Motility Test: Types, Principle, Method, Control & Interpretation

Table of Contents

Motility Test: Types, Principle, Method, Control & Interpretation

Motility of the organisms is an important characteristic to differentiate organism having similar biochemical characteristics e.g Klebsiella pneumoniae is non-motile, whereas Enterobacter cloacae is motile. (Both have similar biochemical reactions). Similarly B. anthracis is non-motile, whereas non-pathogenic Bacillus species are motile. It is useful in preliminary identification of B. anthracis isolates. Two methods are gives:

1. Wet Mount
2. The Tube Motility Test


Procedure :

1. Make suspension of a colony of test organisms in distilled water on a glass slide. Alternatively, a loop of medium from a fresh broth culture can be used. Put a cover glass on it. Examine under the microscope using hypochlorite solution as it contains live organisms.

Wet Mount Slide Test
Wet Mount Slide Test

2. Hanging Drop Method: Clean a cover slip. Apply Vaseline at its 4 corners. Put a drop of distilled water in the center and emulsify a colony of test organisms. Put the glass slide gently on it and hold it up side down. See under microscope with 10X and then 40X objective. Margins of drops are specifically seen. Motile organisms are clearly seen moving rapidly in the field. Non-motile organisms show to-and-fro Brownian motion, but these don’t move in relation to each other.

Hanging Drop Method
Hanging Drop Method



  • Using the sterile inoculating needle, remove suspicious colony of an 18-24 hour culture. Inoculate motility agar medium (Peptone water with 0.2% New Zealand agar, semisolid agar) carefully stabbing the needle 3-4 cm into the medium then withdrawing the needle so that a line of inoculum can be seen.
  • Incubate the tube aerobically at 35-37°C for 18-24 hour.
  • Tetrazolium salts may be added to motility media to make them easier to read.
  • The salt is colorless, but as the organisms grow, the dye gets incorporated into the bacterial cells, where it is reduced to an insoluble red pigment.


Non-motile organisms, such as B. anthracis, from a single line of growth that does not deviate from the original inoculum stab. Motile organisms from a diffuse growth zone around the inoculum stab.

Tube Motility Test
Tube Motility Test


Pseudomonas aeruginosa ATCC 35032 or equivalent is non-motile.


  • Control strains should be assayed on each day of testing.
  • Resolving an out-of-control result needs checking the purity, and identity of the control strains and repeating the test.
  • Tetrasolium salts may be added to motility medium to make them easier to read.
  • The salt is colorless but as the organism grows, the dye is incorporated into the bacterial cells where it is reduced to an insoluble red pigment formazan.


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