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21.4: Materials and Procedures

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    • Student Stock Cultures:
      • Mycobacterium smegmatis
      • Staphylococcus saprophyticus
    • Glass Slides
    • Stain tray and slide holder
    • Stain kit:
      • Kinyoun carbolfuchsin
      • Acid/Alcohol decolorizer
      • Methylene blue
      • Glass bowls with Sanisol


    1. Prepare a heat-fixed smear prep with a mixture of Mycobacterium smegmatis and Staphylococcus saprophyticus. Aseptically place a drop of each onto the same spot on the slide and mix well. Try to break up the waxy Mycobacterium smegmatis so that it can spread evenly across the slide. Air dry, then heat-fix.
    2. Cover the smear with Kinyoun carbolfuchsin and let stand for 5-10 minutes.
    3. Gently wash with DI water.
    4. Gently decolorize with Acid-Alcohol by dropping the decolorizer down the slide until the slide runs clear. Note that this decolorizer is not the same as the Acetone-Alcohol used in the Gram stain.
    5. Gently wash with DI water.
    6. Counterstain by covering the slide with Methylene blue for 1 min.
    7. Gently wash with DI water, blot, and view under oil immersion.
    Figure \(\PageIndex{1}\): Slide Prep, C.S.Ramey, CC-BY-NC-SA


    1. Draw and color (or indicate colors of) your Acid-Fast stain:

    Contributors and Attributions

    This page titled 21.4: Materials and Procedures is shared under a CC BY license and was authored, remixed, and/or curated by Kelly C. Burke.

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