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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 non-carbohydrate carbon source using urease enzyme. NH 2 CONH 2 + H 2 O CO 2 + 2NH 3. Principle cont.

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Urease test

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  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 1.00 • Phenol Red 0.012 • 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) • 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. • Positive complete (full reduction— clear): Pseudomonas aeruginosa. • Negative (pink): Acinetobactercalcoaceticus.

  20. Results con.

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

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