Flagella is a filamentous, unbranched, thread-like structure which is emerging from the bacterial cell wall. Bacterial cell may contain a single polar flagellum (monotrichous), tufts of flagella at each pole (lophotrichous), one or more flagella at both poles (amphitrichous), or flagella surrounding the perimeter of the cell (peritrichous).

Bacterial flagella are made of a protein subunit termed flagellin. In eukaryotic flagella, flagellin has a distinct structure.

In 1930, Leifson introduced a simple flagella stain, using this stain he observed that mutations leading to nonflagellated from flagellated bacteria were common, as well as mutation leading to loss of motility without loss of the flagellum itself.

Aim

The main purpose of flagella staining is to stain bacterial flagella and observe the presence or absence of bacterial flagella as well as their arrangement on bacterial cell.

Principle of Flagella Staining

The Leifson flagella stain method uses tannic acid and a dye. When bacterial flagella absorbs this tannic acid and a dye, it forms a colloidal precipitate as a result the flagella is colorized and as well as increase in diameter, thus amenable to viewing by light microscopy. 

The tannic acid-dye complex is more solvable in alcohol as compared to water, and furthermore soluble with reduced pH. The alcohol concentration in the Leifson solution is enough to maintain the solubility of the ingredients.

When the processed specimen is stained, the alcohol dries faster than the water, and the intensity of the tannic acid and dye rises to create precipitation, leading to staining of the flagella.

Salt concentration also influences the staining, probably by changing charge of the tannic acid-dye complex and the flagellum itself. 

The flagella stain is finicky because numerous variables affect the outcome: age of the bacterial culture, thickness of the culture on the microscope slide, age of the staining solutions, pH, temperature, alcohol concentration, dye concentration, heat, and/or airflow.

Leifson flagella staining Method

Flagella Staining Requirement

•  16 and 20 hours old bacterial culture, because older cultures tend to lose flagella. 
• New microscope slides
• 95% ethanol and Kimwipes to clean slides
• Heat—provided by a Bunsen burner, alcohol lamp, ceramic heater, or other appropriate heat source
• Micropipettors to deliver 5 to 200 ml
• Sterile disposable tips for micropipettors
• Distilled water
• Bibulous paper

Reagents

• Leifson flagella stain
• Solution A: Can be prepared by mixing  1.5 g Sodium chloride with 100 ml Distilled water.
• Solution B: Can be prepared by mixing  3.0 g Tannic acid with 100ml Distilled water.
• Solution C:  Can be prepared by mixing 0.9 g Pararosaniline acetate and 0.3 g Paraosaniline hydrochloride with 100 ml Ethanol, 95% (vol/vol).

Mix equal volumes of solutions A and B; then add 2 volumes of the mixture to 1 volume of solution C. The resulting solution may be kept refrigerated for 1 to 2 months.

Flagella Staining Procedure

Preparation of culture

From Solid Culture

1. From an agar plate or slant cultures, prepare a suspension by removing a small amount of growth, approximately one-fourth of the colony, with an inoculating loop using proper aseptic technique.
2. Emulsify in 100 ml of distilled water in a microcentrifuge tube by gently vortexing.
3. The emulsion should be only slightly cloudy. Using too much inoculum results in the inability to visualize the flagella.

Form Liquide Culture

For staining from liquid cultures, Leifson recommends two rounds of centrifugation and final suspension in distilled water to remove any medium components.

1. Place 100 ml of the liquid culture in a microcentrifuge tube, centrifuge, and remove spent medium.
2. Resuspend in 100 ml of distilled water by gently vortexing, again centrifuge, and remove supernatant.
3. Form a slightly cloudy emulsion by resuspending in ~200 ml of distilled water. Gently vortex. Again, emulsions should be only slightly cloudy prior to proceeding to staining.
4. Optimization of the washing procedure will most likely be necessary to maximize quality of flagella stain.

Slide Preparation

1. Wipe clean a new microscope slide with 95% ethanol and a Kimwipe. Flame to dry thoroughly. Use slides immediately.
2. When the slide is cool enough to handle, label it using tape with the name of the organism you will be staining.
3. Place 5 to 10  ml of the culture emulsion on one end of the slide using a micropipettor and spread the emulsion using the same pipette tip held parallel to the microscope slide.
4. Allow the sample to dry at room temperature. Do not heat fix as this will destroy the proteinaceous flagella structure.

Staining

1. Take a prepared slide and using a wax pencil draw a rectangle around the dried sample. Place slide on staining rack.
2. Flood Leifson dye solution on the slide within the confines of the wax lines. Incubate at room temperature for 7 to 15 minutes. The best time for a particular preparation will require trial and error.
3. As soon as a golden film develops on the dye surface and a precipitate appears throughout the sample, as determined by illumination under the slide, remove the stain by floating off the film with gently flowing tap water. Air dry.
4. View using oil immersion, at 1,000x magnification, by bright-field microscopy. 

Flagella Staining Result

Bacterial bodies and flagella will stain red.

Presque Isle Cultures flagella stain Method

Requirement

1. Solutions are available from Presque Isle Cultures. The components of the flagella stain are proprietary.
2. Flagella mordant (Solution I)
3. Silver stain (Solution II)
4. Solutions I and II can be stored at room temperature for several weeks.

Procedure

1. Prepare slide as described above. Place slide on staining rack.
2. Flood slide with Presque Isle Cultures Solution I, the mordant. Incubate at room temperature for 4 minutes.
3. Gently rinse with distilled water. Shake excess water from slide.
4. Flood with Presque Isle Cultures Solution II, the silver stain.
5. Heat over Bunsen burner by moving slide back and forth, just until steam is emitted. If a Bunsen burner is not available then an alternate heat source can be used, but optimization will be necessary. Be careful not to overheat sample, as excess heat will destroy the flagella. Incubate at room temperature for 4 minutes.
6. Rinse with distilled water. Carefully blot dry with bibulous paper.
7. View using oil immersion, at 1,000x magnification, by bright-field microscopy. 

Result

Bacteria and flagella will appear golden brown.