Tests for Bacterial Motility: Procedure, Results

The motility test is used to determine whether an organism is motile or non-motile. Motile organisms contain flagella which helps them to travel beyond the point of inoculation. Motile bacteria are generally bacilli although a few motile cocci do exist. 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). Motility test helps us to differentiate between genera and species of bacteria.


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. For the wet preparation, a drop of the organism in broth is suspended on a clean glass slide, a coverslip is added, and the culture is observed microscopically for motility.

Occasionally the organism is inoculated in a semisolid motility medium in a straight line down through the center of a tube and is incubated. Motile organisms will cause turbidity throughout the tube as they migrate out from the line of inoculation whereas nonmotile organisms will grow only along the line of inoculation. In semi-solid agar media, motile bacteria ‘swarm’ and give a diffuse spreading growth that is easily recognized by the naked eye.

Quality Control

Test each newly prepared or purchased motility test media for sterility and performance using standard bacterial isolates.

Organism and ATCC no.MotilityIndoleH2S
Escherichia coli 25922++
Klebsiella pneumoniae 13883/27736
Proteus vulgaris 33420+++


  1. Prepare a semisolid agar medium in a test tube. Sulphide indole motility (SIM) medium or motility test medium with or without TTC (triphenyltetrazolium chloride) or motility nitrate medium can be used.
  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). A freshly prepared medium containing 1% glucose can be used for motility tests on anaerobes.


 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)

Motile organisms will migrate out from the line of inoculation, causing visible turbidity throughout the tube (corner tubes are motile). Nonmotile organisms will grow only along the line of inoculation (observe the tube at the center).

Reporting Results

  • Nonfermenting, Gram-negative rods, and Enterobacteriaceae vary in their motilities.
  • Bacillus spp. should be motile. Lack of motility could indicate Bacillus anthracis.
  • Enterococcus casseliflavus and Enterococcus gallinarum are motile.
  • Listeria is motile at 25°C with a characteristic umbrella-shaped growth at the top of the tube. On wet mount, they exhibit tumbling motility. They are non-motile at 35°C.
  • Yersinia enterocolitica is motile at 25°C but not at 35°C.
  • Acinetobacter are nonmotile.


In the laboratory, motility testing using a semi-solid medium is commonly used for the identification of gram-negative bacteria of the 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.

A motility test is also used for the species differentiation of gram-positive cocci, and 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.


  1. Mucoid Klebsiella strains may give a false-positive motile reaction in tube media; this is due to mucoid strains spilling between the medium and the tube, giving a cloudy appearance which is often confused with motility. False-positive results can be avoided by the use of media with adequate tube depth and careful reading with attention to the density of growth in the central stab.
  2. A large number of E. casseliflavus and E. gallinarum have been reported as nonmotile using some tube motility medium.
  3. False-negative reactions may occur if bacterial flagella are damaged due to heating, shaking, or other trauma. Such environmental shock will render the organism nonmotile.
  4. Some microorganisms do not produce flagellar proteins at 35 to 37° do so at 22°C.
  5. Triphenyltetrazolium chloride may be inhibitory to certain fastidious bacteria.

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.


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

References and further readings

  • Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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