Flagella are filamentous cytoplasmic structure which are projecting from the cell wall. These are 12-30 nm in diameter and 5-16 µm in length, unbranched, thread-like structure, and made of the protein flagellin.
Based on distribution they are classified into different classes such as Monotrichous (Single polar flagellum), Amphitrichous (Single flagellum on both sides), Lophotrichous (Tufts of flagella at one or both sides), and Peritrichous (Numerous flagella all over the bacterial body).
Flagella is consists of different parts such as Filament, Hook and Basal Body. Flagella involve in different activities such as Movements, Sensation, Signal transduction, Adhesion, etc.
The staining of bacterial flagella is a very complex process that requires extraordinary care for the slides, stain, and cells. For flagella staining, we use wet mount technique, for this technique a stable stain and regular slides and coverslips are require. This method helps to find the number and arrangement of flagella which is essential for the identification of bacterial cells.
Objective of Flagella Staining
The main purpose of flagella staining is to study the presence and arrangement of flagella in a motile bacteria.
Principle of Flagella staining
Flagella can not be seen under a bright-field microscope by using ordinary stains. A simple and useful method has been used for visualization of flagella is known as wet mount technique. This method is useful when the number and arrangement of flagella are critical to the identification of species of motile bacteria. In this method, a mordant is used to adhere the stain in flagellar layer.
Procedure of Flagella staining
- Grow the organism to be stained at room temperature on blood agar for 16 to 24 hours.
- Add a small drop of water to a microscope slide.
- Dip a sterile inoculating loop into 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 to 10 minutes. This allows the bacterial cells time to adhere either to the glass slide or to the coverslip.
- 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 halfway from the edge of the coverslip to the center of the mount.
- Focusing the microscope on the cells attached to the coverslip rather than on the cells attached to the slide facilitates visualization of the flagella. The precipitate from the stain is primarily on the slide rather than the coverslip.
Result
Observe the slide under a microscope and write down the following characteristics;
- Presence or absence of flagella
- Number of flagella per cell
- Location of flagella per cell
- Peritrichous
- Lophotrichous
- Polar
- The amplitude of wavelength
- Short
- Long
- Whether or not “tufted”
Quality control
- Peritrichous: Escherichia coli
- Polar: Pseudomonas aeruginosa
- Negative: Klebsiella pneumonia
Limitation
Even with a special stain, visualization of flagella needs an expert laboratory scientist and is not considered an entry-level procedure.
