Bacterial spores are stained using a technique that forces dye into their tough outer coating, often involving heat. The most common method is the Schaeffer-Fulton spore stain. Here's a detailed breakdown of the procedure:
Schaeffer-Fulton Spore Stain Procedure
The Schaeffer-Fulton method, also known as the malachite green stain, is the most widely used technique for staining bacterial spores. It involves using heat to drive the primary stain, malachite green, into the spore.
Materials Needed:
- Bacterial culture (preferably an older culture to encourage spore formation)
- Microscope slides
- Inoculating loop
- Bunsen burner or slide warmer
- Malachite green solution (primary stain)
- Water (decolorizer)
- Safranin solution (counterstain)
- Absorbent paper (optional)
- Microscope
- Immersion oil (for microscopy)
Step-by-Step Procedure:
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Prepare a Smear: Create a thin smear of the bacterial culture on a clean microscope slide.
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Air Dry and Heat Fix: Allow the smear to air dry completely. Then, gently heat-fix the smear by passing the slide quickly through a Bunsen burner flame several times. This adheres the bacteria to the slide and kills them.
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Apply Malachite Green: Place a small piece of absorbent paper (optional, but it helps keep the slide moist) over the smear. Saturate the paper with malachite green solution.
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Heat the Slide: Gently heat the slide by steaming it over a Bunsen burner (or on a slide warmer) for approximately 5 minutes. Do not allow the stain to dry out. The heat helps the malachite green penetrate the spore coat. Replenish the stain if necessary.
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Rinse with Water: Remove the absorbent paper (if used) and rinse the slide thoroughly with water for about 30 seconds. The water acts as a decolorizer, removing the malachite green from the vegetative cells but not from the spores.
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Counterstain with Safranin: Apply safranin solution to the smear for approximately 1 minute. Safranin stains the vegetative cells pink or red.
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Rinse and Dry: Rinse the slide gently with water to remove excess safranin. Allow the slide to air dry or blot it gently with bibulous paper.
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Microscopic Examination: Observe the slide under a microscope using oil immersion (1000x magnification).
Expected Results:
- Spores: Appear green due to the malachite green stain.
- Vegetative cells: Appear pink or red due to the safranin counterstain.
Principle:
The malachite green stain is water-soluble and has a low affinity for cellular material. Vegetative cells can be decolorized easily with water, removing the malachite green. Spores, with their tough outer covering, retain the malachite green even after rinsing. The safranin counterstain then stains the decolorized vegetative cells, providing contrast.
Troubleshooting Tips:
- Poor spore formation: Use an older culture or a culture grown under nutrient-limiting conditions to encourage sporulation.
- Weak staining: Ensure adequate heating during the malachite green staining step.
- Over-decolorization: Rinse gently with water and avoid prolonged rinsing.
- Uneven staining: Ensure the smear is thin and evenly distributed.
