Flagellar staining



Flagella are long thin appendages that help the bacteria for motility. They are so thin that can not be visualized by a simple microscope. To make them visible, they are thickened by an extra coating over them by the use of mordants. For flagella staining best is to use fresh young culture.

Materials and equipment

Tannic acid (2.5%), Loeffler's stain, acid alcohol, distilled water, glass slides, inoculation loop, staining rack, Bunsen burner, blotting paper, test tubes, incubator, microscope.

Procedure

  1. Use a young bacterial culture (about 18 holds) 
  2. Mix 0.1 ml of broth with 5 ml of distilled water.
  3. Incubate at 37°C for 30-45 min. this will free the organisms from the slime.
  4. Wash the slide with acid alcohol.
  5. Place a loopful culture on the slide, tilt it spread the culture to the maximum area (to form uniform film).
  6. Air-dry the smear.
  7. Flood the smeared area with tannic acid for 3-4 min
  8. .Wash the slide with tap water.
  9. Flood the slide with Loeffler’s stain and wait for 3-4 min.
  10. Wash the slide and dry by blotting.
  11. Examine under the oil immersion lens of the microscope.
Note: The cell will appear as pink while the outmost boundary will be red. While the flagella will appear as a pink-colored long strand

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