3.5.3 Expected Result
Positive: Red color change.
Negative: No color change.
3.6 Voges–
Proskauer Reaction
The Voges–Proskauer (VP) procedure is used to identify if an
organism develops acetylmethyl carbinol from glucose fermentation. When produced, acetylmethyl carbinol is converted to diacetyl in the presence of naphthol, high alkali (40% KOH), and
atmospheric oxygen. α-Naphthol was not part of the initial protocol, but was identified as a color enhancer by Barritt and must be
applied first. Diacetyl and quanidine-containing complexes present
in the broth peptones are then reduce to form a pinkish red
polymer.
3.6.1 Materials Required
l
Media: MR-VP broth.
l
Reagent: Alpha-naphthol, potassium hydroxide.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.6.2 Procedure
1. Inoculate a pure test organism culture into a tube of MR-VP
broth.
2. Incubate the tube for 24 h at 35
C.
3. At this moment, add 1 ml of broth to a sterile test tube.
4. Add 0.6 ml of 5% alpha naphthol and 0.2 ml of 40% KOH.
Note: It is necessary to add these reagents in this order.
5. Agitate the tube slowly to expose the contents to atmospheric
oxygen.
6. Leave the tube uninterrupted for 10–15 min.
3.6.3 Expected Result
Appearance of pinkish red color is positive.
3.7 Indole
Production
Tryptophan is an amino acid which goes through deamination and
hydrolysis of bacteria which produce tryptophanase enzyme. Indole
is yielded by reductive deamination of tryptophan through the
intermediate molecule of indolepyruvic acid. Tryptophanase catalyzes the deamination process from which the tryptophan molecule
(–NH2) group of amines is removed. The end products of the
reaction are indole, pyruvate, ammonium (NH4
+ ), and energy.
Pyridoxal phosphate is necessary as a coenzyme.
3.7.1 Materials Required
l
Media: Tryptophan broth.
l
Reagent: Kovac’s reagent.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
Biochemical Characterization
125
Positive: Red color change.
Negative: No color change.
3.6 Voges–
Proskauer Reaction
The Voges–Proskauer (VP) procedure is used to identify if an
organism develops acetylmethyl carbinol from glucose fermentation. When produced, acetylmethyl carbinol is converted to diacetyl in the presence of naphthol, high alkali (40% KOH), and
atmospheric oxygen. α-Naphthol was not part of the initial protocol, but was identified as a color enhancer by Barritt and must be
applied first. Diacetyl and quanidine-containing complexes present
in the broth peptones are then reduce to form a pinkish red
polymer.
3.6.1 Materials Required
l
Media: MR-VP broth.
l
Reagent: Alpha-naphthol, potassium hydroxide.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.6.2 Procedure
1. Inoculate a pure test organism culture into a tube of MR-VP
broth.
2. Incubate the tube for 24 h at 35
C.
3. At this moment, add 1 ml of broth to a sterile test tube.
4. Add 0.6 ml of 5% alpha naphthol and 0.2 ml of 40% KOH.
Note: It is necessary to add these reagents in this order.
5. Agitate the tube slowly to expose the contents to atmospheric
oxygen.
6. Leave the tube uninterrupted for 10–15 min.
3.6.3 Expected Result
Appearance of pinkish red color is positive.
3.7 Indole
Production
Tryptophan is an amino acid which goes through deamination and
hydrolysis of bacteria which produce tryptophanase enzyme. Indole
is yielded by reductive deamination of tryptophan through the
intermediate molecule of indolepyruvic acid. Tryptophanase catalyzes the deamination process from which the tryptophan molecule
(–NH2) group of amines is removed. The end products of the
reaction are indole, pyruvate, ammonium (NH4
+ ), and energy.
Pyridoxal phosphate is necessary as a coenzyme.
3.7.1 Materials Required
l
Media: Tryptophan broth.
l
Reagent: Kovac’s reagent.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
Biochemical Characterization
125