Motility Test: Introduction, Principle, Composition, Procedure, Result Interpretation and keynotes

Motility Test: Introduction, Principle, Composition, Procedure, Result Interpretation and keynotes

Introduction of Motility Test

The motility test of bacteria can be tested using SIM medium, MIU medium, liquid medium like peptone water, nutrient broth, tryptone broth, BHI broth, and direct from the specimen (feces)  hanging drop preparation. MIU stands for Motility, Indole, and Urease. In this chapter, the motility test of bacteria can be discussed in detail using MIU.  The test is useful for the identification of gram-negative bacilli especially bacteria of  Enterobacteriaceae. Three tests in a single tube help to differentiate the organisms on the basis of motility, urease, and indole production.

List of some motile bacteria-

Gram positive bacteria-

  • Bacillus species ( except Bacillus anthracis)
  • Enterococcus casseliflavus
  • Enterococcus  gallinarum

Gram Negative bacteria

List of Non-moltile bacteria

Gram positive bacteria

Gram negative bacteria


 Principle of Motility Test

The test organisms in MIU agar after incubation show either diffused growth or turbidity extending away from the stab inoculation line in the case of motile organisms while non-motile organisms appear as restricted growth along the stab-line.

Composition and Function of Constituents

Composition of Motility Indole Urea agar for 100 ml distilled water

Ingredients                                amount

  • Casein enzymic hydrolysate :  1.0 gm
  • Dextrose :   0.1 gm
  • Sodium chloride :  0.5 gm
  • Phenol red: 10 mg
  • Agar:  0.2 gm
  • Final pH ( at 25°C): 6.8±0.2

Peptones of the medium provide carbon and nitrogen required for the growth of bacteria. Urea is responsible a source of nitrogen for those organisms that possess the enzyme, urease. Casein enzymic hydrolysate provides amino acids and other nitrogenous substances. Sodium chloride maintains osmotic equilibrium. Dextrose is a fermentable carbohydrate. Phenol red is the pH indicator that turns pink-red in alkaline conditions. Urease is indicated by a color change of the pH indicator, Phenol Red, from yellow-orange, (pH 6.8) to red-pink, (pH 8.4). The low agar concentration i.e. 0.2% is useful for the demonstration of motility of bacteria.

MIU Agar Preparation

  • The amount of MIU agar preparation is directly dependent on the test workload.
  • For making 100 ml, Suspend 1.8 grams in 50 ml distilled water.
  • Mix properly to dissolve the medium completely and add the remaining 50 ml distilled water.
  • Again mix properly.
  • Dispense in 95 ml amounts into flasks and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  • Cool to about 50-55°C and add aseptically 5 ml sterile 40% urea solution per 95 ml basal medium.
  • Mix well and dispense 5-5 ml into sterile test tubes (12×75 mm).
  • Allow cooling in an upright position.
  • Use loose-fitting cotton plugs in all test tubes.

Requirements for Motility Test

  • Test organisms (Gram-negative bacilli)
  • MIU test medium
  • Inoculating wire
  • Bunsen burner
  • Incubator

Quality control strains

  • Escherichia coli ATCC 25922
  • Klebsiella pneumoniae ATCC 13883

Test Procedure of Motility Test

  1. Take a well-isolated single colony with an inoculating needle and stab the medium leaving 1/3 part from the bottom of a tube.
  2. Use a loose-fitting cotton plugin as a test tube.
  3. Incubate at 37°C for 18-24 hours.

Observation of Motility Test

Observe the tube for growth. Motility test – A positive reaction is shown by clouding of the medium or by growth extension from the inoculating line. A negative reaction is seen when the growth is restricted to the inoculating line.

Result and Interpretation of Motility Test

Motility test positive: A diffuse zone of growth flaring from the line of inoculation

Motility test Negative: restricted growth along the stab line

Escherichia coli ATCC 25922

Motility test: Positive

Klebsiella pneumoniae ATCC 13883

Motility test: Negative

Keynotes on Motility Test

  • Urea test or urease test or urea hydrolyzation test for bacteria is the same thing.
  • The motility and urease reactions are read before testing Indole production.
  • The medium is heat-sensitive. No further sterilization is necessary or desirable.

Further Readings

  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 Handbook Vol. I & II, 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 Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
  5. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
  6. 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.
  7.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
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