Urea Broth is a liquid medium used extensively for the differentiation of microorganisms, especially within the Enterobacteriaceae family, based on urease production. This explanation discusses the composition, application, and significance of Urea Broth in the differentiation of organisms like Proteus from Salmonella and Shigella in enteric infection diagnosis.
Urea Broth is a liquid medium formulated for the differentiation of urease-producing organisms from clinical and non-clinical samples. Its typical composition includes yeast extract, urea, disodium phosphate, monopotassium phosphate, and phenol red, with a final pH of 6.8 ± 0.2 at 25°C. The yeast extract provides essential nutrients, while urea serves as a nitrogen source for organisms producing urease. The phosphate components act as buffering agents, and phenol red is the pH indicator. Urea Broth is particularly effective for differentiating members of the genus Proteus from those of Salmonella and Shigella, making it vital for enteric infection diagnosis.
Composition and Method Principle
Urea Broth consists of 0.1 g/l yeast extract, 20.0 g/l urea, 9.5 g/l disodium phosphate, 9.1 g/l monopotassium phosphate, and 0.01 g/l phenol red. Yeast extract supplies amino acids, nitrogen, carbon, vitamins, and minerals for organism growth. The urea is a source of nitrogen for organisms producing urease. The buffering agents maintain the medium’s pH, and the phenol red acts as an indicator, changing to pink-red upon urease activity and ammonia production.
Preparation and Test Procedure
To prepare Urea Broth, 38.7 g of the powder is suspended in 1 liter of distilled or deionized water and filtered sterilized without boiling or autoclaving. For testing, the medium is heavily inoculated with a pure culture of the microorganism under investigation and incubated at 35 ± 2°C for 8-48 hours.
Microbial growth is indicated by turbidity in the broth. A positive urease reaction is demonstrated by a color change to pink or red, while a negative reaction shows no color change. This reaction is particularly significant for organisms like Proteus, Morganella, and Providencia within the first 24 hours of incubation.
Storage and Shelf Life
The dehydrated medium is very hygroscopic and should be stored at 10-30°C in a dry environment. The shelf life of the dehydrated medium is 4 years, and once prepared in tubes, it lasts for 1 year when stored at 2-8°C away from light.
Quality control tests involve inoculating the broth with specific microbial strains such as Proteus mirabilis, Salmonella Typhimurium, and Escherichia coli. The expected results include good growth for all these organisms and a positive urease reaction for Proteus mirabilis, with negative reactions for Salmonella Typhimurium and Escherichia coli.
Urea Broth is an essential medium in microbiology for differentiating urease-producing bacteria, especially in the context of diagnosing enteric infections. Its specific formulation, ease of use, and reliable interpretation make it a critical tool in clinical and non-clinical microbiological analysis.