The flagella stain procedure is a specialized staining technique used in microbiology to visualize bacterial flagella under a microscope. Flagella are thin and not visible with standard staining, this procedure employs stains that adhere to and increase their thickness.
Materials Needed
- Bacterial culture (freshly grown, motile bacteria)
- Microscope slides
- Flagella stain
- Distilled water
- Inoculating loop
- Microscope
Procedure
- Clean the microscope slide thoroughly to remove any grease or oil. This ensures that the stain adheres properly.
- Place a small drop of distilled water on the center of the slide.
- Using an inoculating loop, gently transfer a small amount of the bacterial culture into the drop of water. Be careful to avoid shearing the flagella.
- Gently mix and spread the bacteria into a thin smear without pressing hard.
- Let the smear air dry completely.
- Avoid using heat, as it can destroy the flagella.
- Cover the smear with the mordant solution provided in the staining kit (e.g., tannic acid or another mordant). This step helps to coat the flagella, making them thicker and more visible.
- Allow the mordant on the slide for the time specified by the manufacturer (typically 4-5 minutes).
- Rinse the slide gently with distilled water to remove excess mordant.
- Flood the slide with the flagella stain (e.g., crystal violet or carbol fuchsin) and allow it to sit for the specified time (usually 4-5 minutes).
- Rinse the slide gently with distilled water to remove the excess stain.
- Allow the slide to air dry. Avoid blotting, as this can damage the flagella.
- Use oil immersion at 1000x magnification to see the stained flagella.
KEY POINTS
- Use fresh cultures: Older cultures may have damaged or fewer flagella.
- Handle gently because Excessive agitation during smear preparation can break off the flagella.
- Optimize timing: Make sure the mordant and stain are left on the slide for the recommended duration. This will help achieve the best results.