To spore stain, you'll use a technique to differentiate bacterial spores from vegetative cells, typically using malachite green to stain the spores and safranin to counterstain the vegetative cells. Here's a basic spore staining procedure using the Schaeffer-Fulton method:
Materials:
- Bacterial smear on a microscope slide
- Malachite green stain
- Safranin stain
- Distilled water
- Paper towels
- Slide warmer or Bunsen burner (optional)
- Microscope
Procedure:
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Prepare a Bacterial Smear: Prepare a bacterial smear on a clean microscope slide. Allow the smear to air dry completely and then heat-fix it by passing it quickly through a flame a few times. This adheres the bacteria to the slide.
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Apply Malachite Green: Cover the smear with a piece of absorbent paper (like a small square of paper towel) cut to fit within the slide's borders. The paper helps keep the stain moist during the steaming process.
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Steam the Slide: Place the slide on a slide warmer set to a low temperature, or carefully hold it over a beaker of steaming water (or a low Bunsen burner flame, being extremely cautious). Flood the paper towel with malachite green stain and allow it to steam for 3-5 minutes. Keep the paper towel moist by adding more malachite green as needed. The steaming process helps the malachite green penetrate the resistant spore coat. Do not let the slide dry out.
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Wash with Water: Remove the slide from the heat source. Carefully remove and dispose of the paper towel. Rinse the slide thoroughly with distilled water for about 30 seconds. This step removes the malachite green from the vegetative cells but not from the spores.
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Counterstain with Safranin: Flood the slide with safranin stain for 1 minute. Safranin will stain the vegetative cells, providing a contrast to the green-stained spores.
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Rinse and Dry: Rinse the slide gently with distilled water to remove excess safranin.
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Blot Dry: Carefully blot the slide dry with bibulous paper or a clean paper towel.
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Observe Under Microscope: Observe the slide under a microscope, starting with low power and then switching to higher magnification (e.g., 100x with oil immersion) to clearly view the spores and vegetative cells.
Expected Results:
- Spores will appear green.
- Vegetative cells will appear pink or red (from the safranin counterstain).
Important Considerations:
- Heat is crucial for driving the malachite green into the spores.
- Avoid overheating the slide, which can distort the cells.
- The age of the bacterial culture can affect sporulation. Older cultures are more likely to contain spores.
- Dispose of used materials properly according to laboratory safety guidelines.
