Urease Test

Objective:

To determine the ability of microorganisms to degrade urea by the enzyme urease.

Principle:

 Urea is a nitrogen containing compound that is produced during decarboxylation of the amino acid arginine in the urea cycle. Urea is highly soluble in water and is therefore an efficient way for the human body to discharge excess nitrogen. This excess urea is then taken out of the body through the kidneys as a component of urine. Some bacteria have the ability to produce an enzyme urease as part of its metabolism to break down urea to ammonia and carbon dioxide.
 Many enteric bacteria have the ability to hydrolyze urea as part of their metabolism, members of the genus Proteus are considered rapid urease producers due their efficiency in carrying out this process. Therefore, this experiment is useful in distinguishing members of Proteus, a urinary tract pathogen, from other enterics based on their ability to rapidly hydrolyze urea. Many enterics can hydrolyze urea but only a few can degrade  it rapidly. These are commonly referred as "rapid urease-positive" organisms. Members of the genus Proteus have the ability to hydrolyze urea rapidly.

  

Urea Hydrolysis:  Urea is waste product excreted in urine by animals. Some enteric bacteria produce the enzyme urease, which splits the urea molecule into carbon dioxide and ammonia. 

Urease, which is produced by some micro organisms, is an enzyme that is especially helpful in the identification of Proteus vulgaris, although other organisms may produce urease, their action on the substrate urea tends to be slower than that seen with Proteus species. Therefore this test serves to rapidly distinguish members of this genus from other lactose non fermenting enteric microorganisms.Urea is unstable and is broken down at 15 psi or pressure. It cannot be added to the medium for autoclaving and is therefore filter sterilized and added to the medium after autoclaving.

 Materials Required:

24 hours broth cultures (Proteus sp. and E.coli). 

Urease both / Urease Slant

Procedure: 

Using a sterile technique, inoculate each experimental organism into its appropriately labeled tube by means of loop inoculation. Incubate cultures 24-48 hours at 37°C.

Urea Broth method: -Inoculate the urea broth with the inoculation loop containing the organism from the broth cultures.-Incubate for 24-48 hours at 37°C. –-Obtain the broths from the incubator and observe the colour.

Urea Slant method:-Inoculate the urea slant (slope) with the inoculation loop containing the organism from the broth cultures. Do not stab the butt.-Incubate for 24-48 hours at 37°C.-Obtain the broths from the incubator and observe the color.

Result: -Proteus sp. gives urease positive reaction  -E.coli gives urease negative reaction

 Interpretation: Proteus sp. produces urease enzyme to degrade the urea to release the ammonia, it leads to alkaline condition of the medium. The phenol red indicator in the medium turns red pink color in alkaline condition and gives positive result.  E.coli does not produces urease enzyme and giving negative reaction.

Urea Broth Composition:

Urea      20g

Dibasic Sodium phosphate    9.5 g

Monopotassium phosphate    9.1 g of

Phenol red      0.01 g

The pH is made to   6.8±0.2 at 25°c.

Distilled water   1000 ml

  

Urea Slant Composition:

Same as above composition, but add agar as one of the ingredient.