Urease test is used screen lactose negative gramnegative 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 nonpathogen Proteus and some ID: 316358
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Slide1Slide2
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.
Ureas
TestSlide3
Urease-Producing by some Enterobacteriaceae
like:
Proteus
Klebsiella pneumoniae Enterobacter
cloacae
Yersinia enterocoliticaSlide4
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.
PrincipleSlide5
Dextrose are presents in a small amount in media, so bacteria have to find another carbon source or it will stop growing.
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.Slide6
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.
ProcedureSlide7Slide8
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.
Indole TestSlide9
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.
ProcedureSlide10
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
(-).Slide11Slide12
Nitrate
reductase
test : is a test to differentiate between bacteria based on their ability or inability to reduce nitrate (NO
3−) 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.
Nitrate Reduction TestSlide13
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.
PrincipleSlide14
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).
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. Slide15
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
ProcedureSlide16
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 Slide17Slide18Slide19Slide20Slide21
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.
Significance of Nitrate Reduction TestSlide22
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. Slide23