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3 Materials and Methods
3.4.1.1 Determination of Protein Concentration [3, 4]
Apparatus: UV–Vis spectrophotometer, conical flask, glass beaker and pipette.
Reagents
(a) 5% Na 2 CO 3 solution (5 g of Na 2 CO 3 per 100 mL of distilled water).
(b) CuSO 4 solution (0.5 mg of CuSO 4 , 5H 2 0 is dissolved in 100 mL of 1% Na-Ktartrate solution).
(c) Alkali copper reagent (50 mL of reagent a + 2 mL of reagent b). The reagent
should be prepared freshly just before use.
(d) Folin reagent solution (Folin reagent: distilled water = 1:1).
(e) 1 (N) NaOH solution (4 g NaOH is dissolved in 100 mL distilled water).
(f) Standard bovine serum albumin (BSA) solution of concentration 200 mf/l
(20 mg of BSA, Sigma USA brand is dissolved in 100 mL distilled water).
Procedure
The cell suspension of 0.5 mL was mixed with 0.5 mL 1 (N) NaOH solution and
kept in boiling water bath for 5 min. The content was then cooled in cold water. A
5 mL of freshly prepared reagent C was added to the content and allowed to stand
for 10 min. Now, 0.5 mL of Folin–Ciocalteu’s reagent solution was added and the
whole mixture was allowed to stand for 30 min for color development. The blank
was prepared taking 0.5 mL of distilled water instead of bacterial suspension and
treating it in the same way. The absorbance value was measured at 750 nm wavelength
using a visible spectrophotometer against the distilled water blank. BSA standards
ranged between 0 and 200 µg/mL were processed in the same manner as samples
for developing the calibration curve.
3.4.1.2 Biological Sample for Observation in Scanning Electron
Microscope Was Prepared in Following Ways
Fixation: Biofilm is attached to the perspex sheet (1 cm × 1 cm, 3 mm thickness) in
the treatment process.
• Dehydration: Successive immersion of the biofilm surface in ethanol solution of
varying strengths like 10, 25, 50, 75, 96 and 99% for 30 min in each case.
• Drying: At 37 degree centigrade for 1 h.
• Coating: The prepared samples are to be coated with a thin gold film (<10 nm).
• Acceleration voltage maintained: <1 kV.
• Working distance: <1 mm.
3 Materials and Methods
3.4.1.1 Determination of Protein Concentration [3, 4]
Apparatus: UV–Vis spectrophotometer, conical flask, glass beaker and pipette.
Reagents
(a) 5% Na 2 CO 3 solution (5 g of Na 2 CO 3 per 100 mL of distilled water).
(b) CuSO 4 solution (0.5 mg of CuSO 4 , 5H 2 0 is dissolved in 100 mL of 1% Na-Ktartrate solution).
(c) Alkali copper reagent (50 mL of reagent a + 2 mL of reagent b). The reagent
should be prepared freshly just before use.
(d) Folin reagent solution (Folin reagent: distilled water = 1:1).
(e) 1 (N) NaOH solution (4 g NaOH is dissolved in 100 mL distilled water).
(f) Standard bovine serum albumin (BSA) solution of concentration 200 mf/l
(20 mg of BSA, Sigma USA brand is dissolved in 100 mL distilled water).
Procedure
The cell suspension of 0.5 mL was mixed with 0.5 mL 1 (N) NaOH solution and
kept in boiling water bath for 5 min. The content was then cooled in cold water. A
5 mL of freshly prepared reagent C was added to the content and allowed to stand
for 10 min. Now, 0.5 mL of Folin–Ciocalteu’s reagent solution was added and the
whole mixture was allowed to stand for 30 min for color development. The blank
was prepared taking 0.5 mL of distilled water instead of bacterial suspension and
treating it in the same way. The absorbance value was measured at 750 nm wavelength
using a visible spectrophotometer against the distilled water blank. BSA standards
ranged between 0 and 200 µg/mL were processed in the same manner as samples
for developing the calibration curve.
3.4.1.2 Biological Sample for Observation in Scanning Electron
Microscope Was Prepared in Following Ways
Fixation: Biofilm is attached to the perspex sheet (1 cm × 1 cm, 3 mm thickness) in
the treatment process.
• Dehydration: Successive immersion of the biofilm surface in ethanol solution of
varying strengths like 10, 25, 50, 75, 96 and 99% for 30 min in each case.
• Drying: At 37 degree centigrade for 1 h.
• Coating: The prepared samples are to be coated with a thin gold film (<10 nm).
• Acceleration voltage maintained: <1 kV.
• Working distance: <1 mm.