Flagella Staining: Principle, Procedure and Results

Aim: To study flagella staining.

Introduction:

The flagellum is a locomotory organ of bacterial cells. Bacterial flagella have a narrow diameter so they cannot be seen with a light microscope. Flagella require a special staining technique. Flagella are complex filamentous cytoplasm structures protruding through cell walls. Flagella’s thread-like structure is mostly composed of the protein flagellin. Flagella play an important role in motility.

Principle:

The bacterial flagella are thin and delicate, so the special stain called flagella stain containing a mordant is prepared. This mordant allows piling of stain on flagella and increases the thickness to make the flagella visible. This helps to observe the various arrangements of flagella.

Requirements:

• Bacterial culture: Flagellated cell culture slant (18 hours culture of Proteus vulgaris).
• Chemicals: Carbol fuchsin stain, 95% ethanol, distilled water, flagella mordant.
• Apparatus: Inoculating loop, glass slide, and microscope.

Procedure:

1. Suspend the loopful of bacteria in distilled water and obtain a suspension.
2. Keep the suspension for 15-20 minutes till flagella are regenerated and extended.
3. Select the slide which is cleaned with cleaning powder and flame the slide, allow to cool.
4. Prepare smear, lay the loopful of a bacterial suspension at one end of the slide.
5. Place the slide on the staining rack.
6. Flood the slide with flagella stain for 10-15 minutes.
7. Rinse the stain on the slide and flood the slide with flagella stain.
8. Drain and flood the slide with Carbol fuchsin for 5 minutes.
9. Focus the image under the oil immersion lens.
10. Flagella are much longer than the cell and often look like faint hairs.

Observation:

Examine the slide under the oil immersion lens. Record the number of flagella present on the cell surface and also determine the arrangements of flagella.

Result:

Bacterial cells appear pink which are surrounded by deeply stained flagella; the flagella may be monotrichous, amphitrichous, peritrichous, or lophotrichous.

Application:

Flagellum staining is used for

i) Identification of motile bacteria from non motile bacteria.

ii) Study of the pattern and arrangement of flagella on the surface of the bacterial cells.

Key Points:

• Flagella staining must require mordant.
• Less illumination is necessary for the visualization of bacterial flagella.
• Always use the specially cleaned grease-free slide.
• Do not blot dry the smear.

---

YOU MAY READ:

1. Negative Staining: Principle, Procedure, Results and Application
2. Endospore Staining: Principle, Procedure, Results and Example
3. Gram Staining: Principle, Theories, Procedure and Examples