6.3 Disk Diffusion
Method (Kirby–Bauer
Disc Diffusion Method)
[204]
6.3.1 Materials Required
l
Organisms: Bacterial pathogens.
l
Media: Mueller–Hinton agar (MHA).
l
Chemicals: Dimethyl sulfoxide (DMSO).
l
Sample: Crude extract/fractions/compounds of symbiotic
bacteria.
l
Others: Sterile cotton swabs, standard antibiotic disk/sterile
filter disc.
6.3.2 Procedure
1. Dissolve bacterial extracts in 1 ml dimethyl sulfoxide (DMSO).
2. Drop 10 μl solution onto a sterile 6 mm disc, which is placed on
the MHA plate inoculated with drug-resistant bacteria.
3. Use standard antibiotic (gentamicin 10 μg/disc) discs as positive control and disc with DMSO as a negative control.
4. Incubate the plates at 37
C for 24 h.
5. Measure zone of inhibition (mm) using a ruler.
6.4 Minimum
Inhibitory
Concentrations (MIC)
and Minimal
Bactericidal
Concentrations (MBC)
1. MIC and MBC are demarcated as the lowest concentration of
extract in which there is no observable growth of testing bacteria in the well.
2. Resolve the MIC of symbiotic bacterial extract by using the
broth twofold serial dilution methods in 96-well microtiter
plates.
3. Dissolve the initial concentrations of extract in DMSO to
generate 500 and 220 mg/ml solutions, respectively.
4. Mix 100 μl of extract with 100 μl MH broth in a well of the
microtiter plates followed by twofold serial dilution.
5. Add 100 μl bacterial suspension (1 Â 10
8 cell/ml) to each well
and mix.
6. Incubate plates at 37
C for 24 h.
7. Observe visually the turbidity of each well.
8. The clear wells in the microtiter plates with the lowest concentration from each extract are considered as representing the
MIC and MBC.
6.5 Mosquitocidal
activity
A biological assay is executed under two conditions: fed condition
(larvae exposed to symbiotic bacteria are fed with pulverized pet
food) and unfed condition (larvae exposed to symbiotic bacteria are
not given food). Symbiotic bacteria are tested as a larvicide against
mosquitoes.
6.5.1 Materials Required
l
Aedes spp. or Culex spp. mosquito larvae.
l
Dechlorinated water.
Biological Assay
141
Method (Kirby–Bauer
Disc Diffusion Method)
[204]
6.3.1 Materials Required
l
Organisms: Bacterial pathogens.
l
Media: Mueller–Hinton agar (MHA).
l
Chemicals: Dimethyl sulfoxide (DMSO).
l
Sample: Crude extract/fractions/compounds of symbiotic
bacteria.
l
Others: Sterile cotton swabs, standard antibiotic disk/sterile
filter disc.
6.3.2 Procedure
1. Dissolve bacterial extracts in 1 ml dimethyl sulfoxide (DMSO).
2. Drop 10 μl solution onto a sterile 6 mm disc, which is placed on
the MHA plate inoculated with drug-resistant bacteria.
3. Use standard antibiotic (gentamicin 10 μg/disc) discs as positive control and disc with DMSO as a negative control.
4. Incubate the plates at 37
C for 24 h.
5. Measure zone of inhibition (mm) using a ruler.
6.4 Minimum
Inhibitory
Concentrations (MIC)
and Minimal
Bactericidal
Concentrations (MBC)
1. MIC and MBC are demarcated as the lowest concentration of
extract in which there is no observable growth of testing bacteria in the well.
2. Resolve the MIC of symbiotic bacterial extract by using the
broth twofold serial dilution methods in 96-well microtiter
plates.
3. Dissolve the initial concentrations of extract in DMSO to
generate 500 and 220 mg/ml solutions, respectively.
4. Mix 100 μl of extract with 100 μl MH broth in a well of the
microtiter plates followed by twofold serial dilution.
5. Add 100 μl bacterial suspension (1 Â 10
8 cell/ml) to each well
and mix.
6. Incubate plates at 37
C for 24 h.
7. Observe visually the turbidity of each well.
8. The clear wells in the microtiter plates with the lowest concentration from each extract are considered as representing the
MIC and MBC.
6.5 Mosquitocidal
activity
A biological assay is executed under two conditions: fed condition
(larvae exposed to symbiotic bacteria are fed with pulverized pet
food) and unfed condition (larvae exposed to symbiotic bacteria are
not given food). Symbiotic bacteria are tested as a larvicide against
mosquitoes.
6.5.1 Materials Required
l
Aedes spp. or Culex spp. mosquito larvae.
l
Dechlorinated water.
Biological Assay
141