Motility Test

Motility test can consider as the biochemical or microscopic examination of an organism to check the existence of cellular-motility. By performing this test, one can differentiate between the two major groups of bacteria, namely motile and non-motile, based on their cellular movement.

Few organisms are motile, whereas few are non-motile, but all the living organisms tend to show certain kind of movement in search of food, against environmental stress etc., for which locomotion is a common phenomenon. An organism’s locomoting through various modes of locomotion; some show flagellar movement while few shows non-flagellar locomotion.

Contents: Motility Test

  1. Definition
  2. Purpose
  3. Principle
  4. Test Media
  5. Methods
  6. Uses
  7. Limitations


Motility test can define as the analytical method, which examines whether the bacteria are motile or non-motile through biochemical and microscopic analysis. Tube test is a biochemical method giving macroscopic and cumulative results obtained by the reduction of the biological media (triphenyl tetrazolium chloride) by the positive or motile organisms. Slide test is a microscopic analysis of a microorganism that involves direct examination through hanging drop and wet mount slide test, based on the cell’s microscopic features.


  • Motility test aims to check the cellular motility of a test organism.
  • It provides a valuable tool to determine an organism’s ability to locomote within the liquid or semi-solid media.
  • Motility test also distinguishes different kinds of bacteria.
    Example: Like Yersinia enterocolitica tends to be motile at a temperature of 20-25 degrees Celsius. Capnocytophyta is the bacteria that move via gliding motility, whereas few bacteria like Campylobacter species are non-motile.

Principle of Motility Test

Motility test depends upon the organism’s tendency to move either in liquid or semisolid media. Eukaryotes locomote through the flagella, cilia, pseudopodia, whereas prokaryotes locomote through the propeller-like flagella and sometimes glide through the protein fibrils. Bacteria also show chemotaxis property, by surrounding themselves near to the chemical compounds like sugar, amino acid etc.

Motility test makes the use of triphenyl tetrazolium chloride media, which in oxidized form remains colourless. When the bacteria grow in this medium, they absorb the dye by causing reduction of the media. In a reduced state, the bacterial growth is indicated by the formation of an insoluble red coloured complex (Formazon). The motility is detected by the immense red colour formation around the line of inoculation.

Test Media

Motility test requires the use of a standard media like TTC (triphenyl tetrazolium chloride) that contains the following ingredients like:

Beef extract: 3 grams
Pancreatic digest of casein: 10 grams
Sodium chloride: 5 grams
Agar: 4 grams
Distilled water: 1 litre

TTC is the most popular media to check whether the test organism is motile or non-motile, and also available as pre-poured tubes by the biological company suppliers. The motility of an organism can also be tested by the use of multitest media like (MIL) motility indole lysine, (MIO) motility indole ornithine, (SIM) Sulfide indole motility test, and Mannitol motility media etc.

Methods for Motility Test

There are two standard methods to perform motility test:
Methods for motility test

Tube Test

It involves the following steps:

Media preparation: Take all the ingredients required and weigh accurately. Dissolve the contents in a sterile flask by adding the desired volume of sterile water. Autoclave the solution for 15-20 minutes at 121 degrees Celsius. After that, pour 5ml of the TTC media to each test tubes, and allow it to solidify.

Bacterial inoculation: Take sterile inoculating loop, and take out a well-isolated colony of the test organism straight into the media.

Incubation: Allow the culture tubes to incubate at 35-37 degrees Celsius for at least 24-48 hours.
Tube motility test
Observation: Observe the tube for the diffusion of a red coloured zone around the line of inoculation.
Test results: Diffused growth occurs around the stab line indicates a positive outcome, whereas no growth around the route of inoculation represents a negative test result.
Accuracy: The accuracy of the tube motility test depends upon the inoculating needle (should be straight) and handling of the process.
Test results of tube motility test

Slide Test

It includes two methods, namely hanging drop and wet mount slide test.

Hanging drop method: It is a method of microscopic examination that confirms the motility of the organism. Hanging drop is a direct method that includes the following steps:
hanging drop slide test

  1. First, take a clean microscopic depression slide.
  2. Then spread the vaseline petroleum jelly towards the vicinity of the depression wall.
  3. Then take a clean, dust-free coverslip, and add a loopful of bacterial suspension towards the centre of the coverslip.
  4. After that, place the cavity region of the glass slide facing towards the bacterial suspension on the coverslip.
  5. Invert the glass slide and press the coverslip and cavity slide simultaneously.
  6. Finally, observe the glass slide under the microscope to check the presence of motility.

Wet mount method: It is also a direct method to identify the motility in bacteria. Wet mount method involves the following steps:

wet mount slide test

  1. Take a clean, dust-free microscopic glass slide.
  2. Add a drop of saline at the middle of the slide.
  3. Inoculate a well-isolated colony of the test organism and mix the inoculum with a drop of saline.
  4. Place coverslip to the one end of a glass slide, and slowly keep it over the test sample with the help of a needle.
  5. Press the coverslip to remove excess moisture or water bubbles by the use of blotting paper.
  6. At last, observe the glass slide under the microscope to check the motility of the test organism.


Motility test distinct microorganisms as motile or non-motile, based on their motility. It also helps in the taxonomic classification of bacteria focussing on macroscopic and microscopic characteristics.

Motility test aids in characterization or identification of pathogens or non-pathogens (as motility is one of virulence factor), and also facilitates species-level differentiation.
Example: Distinguishes between Enterococcus faecalis (non-motile) and Enterococcus gallinarum (motile).


In tube motility test, the inoculating needle should be taken out along the same line of inoculation otherwise, it may give false-positive growth. The water condensation in the tube motility test may lead to the false-positive results, and weakly motile organisms may give contrast result interpretation.

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