Abbreviated (MIU), Motility Indole Urea Medium base is used for the differentiation of Enterobacteriaceae. This medium is based on motility, urease and indole production. This medium contains peptones that provide carbon and nitrogen required for growth of bacteria. Urea provides a source of nitrogen for those microorganisms producing urease. This is indicated by a color change of the pH indicator, Phenol Red, from yellow – orange, (pH 7,0) to red – pink, (pH 8.1). The low agar concentration allows the demonstration of motility. The indole Cap is impregnated with indole Reagent for the detection of indole production, shown by the appearance of a pink – mauve colour in the bottom of the cap.
Principle and Interpretation
As earlier said, it is formulated to detect motility, urease and indole production in single tube.
- 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.
The test cultures are stab-inoculated. Motile organisms show either diffused growth or turbidity extending away from stab inoculation line while non-motile organisms grow along the stab-line. Those organisms able to utilize urea, produces ammonia which change the pH of the medium alkaline, showing pink-red colour by change in the phenol red indicator. Indole is produced from tryptophan present in casein enzymic hydrolysate .The indole produced combines with the aldehyde present in the Kovac’s reagent to form a red complex.
Composition of Motility Indole Urea
|Casein enzymic hydrolysate||10.000|
|Final pH ( at 25°C)||6.8±0.2|
How to Prepare Motility Indole Urea Agar
The preparation of MIU Agar depends on each laboratory workload or the numbers of culture plates available. Anyway, note that a standard Petri dish is equivalent to 20ml when poured completely.
- Suspend 18 grams in 950 ml distilled water.
- Heat to boiling to dissolve the medium completely.
- 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 into sterile test tubes.
- Allow to cool in an upright position.
NB: It is also advisable to incubate a petri dish for 24hour for quality control in order to avoid wastage of resources and ease early detection of contamination after the preparation.
NB: The medium is heat sensitive. No further sterilization is necessary or desirable.
Appearance of Motility Indole Urea after preparation
Yellowish orange colored clear to slightly opalescent gel is obtained in tubes as butts after the addition of urea solution.
Type of Inoculums
- water samples
- Pick isolated colonies with an inoculating needle and stab the medium to the bottom of the tube.
- Discard the plastic cap and recap the tube with the Indole Lignin Cap
- Incubate at 35±2ºC for 18-24 hours.
Reading of Colonies on Motility Indole Urea
Cultural characteristics observed after an incubation at 35-37°C for 18 – 24 hours.
- Examine the tubes for growth, motility and color change of the medium and of the cap, as follows :
Urea – A color change from yellow –orange to pink-red indicates a positive reaction. No color change indicates a negative reaction.
Motility – 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 – Record as Indole Positive Reaction if a pink-mauve color appears on the bottom of the lignin cap and as Indole Negative if there is no color on the cap.
NB : The motility and urease reactions are read before testing Indole production.
|Escherichia coli||Positive reaction, red ring at the interface of the medium||Positive, growth away from stabline causing turbidity||Negative reaction,no change|
|Klebsiella pneumoniae||Negative reaction no colour development / cloudy ring||Negative growth along the stabline,surrounding medium remains clear||Weakly positive|
|Proteus mirabilis||Negative reaction no colour development / cloudy ring||Positive, growth away from stabline causing turbidity||Positive reaction, cerise colour|
|Proteus vulgaris||Positive reaction, red ring at the interface of the medium||Positive, growth away from stabline causing turbidity||Positive reaction, cerise colour|
|Salmonella typhimurium||Negative reaction no colour development / cloudy ring||Positive, growth away from stabline causing turbidity||Negative reaction,no change|
• Rustigian and Stuart (1941) Proc. Soc. Exp. Biol. Med., 47:108.
• McFaddin J.F. (1985) Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams and Wilkins, Baltimore
• Ewing (1986) Edwards and Ewings ‘Identification of Enterobacteriaceae’, 4th ed. Elsevier Science Publishing Co., Inc., New York.