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Biology LibreTexts

18.4: Materials and Procedures

  • Page ID
    40270
  • Materials

    • 1 broth culture per table:
      • Streptococcus mutans
    • Student Stock Cultures-use broths
    • Glass Slides
    • Stain tray and slide holder
    • Nigrosin
    • Glass bowls with Sanisol
    • Lens cleaner/lens cleaning kits

    Procedures

    Suggested bacteria for staining (any broth from the Stock Cultures may be used)—

    • Moraxella catarrhalis
    • Bacillus cereus
    • Streptococcus mutans
    • Staphylococcus epidermidis
    • Escherichia coli
    • Micrococcus luteus
    • Rhodospirillum rubrum
    1. Clean all slides with lens cleaner prior to doing the stain. The Nigrosin will spread more readily and evenly on cleaned slides; it may be repelled by dirt and fingerprints and thus bead up on the slide.
    2. Refer to Figure \(\PageIndex{1}\) when preparing for and performing the stain.
    3. Place a small drop of Nigrosin at one end of the slide.

    Caution

    Do not touch the slide with the eyedropper; this can contaminate the stain container.

    1. Using your loop aseptically transfer a loopful of culture into the drop of Nigrosin and mix gently without spreading the stain. Do not let the slide dry at this step.
    2. Back up the end of a second slide to the mixture so that the drop grabs onto the back of the slide. Gently PULL the drop forward with the second slide to produce a smear across the entire slide. The smear will appear very dark at the starting end and should thin out towards the other end.
    3. Let the slide air-dry.
    4. Observe dried slides using oil-immersion.

    Cautions

    1. Remember that the working distance is very small with oil-immersion and that the cells may be viable. Do not run the slide into the objective as this can contaminate the lens with bacteria. Focus very carefully.
    2. When viewing the slide, where the stain is very dark and thick, you may see star shaped cracks. The stain may crackle as it dries in very thick areas. If you see this, simply scan to another area with a lighter gray background and look for cells.
    3. Do not mistake air bubbles, which may form, for the bacterial cells. Practice and experience will help you with this; however, air bubbles are usually very round, very clear, and larger than cocci.
    4. If you are observing known rod shaped bacteria, you may see some that look like cocci. These could be cells that are “on end”, and short cocco-baccilli could be young recently divided cells. Or, they could be air bubbles.
    1. Place used slides into the glass bowl with Sanisol.
    clipboard_e4c880c13660021db92861cb38940ad9a.png
    Figure \(\PageIndex{1}\): Negative Stain Procedure

    Results

    1. Draw your observations in the circles below:
    clipboard_e1142b69a641114ee83764d45e8d1b6c0.png
    1. Fill in the table with the organisms you stained:
    Table \(\PageIndex{1}\): Negative stained organisms

    Organism

    Cell Morphology

    Cell Arrangement

    Size (\(\mu m\))

    Contributors and Attributions

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