Motility Tests for Bacteria: Principles, Procedures And Results

By Prof Walter Jaoko


Motility is one characteristic used in the identification of bacteria.The mobility in bacteria is largely credited to presence of large hair-like appendages over the body called as “flagella.” These are the locomotor organ of bacteria, though some move using axial filaments.Bacteria are classified into two: motile and non-motile. A motile bacteria have the ability to move in the surrounding medium while non-motile do not have the ability to move in the surrounding medium. However, the presences of bacteria/activity of flagella can be visualized by the ability of microbe to move in growth medium or by hanging drop technique.

Motility is determined by using a motility medium. The ingredients include motility test medium, nutrient broth powder, NaCl and distilled water. An inoculating needle (not a loop) is used to insert the bacterial sample. The needle is inserted through the medium for a length of one inch. The media tube incubated at 38 °C (100 °F). Bacteria that are motile grow away from the stab, and toward the sides and downward toward the bottom of the tube. Growth should be observed in 24 to 48 hours. With some species, the bacterium is inconsistent related to its motility. Coliform and Streptococci are examples of non-motile bacteria as are Klebsiella pneumoniae, and Yersinia pestis.

Principle Of Motility Test

Motility is the ability of an organism to move by itself by means of propeller-like flagella unique to bacteria or by special fibrils that produce a gliding form of motility. Motile  bacteria  move  using   flagella, thread like  locomotor  appendages  extending  outward  from  the plasma membrane and cell wall either single flagellum or multiple flagella. Each flagellum has a very rigid, helical structure and actual motility results from the rotation of the flagellum in a manner similar to that of a boat propeller. Motility by bacterium is demonstrated in semi solid agar medium.

The medium mainly used for this purpose is SIM medium (Sulphide Indole Motility medium) which is a combination differential medium that tests three different parameters, Sulfur Reduction, Indole Production and Motility. This media has a very soft consistency that allows motile bacteria to migrate readily through them causing cloudiness. The inoculum is stabbed into the center of a semisolid agar deep using a sterile inoculating needle.  Bacterial motility is evident by a diffuse zone of growth extending out from the line of inoculation. Some organisms grow throughout the entire medium, whereas others show small areas or nodules that grow out from the line of inoculation. The non-motile bacteria will only grow in the soft agar tube  and only the area where they are inoculated.

Composition Of SIM Medium (Sulphide Indole Motility Medium)

Composition Grams Per Litre
Pancreatic digest of casein20.0g
Peptic digest of animal tissue6.1g
pH7.3 ± 0.2 at 25°C


Requirements for Motility tests

  • Organisms tested-Campylobacter; Legionella; enterococci; Enterobacteriaceae; Listeria; Bacillus; other gram-positive rods; non-glucose-fermenting, gram-negative rods; and any other organism where motility is useful for identification
  • Broth medium likeTSB or BHI or Nitrate broth
  • Semi solid medium like SIM, MIU or MIO
  • 22- by 22-mm cover slips and microscope slides
  • Bright-field microscope or phase-contrast
  • Sterile inoculating needle or sticks


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

Results Interpretation

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

Limitations of Motility tests

  • Excessive heat on a microscope slide can affect the test results.
  • False-negative reactions may occur if bacterial flagella are damaged due to heating, shaking, or other trauma and such environmental shock will render the organism non-motile.
  • Some microorganisms do not express flagellar proteins at 35 to 37°C but do so at 22°C.
  • When inoculating semi-solid media, it is important that the inoculating needle be removed along the exact same line used to inoculate the medium. A fanning motion may result in growth along the stab line that may result in false-positive interpretation.
  • It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

Uses Of Motility Test

  • Motility test gives a distinct difference between motile and nonmotile bacteria. It also helps in the taxonomic classification of bacteria focusing on macroscopic and microscopic characteristics.
  • Motility helps in characterization or identification of pathogens or non-pathogens (as motility is one evidence of virulence factor), and also facilitates species-level differentiation. Example: Distinguishes between Enterococcus faecalis (non-motile) and Enterococcus gallinarum (motile).

Different Motility Test Methods

Motility-indole-lysine (MIL) medium


  • Peptone
  • Tryptone
  • Dextrose
  • L-lysine hydrochloride
  • Yeast extract
  • Ferric ammonium citrate
  • Agar
  • Bromcresol purple


  1. Dissolve the agar by boiling a liter of distilled water.
  2. Dispense 5ml aliquots in a test tube.
  3. Autoclave for 15 minutes at 121 degree Celsius under 15 psi pressure.
  4. Check for motility by using a sterile needle.
  5. Pick an isolated colony and stab the medium to within 1 cm of the bottom of the test tube.
  6. Incubate for 18 hours at 35 degree Celsius or until such time that growth is noticeable. 

Results Interpretation

Positive – A diffuse cloud of growth away from the inoculation line is evident. What is good about this procedure is it does not only check for bacteria’s motility, but it also helps determine other metabolic characteristics. 

Hanging drop method


  • Cavity slide
  • Coverslip
  • Lubricant (petroleum jelly)
  • Broth culture of bacteria
  • Immersion oil
  • Loop
  • Microscope


  1. Petroleum jelly is applied around the clean and dry cavity slide.
  2. The loop is put over the flame and allow to cool. Using the sterilized loop, a bacteria is taken from the broth culture.
  3. A drop of suspension is put at the coverslip’s center.
  4. The slide is inverted and put on the coverslip. The two are pressed gently to seal the cavity. Make sure that not a single part touches the drop.
  5. The slide is inverted so that the drop hangs into the cavity.
  6. The slide is then clipped to the stage and will be examined under the low power objective of the microscope.

Result Interpretation

With the hanging drop method, the bacteria will be checked for its motility, shape, arrangement, and size.

Observations (Under Oil-immersion Objective)


  • Motile or non-motile

Shape of bacteria

  • Spherical (coccus)
  • Rod-shaped (bacilli)
  • Comma-like (vibrio)
  • Spiral (spirochetes)

Arrangement of bacteria

  • Pairs (diplobacillus/diplococcus)
  • In fours (tetrads)
  • In chains (streptococcus/streptobacillus)
  • Grape-like clusters (staphylococcus)
  • Cuboidal (sarcinae or octet).

Sulfide-indole-motility (SIM) medium


  • Beef extract
  • Peptone
  • Agar
  • Ferrous ammonium sulphate
  • Sodium thiosulfate


  1. Bring to boil a liter of distilled water and dissolve the agar.
  2. Dispense 5 ml aliquots in a test tube.
  3. Autoclave for 15 minutes at 121°C under 15 psi pressure.
  4. Test for motility using a sterile needle. Pick an isolated colony and stab the medium to within 1 cm of the bottom of the test tube.
  5. Keep the needle in the same line as it is inserted and removed from the medium.
  6. Incubate for 18 hours at 37 degree Celsius or until you see noticeable growth. (9, 10)

Results Interpretation

The test is positive if there is a diffuse cloud of growth away from the inoculation line. This procedure does not only check for bacteria’s motility but also other metabolic characteristics.

Motility-indole-ornithine (MIO) medium


  • Yeast extract
  • Tryptone
  • Peptone
  • Dextrose
  • L-ornithine HCl
  • Bromcresol purple
  • Agar


  1. Dissolve agar by bringing to boil a liter of distilled water.
  2. Dispense 5ml aliquots in a test tube.
  3. Autoclave for 15 minutes at 121°C under 15 psi pressure.
  4. Use a sterile needle to collect an isolated colony of bacteria.
  5. Stab the medium to within 1 cm of the bottom of the test tube.
  6. See to it that the needle is aligned as it is inserted and removed from the medium.
  7. Incubate for at least 18 hours at 35 degree Celsius or until such time that growth is noticeable. 

Results Interpretation

The test is positive if a diffuse cloud is formed away from the inoculation line. This type of bacteria motility test does not only determine the motility of bacteria but also determines other metabolic characteristics of bacteria.

Further References

  1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University press.
  2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  3. Clinical Microbiology Procedure Hand book, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  4. Colour Atlas and Text book of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
  5. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  6.  Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier