Skip to main content

Urease Test

By

Urease Test
The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme urease.
Image result for bacteriology

Principle 

Urea is the product of decarboxylation of amino acids. Hydrolysis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid urease-positive organisms turn the entire medium pink within 24 hours.
Weakly positive organisms may take several days, and negative organisms produce no color change or yellow as a result of acid production.

Uses 
1.    This test is used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease.
2.    This test can be used as part of the identification of several genera and species of Enterobacteriaceae, including Proteus, Klebsiella, and some Yersinia and Citrobacter species, as well as some Corynebacterium species.
3.    It is also useful to identify Cryptococcus spp., Brucella, Helicobacter pylori, and many other bacteria that produce the urease enzyme.
4.    Directly, this test is performed on gastric biopsy samples to detect the presence of H. pylori.
Image result for urease test principle
Media 
Composition
Urea                                       20.0 gm

Sodium Chloride                   5.0 gm
Monopotassium Phosphate   2.0 gm
Peptone                                 1.0 gm
Dextrose                                1.0 gm
Phenol Red                            0.012 gm
Agar                                      15.0 gm
Final pH 6.7 +/- 0.2 at 25 degrees C.

Preparation
1.    Dissolve the ingredients in 100 ml of distilled water and filter sterilize (0.45-mm pore size).
2.    Suspend the agar in 900 ml of distilled water, boil to dissolve completely.
3.    Autoclave at 121 degree C and 15 psi for 15 minutes.
4.    Cool the agar to 50 to 55 degree C.
5.    Aseptically add 100 ml of filter-sterilized urea base to the cooled agar solution and mix thoroughly.
6.    Distribute 4 to 5 ml per sterile tube (13 x 100 mm) and slant the tubes during cooling until solidified.
Procedure 
1.    Streak the surface of a urea agar slant with a portion of a well-isolated colony or inoculate slant with 1 to 2 drops from an overnight brain-heart infusion broth culture.
2.    Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air for 48 hours to 7 days.
3.    Examine for the development of a pink color for as long as 7 days.
Result 
Image result for urease test principle
Positive Reaction: Development of an intense magenta to bright pink color in 15 min to 24 h.
Examples: Proteus spp, Cryptococcus spp, Corynebacterium spp, Helicobacter pylori, Yersinia spp, Brucella spp, etc.
Negative Reaction:  No color change.
Examples: Escherichia, Shigella, Salmonella, etc.
Quality Control 
Positive: Proteus vulgaris (ATCC13315)
Weak positive: Klebsiella pneumoniae (ATCC13883)
Negative: Escherichia coli (ATCC25922)

References
www.time2026end.com

Labels:

Comments

Post a Comment

Popular posts from this blog

Mueller Hinton Agar

By
Mueller Hinton Agar (MHA)       The major use of Mueller Hinton Agar is for antimicrobial susceptibility testing. Composition of MHA Ingredients   Beef Extract                                  2.00 gm Acid Hydrolysate of Casein         17.50 gm Starch                                           1.50 gm Agar                                             17.00 gm Distilled Water                             1000 ml Final pH 7.3 ± 0.1 at 25ºC Why Mueller Hinton agar is used for antibiotic susceptibility testing? 1.     It is a non-selective, non-differential medium. This means that almost all organisms plated on here will grow. 2.     It contains starch. Starch is known to absorb toxins released from bacteria, so that they cannot interfere with the  antibiotics . It also mediates the rate of diffusion of the antibiotics through the agar. 3.     It is a loose agar. This allows for better diffusion of the antibiotics than most other plates. A bette
Post a Comment
Read more

Simmons Citrate Test

By
Simmons Citrate Reaction It  used to differentiate among the Gram-Negative bacilli in the family Enterobacteriaceae. Principle  Citrate agar is used to test an organism’s ability to utilize citrate as a source of energy. The medium contains  citrate  as the sole  carbon source  and  inorganic ammonium salts (NH4H2PO4)  as the sole source of  nitrogen . Bacteria that can grow on this medium produce an enzyme,  citrate-permease , capable of converting  citrate  to  pyruvate .  Pyruvate  can then enter the organism’s metabolic cycle for the production of  energy . Growth is indicative of utilization of citrate, an intermediate metabolite in the  Krebs cycle . When the bacteria metabolize  citrate , the  ammonium salts  are broken down to  ammonia , which increases  alkalinity . The shift in pH turns the  bromthymol blue  indicator in the medium from  green to blue  above pH 7.6. Christensen  developed an alternative citrate test medium that does not require the organism
Post a Comment
Read more