5. Check for zone of hydrolysis around the well.
3.3.3 Expected Result
Positive: Clear zone.
Negative: No clear zone.
3.4 Hemolysin
Blood agar is a universal purpose supplemented medium used to
grow particular organisms and to identify bacteria based on their
hemolytic characteristics. It is an exceedingly nutritious medium
commonly used as a basal medium for the preparation of blood agar
by supplementation with blood.
3.4.1 Materials Required
l
Media: Nutrient agar.
l
Others: Defibrinated sheep blood.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
l
Distilled water.
3.4.2 Procedure
1. Add 2.8 g of nutrient agar powder to 100 ml of distilled water
and autoclave at 121
C for 15 min.
2. Permit the nutrient agar to cool at 50
C but not solidify.
3. Dispense 5% (vol/vol) sterile defibrinated blood that has been
warmed to room temperature and blend softly, prevent air
bubbles.
4. Pour the liquid onto sterile plates.
5. Inoculate the organism in the blood agar plate.
6. Incubate the plates in a carbon dioxide rich atmosphere at
37
C overnight.
3.4.3 Expected Result
Zone of the hemolysis is positive for hemolysin.
3.5 Methyl Red
Methyl red (MR) test defines whether the microbe achieves fermentation of mixed acids when glucose is supplied.
3.5.1 Materials Required
l
Media: MR-VP broth.
l
Reagent: Methyl red indicator.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.5.2 Procedure
1. Inoculate tubes comprising MR-VP broth with culture.
2. Incubate at 35
C for up to 4 days.
3. Dispense around five drops of the methyl red indicator solution
to the first tube.
4. If the color of the medium varies to red after a few minutes, a
favorable reaction is indicated.
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Laboratory Techniques for Symbiotic Bacteria
3.3.3 Expected Result
Positive: Clear zone.
Negative: No clear zone.
3.4 Hemolysin
Blood agar is a universal purpose supplemented medium used to
grow particular organisms and to identify bacteria based on their
hemolytic characteristics. It is an exceedingly nutritious medium
commonly used as a basal medium for the preparation of blood agar
by supplementation with blood.
3.4.1 Materials Required
l
Media: Nutrient agar.
l
Others: Defibrinated sheep blood.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
l
Distilled water.
3.4.2 Procedure
1. Add 2.8 g of nutrient agar powder to 100 ml of distilled water
and autoclave at 121
C for 15 min.
2. Permit the nutrient agar to cool at 50
C but not solidify.
3. Dispense 5% (vol/vol) sterile defibrinated blood that has been
warmed to room temperature and blend softly, prevent air
bubbles.
4. Pour the liquid onto sterile plates.
5. Inoculate the organism in the blood agar plate.
6. Incubate the plates in a carbon dioxide rich atmosphere at
37
C overnight.
3.4.3 Expected Result
Zone of the hemolysis is positive for hemolysin.
3.5 Methyl Red
Methyl red (MR) test defines whether the microbe achieves fermentation of mixed acids when glucose is supplied.
3.5.1 Materials Required
l
Media: MR-VP broth.
l
Reagent: Methyl red indicator.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.5.2 Procedure
1. Inoculate tubes comprising MR-VP broth with culture.
2. Incubate at 35
C for up to 4 days.
3. Dispense around five drops of the methyl red indicator solution
to the first tube.
4. If the color of the medium varies to red after a few minutes, a
favorable reaction is indicated.
124
Laboratory Techniques for Symbiotic Bacteria