General Microbiology Laboratory Biochemical Test By Mahmoud W

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

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

Principle �The ability to degrade amino acids to identifiable end products is often used to differentiate among bacteria. �Tryptophan, Tryptophan for example, is hydrolyzed to indole, pyruvic acid and ammonia by tryptophanase. By: Mahmoud W El-Hindi 3

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 (Paradimethylaminobenzaldehyde in alcohol) alcohol to produce a red color. By: Mahmoud W El-Hindi 4

Cont. By: Mahmoud W El-Hindi 5

Cont. Kovac’s Reagent By: Mahmoud W El-Hindi 6

Control POSITIVE CONTROL: E. coli. By: Mahmoud W El-Hindi NEGATIVE CONTROL: Enterobacter aerogenes 7

Materials �Trypton Broth 1%. �Kovac’s Reagent. �Test Tube. �Culture from bacteria. By: Mahmoud W El-Hindi 8

Procedure � 1. Inoculate tryptone broth (1%) with the organism. � 2. Incubate at 37 o. C 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 9

Result By: Mahmoud W El-Hindi 10

Cont. By: Mahmoud W El-Hindi 11

Cont. By: Mahmoud W El-Hindi 12

Cont. By: Mahmoud W El-Hindi 13

Exercise 12 Methyl Red and voges- proskauer tests MR-VP Test By: Mahmoud W El-Hindi 14

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. By: Mahmoud W El-Hindi 15

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 p. H indicator, which remains red in color at a p. H of 4. 4 or less. By: Mahmoud W El-Hindi 16

IMVi. C test Methyl Red-Voges Proskauer (MR-VP) Tests Principle Glucose Acidic pathway Or Acety methyl carbinol (ACETOIN) Mixed acids p. H less than 4. 4 Barrit’s A Barrit’s B Methyl Red indicator Red color Neutral pathway MR positive E. coli By: Mahmoud W El-Hindi VP positive Klebsiella Pink color 17

Control of MR test Positive Control: E. Coli By: Mahmoud W El-Hindi Negative Control: Klebsiella pneumoniae 18

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 24 -48 hours. � 4. Test the presence of mixed acids by the addition of methyl red indicator. By: Mahmoud W El-Hindi 19

Result of MR test By: Mahmoud W El-Hindi 20

Cont. By: Mahmoud W El-Hindi 21

Cont. By: Mahmoud W El-Hindi 22

Cont. By: Mahmoud W El-Hindi 23

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 24

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 25

Cont. By: Mahmoud W El-Hindi 26

Control of VP test Positive Control: Klebsiella pneumoniae By: Mahmoud W El-Hindi Negative Control: E. Coli 27

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 24 -48 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 28

Result of VP test By: Mahmoud W El-Hindi 29

Cont. By: Mahmoud W El-Hindi 30

Cont. By: Mahmoud W El-Hindi 31

Cont. By: Mahmoud W El-Hindi 32

Cont. By: Mahmoud W El-Hindi 33

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

Cont. GOOD LUCK GIRLS