UREA BROTH: FOUNDATION, PREPARATION AND USES - BIOLOGY - 2025

The urea broth is a liquid culture medium, used to detect the presence of urease enzyme in certain microorganisms. Urease is a microbial enzyme that is produced in a constitutive way, that is, it is synthesized regardless of whether or not the substrate on which it acts is present.

The function of urease is related to the decomposition of organic compounds. Not all microorganisms are capable of synthesizing this enzyme, therefore its determination in the laboratory allows identifying certain bacterial strains and even differentiating between species of the same genus.

Negative and positive urea test. Source: Photo taken by the author MSc. Marielsa Gil.

There are two types of urea test: Stuart and Christensen. They differ in composition and sensitivity. The first is special to show a large amount of urease produced by species of the genus Proteus.

The second is more sensitive and can detect small amounts of urease generated late by other bacterial genera, such as Klebsiella, Enterobacter, Staphylococcus, Brucella, Bordetella, Bacillus, Micrococcus, Helicobacter, and Mycobacterium.

Stuart's Urea Broth is composed of Urea, Sodium Chloride, Dipotassium Phosphate, Monopotassium Phosphate, Yeast Extract, Phenol Red, and Distilled Water.

Meanwhile, Christensen's urea broth or agar is composed of peptones, sodium chloride, monopotassium phosphate, glucose, urea, phenol red, distilled water, and agar-agar. The latter only if it is the solid medium.

Basis

The enzyme urease hydrolyzes urea to form carbon dioxide, water, and two molecules of ammonia. These compounds react to form the end product called ammonium carbonate.

Stuart's Urea Broth

Stuart's urea broth is more buffered with a pH of 6.8. Therefore, the microorganism must be able to form large amounts of ammonia to turn phenol red. The pH should rise above 8.

Therefore, Stuart's urea broth is selective for Proteus species, giving positive results within 24 to 48 hours of incubation, and it is not effective for bacteria that produce low amounts of urease or that slowly hydrolyze urea.

This is because Proteus species are able to use urea as a nitrogen source. Instead, other urease-producing bacteria need an additional source.

However, Pérez et al. (2002) determined that Stuart's urea broth was as efficient as Christensen's urea agar in determining urease in yeast strains of the genera Candida, Cryptococcus, Rhodotorula, Trichosporon and Saccharomyces.

The study authors claim to have achieved 100% agreement with both media (Stuart and Christensen) when incubating for 24 and 48 hours; with the exception that the strains that managed to turn the media to a strong pinkish-fuchsia color were taken as positive.

This clarification is necessary, since Lodder (1970) stated that almost all yeasts manage to turn the Christensen urea agar bevel to pale pink. This is due to the fact that they can hydrolyze urea in minimal amounts, and the formation of amines by oxidative decarboxylation of amino acids on the surface. This should not be construed as positive.

Christensen's Urea Agar or Broth

Christensen's urea broth or agar is less buffered, being able to detect small amounts of ammonia. Furthermore, this medium is enriched with peptones and glucose. These compounds cause other urease-producing microorganisms to grow that do not grow in Stuart broth.

Likewise, the Christensen urea test offers faster results, especially for Proteus, being able to give strongly positive in just 30 minutes as a minimum time and up to 6 hours as a maximum time.

The rest of the urease-producing microorganisms manage to turn the color of the medium slightly after 6 hours, and strongly after 24, 48, 72 hours or more, and even some strains can give weak reactions after 5 or 6 days.

Interpretation of both media (Stuart and Christensen)

The medium is originally yellow-orange in color and a positive reaction will turn the color of the medium to pinkish-fuchsia. The intensity of the color is directly proportional to the amount of ammonia produced.

A negative reaction will leave the medium of the original color with the exception of yeasts, which may turn pale pink on Christensen's urea agar medium.

Preparation

Stuart's Urea Broth

Weigh the necessary grams according to the indications of the commercial company. Dissolve in preferably sterile distilled water. Do not use heat to dissolve, as urea is sensitive to heat.

Membrane filtration is used to sterilize. For this, a Millipore filter with pores of 0.45 µ diameter is used. Do not use an autoclave. Once the solution is filtered, it is distributed into sterile tubes. To obtain reliable results, it should be transferred between 1.5 ml as a minimum quantity and 3 ml as a maximum quantity per tube.

Store in a refrigerator and warm before use.

If the filtration method is not available, the medium should be used immediately to obtain reliable results.

Another way to prepare Stuart's Urea Broth is as follows:

Some commercial houses sell the base medium for the urea test, not including urea.

The quantity indicated by the commercial company is weighed. It is dissolved in distilled water and sterilized in the autoclave at 121 ° C for 15 minutes. It is left to rest a little and when the medium is warm, add 100 ml of a urea solution prepared at 20% and sterilized by filtration.

It is distributed in sterile tubes, as previously described.

Christensen's Urea Agar or Broth

-Preparation of the urea solution

Weigh out 29 g of dehydrated urea and dissolve in 100 ml of distilled water. Use the filtration method to sterilize. Do not autoclave.

-Urea base agar

Dissolve 24 g of the dehydrated base agar in 950 ml of distilled water. Sterilize in the autoclave at 121 ° C for 15 minutes. Let it rest until it reaches a temperature of 50 ° C and add the previously prepared urea aseptically.

Pour 4-5 ml into sterile tubes and tilt until solid. There should be a long flute beak.

This medium can also be prepared in liquid form.

Applications

The urea test is extremely effective in distinguishing the genus Proteus from other genera in the Family Enterobacteriaceae, given the rapid reaction provided by Proteus.

Using the Christensen composition, the test helps to differentiate between species of the same genus. For example, S. haemolyticus and S. warneri are coagulase-negative and beta-hemolytic Staphylococcus, but differ in that S. haemolyticus is urea negative and S. warneri is urea positive.

On the other hand, McNulty successfully used Christensen's 2% urea broth to study the presence of Helicobacter pylori in biopsy samples taken from the gastric mucosa (antral region).

The presence of H. pylori is evidenced by a positive urea test. The duration time to observe the results is directly proportional to the amount of microorganisms present.

As can be seen, it is a simple method for the diagnosis of Helicobacter pylori in gastric biopsies.

Lastly, this test is also useful for differentiating species from the genera Brucella, Bordetella, Bacillus, Micrococcus, and Mycobacteria.

Sowing the urea test

Both methods require a strong microbial inoculum to optimize results. Bacterial colonies are preferably taken from blood agar and yeasts from Sabouraud agar, with a few exceptions. The inoculum is emulsified in the liquid medium.

For Stuart's urea broth, incubate at 37 ºC for 24 to 48 hours, knowing that you are only looking for strains of the genus Proteus when the strain is a bacterium. For yeasts, it can be incubated at 37 ° C or at room temperature for 24 to 48 hours of incubation.

In the case of Christensen's urea broth, it is incubated at 37 ºC for 24 hours. If the test is negative, it can be incubated for up to 6 days. If the test is positive before 6 hours, it indicates that it is a strain of the genus Proteus.

In the case of Christensen's urea agar, the agar bevel is strongly inoculated, without puncturing. The broth is incubated and interpreted in the same way.

QA

Control strains such as Proteus mirabilis ATCC 43071, Klebsiella pneumoniae ATCC 7006003, Escherichia coli ATCC 25922 and Salmonella typhimurium can be used to test the medium. The first two should give positive results and the last two negative results.

Source: Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.

References

1. Pérez C, Goitía K., Mata S, Hartung C, Colella M, Reyes H. et al. Use of Stuart's urea broth for the urease test, as a test in the diagnosis of yeast. Rev. Soc. Ven. Microbiol. 2002; 22 (2): 136-140. Available at: Scielo.org.
2. Mac Faddin J. (2003). Biochemical tests for the identification of bacteria of clinical importance. 3rd ed. Editorial Panamericana. Buenos Aires. Argentina.
3. Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana SA Argentina.
4. Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.
5. Britannia Laboratories. Christensen Medium (Urea agar base) 2015.Available at: britanialab.com