2.4.2: Acid-Fast Stain Activity

OER - BIOL 2420 Laboratory Manual · LibreTexts

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    From LibreTexts, under CC BY 4.0, as the source states it; its equations are written in TeX, which the PDF shows typeset. This page at LibreTexts.

    In this activity you will prepare five bacterial samples using the Acid-fast stain procedure. You will be using Bacillus subtilis (Bs), Mycobacterium smegmatis (Ms), Escherichia coli (Ec), Streptococcus salivarius (Ss) and Staphylococcus aureus (Sa) as your samples. Please utilize the proper precautions as you work with these samples.

    ***NOTE*** Staphylococcus aureus and Mycobacterium smegmatis are BLS-2 organisms

    1. Obtain five microscope slides, clean and dry the microscope slides using lens cleaner and Kim wipes
    2. Label the slides using a China marker as seen below

      ***NOTE*** the circle must be placed on the bottom of the slide to prevent the marker from being mixed with the bacterial sample

      Labeled microscope slide showing the specimen circle positioned on the bottom of the slide.
      Credit: Sherry Smith
    3. Obtain 1-2 loopfuls of bacteria from the appropriate broth culture and introduce the inoculum to the appropriate microscope slide, spreading it into a thin film
    4. Allow the slides to air dry completely
    5. Fix the smears by passing the air-dried samples 2-3 times through the flame of the Bunsen burner smear side up
    6. Use a clothes pin to place the slides smear side up on the rack above the steam pot
    7. Place a steaming towel (small piece of paper towel) over the smear and cover with carbolfuchsin. Allow the slides to remain over the steam for 5 minutes. Add more dye as needed to prevent the steaming towel from drying.
    8. Remove the steaming towel using the provided forceps and place it in the container provided for disposal. Remove the slides from the steaming rack and place them on your staining rack. Allow the slides to cool completely.
    9. Rinse the slides slowly with DI water, aiming the water above the smears.
    10. Decolorize the smears with acid-alcohol, applying slowly until dye does not run off the slides.

      ***NOTE*** Acid-alcohol is a mixture of ethyl alcohol and hydrochloric acid, which is highly flammable!!!

    11. Rinse the slides slowly with DI water to remove the decolorizing agent.
    12. Cover the smears with methylene blue for 1 minute
    13. Rinse the slides slowly with DI water until the water runs clear
    14. Use Kim wipes to blot the slides, smear side down. Blot, do not rub!!!
    15. Use your compound microscope to visualize your stained slides.

    This page titled 2.4.2: Acid-Fast Stain Activity was last modified on Sat, 25 Jul 2026 03:43:02 GMT and is shared under a CC BY 4.0 license and was authored, remixed, and/or curated by Sherry Smith.