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2.3: Acid-fast Stain Procedure

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  • Acid-fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids. These acids resist staining by ordinary methods such as a Gram stain. It can also be used to stain a few other bacteria, such as Nocardia.

    Preparation of microscope slide:

    1. Clean slide with a Kimwipe and alcohol to remove any fingerprints.
    2. Draw two circles with your Sharpie on the bottom of the slide.
    3. Using your inoculation loop, put two small drops of water in each circle.
    4. Using aseptic technique, remove a very small amount of bacteria from the culture tube. Make sure you flame the tube before and after you enter.
    5. Smear the bacteria in the drop of water on your slide. You may go out of the perimeter of your circles!
    6. Let the slide air dry completely.
    7. Heat-fix the slide by running it through the flame 3-4 times with the ‘smear’ side up. Do not flame the side with the bacteria!
    8. Let the slide cool completely and you are ready to stain it.

    Staining procedure:

    1. Cover the smears with a piece of paper towel within the border of the slide.
    2. Place the slide over a beaker of steaming water. Do not let the beaker boil dry
    3. Flood the paper towel with carbolfuchsin and let the slide steam for 3-5 minutes.
    4. Complete the rest of the procedure at the back sinks.
    5. Remove the stained paper towel and discard it in the trash can, not in the sinks.
    6. Gently rinse the slide with water to remove any pieces of a loose paper towel and tap dry.
    7. Apply acid-alcohol for 15-30 seconds.
    8. Rinse off and tap dry.
    9. Counterstain with methylene blue for 1.5 minutes.
    10. Rinse with water and tap dry.
    11. Blot gently with bibulous paper.
    12. Dry the bottom of the slide before placing it on the stage of the microscope and view with the oil immersion lens.
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