Tests for Bacterial Motility: Procedure and Results

Test for Bacterial Motility: Center (Non-motile); Corners (Motile)

There are a variety of ways to determine the motility of a bacterium—biochemical tests as well as microscopic analysis. If a fresh culture of bacteria is available, microscopy is the most accurate way to determine bacterial motility, and  hanging drop method is a commonly used microscopic technique.

Motile bacteria move with structures called flagella (a few exceptional bacteria move with the help of axial filaments, which cannot be seen in the microscope). In semi-solid agar media, motile bacteria ‘swarm’ and give a diffuse spreading growth that is easily recognized by the naked eye.


  1. Prepare a semisolid agar medium in a test tube.
  2. Inoculate with a straight wire, making a single stab down the center of the tube to about half the depth of the medium.
  3. Incubate under the conditions favoring motility.
  4. Incubate at 37°C
  5. Examine at intervals, e.g. after 6 h, and 1 and 2 days  (depends on generation time of bacteria). Freshly prepared medium containing 1% glucose can be used for motility tests on anaerobes.

Results: Hold the tube up to the light and look at the stab line to determine motility.

  1. Non-motile bacteria generally give growths that are confined to the stab-line, have sharply defined margins, and leave the surrounding medium clearly transparent.
  2. Motile Bacteria typically give diffuse, hazy growths that spread throughout the medium rendering it slightly opaque.

    Test for Bacterial Motility: Center (Non-motile); Corners (Motile)
    Test for Bacterial Motility: Center (Non-motile); Corners (Motile)

Laboratory Use

In the laboratory, motility testing using a semi-solid medium is commonly used for the identification of gram-negative bacteria of Enterobacteriaceae family.  Motility testing is done in conjunction with other biochemical testing using special biochemical media.

  1.  Sulfide Indole Motility (SIM) Medium: It is a semisolid agar used to determine hydrogen sulfide (H₂S) production, indole formation, and motility.
  2. Motility Indole urease (MIU) test: It is used to determine motility, indole formation, and urease production (Urease Test.)

Motility test is also used for the species differentiation of gram-positive cocci, enterococci.  Enterococcus faecium and E. faecalis are non-motile, whereas E. gallinarum and E. casseliflavus/E. flavescens generally are motile.

Note: Incorporation of tetrazolium chloride at a final concentration of 0.005% in the medium is helpful. The tetrazolium makes the motility agar much easier to read for motility. Tetrazolium is colorless in oxidized form, but the reduced salt is red (occurring as a result of bacterial metabolism) and indicates where bacterial growth has occurred.

Specific tests for bacterial motility determination

  1. Distilled water motility test: It is a simple and very useful test to differentiate Vibrio species (gram-negative motile curved rod) and Aeromonas species (gram-negative motile rod). Aeromonas species will grow on MacConkey agar and sometimes on TCBS, producing yellow colonies.  Both of them are oxidase positive.


  1. Mix a loopful of growth from a nutrient agar subculture in a drop of sterile distilled water on one end of a slide. On the other end of the slide, mix another loopful of growth in a drop of peptone water.
  2. Cover each preparation with a cover glass.
  3. Examine microscopically using the 40x objective.

Results: All Vibrio species are immobilized in distilled water but remain motile in peptone water. Aeromonas species remain motile in distilled water and peptone water.

About Acharya Tankeshwar 460 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.


  1. Dear Tankeshwar,

    Please could you let me know if it possible for one to use the picture you have for showing motility testing in semi solid agar?

    Many thanks,

  2. Please can the motility test using sterile distilled water and peptone also be used for pseudomomas species?

  3. Hello I need help with this question, but mostly the last part, about if should the motility results be identical for boths tests?
    here is que complete questions:Why are we inoculating a separate motility tube, if we are already checking for motility with the SIM tubes? Should the motility results be identical for both tests?
    Thanks for your help
    Sandra Head

    • Dear Sandra Head,
      As you already know there are various methods available to test the bacterial motility from microscopic methods to inoculation in semi-solid media; the purpose always being differentiation of two or more than two test isolates on the basis of “motility” property. So we choose one of the available methods which help us to serve that purpose well

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