3. Incubate the test medium at temperature of 37
C for
overnight.
4. Dispense 4–5 drops of 10% aqueous ferric chloride (FeCl 3 )
solution to the slant.
3.12.3 Expected Result
Positive: Production of green color.
Negative: Absence of color (medium remains straw/yellow color).
3.13 Nitrate
Reduction
Reduction of nitrate to nitrite is a metabolic capability of symbionts
that separate oxygen from nitrate to form nitrite. The nitrites react
with the reagents sulfanilic acid and alpha-naphthylamine with the
production of a red compound. In the positive reaction, symbionts
break nitrates to nitrites and after the addition of reagents in the
culture medium; a red-orange color develops.
3.13.1 Materials
Required
l
Media: Nitrate broth with inverted Durham’s tube.
l
Reagents: Sulfanilic acid reagent, alpha naphthylamine reagent,
zinc dust.
l
Others: Inoculating loop, burner, dropper.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.13.2 Procedure
1. Employ aseptic technique to inoculate a heavy growth of test
organisms to the nitrate broth.
2. Incubate at a suitable temperature for 24–48 h.
3. Dispense one dropper full of sulfanilic acid and one dropper
full of α-naphthylamine to each broth.
4. Color change to red specifies a positive nitrate reduction test.
5. Absence of color is a negative reaction, in such case add a small
quantity of zinc powder (a toothpick full) and visualize the
development of the red-orange coloring.
3.13.3 Expected Result
Positive: Formation of red pigment (after sulfanilic acid + -
α-naphthylamine; no color after zinc).
Negative: Absence of color (after sulfanilic acid + α-naphthylamine
followed by red after zinc).
3.14 Amylase
(Starch Hydrolysis)
Symbionts are capable of catabolizing down starch into maltose by
the activity of the extracellular α-amylase enzyme.
3.14.1 Materials
Required
l
Media: Starch agar.
l
Reagent: Iodine solution.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
Biochemical Characterization
129
C for
overnight.
4. Dispense 4–5 drops of 10% aqueous ferric chloride (FeCl 3 )
solution to the slant.
3.12.3 Expected Result
Positive: Production of green color.
Negative: Absence of color (medium remains straw/yellow color).
3.13 Nitrate
Reduction
Reduction of nitrate to nitrite is a metabolic capability of symbionts
that separate oxygen from nitrate to form nitrite. The nitrites react
with the reagents sulfanilic acid and alpha-naphthylamine with the
production of a red compound. In the positive reaction, symbionts
break nitrates to nitrites and after the addition of reagents in the
culture medium; a red-orange color develops.
3.13.1 Materials
Required
l
Media: Nitrate broth with inverted Durham’s tube.
l
Reagents: Sulfanilic acid reagent, alpha naphthylamine reagent,
zinc dust.
l
Others: Inoculating loop, burner, dropper.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.13.2 Procedure
1. Employ aseptic technique to inoculate a heavy growth of test
organisms to the nitrate broth.
2. Incubate at a suitable temperature for 24–48 h.
3. Dispense one dropper full of sulfanilic acid and one dropper
full of α-naphthylamine to each broth.
4. Color change to red specifies a positive nitrate reduction test.
5. Absence of color is a negative reaction, in such case add a small
quantity of zinc powder (a toothpick full) and visualize the
development of the red-orange coloring.
3.13.3 Expected Result
Positive: Formation of red pigment (after sulfanilic acid + -
α-naphthylamine; no color after zinc).
Negative: Absence of color (after sulfanilic acid + α-naphthylamine
followed by red after zinc).
3.14 Amylase
(Starch Hydrolysis)
Symbionts are capable of catabolizing down starch into maltose by
the activity of the extracellular α-amylase enzyme.
3.14.1 Materials
Required
l
Media: Starch agar.
l
Reagent: Iodine solution.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
Biochemical Characterization
129