smear, which may take several drops. Immediately after, rinse the slide again and shake off the excess water.

The final staining step involves methylene blue. Cover the smear with methylene blue and leave it on for 1 minute. After 1 minute, rinse it off the slide, shake off the excess water, and blot it gently with a Kimwipe until dry.

Finally, examine the slide under a microscope, starting with low power, then high dry. It is essential to use oil immersion to see the small acid-fast bacilli, which will appear reddish. Non-acid-fast bacilli and all background material will appear blue.