MIU test: Uses, principle, composition, procedure, result interpretation and key notes

MIU test

Use of MIU test

MIU stands for Motility Indole Urea. MIU  test is useful for the identification of gram-negative bacilli specially bacteria  of Enterobacteriaceae. Three tests in a single tube that helps to differentiate the organisms on  the basis of motility, urease and indole production.

MIU test Principle

The test organisms in MIU agar after incubation show either diffused growth or turbidity extending away from stab inoculation line in case of motile organisms while non-motile organisms appear as restricted growth along the stab-line. Organisms  possess urease, hydrolyses urea and releases ammonia and carbon dioxide. Ammonia reacts in solution to form ammonium carbonate ,which is alkaline leading to an increase in pH of the test medium. Phenol red  present  in the medium changes its color from yellow to pink- red in alkaline pH. Indole, skatole and indole acetic acid are  produced from tryptophan present in casein enzymic hydrolysate by the enzyme, tryptophanase .The indole formed reacts with p-dimethyl amino benzaldehyde present in Kovac’s reagent to form quinoidal red-violet compound.

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 growth of bacteria. Urea is responsible for a source of nitrogen for those organisms possess enzyme, urease. Casein enzymic hydrolysate provides amino acids and other nitrogenous substances. Sodium chloride maintains osmotic equilibrium. Dextrose is the 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. The indole production from Casein enzymic hydrolysate by the tryptophanase present in test organisms is achieved upon addition of aldehyde present in Kovac’ reagent; shown by the appearance of a pink –red color ring in the tube.

MIU Agar preparation

  • The amount MIU agar preparation is directly depend on test workload.
  • For making 100 ml, Suspend 1.8 grams in 50 ml distilled water.
  • Mix properly to dissolve the medium completely and add 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 to cool in an upright position.
  • Use loose-fitting cotton plugs in all test tubes.

Requirements for MIU test

Test organisms (Gram negative bacilli)

MIU test medium

Inoculating wire

Bunsen burner


Quality control strains

Escherichia coli ATCC 25922

Klebsiella pneumoniae ATCC 13883

Proteus mirabilis ATCC 25933

Test Procedure for MIU test

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


Observe  the tube for growth, motility and color change of the medium. Only check indole formation taking reading of motility and urease reaction. Urease test positive A color change from yellow –orange to pink-red. No color change indicates a negative reaction. 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. Indole test positive Record as Indole Positive Reaction if a pink-red color ring appears in a test tube upon addition of Kovac’s reagent and as Indole Negative if there is no color.

Result and Interpretation of MIU test

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

Motility test Negative: restricted growth along the stab line

Indole test positive:  a pink to red color ring  is formed at the top of the medium after addition of Kovac’s reagent

Indole test negative: A yellow color denotes a negative indole test after addition of Kovac’s reagent

Urease test positive: A color change from yellow –orange to pink-red

Urease test negative: No color change indicates a negative reaction

Escherichia coli ATCC 25922

Motility test: Positive, Indole test: positive and Urease test: negative

Klebsiella pneumoniae ATCC 13883

Motility test: Negative, Indole test: negative and Urease test: weakly positive

Proteus mirabilis ATCC 25933

Motility test:positive, Indole test: negative and Urease test:  positive

Key notes

  • Urea test or urease test or urea hydrolization test for bacteria is 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.
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