Flagella Stain- Principle, Procedure and Result Interpretation

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Objective of Flagella Stain

This technique is used to visualize the presence and arrangement of flagella for the presumptive identification of motile bacterial species.

Principle of Flagella Stain

Flagella are too thin to be visualized using a bright field microscope with ordinary stains, such as the Gram stain, or a simple stain. A wet mount technique is used for staining bacterial flagella, and it is simple and useful when the number and arrangement of flagella are critical to the identification of species of motile bacteria. The staining procedures require the use of a mordant so that the stain adheres in layers to the flagella, allowing visualization.

The procedure of Flagella Stain (Wet Mount Technique)

  1. Grow the organism to be stained at room temperature on blood agar for 16 to 24 hours.
  2. Add a small drop of water to a microscope slide.
  3. Dip a sterile inoculating loop into sterile water.
  4. Touch the loopful of water to the colony margin briefly (this allows motile cells to swim into the droplet of water).
  5. 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.
  6. 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.
  7. Examine the slide immediately under 40× to 50× for motile cells. If motile cells are not seen, do not proceed with the stain.
  8. 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.
  9. Gently apply 2 drops of RYU flagella stain (Remel, Lenexa, Kansas) to the edge of the coverslip. 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.
  10. After 5 to 10 minutes at room temperature, examine the cells for flagella.
  11. 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.
  12. 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 Interpretation of Flagella Stain

  1. Presence or absence of flagella
  2. Number of flagella per cell
  3. Location of flagella per cell
    1. Peritrichous
    2. Lophotrichous
    3. Polar
  4. The amplitude of wavelength
    1. Short
    2. Long
  5. Whether or not “tufted”

Limitation

Even with a specific stain, visualization of flagella requires an experienced laboratory scientist and is not considered an entry-level technique.

Quality Control

Peritrichous: Escherichia coli
Polar: Pseudomonas aeruginosa
Negative: Klebsiella pneumoniae

About Author

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Sagar Aryal, PhD

Dr. Sagar Aryal is a microbiologist, researcher, and science communicator from Nepal. He is also co-founder and Research Head of the Department of Natural Products at the Kathmandu Research Institute for Biological Sciences (KRIBS), where he leads research on natural product discovery and microbial biodiversity. Dr. Aryal earned his Ph.D. in Microbiology from Tribhuvan University, Kathmandu, Nepal, in 2025, supported by a DAAD Research Grant and a University Grants Commission (UGC) Nepal Ph.D. Fellowship. His research interests include Actinomycetes, Myxobacteria, microbial taxonomy, and natural products, particularly the discovery of bioactive compounds with potential pharmaceutical applications. He isolated and identified Nocardia nepalensis, a novel bacterial species from Nepal, contributing to understanding microbial diversity in the region. Dr. Aryal has published more than 16 research articles in international journals and contributed three book chapters to established academic publications. He has presented scientific research findings and developed international research collaborations through visits to more than 18 countries across Asia, Europe, North America, and Oceania. From 2023 to 2025, he served as the American Society for Microbiology (ASM) Young Ambassador to Nepal. Since December 2025, he has served as an Editorial Board Member of Scientific Reports, a Nature Portfolio journal. In recognition of his scientific contributions, Dr. Aryal received the NAST Science and Technology Youth Award 2025 from the Nepal Academy of Science and Technology. He previously received the Frs. Locke and Stiller Research Award (LSRA) for the 2017-2018 cycle from St. Xavier's College, Kathmandu, and a Nepal Academy of Science and Technology (NAST) Research Grant for 2017-2018. Alongside his scientific career, Dr. Aryal has more than 15 years of experience in blogging, content writing, and search engine optimization (SEO).