2.7.2 Central composite
design (CCD) of
response surface methodology
The most influencing factors such as magnesium
sulphate, peptone and pH with low and high values and 20 runs were generated as
shown in table 3. The RSM model was validated for further analysis. Three dimensional
response surface plots were also constructed to find out the interaction among significant
factors on depolymerase production yield.
2.8 Partial purification of the
cell-free supernatant after centrifugation (200ml) was precipitated using
fractional (50–80%) ammonium sulphate saturation. The precipitate containing
the concentrated enzyme was dialyzed and the protein thus obtained was treated
as partially purified depolymerase enzyme and used for further experiments.
Visualization of bacterial capsule after depolymerase treatment
K. pneumoniae B5055
was treated with depolymerase (2 IU/ml) and incubated overnight at 37°C where
as in control tube only buffer was added. Next day, bacterial depolymerase treated
and untreated cultures were taken on a glass slide. A drop of Indian ink dye
was mixed with culture and spread uniformly on the slides. Slides were then
observed in a light microscope (100X), clear hallow depicts presence of capsule
around bacterial cells against a dark background.
Antibiotic susceptibility testing
Lawn of K.
pneumoniae B5055 was made by spreading 100µl of culture from the treated
and untreated cultures on nutrient agar plates as described in above section. The
hexa-disks(Himedia) of different antibiotics including ampicillin(10µg), ampicillin/sublactam(10/10µg),
amoxicillin/clavulanic acid(20/10µg), piperacillin/tazobactum(100/10µg), ticarcillin/clavulanic
acid(75/10µg), cefoparazone(175/10µg), cefuroxime(30µg), cefotaxime(30µg), amikacin(30 µg),
carbenicillin(100µg), ceftadizime(30µg), ceftriaxzone(30µg), netillin (30µg),
piperacillin(100µg), tobramicin(10µg) and gentamicin (10µg) were put on the
plate. After overnight incubation, the zone of inhibition around each
antibiotic disc was measured and compared.