Chapter 4 A LGINATE MEDIATED ‘G REEN ’ S YNTHESIS OF S ILVER
4.3. Experimental Section
4.3.1. Chemicals, Bacterial strains, Growth media and conditions
Alginate was obtained from Loba Chemie Pvt. Ltd., Mumbai, India. Acetone was purchased from Merck India Ltd, Mumbai, India. The growth media and conditions of GFP expressing recombinant Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa,Bacillus cereus and Enterococcus faecalis have been described in detail in Experimental Section in Chapters 2 and 3.
Experimental Section Chapter 4
60 4.3.2. Preparation of alginate capped Ag NPs (Alg-Ag NPs) composite
Alg-Ag NPs composite was synthesized by adding 2 mL of freshly prepared 1.0 × 10-2 M AgNO3 solution to 50 mL of 0.2 % (w/v) alginate solution, with constant stirring at 90 °C. The appearance of brown colour after an hour indicated formation of Ag NPs.
4.3.3. Characterization of the composite
The formation of Ag NPs was monitored by making UV-Vis spectroscopic measurements using a spectrophotometer (Lambda 45; Perkin-Elmer, Fremont, CA).
For transmission electron microscopy (TEM), 5 µL of the sample was drop-cast onto a carbon-coated copper TEM grid, which was subsequently air-dried. The drop-coated grid was analyzed by a high resolution transmission electron microscope (JEM 2100;
Jeol, Peabody, MA, USA) operating at an accelerating voltage of 200 kV. The selected area electron diffraction (SAED) pattern of Ag NP was also recorded using the same instrument. The histograms for particle size distribution were constructed analyzing several frames of similar images. The zeta potential of Ag NPs was measured using Delsa ™ Nano C particle analyzer.
4.3.4. Bactericidal activity of the composite
The antibacterial activity of Alg-Ag NPs was tested against Escherichia coli and Bacillus cereus MTCC 1305 as representative strains of Gram negative and Gram positive bacteria, respectively by well diffusion method. The suspensions of bacterial cultures in the range of 105-106 CFU mL-1 were made and streaked over the surface of agar plates to obtain uniform growth. After the plates were dried for 5 min, 4 distinct wells were punctured into the agar plates and filled with Alg-Ag NPs solution and alginate solution as control. The zones of inhibition were observed after incubation at 37°C overnight.
4.3.5. Preparation of alginate-Ag NPs-Chitosan (Alg-Ag NPs-Chi) blended Films Films were prepared by varying the ratio of alginate and chitosan to 1:1, 2:1, 4:1. In detail, 1:1 film was prepared by adding 6 mL of chitosan solution (0.1 % w/v in a 1:1 v/v solvent mixture of 2 % aqueous acetic acid and acetone) to 3 mL of above Alg-Ag NPs solution under vigorous stirring at room temperature for 20 min. This was repeated five times; the resultant suspension (45 mL after pooling) was cast into a polyethylene
Experimental Section Chapter 4
61 petri dish and dried for 24 h at 37 °C in an incubator (Reico Equipment & Instrument Pvt. Ltd.). By a similar casting method, control films of chitosan and sodium alginate were also prepared. All films were stored in dessicator at room temperature until use.
4.3.6. Characterization of films
The surface morphology of the films was examined by field emission scanning electron microscopy (FESEM) and light microscopy. For FESEM, the films were sputter-coated with gold film using a sputter coater (SC7620“Mini”, Polaron Sputter Coater, Quorum Technologies, Newhaven, England) and imaged under FESEM (Carl Zeiss, SIGMA VP). Optical microscopy images were acquired with a Carl Zeiss Axioskop 2 Mat Microscope fitted with a digital camera. The Fourier transform infrared (FTIR) spectra of the films were recorded using a Perkin-Elmer Spectrum one spectrometer. X-ray diffraction (XRD) patterns of the solid product of Alg-Ag NPs obtained after drying the colloidal dispersion and films were recorded with a Bruker D8 ADVANCE (Bruker AXS Inc.) XRD machine using Cu Kα (λ= 1.54 Å) source.
4.3.7. Water uptake studies
Triplicate samples of each film were weighed by using a weighing balance (GR-202, A&D Company Ltd.) and equilibrated in 15 mL of PBS (pH 7.4) at room temperature.
After 24 h, the films were retrieved, blotted of excess water and weighed immediately.
The procedure was repeated until the membrane attained constant weight. The % uptake was determined by following equation:
% uptake = [(wet weight - dry weight)/dry weight] x 100
4.3.8. Mechanical properties
The mechanical properties of films were measured using a Deben MICROTEST (Gatan) machine running at a speed of 5 mm min-1. Tensile strength was calculated by dividing the maximum force at break by the cross-sectional area of film (Newton m-2 = Pascal). Percent elongation at break was calculated on the basis of length extended as compared to the original length of the film. The reported film properties were average of measurements on three different films. The film thickness was measured by FESEM (Carl Zeiss, SIGMA VP) and the average of eight measurements was reported.
Results and Discussion Chapter 4
62 4.3.9. Antibacterial activity of films
The agar diffusion method was used to determine the antimicrobial effects of films on bacterial strains. The suspensions of bacterial cultures in the range of 105 - 106 CFU mL-1 were made and streaked over the surface of agar plates to obtain uniform growth.
After the plates dried for 5 min, films of diameters 1 cm, 1.5 cm and 2 cm were placed on the top of swabbed agar. The zones of inhibition were measured after incubating plates at 37 °C overnight. The reported values were average of three independent experiments carried out with each strain.