1. Biochemical Test By: Mahmoud W El-Hindi 2013
General Microbiology
Laboratory
Biochemical Test By: Mahmoud W El-Hindi 2013
By: Mahmoud W El-Hindi

2. Tryptophan hydrolysis
Exercise 11
Tryptophan hydrolysis
“Indole Production
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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.
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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.
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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.
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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
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15. Introduction
MR-VP medium/broth is two tests in one, the Methyl Red Test and the Voges-Proskauer test.
It is very useful in separating members of the family Enterobacteriaceae and some Streptococcus.
Most members of the Enterobacteriaceae give either a positive MR or a positive VP test.
Several species are positive for both, such as Proteus mirabilis.
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16. METHYL RED (MR) TEST Principle
This is to detect the ability of an organism to produce and maintain stable acid end products from glucose fermentation.
Some bacteria produce large amounts of acids from glucose fermentation that they overcome the buffering action of the system.
Methyl Red is a pH indicator, which remains red in color at a pH of 4.4 or less.
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17. 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
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18. Control of MR test Positive Control: E.Coli
Negative Control: Klebsiella pneumoniae
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19. 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.
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20. Result of MR test
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21. Cont.
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22. Cont.
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23. Cont.
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24. 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.
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25. 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.
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26. Cont.
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27. Control of VP test Positive Control: Klebsiella pneumoniae
Negative Control: E.Coli
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28. 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
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29. Result of VP test
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30. Cont.
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31. Cont.
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32. Cont.
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33. Cont.
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34. 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
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35. Cont.
GOOD LUCK GIRLS