Ureas Test Some bacteria are able to produce an enzyme called urease that attacks the nitrogen and carbon bond in amide compounds such as urea, forming the end products ammonia, CO2, and water. Urease test is used screen lactose negative gram-negative Enterobacteriaceae on differential media plated with materials from stool specimen, helping to differentiate Salmonella and Shigella species which are urease negative from the urease positive non-pathogen. Proteus, and some Citrobacter species and some Haemophilus species are urease positive. P. mirabilis is a major cause of human urinary tract infections.

Urease-Producing by some Enterobacteriaceae like: Proteus Klebsiella pneumoniae Enterobacter cloacae Yersinia enterocolitica

Principle To differentiate between urease positive and urease negative bacteria using Christensen urea agar, that contains Urea (20.00 g/l), Gelatin Peptone (1.00 g/l), Sodium Chloride (5.00 g/l), Dextrose (1.00 g/l), Phenol Red (0.012 g/l) and Monopotassium Phosphate (2.00 g/l). Some bacteria can utilize urea as a non-carbohydrate carbon source using urease enzyme.

Urease activity (the urease test) is detected by growing bacteria in medium containing urea and using a pH indicator such as phenol red. When urea is hydrolyzed, ammonia accumulates in the medium and makes it alkaline. This increase in pH causes the indicator to change from orange-red to deep pink or purplish red and is a positive test for urea hydrolysis. Dextrose are presents in a small amount in media, so bacteria have to find another carbon source or it will stop growing.

Procedure Streak the slant of Christensen`s urea medium with the test organism. Incubate at 35 oC (or the appropriate temperature for the organism) for 24 hours to four days. Some bacteria have a delayed urease reaction that may require an incubation period longer than 48 hours. Positive: A bright pink colour develops on the slant and may extends throughout the medium Negative: No change in the original colour of the medium.

Indole Test The ability to degrade amino acids to identifiable end products is often used to differentiate among bacteria. Tryptophan, for example, is hydrolyzed to Indole, pyruvic acid and ammonia by tryptophanase. The pyruvic acid can be further metabolized to produce large amounts of energy. The ammonia is available for use in synthesis of new amino acids. Indole can be detected by reaction with Kovac's reagent (para-dimethylaminobenzaldehyde in alcohol) to produce a red color.

Procedure Inoculate Tryptone broth or SIM media {contains tryptophan} with inoculating loop. Incubate at 37°C for 24 hours . After incubation interval, add 1 ml Kovacs reagent, shake the tube gently and read immediately.

Result A red color in the top layer indicates the presence of indole The absence of color means that indole was not produced. Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella, Enterobacter aerogenes (-).

Nitrate Reduction Test Nitrate reductase test : is a test to differentiate between bacteria based on their ability or inability to reduce nitrate (NO3−) to nitrite (NO2−) using anaerobic respiration. Some of these bacteria possess the enzymes to further reduce the nitrite to either the ammonium ion or molecular nitrogen.

Principle Organisms that possess the enzyme, nitrate reductase reduces nitrate to nitrite. The nitrite ions are detected by the addition of Sulfanilic acid and N,N-dimethyl-1-naphthylamine to the culture. Any nitrite in the medium will react with these reagents to produce a pink or red color.

If a culture does not produce a color change, several possibilities exist: the bacteria possess nitrate reductase and also reduce nitrite further to ammonium or molecular nitrogen; they possess other enzymes that reduce nitrite to ammonium; nitrates were not reduced by the bacteria. To determine if nitrates were reduced past nitrite: a small amount of zinc powder is added to the culture containing the reagents. Since zinc reduces nitrates to nitrites, a pink or red color will appear and verifies the fact that nitrates were not reduced to nitrites by the bacteria (nitrate unreacted). If a red color does not appear, the nitrates in the medium were reduced past the nitrite stage to either ammonium or nitrogen gas (nitrate reacted).

Procedure Inoculate a nitrate broth (0.5% potassium nitrate (KNO3)) with the test organism. Incubate at 37C for 24 hr. Add 5 drops of reagent A (Sulfanic acid) and 5 drops of reagent B (naphthylamine ) to the broth. If nitrate is present in the medium, it will turn red within 1 to 2 minutes; if it is absent, there will be no color change. Positive: A red color. Negative: Colorless. Negative tests should be confirmed by adding several grains of zinc powder and gently shaking the tube. Positive: Colorless Negative: Red color

Color After Adding Reagents Color After Adding Zinc Result Reaction N2 Gas Color After Adding Reagents Color After Adding Zinc NO3 To NO2  None Red (Not Added) NO3 To N2 Yes No Color NO3 To Ammonia NO3-No Reaction Pink-red

Significance of Nitrate Reduction Test Three different bacteria that give three different nitrate reduction results will be learned. Staphylococcus epidermidis is unable to use nitrate as a terminal electron acceptor; therefore, it cannot reduce nitrate. Escherichia coli can reduce nitrate only to nitrite. Pseudomonas fluorescens are characterized by excretion of diffusible yellow-green pigments that fluoresce in ultraviolet light) often reduces nitrate completely to molecular nitrogen.

Safety consideration Since N, N-dimethyl-1-naphthylamine might be carcinogenic (nitrite test reagent B), wear disposable gloves and avoid skin contact or aerosols. The acids in nitrite test reagent A are caustic. Avoid skin contact and do not breathe the vapors. Be careful when working with zinc. Do not inhale or allow contact with skin. No mouth pipetting.