Materials and Methods

83

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.

Materials and Methods

84

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,

Materials and Methods

85

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-(4,5-

dimethylthiazol-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-water-

soluble 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

Materials and Methods

86

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.

Materials and Methods

87

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.

Materials and Methods

88

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,

Materials and Methods

89

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.

Materials and Methods

90

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 micro-

organisms

Retrieval of essential genes and their products in selected micro-

organisms was performed using Database of Essential genes (DEG)

(Figure 3.1) and The National Microbial Pathogen Database Resource

(NMPDR) (Figure 3.2).

Materials and Methods

91

Figure 3.1 Homepage of DEG.

Figure 3.2 Homepage of NMPDR.

Materials and Methods

92

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

Materials and Methods

93

Figure 3.3 Homepage of UniProt.

Figure 3.4 Homepage of UniProt showing protein data.

Materials and Methods

94

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.

Materials and Methods

95

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.

Materials and Methods

96

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.

Materials and Methods

97

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.

Materials and Methods

98

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

Materials and Methods

99

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 Gram-

negative 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.

Materials and Methods

100

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

Materials and Methods

101

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

Materials and Methods

102

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.