Urease Test

Background

The purpose of this experiment is to successfully perform the Urease test on a selected microorganism. Urease is an enzyme that breaks the bond between nitrogen and hydrogen in amide compounds which is commonly found in urea. This test is ideal when you are trying to identify P. vulgaris. This organism produces urease at a much higher rate than other organisms that also produce urease. The test works by changing color in the presence of ammonia, which is a byproduct of the breakdown of urea by urease. The ammonia changes the pH of the urea broth. This causes phenol red that is in the urea broth to change the broth color from yellow to a deep pink. The Urease test is used in a hospital setting to differentiate between types of enteric bacteria. Urease positive enteric bacteria are generally more pathogenic (Cappuccino 187).

Materials and Methods

The materials for this experiment include: three tubes of Trypticase soy broth cultures each containing one of the following: E. coli, P. vulgaris and E. faecalis; three tubes containing urea broth; a Bunsen burner, inoculating loop, tube rack, tape, and a marker.

The procedure consist of the following steps. We received the urea broth from the group that made the broths for this experiment. We then turned on the Bunsen burner and put the test tube rack beside it. With the inoculation loop in one hand and a tube of organism in the other hand we flamed the loop to sterilize it, uncaped the organism and flamed the neck of the tube. After this step we used the loop to pick up some of the organism from the tube and move it to the urea broth. We then flamed the neck of the tube and capped the organism, put it back in the test tube rack, then the neck of the newly inoculated urea broth was flamed and caped. After capping both, we then labeled the urea broth with the organism inoculated within it using tape and a marker.

After completing this step, we then did this same procedure with another organism until all three organisms were inoculated into a broth. Then we placed the test tube rack in the 37 degree incubator until the next time we had class.

Results

The broth cultures were visually examined for color changes and the results can be seen in the pictures below. From the results we seen, it appears that all three organisms tested negative for the urease test. When referring to the text in the lab manual and it stated that the P. vulgaris should have tested positive. From our results it seems that all tested negative.

Labeled urea test

Results

 

 

 

 

 

 

 

Conclusions

The results from the urease test indicate that something went wrong with our test. We should have seen two negative and one positive test. We are not sure what went wrong with our test, other students’ test also show abnormal test results. I think this could do with time. We waited a couple days after the test to look at the results and in that amount of time the pH could have normalized from the positive test. Also perhaps we could have not inoculated the tubes correctly. We believe that a blank urease test should have been run along side the inoculated tubes to see if it turned this same color.

 Review Questions:

1. Explain the mechanism of urease activity.

Urease is an enzyme that hydrolizes amide compounds such as urea.  It attacks the carbon and nitrogen bonds of these compounds and forms ammonia.  Ammonia is alkaline.

2. Explain the function of phenol red in the urea broth medium.

The function of phenol red in the urea broth medium is as a pH indicator.  When the catalytic reaction of urease on urea takes place, the medium will change color to a deep pink color due to the change in pH of the broth to alkaline.

3. Explain how the urease test is useful for identifying members of the genus Proteus.

The urease reaction on urea in the genus Proteus organisms occurs rapidly and helps to differentiate these organisms from other non-lactose-fermenting enteric microorganisms.

4. A swollen can of chicken soup is examined by the public health laboratory and found to contain large numbers of gram-negative, H2S-positive bacilli.  Which biochemical tests would your perform to identify the genus of the contaminant?  Justify your test choices.

If the morphology of the organism indicates that it is a bacillus, then an oxidase test should be run.  If the oxidase test is negative then a test for lactose fermentation should be run.  If the lactose fermentation test is negative then the urease test should be run.  If the urease test is positive then the organism belongs to the genus Proteus.

 

Works Cited

Cappuccino, James, and Natalie Sherman. Microbiology: A Laboratory Manual. Tenth Edition. Boston: Pearson, 2014. Print.

 

Last updated on 7-April-2014 at 6:50 PM