Urease test

objective

• To differentiate between urease positive and urease negative bacteria using Christensen urea agar.

principle

• Some bacteria can utilize urea as a non-carbohydrate carbon source using urease enzyme.

NH2CONH2 + H2O CO2 + 2NH3

Christensen's urea agar composition g/l

• Urea 20.00• Gelatin Peptone 1.00 • Sodium Chloride 5.00 • Dextrose 1.00 • Phenol Red 0.012 • Monopotassium Phosphate 2.00

Principle cont.

• Dextrose are presents in a small amount in media, so bacteria have to find another carbon source or it will stop growing.

• Urease positive bacteria will breakdown urea producing ammonia which in turn will rise the pH above 8.4.

• Phenol red indicator will turn to pink at this pH

Principle cont.

Procedure

1. Streak the slant of Christensen`s urea medium with the test organism.

2. Incubate at 35 oC (or the appropriate temperature for the organism) for 24 hours to four days.

Results

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.

Results cont.

To the left : +veTo the right : -ve

Significance

• Used to screen Salmonella and Shigella species after routine stool culture, both will give –ve result, this will differ them from Proteus (UTI causative agent) which will arise +ve result.

• Used to differ E.coli (-ve) from Klebsilla (+).

Indole test

Principle

• Certain microorganisms can metabolize tryptophan by tryptophanase

• The enzymatic degradation leads to the formation of pyruvic acid, indole and ammonia

• The presence of indole is detected by addition of Kovac's reagent.

Tryptophaneamino acids

Tryptophanase Indole + Pyurvic acid + NH3

Kovac’s Reagent

Red color in upper organic layer`

Method

Inoculate tryptone water with the tested

microorganism.

Incubate at 37°C for 24 hours .

After incubation interval, add 1 ml Kovacs reagent

(Para-dimethylaminobenzaldehyde in isoamyle

alcohol), shake the tube gently and read immediately.

Result

A bright pink color in the top layer indicates the presence of indole

The absence of color means that indole was not produced i.e. indole is negative

Special Features: Used in the differentiation of

genera and species. e.g. E. coli (+) from Klebsiella (-).

Positive teste.g. E. coli

Negative teste.g. Klebsiella

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.

• In order to determine if a bacteria can reduce nitrate, the test organism is inoculated into nitrate reduction broth, an undefined medium that contains an amounts of nitrate 0.5% (KNO3).

• After incubation, 0.6%N,N-dimethyl-1-napthylamine and 0.8%sulfanilic acid are added.

• These two compounds react with nitrite and turn red in color, indicating a positive nitrate reduction test.

• If there is no color change at this step, nitrite is absent.

Principle

• If the nitrate is unreduced and still in its original form, this would be a negative nitrate reduction result.

• However, it is possible that the nitrate was reduced to nitrite but has been further reduced to ammonia or nitrogen gas. This would be recorded as a positive nitrate reduction result.

• To distinguish between these two reactions, zinc dust -which reduces nitrate to nitrite- must be added.

Principle cont.

• If the test organism did not reduce the nitrate to nitrite, the zinc will change the nitrate to nitrite. The tube will turn red because alpha-napthylamine and sulfanilic acid are already present in the tube.

• Thus a red color after the zinc is added indicates the zinc found the nitrate unchanged(-ve)

Principle cont.

Methodology• Inoculate a nitrate broth 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 no colour appears, add several grains of zinc

powder and gently shaking the tube.

Results

Results con.

REACTION Color after adding reagents

Color after adding zinc

NO3 to NO2 red -- (not added)

NO3 to N2 no color no color

NO3 - no reaction no color pink-red

Results con.

• All Enterobactriacae reduce nitrate to nitrite.

• Positive complete (full reduction— clear): Pseudomonas aeruginosa.

• Negative (pink): Acinetobacter calcoaceticus.

Results con.

IndoleIndole MRMR VPVP CitrateCitrate UreaseUrease MotilityMotility

E. coliE. coli +ve+ve +ve+ve -ve-ve -ve-ve -ve-ve MotileMotile

Citrobacter Citrobacter freundiifreundii

+ve+ve +ve+ve -ve-ve +ve+ve -ve-ve MotileMotile

Klebsiella Klebsiella pneumoniaepneumoniae

-ve-ve -ve-ve +ve+ve +ve+ve +ve+ve Non Non motilemotile

Enterobacter Enterobacter cloacaecloacae

-ve-ve -ve-ve +ve+ve +ve+ve +ve+ve MotileMotile

Salmonella Salmonella typhityphi

-ve-ve +ve+ve -ve-ve +ve+ve -ve-ve MotileMotile

Shigella Shigella boydiiboydii

-ve-ve +ve+ve -ve-ve -ve-ve -ve-ve Non Non motilemotile

ProteusProteus mirabilismirabilis

-ve-ve +ve+ve -ve-ve +ve+ve +ve+ve MotileMotile

SwarmingSwarming

Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae