Most motile bacteria possess flagella, the shape, number, and position of which are important characteristics in the differentiation of genera and species identification. Staining bacterial flagella differs from staining other bacterial structures because it usually requires extraordinary care for the slides, stain, and cells. Flagellar stains are painstakingly prepared to coat the surface of the flagella with dye or a metal such as silver.
The number and arrangements of flagella are critical in identifying species of motile bacteria.
Two techniques for staining flagella are in use:
- A wet-mount procedure (Ryu method)
- Dried-smear preparation (Leifson staining technique)
A wet-mount technique for staining bacterial flagella is highly successful when a stable stain and regular slides and coverslips are used. This technique is simple for routine use when the number and arrangement of flagella are critical in identifying species of motile bacteria.
The preparations are not permanent because the stain precipitates as the wet mount dry.
- Grow the organism to be stained at room temperature on blood agar for 16-24 hours.
- Add a small drop of water to a microscope slide.
- Dip a sterile inoculating loop into the sterile water
- Touch the loopful of water to the colony margin briefly (this allows motile cells to swim into the droplet of water)
- Touch the loopful of motile cells to the drop of water on the slide. NOTE: Agitating the loop in the droplet of water on the slide causes the flagella to shear off the cell.
- Cover the faintly turbid drop of water on the slide with a coverslip. A proper wet mount has barely enough liquid to fill the space under a coverslip. Small air spaces around the edge are preferable.
- Examine the slide immediately under 40× to 50× for motile cells. If motile cells are not seen, do not proceed with the stain.
- If motile cells are seen, leave the slide at room temperature for 5 to10 minutes. This allows time for the bacterial cells to adhere to either the glass slide or the cover slip.
- Apply 2 drops of RYU flagella stain gently to the edge of the cover slip. The stain will flow by capillary action and mix with the cell suspension. Small air pockets around the edge of the wet mount are useful in aiding the capillary action.
(Note: The Ryu stain has two components. Solution I, the mordant, contains 10 ml of 5% aqueous solution of phenol, 2 g of tannic acid, and 10 ml of saturated aqueous solution of aluminum potassium sulfate-12 hydrate. Solution II, the stain, is a saturated ethanolic solution of crystal violet (12 g in 100 ml of 95% ethanol). The final stain was prepared by mixing 1 part solution Il with 10 parts solution I and then filtering the mixture through filter paper to remove coarse precipitate)
- After 5 to 10 minutes at room temperature, examine the cells for flagella.
- Cells with flagella may be observed at 100× (oil) in the zone of optimum stain concentration, about half way from the edge of the coverslip to the center of the mount.
- Focusing the microscope on the cells attached to the coverslip rather than the cells attached to the slide facilitates visualization of the flagella. The precipitate from the stain is primarily on the slide rather than the cover slip.
Observe the slide and note the following: