CHAPTER 3 MATERIALS AND METHODS 3.1. Fungal ... - Shodhganga

Materials and Methods

CHAPTER 3

MATERIALS AND METHODS

3.1. Fungal growth studies

3.1.1 Fungus and fungal culture The fungus (Fusarium oxysporum NCIM No. 1072) was obtained from National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune. The culture of fungus was maintained in petri plates on potato dextrose agar (PDA) medium. The PDA medium was obtained from Hi Media laboratories. Kanamycin was used as an antimicrobial agent. All stock cultures were maintained on PDA slants and subcultured. The slants were incubated at 25°C for 7 days and then stored at 4°C.

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3.1.2 Inoculum preparation A part of the fungal colony was then transferred into the potato dextrose broth (PDB) by aseptically punching out 5 mm of the agar plate culture with a cutter. A shake flask culture was carried out in 250-ml flasks containing 50 ml of the medium at 130 rpm and incubated at room temperature over a period of time. The inoculum was periodically tested at every 24 hours for various growth parameters as provided below.

3.1.3 Dry weight of the fungus/ Biomass production The fungal mycelium was harvested after every 24 hours of growth, separated from the culture liquid by filtration through a Whatman No. 1 filter paper. The mycelial pellet was repeatedly washed with distilled water and dried at 70°C overnight. The dry weight of the fungus was calculated by using the following formula: Dry weight = (weight of filter paper + mycelium) - (weight of filter paper) The standard curve was prepared using the data collected above.

3.1.4 Glucose utilization Glucose utilization was periodically measured using the DNS reagent method as suggested by Miller (1959). Glucose estimation was carried out in the culture filtrate (media) at different time interval spectrophotometrically at 540 nm by measuring the reduction of DNS (Dinitrosalicylic acid). 3 ml of DNS was added to 1 ml of diluted sample, 84


it was then incubated in a boiling water bath for 10 min. After incubation 1ml of 40% sodium potassium tartarate (Rochelle salt) was added. Final volume of the reaction mixture was made to 10 ml by adding 5 ml of distilled water. Absorbance was measured at 540 nm. The glucose concentration was calculated by plotting a standard curve for glucose using DNS reagent method.

3.1.5

MTT

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide] assay The MTT assay was performed as suggested by Denizot and Lang (1986) and later modified by Freimoser et al. (1999). The colorimetric method for the determination of cell densities using MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is more accurate and timesaving. The respiratory chain and other electron transport systems reduce MTT and other tetrazolium salts and form non-watersoluble violet formazan crystals within the cell. The amount of these crystals can be determined spectrophotometrically and serves as the number of living cells in the sample. An MTT stock solution (5 mg of MTT/ml of distilled water) was filter sterilized and kept at 4°C. To start the reaction, stock solution was added to growing cultures having a final concentration of 0.5 mg/ml. The mixture was incubated for 16 hours on a shaker (160 rpm at 20°C). Cells were pelleted by centrifugation in Eppendorf tubes at 15,000g for 5 min. The medium was then removed and 500 ml of 1-propanol was added to the cells followed by vortexing. Lysed cells and debris were pelleted at 15,000g for 5 min. 100 ml of the 85


supernatant was transferred into a 96-well plate. The optical density (OD) was measured with the spectrophotometer at 560 nm. A blank with propanol alone was measured and subtracted from all values.

3.1.6 pH variation over time The pH of the culture filtrate was taken at every 24 hours using a pH meter. A standard curve was prepared for the pH of culture filtrate over time.

3.1.7 Protein estimation Total protein contents in the pellet and supernatant were determined by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as standard. The fungal mycelia present in the PD broth were centrifuged at 5000 rpm at 4oC for 10 min. The supernatant was taken for protein estimation by the method proposed by Lowry et al. (1951). The pellet was thoroughly washed and suspended in a mixture of 100mM TRIS (pH 6.8) and 30 μl 1% Triton X 100. The fungal cells were harvested by centrifugation at 10,000 rpm for 10 min at 4oC. The pellet was discarded and the supernatant taken for protein estimation of the pellet.

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3.2 Production of silver nanoparticles Two types of experiments were conducted parallel in duplicate: one with the washed fungal mycelium and other with the growth medium of the fungus in which it was harvested. The biomass of F. oxysporum was prepared by growing the fungus in a liquid medium (potato dextrose broth). The flasks were inoculated and put on orbital shaker at 25oC and agitated at 120 rpm for 72 h. The biomass was harvested after complete incubation by filtering through filter paper followed by repeated washing with distilled water to remove any medium component from the biomass. The fungus was then brought in contact with 100 ml of sterilized double distilled water for 24 h at 25oC in a 250 ml Erlenmeyer flask and agitated again at 120 rpm. After the incubation, the cell filtrate was obtained by passing it through Whatman filter paper No. 1. The silver nanoparticles were produced by the following two methods (i) The washed mycelium was challenged with 100 ml of 1 mM silver nitrate (AgNO3) solution (prepared in deionized water) and incubated in shaker at 200 rpm in dark condition at 37°C. Simultaneously, a positive control of incubating the fungus mycelium with deionized water and a negative control containing only silver nitrate solution were maintained under same conditions. (ii) The filtrate was treated with aqueous 1 mM silver nitrate (AgNO3) solution in an Erlenmeyer flask and incubated at room temperature.

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3.3 Detection and characterization of silver nanoparticles The characterization of silver nanoparticles was carried out by different instruments and techniques. It included visual observation, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis.

3.3.1 Detection of silver nanoparticles The preliminary detection of the formation of silver nanoparticles was carried out by observing the colour change of the cell filtrate after treatment with silver nitrate solution (1 mM).

3.3.1.1 Scanning electron microscope (SEM) analysis SEM was used to obtain images of silver nanoparticle. The first step involved dehydration of the material utilizing the principal of critical point drying (CPD). Processing of CPD was initiated by cleaning the surface of sample with ethanol. Further fixation of the sample was carried out in gluteraldehyde at room temperature for 24 hrs. After fixation, dehydration of sample with different concentrations of ethanol (10, 20, 30, 50, 70, 90 and 100%) was carried out. After dehydration some drops of acetone were added and kept in CPD machine for complete drying of the sample. After the completion of CPD process, mounting of sample on aluminium stub was carried out. For coating of the sample, 88


gold/palladium metal was used. The micrographs were taken with scanning

electron

microscopy

(Leo

Electron

Microscopy

Ltd.,

Cambridge).

3.3.1.2 Transmission electron microscope (TEM) analysis For TEM, a drop of aqueous solution containing the silver nanomaterials was placed on the carbon coated copper grids and dried under infrared lamp. Micrographs were obtained using a FEI-Philips Tecnai 12 operating at 200 kV. The size distribution of the silver nanoparticles was based on diameter of >200 particles on TEM micrographs.

3.3.1.3 Zetasizer Particle size and size distribution of the sample was performed using Zetasizer Nano-ZS, Model ZEN3600 equipped with 4.0 mW, 633 nm laser (Malvern Instruments, Worcestershire, United Kingdom). Water was used as a dispersant. Two experiments were carried out simultaneously. The aqueous solution of AgNO3 without the fungus served as control, while the treated sample comprised of AgNO3 and fungus filtrate.

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3.4 In silico studies

3.4.1 Selection of microorganisms The selection of microorganisms for further study was carried out by using the following paths: (a) Literature survey (b) Screening of water pathogenic bacteria from literature After exhaustive surveys, the following two microorganisms viz. Escherichia coli and Pseudomonas aeruginosa were selected. This selection was based on criteria like prominence in water samples, easy availability, ease of culture and economic importance.

3.4.2 Retrieval of essential genes and their products in selected microorganisms Retrieval of essential genes and their products in selected microorganisms was performed using Database of Essential genes (DEG) (Figure 3.1) and The National Microbial Pathogen Database Resource (NMPDR) (Figure 3.2).

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Figure 3.1 Homepage of DEG.

Figure 3.2 Homepage of NMPDR.

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3.4.3 In-silico screening of membrane proteins of selected microbes In-silico screening of membrane proteins (Target Identification) of selected microbes was done using Universal Protein Resource (UniProt) (Figures 3.3 and 3.4). The Universal Protein Resource (UniProt) is a comprehensive resource for protein sequence and annotation data. The UniProt databases are the UniProt Knowledge base (UniProtKB), the UniProt Reference Clusters (UniRef), and the UniProt Archive (UniParc). The UniProt Metagenomic and Environmental Sequences (UniMES) database is a repository specifically developed for metagenomic and environmental data. A total of 5 proteins were selected from 2 microorganisms namely: 

Outer Membrane Protein (trans membrane domain) (1QJ8)- E. coli



Outer Membrane Protein (trans membrane domain) (1QJP)- E. coli



Drug Discharge outer membrane Protein (1WP1)- P. aeruginosa



Outer membrane Protein (3D5K)- P. aeruginosa



Outer Membrane Protein (Modelled) (Opr 86)- P. aeruginosa

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Figure 3.3 Homepage of UniProt.

Figure 3.4 Homepage of UniProt showing protein data.

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3.4.4 Search of 3D structure of screened membrane proteins The 3-dimensional structure of proteins was downloaded from Research Collaboratory for Structural Bioinformatics (RCSB Protein Data Bank- PDB; http://www.rcsb.org) (Figures 3.5 and 3.6).

Figure 3.5 Homepage of PDB.

Figure 3.6 Homepage of PDB showing protein data. 94


3.4.5 Search for ligand structure The ligand structure was searched at Pubchem (Figure 3.7), a free database of chemical molecules and their activities against biological assays.

Figure 3.7 Homepage of Pubchem. Ag-Commercial Item Description (CID) No. 23954 obtained from Pubchem was used for further studies.

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3.4.6 Ligand binding site prediction Identifying the location of ligand binding sites on a protein is of fundamental importance for a range of applications including molecular docking, de novo drug design and structural identification and comparison of functional sites. Q-Site Finder (Figure 3.8) and Pocket-Finder were utilized for prediction of functional site amino acids using the online tools available

at

http://www.bioinformatics.leeds.ac.uk/qsitefinder

and

http://www.bioinformatics.leeds.ac.uk/pocketfinder

Figure 3.8 Homepage of Q-SiteFinder.

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3.4.7 Target protein preparation Before docking the ligand into the protein active site, all water molecules and hetero atoms were removed. Hydrogen atoms were added to the protein to define the correct ionization and tautomeric states of amino acid residues.

3.4.8 Protein-Ligand docking Metal docking was carried out using the licensed software SYBYL X 1.1.1. The FlexX score was used to guide the growing of the ligand and was assigned to the successfully docked compound to measure the goodness of its fit with the receptor. FlexX is a fast automated docking program that considers ligand conformational flexibility into a rigid protein structure by an incremental fragment placing technique (Rarey et al., 1996; Kramer et al., 1999). The FlexX scoring function includes both polar (hydrogen bond and charge-charge) and non-polar (hydrophobic) interactions that are used to dock the ligand into the active site. The scoring function, which was optimized to reproduce experimental binding affinities, was used to estimate the binding free energy (ΔG bind) of the protein-ligand complex. SYBYL was also used to generate dynamic hydrogen bonds between the docked ligand and the amino acid residues in the active site of the protein.

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3.5 Antibacterial effects of silver nanoparticles

3.5.1 E. coli

3.5.1.1 Isolation and preparation of E.coli inoculum Running water from Indira canal (Gomti Nagar, Lucknow) was taken and tested for presence of Escherichia coli (Gram negative bacteria). Escherichia coli were identified, isolated and sub-cultured. LB medium was used for growing the bacterial strains at 37°C until approximately 105-106 colony forming units (CFU) of bacteria were obtained. All the glassware and media used for the assays were sterilized in autoclave at 121oC for 20 min.

3.5.1.2 Assessment of minimum inhibitory concentration of E. coli inoculums Minimum inhibitory concentration is the minimum concentration of silver nanoparticles that completely inhibit the growth of the E. coli cells. The inocula of E. coli was prepared by growing strains in Luria– Bertani (LB) medium at 37◦C and shaken at 150 rpm until approximately 108 CFU/ml of bacteria was reached. Then 75 μl 108 CFU/ml bacterial suspension and 25 ml LB medium with 5, 10, 20, 50 and 100 μg/ml of silver nanoparticles was added to a series of flasks. The flasks were incubated at 37◦C on a rotary shaker (100 rpm) for 24 hours. Growth rates 98


and bacterial concentrations were determined by measuring optical density (OD) at 600 nm (O.D. of 0.1 corresponds to a concentration of 108 cells per milliliter).

3.5.1.3 Antibacterial activity of silver nanoparticles in liquid media Inoculations were given from fresh E. coli colonies on agar plates into 50 ml LB culture medium. 108 CFU from above were added to 100 ml liquid LB media supplemented with 5, 10, 20, 50 and 100 μg/ml of silver nanoparticles. The flasks were incubated at 37◦C on a rotary shaker (100 rpm) for 24 hours. Growth rates and bacterial concentrations were determined by measuring optical density (OD) at 600 nm (O.D. of 0.1 corresponds to a concentration of 108 cells per milliliter). Control broths were used without nanoparticles.

3.5.1.4 Antibacterial activity of silver nanoparticles on solid media To examine the bactericidal effect of silver nanoparticles on Gramnegative bacteria, approximately 108 colony forming units (CFU) of E. coli were cultured on LB agar plates. LB agar plates containing 108 colony forming units (CFU) of E. coli were supplemented with silver nanoparticles at concentrations of 5, 10, 20, 50 and 100 μg/ml. Silver nanoparticle free LB plates cultured under the same conditions were used as a control. Plates were incubated for 24 h at 37◦C.

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The bacterial cell colonies on agar-plates were detected by viable cell counts. Viable cell counts are the counted number of colonies that are developed after a sample has been diluted and spread over the surface of a nutrient medium solidified with agar in a petri dish. The number of CFU were counted. The counts on the three plates corresponding to a particular sample were averaged.

3.5.2 Pseudomonas aeruginosa

3.5.2.1 Isolation and preparation of P. aeruginosa inoculum P. aeruginosa was obtained from National Collection of Industrial Microorganisms (NCIM), NCL, Pune. Cultures of the bacterium were sub-cultured in Muller Hinton broth and agar (Hi Media laboratories) for 24 h at 37oC. All the glassware and media used for the assays were sterilized in autoclave at 121oC for 20 min.

3.5.2.2 Assessment of minimum inhibitory concentration of P. aeruginosa inoculum

Minimum inhibitory concentration is the minimum concentration of silver nanoparticles that completely inhibit the growth of the P. aeruginosa cells. For determining the minimum concentration of silver nanoparticles required for the inhibition of P. aeruginosa growth, broth dilution method was used which facilitated the testing of inhibitory activity at various nanoparticle concentrations. Nutrient broth was 100


supplemented with different concentration of nanoparticles (5, 10, 15, 20, 25 µg/ml) and inoculated with bacterial suspension to obtain 106 colony forming units (CFU)/ml. Control tubes were maintained without silver nanoparticles. The flasks were incubated at 37◦C on a rotary shaker (100 rpm) for 24 h. The MIC was determined after 24 h of incubation at 37oC by observing the visible turbidity and measuring the optical density (OD) of these culture broths at 600 nm.

3.5.2.3 Antibacterial activity of silver nanoparticles on P. aeruginosa in liquid media

Inoculations were given from fresh E. coli colonies on agar plates into 50 ml LB culture medium. 108 CFU from above were added to 100 ml liquid LB media supplemented with 1, 2, 5, 10 and 20 μg/ml of silver nanoparticles. The flasks were incubated at 37◦C on a rotary shaker (100 rpm) for 24 hours. Growth rates and bacterial concentrations were determined by measuring optical density (OD) at 600 nm (O.D. of 0.1 corresponds to a concentration of 108 cells per milliliter). Control broths were used without nanoparticles.

3.5.2.4 Antibacterial activity of silver nanoparticles on P. aeruginosa on solid media Agar dilution method was used to study the bactericidal activity of silver nanoparticles on P. aeruginosa. MHA was supplemented with 101


various concentrations of nanoparticles (1-25 µg/ml) and each plate was inoculated with 106 colony forming units (CFU) by spread plating. Plates without nanoparticles were used as control. Number of surviving bacteria in agar plates was counted after 18 h of incubation at 37◦C. The bacterial cell colonies on agar-plates were detected by viable cell counts. The numbers of CFU were counted. The counts on the three plates corresponding to a particular sample were averaged.

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