3.1.2 Procedure
Tube Method
1. Dispense 1–2 ml of hydrogen peroxide solution into a
test tube.
2. Place a colony of the 18–24 h test organism using a sterile and
clean wooden stick or a glass rod and dip in the hydrogen
peroxide solution.
3. Visualize for immediate bubbling.
Slide Method
1. Take a small amount of colony growth using a loop onto the
surface of a sterile, clean, desiccated glass slide.
2. Dispense a drop of 3% H 2 O 2 in the glass slide.
3. Visualize for immediate bubbling.
3.1.3 Expected Result
Positive: Abundant bubbles produced, active bubbling.
Negative: No bubbles at all or very few bubbles produced.
3.2 Proteolysis
3.2.1 Materials Required
l
Media: Casein agar.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
l
Petri dish.
3.2.2 Procedure
1. Prepare casein agar and autoclave at 121
C and pour into
sterile petri dish plate.
2. Inoculate with culture into the well and incubate the plates for
24–48 h.
3. Detect the clear zone surrounding the growth of proteolysis on
the plates.
3.2.3 Expected Result
Positive: Clear zone surrounding the growth.
Negative: No clear zone.
3.3 Lipase Activity
The method is established on the rate at which the enzyme hydrolyzes tributyrin at pH 7.5. The butyric acid which is obtained is
titrated with sodium hydroxide and the utilization of the latter is
recorded as a function of time.
3.3.1 Materials Required
l
Media: Tributyrin agar.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.3.2 Procedure
1. Prepare tributyrin agar and autoclave at 121
C.
2. Inoculate the culture after the solidification of agar.
3. Incubate at 37
C for 24–48 h.
4. Subject the plate to agar diffusion method.
Biochemical Characterization
123
Tube Method
1. Dispense 1–2 ml of hydrogen peroxide solution into a
test tube.
2. Place a colony of the 18–24 h test organism using a sterile and
clean wooden stick or a glass rod and dip in the hydrogen
peroxide solution.
3. Visualize for immediate bubbling.
Slide Method
1. Take a small amount of colony growth using a loop onto the
surface of a sterile, clean, desiccated glass slide.
2. Dispense a drop of 3% H 2 O 2 in the glass slide.
3. Visualize for immediate bubbling.
3.1.3 Expected Result
Positive: Abundant bubbles produced, active bubbling.
Negative: No bubbles at all or very few bubbles produced.
3.2 Proteolysis
3.2.1 Materials Required
l
Media: Casein agar.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
l
Petri dish.
3.2.2 Procedure
1. Prepare casein agar and autoclave at 121
C and pour into
sterile petri dish plate.
2. Inoculate with culture into the well and incubate the plates for
24–48 h.
3. Detect the clear zone surrounding the growth of proteolysis on
the plates.
3.2.3 Expected Result
Positive: Clear zone surrounding the growth.
Negative: No clear zone.
3.3 Lipase Activity
The method is established on the rate at which the enzyme hydrolyzes tributyrin at pH 7.5. The butyric acid which is obtained is
titrated with sodium hydroxide and the utilization of the latter is
recorded as a function of time.
3.3.1 Materials Required
l
Media: Tributyrin agar.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.3.2 Procedure
1. Prepare tributyrin agar and autoclave at 121
C.
2. Inoculate the culture after the solidification of agar.
3. Incubate at 37
C for 24–48 h.
4. Subject the plate to agar diffusion method.
Biochemical Characterization
123