1. Biochemical Test biology and biotechnology department
General Microbiology
Laboratory
Biochemical Test biology and biotechnology department

2. Tryptophan hydrolysis
Exercise 11
Tryptophan hydrolysis
“Indole Production
By: Mahmoud W El-Hindi

3. Principle
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.
By: Mahmoud W El-Hindi

4. Cont.
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.
By: Mahmoud W El-Hindi

5. Cont.
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6. Cont.
Kovac’s Reagent
Red Color Ring
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7. Control POSITIVE CONTROL: E.coli.
NEGATIVE CONTROL: Enterobacter aerogenes
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8. Materials Trypton Broth 1%. Kovac’s Reagent. Test Tube.
Culture from bacteria.
By: Mahmoud W El-Hindi

9. Procedure 1. Inoculate tryptone broth (1%) with the organism.
2. Incubate at 37 oC for 48 hours.
3. Add 10 drops of Kovac's reagent. A red color in the alcohol (upper) layer is a positive result.
By: Mahmoud W El-Hindi

10. Result
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11. Cont.
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12. Cont.
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13. Cont.
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14. Methyl Red and voges- proskauer tests
Exercise 12
Methyl Red and voges- proskauer tests
MR-VP Test
By: Mahmoud W El-Hindi

15. Introduction
These fermentation tests are used to differentiate between certain intestinal bacteria called coliforms.
The medium contains dextrose as the carbohydrate source.
Some coliforms will ferment the dextrose to acid products that will cause the pH to drop below pH 5.

16. This is called a mixed acid fermentation
This is called a mixed acid fermentation. After incubation the addition of methyl red, a dye which turns red below pH 4.4, will indicate whether such fermentation has occurred.
Other coliforms will convert dextrose to less acidic products such as ethanol or butanediol.
These bacteria are negative in the methyl red test.

17. Butanediol fermentation is demonstrated by the Voges-Proskauer test which measures the presence of acetoin (acetyl methyl carbinol), a precursor to butanediol.
This test uses the same medium as the methyl red test and both tests are usually performed in parallel

18. Barritt's reagents, alpha-naphthol and potassium hydroxide, are added to a 48 hour culture and the tube is shaken to aerate the solution.
The development of a pink or red color after agitation is a positive reaction for the production of acetoin.

19. CONTROLS:
E. coli (positive MR for negative for VP);
E. aerogenses (positive for VP negative for MR)

20. IMViC test Methyl Red-Voges Proskauer (MR-VP) Tests
Principle
Glucose
Acidic pathway Or
Neutral pathway
Acety methyl carbinol
(ACETOIN)
Mixed acids
 pH less than 4.4
Barrit’s A
Barrit’s B
Methyl Red
indicator
VP positive
Klebsiella
MR positive
E. coli
Red color
Pink color
By: Mahmoud W El-Hindi

21. Control of MR test Positive Control: E.Coli
Negative Control: Klebsiella pneumoniae
By: Mahmoud W El-Hindi

22. Protocol of MR test
1. The tubes of MR-VP broth were inoculated using sterile technique with given bacterial culture.
2. One tube was served as control.
3. Incubate at 37 0 C for hours.
4. Test the presence of mixed acids by the addition of methyl red indicator.
By: Mahmoud W El-Hindi

23. Result of MR test
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24. Cont.
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25. Cont.
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26. Cont.
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27. Test (VP) Voges- Proskauer
Principle
VP test detects butylene glycol producers.
Acetyl-methyl carbinol (acetoin) is an intermediate in the production of butylene glycol.
In this test two reagents, 40% KOH and alpha-naphthol are added to test broth after incubation and exposed to atmospheric oxygen.
By: Mahmoud W El-Hindi

28. Cont.
If acetoin is present, it is oxidized in the presence of air and KOH to diacetyl.
Diacetyl then reacts with guanidine components of peptone, in the presence of alpha naphthol to produce red color.
Role of alpha-naphthol is that of a catalyst and a color intensifier.
By: Mahmoud W El-Hindi

29. Cont.
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30. Control of VP test Positive Control: Klebsiella pneumoniae
Negative Control: E.Coli
By: Mahmoud W El-Hindi

31. Protocol of VP test
1. The tubes of MR-VP broth were inoculated using sterile technique withgiven bacterial culture.
2. One tube was served as control.
3. Incubate at 37 0 C for hours.
4. Test the presence of acetyl-methyl carbinol by the addition of Barrit’s reagent.
5. Shacked well and allowed for 15 minutes. The presence of rose colouration is a positive result
By: Mahmoud W El-Hindi

32. Result of VP test
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33. Cont.
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34. Cont.
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35. Cont.
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36. Cont.
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37. Conclusion
IMVC test Indole Methyl Red-Voges Proskauer (MR-VP) and Citrate Tests.
This Four test used to differentiation between of Enterobacteriacea member. C V M I
Name test - +
E. Coli
Enterobacter
By: Mahmoud W El-Hindi

38. Cont.
GOOD LUCK GIRLS