Lab Report: Urea Hydrolysis Test

PURPOSE:

The purpose of the urea hydrolysis test is to determine if a bacterium is able to degrade urea with the enzyme urease. 

INTRODUCTION:

Biochemical tests serve as an important resource in identifying bacteria. With less than 1% of bacteria on Earth classified (Tortora et al., 2009), narrowing down various chemical components of bacterial life proves to be instrumental in the identification of specific bacteria. Urea, which is produced by specific amino acids through decarboxylation, can be hydrolyzed to ammonia and carbon dioxide by bacteria that contain the enzyme urease (Leboffe & Pierce, 2008). Proteus, Morganella, and Providencia bacteria are able to metabolize urea rapidly, which is a crucial factor in the test (Leboffe & Pierce, 2008). There are other bacteria, however, that are able to metabolize urea, specifically enteric bacteria, but they are not able to do this quickly enough to classify as rapid-urease positive organisms (Leboffe & Pierce, 2008). Urea Broth contains nutrients, including urea and .0001% yeast extract, as well as buffers that inhibit alkalization of the medium in all organisms except for those that are able to rapidly hydrolyze urea (Leboffe & Pierce, 2008). Phenol Red serves as the indicator in the test to show a change in the pH of the broth caused by alkalization (Leboffe & Pierce, 2008). A pH below 8.4, in phenol red, is yellow or orange and indicates that an organism is a rapid-urease negative organism (Leboffe & Pierce, 2008). With any pH above 8.4, the phenol red will reflect the change with red or pink coloration of the broth (Leboffe & Pierce, 2008).

MATERIALS AND METHODS: 

To perform the Urea Hydrolysis test, my lab group of four students inoculated eight test tubes that contained Urea Broth, each with a specific bacterium. Additionally we kept a ninth test tube containing Urea Broth, that was not inoculated, to serve as a control for the test. The eight bacteria used in the test were Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, Klebsiella pneumoniae, Proteus vulgaris, and Serratia marcesans. First, after labeling each test tube indicating the name of the test and the specific bacterium being tested, my group inoculated the tubes using the sterile loop technique, by flaming the laboratory loop and the rim of both the bacterial broth culture tube and the test tube. We then placed the sample of each of the bacteria into the test tube, thoroughly mixing it into the broth, and then heat fixed the rim of the tube before putting on the top. In continuation of the sterile loop technique, the loop used was heat fixed before and after each use. The test tubes were then placed in a 37˚C aerobic incubator for 48 hours. After waiting the allotted incubation time, my group examined the results of our test. 

RESULTS:

According to my group’s test results, K. pneumoniae and P. vulgaris were the only rapid-urease positive organisms. The color of the Urea Broth changed from its original orange-yellow, to red in the tube inoculated with K. pneumoniae, and turned magenta in the tube inoculated with P. vulgaris. Our test results also concluded that all other tested bacteria, were rapid-urease negative organisms. The rapid-urease negative organisms include E. coli, E. aerogenes, P. aeruginosa, S. typhimurium, S. flexneri, and S. marcesans. The coloration of the Urea Broth for all of the urease-negative organisms remained an orange-yellow hue. This information can be found in Table 1 and Figure 1 located below.

Table 1. The bacteria that were able to turn the color of the Urea Broth pink, from the original orange-yellow, are rapid-urease positive organisms. 

Organism	Reaction (+ or -)
E. coli	-
E. aerogenes	-
P. aeruginosa	-
S. typhimurium	-
S. flexneri	-
K. pneumoniae	+
P. vulgaris	+
S. marcesans	-

Figures 1-8. 

Figure 1. Urea Hydrolysis test tubes five days after inoculation. E. aerogenes, the color of the broth remained orange-yellow indicating a negative reaction to the test.

Figure 2.  Urea Hydrolysis test tubes five days after inoculation. E. coli, the color of the broth remained orange-yellow indicating a negative reaction to the test.

Figure 3. Urea Hydrolysis test tubes five days after inoculation. K. pneumoniae, the color of the broth turned red indicating a positive reaction to the test.

Figure 4. Urea Hydrolysis test tubes five days after inoculation. P. aeruginosa, the color of the broth remained orange-yellow indicating a negative reaction to the test.

Figure 5. Urea Hydrolysis test tubes five days after inoculation. P. vulgaris, the color of the broth changed from the original orange-yellow to magenta, indicating a positive reaction to the test.

Figure 6. Urea Hydrolysis test tubes five days after inoculation. S. flexneri, the color of the broth remained orange-yellow indicating a negative reaction to the test.

Figure 7. Urea Hydrolysis test tubes five days after inoculation. S. marcesans, the color of the broth remained orange-yellow indicating a negative reaction to the test.

Figure 8. Urea Hydrolysis test tubes five days after inoculation. S. typhimurium, the color of the broth remained orange-yellow indicating a negative reaction to the test.

DISCUSSION:

The Urea Hydrolysis test results from my group were reflective of the results provided in Bergey’s Manual, which most probably means that few errors occurred in the testing (Garrity, 2005). The organisms that did not change the color of the Urea Broth were identified as rapid-urease negative organisms. The rapid-urease negative organisms include E. coli, E. aerogenes, P. aeruginosa, S. typhimurium, S. flexneri, and S. marcesans. These organisms do not produce the enzyme urease to break down urea, which was able to be concluded by the lack of color change in the broth. The color change did not occur in the broths inoculated with these bacteria, because the pH of these organisms remained below 8.4. 

In the tubes containing K. pneumoniae and P. vulgaris, the color of the phenol red dye in the broth changed because of a rise in pH to levels above 8.4. This change of  in pH was raised as a result of alkalization that occurs in the broth during the breakdown of urea. The indication of this was reflected by the pink coloration of the P. vulgaris broth, and the red coloration of the K. pneumoniae broth. The Urea Hydrolysis test is a relatively simple and effective way to accurately identify certain species of bacteria.

References:

Garrity GM. 2005. Bergey’s Manual of Systemic Bacteriology. Bergey’s Manual Trust. 2(2).

Leboffe MJ, Pierce BE. 2008. Microbiology Laboratory Theory & Application. Brief Edition. 285-286

Tortora GJ, Funke BR, Case CL. 2009. Microbiology. An Introduction 1(10):282