MIU Test: Introduction, Principle, Composition, MIU Agar Preparation, Test Procedure, Result-Interpretation, and Keynotes

Introduction of MIU Test

MIU test stands for motility indole urease test and it is used for three tests among them one for motility of bacteria, second for indole formation, and third for urea hydrolyzation test of bacteria. This assay is useful for the identification of gram-negative bacilli especially bacteria of  Enterobacteriaceae. Three tests in a single test tube assist to differentiate the organisms on the basis of motility,  indole, and urease production.

Principle of Motility, Urease, Indole 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. Bacteria possess urease, hydrolyze urea, and release ammonia and carbon dioxide. Ammonia reacts in solution to form ammonium carbonate, which is alkaline leading to an increase in the 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 a 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
Distilled Water	100 ml
Final pH ( at 25°C)	6.8±0.2

Peptones of the medium provide the carbon and nitrogen required for the growth of bacteria. Urea is responsible as 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 the motility of bacteria. The indole production from Casein enzymic hydrolysate by the tryptophanase present in test organisms is achieved upon the addition of aldehyde present in Kovac’s reagent; shown by the appearance of a pink-red color ring in the tube.

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 of distilled water.
• Mix properly to dissolve the medium completely and add the remaining 50 ml of 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 MIU 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
• Proteus mirabilis ATCC 25933

Test Procedure for MIU 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.
4. Repeat steps 1 to 3 for all quality control strains i.e. Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, and Proteus mirabilis ATCC 25933.

Observation of MIU Test

Observe the tube for growth, motility, and color change of the medium. Only check indole formation taking a 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 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 the addition of Kovac’s reagent
• Indole test negative: A yellow color denotes a negative indole test after the 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

Keynotes on MIU Test

1. Urea test or urease test or urea hydrolyzation test for bacteria is the same thing.
2. The motility and urease reactions are read before testing Indole production.
3. 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: Betty 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.