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:
1) Proteus
2) Klebsiella pneumoniae
3) Enterobacter cloacae
4) 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
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.
 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 (paradimethylaminobenzaldehyde 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.
1)
If a culture does not produce a color
change, several possibilities exist:
the bacteria possess nitrate reductase and also
reduce nitrite further to ammonia or molecular
nitrogen;
2)
they possess other enzymes that reduce nitrite to ammonia;
3)
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 ammonia or nitrogen gas (nitrate reacted).
Procedure
1) Inoculate a nitrate broth (0.5% potassium nitrate (KNO3))
with the test organism.
2) Incubate at 37C for 24 hr.
3) 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.
4) Negative tests should be confirmed by adding several grains
of zinc powder and gently shaking the tube.
Positive: Colorless
Negative: Red color
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
No Color
NO3 To Ammonia
None
No Color
No Color
NO3-No Reaction
None
No Color
Pink-red
Significance of Nitrate Reduction Test
Three different bacteria that give three different nitrate
reduction results will be learned.
1) Staphylococcus epidermidis is unable to use nitrate as a
terminal electron acceptor; therefore, it cannot reduce
nitrate.
2) Escherichia coli can reduce nitrate only to nitrite.
3) 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.