1. Urease test

2. 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

3. Principle cont. Christensen's urea agar composition g/l Urea 20.00
Gelatin Peptone 1.00
Sodium Chloride 5.00
Dextrose
Phenol Red
Monopotassium Phosphate 2.00

4. 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

5. Procedure
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.

6. Results cont.
To the left : +ve
To the right : -ve

7. 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 (+).

8. Indole test

9. 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.
Tryptophanase
Tryptophane
amino acids
Indole + Pyurvic acid + NH3
Kovac’s Reagent
Red color in upper organic layer`

10. 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.

11. Result
Negative test
e.g. Klebsiella
Positive test
e.g. E. coli
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 (-).

12. NITRATE REDUCTION TEST

13. 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.

14. 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.

15. 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.

16. Principle cont. Methodology
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.

17. Results

18. Results con.

19. Results con. All Enterobactriacae reduce nitrate to nitrite.
REACTION
Color after adding reagents
Color after adding zinc
NO3 to NO2
red
-- (not added)
NO3 to N2
no color
NO3 - no reaction
pink-red
All Enterobactriacae reduce nitrate to nitrite.
Positive complete (full reduction— clear): Pseudomonas aeruginosa.
Negative (pink): Acinetobacter calcoaceticus.

20. Results con.

21. Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
Motility
Urease
Citrate VP MR
Indole
Motile
-ve
+ve
E. coli
Citrobacter freundii
Non motile
Klebsiella pneumoniae
Enterobacter cloacae
Salmonella typhi
Shigella boydii
Swarming
Proteus mirabilis