Protocol for Urease Test

Protocol for Urease Test:

Stuart’s Urea Broth :

Ingredient:

Yeast extract:  0.1 g

Potassium phosphate, monobasic:  9.1 g

Potassium phosphate, dibasic: 9.5 g

Urea: 20 g

Phenol red: 0.01 g

Ø Dissolve all ingredients in 1 liter of distilled water and filter sterilize (0.45-mm pore size). Distribute 3 ml of prepared broth per sterile tube (13 x 100 mm). Prepared media will have a yellow-orange color. Store the prepared broth in the refrigerator at 4 to 8^oC until needed. Do not heat the medium as the urea will decompose.

Procudure:

• Use a heavy inoculum from an 18- to 24-hour pure culture to inoculate the broth. Shake the tube gently to suspend the bacteria. Incubate tubes with loosened caps at 35^oC. Observe the broth for a color change at 8, 12, 24, and 48 hours. Urease production is indicated by a bright pink (fuchsia) color throughout the broth.

• Rapidly urease-positive Proteeae (Proteus spp., Morganella morganii, and some Providencia stuartiistrains) for which this medium is differential, will produce a strong positive reaction as early as 8 hours, but always within 48 hours of incubation. Delayed-positive organisms (e.g., Enterobacter) will not produce a positive reaction due to the high buffering capacity of this medium.

  

  Urease Test