3.7.2 Procedure
1. Use sterilized test tubes, transfer 4 ml of tryptophan broth.
2. Inoculate the tube in sterile conditions by taking the growth
from 18 to 24 h culture.
3. Incubate the tube at a temperature of 37
C for 24–28 h.
4. Dispense 0.5 ml of Kovac’s reagent to the broth culture.
5. Visualize for the presence or absence of ring.
3.7.3 Expected Result
Positive: Appearance of a pink to red color (“cherry-red ring”) in
the reagent layer on the medium surface.
Negative: No color change.
3.8 Simmons’ Citrate
Citrate agar is employed to test an organism’s capability to utilize
citrate as a source of energy. The medium contains citrate as the sole
carbon source and inorganic ammonium salts (NH 4 H 2 PO 4 ) as the
individual source of nitrogen. Bacteria that can grow on this
medium yield an enzyme, citrate-permease, capable of converting
citrate to pyruvate. Pyruvate can then pass in the organism’s metabolic cycle for the production of energy. Growth is symptomatic of
utilization of citrate, an intermediate metabolite in the Krebs cycle.
When the bacteria metabolize citrate, the ammonium salts are
catabolized to ammonia, which increases alkalinity. The shift in
pH turns the bromothymol blue indicator in the medium from
green to blue above pH 7.6.
3.8.1 Materials Required
l
Media: Citrate agar.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.8.2 Procedure
1. Dissolve citrate agar in deionized water and adjust pH to 6.9
and add 5 ml into tubes and autoclave at 121
C.
2. Allow the tubes to cool in slanting position (long slant,
shallow butt).
3. The uninoculated medium may appear to be deep forest green
due to the pH of the sample and the bromothymol blue.
4. Pick the organisms from a well-isolated colony and streak the
slant back and forth with the light inoculum.
5. Incubate aerobically at 37
C for up to 4–7 days.
6. Visualize a color change from green to blue along the slant.
3.8.3 Expected Result
Positive: Growth (i.e., citrate utilization) and bright blue color.
Negative: No growth and no color change.
3.9 Urease
Urea is yielded by the decarboxylation of amino acids. Hydrolysis of
urea generates ammonia and CO 2 . The generation of ammonia
126
Laboratory Techniques for Symbiotic Bacteria
1. Use sterilized test tubes, transfer 4 ml of tryptophan broth.
2. Inoculate the tube in sterile conditions by taking the growth
from 18 to 24 h culture.
3. Incubate the tube at a temperature of 37
C for 24–28 h.
4. Dispense 0.5 ml of Kovac’s reagent to the broth culture.
5. Visualize for the presence or absence of ring.
3.7.3 Expected Result
Positive: Appearance of a pink to red color (“cherry-red ring”) in
the reagent layer on the medium surface.
Negative: No color change.
3.8 Simmons’ Citrate
Citrate agar is employed to test an organism’s capability to utilize
citrate as a source of energy. The medium contains citrate as the sole
carbon source and inorganic ammonium salts (NH 4 H 2 PO 4 ) as the
individual source of nitrogen. Bacteria that can grow on this
medium yield an enzyme, citrate-permease, capable of converting
citrate to pyruvate. Pyruvate can then pass in the organism’s metabolic cycle for the production of energy. Growth is symptomatic of
utilization of citrate, an intermediate metabolite in the Krebs cycle.
When the bacteria metabolize citrate, the ammonium salts are
catabolized to ammonia, which increases alkalinity. The shift in
pH turns the bromothymol blue indicator in the medium from
green to blue above pH 7.6.
3.8.1 Materials Required
l
Media: Citrate agar.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.8.2 Procedure
1. Dissolve citrate agar in deionized water and adjust pH to 6.9
and add 5 ml into tubes and autoclave at 121
C.
2. Allow the tubes to cool in slanting position (long slant,
shallow butt).
3. The uninoculated medium may appear to be deep forest green
due to the pH of the sample and the bromothymol blue.
4. Pick the organisms from a well-isolated colony and streak the
slant back and forth with the light inoculum.
5. Incubate aerobically at 37
C for up to 4–7 days.
6. Visualize a color change from green to blue along the slant.
3.8.3 Expected Result
Positive: Growth (i.e., citrate utilization) and bright blue color.
Negative: No growth and no color change.
3.9 Urease
Urea is yielded by the decarboxylation of amino acids. Hydrolysis of
urea generates ammonia and CO 2 . The generation of ammonia
126
Laboratory Techniques for Symbiotic Bacteria