3.14.2 Procedure
1. Pick a few colonies using a sterile loop or swab from pure
culture plate.
2. Take a starch plate and streak in the form of a line transversely
along the width of the plate.
3. Incubate plate at 37
C for 48 h.
4. Dispense 2-–3 drops of 10% iodine solution directly onto the
edge of colonies.
5. Leave 10–15 min for starch hydrolysis.
3.14.3 Expected Result
Positive: Clear zone around the colonies.
Negative: No clear zone.
3.15 Cytochrome
Oxidase
The oxidase test is employed to ascertain symbiotic bacteria that
generate cytochrome c oxidase, an enzyme of the bacterial electron
transport chain. When present, the cytochrome c oxidase oxidizes
the reagent (tetramethyl-p-phenylenediamine) to (indophenols)
purple color end product.
3.15.1 Materials
Required
l
Reagent: 1% tetramethyl-p-phenylenediamine dihydrochloride.
l
Others: Filter paper, commercially paper disk.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.15.2 Procedure
1. Soak a filter paper with the substrate tetramethyl-p-phenylenediamine dihydrochloride.
2. Humidify the paper with sterile distilled water.
3. Smear a colony on the filter paper by picking with a help of
a loop.
4. Visualize the inoculated area of paper for a color change to
deep blue or purple within 10–30 s.
3.15.3 Expected Result
Positive: Dark purple color.
Negative: No color.
3.16 API 50 CH Test
Physiological and biochemical categorization of the bacteria are
established by using API 50 CH (BioMerieux). API 50 CH is a
standardized system combining 50 biochemical tests to study the
carbohydrate metabolism (fermentation of substrates belonging to
the carbohydrate family and its derivatives which includes heterosides, polyalcohols, and uronic acids) of symbionts. API 50 CHL
medium permits the fermentation of carbohydrates on the API
50 CH strip to be premeditated and recognizes the bacteria.
130
Laboratory Techniques for Symbiotic Bacteria
1. Pick a few colonies using a sterile loop or swab from pure
culture plate.
2. Take a starch plate and streak in the form of a line transversely
along the width of the plate.
3. Incubate plate at 37
C for 48 h.
4. Dispense 2-–3 drops of 10% iodine solution directly onto the
edge of colonies.
5. Leave 10–15 min for starch hydrolysis.
3.14.3 Expected Result
Positive: Clear zone around the colonies.
Negative: No clear zone.
3.15 Cytochrome
Oxidase
The oxidase test is employed to ascertain symbiotic bacteria that
generate cytochrome c oxidase, an enzyme of the bacterial electron
transport chain. When present, the cytochrome c oxidase oxidizes
the reagent (tetramethyl-p-phenylenediamine) to (indophenols)
purple color end product.
3.15.1 Materials
Required
l
Reagent: 1% tetramethyl-p-phenylenediamine dihydrochloride.
l
Others: Filter paper, commercially paper disk.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.15.2 Procedure
1. Soak a filter paper with the substrate tetramethyl-p-phenylenediamine dihydrochloride.
2. Humidify the paper with sterile distilled water.
3. Smear a colony on the filter paper by picking with a help of
a loop.
4. Visualize the inoculated area of paper for a color change to
deep blue or purple within 10–30 s.
3.15.3 Expected Result
Positive: Dark purple color.
Negative: No color.
3.16 API 50 CH Test
Physiological and biochemical categorization of the bacteria are
established by using API 50 CH (BioMerieux). API 50 CH is a
standardized system combining 50 biochemical tests to study the
carbohydrate metabolism (fermentation of substrates belonging to
the carbohydrate family and its derivatives which includes heterosides, polyalcohols, and uronic acids) of symbionts. API 50 CHL
medium permits the fermentation of carbohydrates on the API
50 CH strip to be premeditated and recognizes the bacteria.
130
Laboratory Techniques for Symbiotic Bacteria