Download Urease test

Urease test
objective
• To differentiate between urease positive and urease
negative bacteria using Christensen urea agar.
principle
• Some bacteria can utilize urea as a noncarbohydrate carbon source using urease enzyme.
NH2CONH2 + H2O
CO2 + 2NH3
Principle cont.
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
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 : +ve
To 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
•
•
•
Tryptophane
amino acids
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.
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 (-).
Negative test
e.g. Klebsiella
Positive test
e.g. E. coli
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
• 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 cont.
• 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)
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.
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
• All Enterobactriacae reduce nitrate to nitrite.
• Positive complete (full reduction— clear):
Pseudomonas aeruginosa.
• Negative (pink): Acinetobacter calcoaceticus.
Results con.
Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
Indole
MR
VP
Citrate
Urease
Motility
E. coli
+ve
+ve
-ve
-ve
-ve
Motile
Citrobacter
freundii
+ve
+ve
-ve
+ve
-ve
Motile
Klebsiella
pneumoniae
-ve
-ve
+ve
+ve
+ve
Non
motile
Enterobacter
cloacae
-ve
-ve
+ve
+ve
+ve
Motile
Salmonella
typhi
-ve
+ve
-ve
+ve
-ve
Motile
Shigella
boydii
-ve
+ve
-ve
-ve
-ve
Non
motile
Proteus
mirabilis
-ve
+ve
-ve
+ve
+ve
Motile
Swarming