Comparative study of well diffusion and disc diffusion method to investigate the antibacterial properties of silver nanoparticles synthesized from Curcuma longa extracts

. Green synthesis method for the preparation of silver nanoparticles (AgNPs) using Curcuma longa extracts as reducing and stabilizing agents was studied. The optimization of the synthesis was determined by varying the weight of the extracts, the AgNO 3 concentration, and the reaction time. The results revealed that 3 g of extracts, 0.005 M AgNO 3 , and 30 min of incubation time were the ideal conditions to synthesize AgNPs-Cur. The characterization was assessed by UV-Visible spectroscopy, FTIR, and TEM. UV-Vis spectra showed a surface plasmon resonance (SPR) peak at 428 nm with the stability after 5 months of storage time. FTIR analysis revealed the formation of AgNPs using turmeric extracts and the synthesized AgNPs-Cur had the average size of 22.12 ± 0.60 nm (analyzed by TEM). The well diffusion and disc diffusion method were performed to investigate the antibacterial activity of AgNPs-Cur against Bacillus subtilis, Staphylococcus aureus , Escherichia coli and Pseudomonas aeruginosa . The results concluded that AgNPs-Cur exhibited better

bacterial growth inhibitory activities with well diffusion method than disc diffusion method.
Nanoparticles have been globally studied due to their various applications.The synthesis of silver nanoparticles (AgNPs) using plant extracts is one of the important nanotechnology branches that has been widely developed.Biosynthesis of AgNPs is a type of a bottom up approach that has advantages, such as inexpensive, clean, nontoxic, and eco-friendly [1].Plants extracts possessed the ability to reduce the silver ions, hence they could be used in the biologically production of AgNPs [1][2][3].It was reported that the optimization of plant extracts concentration, AgNO3 concentration, and reaction time had an influence in forming an optimum size of AgNPs [2].
Curcumin is one of natural extracts that is derived from Curcuma longa (turmeric), usually applied as a food coloring agent.Curcumin contains diverse properties, such as antioxidants and anti-inflammatory, leading to a wide utilization in medical field.It also has been applied as an eco-friendly reducing agent in the AgNPs preparation [4].Green synthesis of AgNPs offers diverse applications, one of them is an alternative bactericidal agent [5][6][7].Previously, AgNPs were synthesized from Curcuma longa extracts to assess their antibacterial potential against Bacillus subtilis (B.subtilis), Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P.aeruginosa), and Escherichia coli (E.coli) [8].Nayak et al. synthesized AgNPs from turmeric extracts (AgNPs-Cur) against B. subtilis and P. aeruginosa using disc diffusion method [9].Another study reported that the antibacterial effect of AgNPs-Cur was investigated using well diffusion assay against S. aureus, E. coli, P. aeruginosa, Streptococcus pyogenes (S. pyogenes), and Candida albicans (C.albicans) [10].
The present study focused in the optimizing synthesis of silver nanoparticles via green synthesis using turmeric extracts, followed by the stability test and characterization with spectrophotometer UV-Vis, FT-IR spectroscopy, and Transmission Electron Microscope (TEM).The antibacterial application of AgNPs-Cur was examined against B. subtilis, S. aureus, E. coli, and P. aeruginosa.The assay was carried out by well diffusion technique and disc diffusion technique.Both techniques were compared to determine the method that yielded a better antibacterial effect.AgNO3 (Merck), turmeric powder from local market, tetracycline (Sigma), E. coli strain ATCC 8739, P. aeruginosa strain ATCC 15442, B. subtilis strain ATCC 19659, S. aureus strain ATCC 6538, media Mueller-Hinton Broth (MHB) and Mueller-Hinton Agar (MHA) were purchased from Oxoid.
The UV-Vis spectra of AgNPs-Cur were assessed by spectrophotometer UV-Vis Agilent Cary 60 in the wavelength range of 200-800 nm.FTIR spectra were determined by Shimadzu IR Prestige 21 in the 400-4000 cm −1 range.Transmission electron microscope (TEM) Tecnai G2 20S-Twin Function was used to examine the morphology and size of AgNPs-Cur.
The antibacterial activity test was carried out using well diffusion method.Each bacterial cultures were tested on MHB media overnight (24-h at 37 °C and shaken at 120 rpm).The bacterial culture (2 mL) was then inoculated to 100 mL MHA media.After the MHA plates were in solid condition, 6 mm diameters of wells were created on the plates aseptically.Next, 50 μL of AgNPs-Cur, turmeric extract solution, and AgNO3 were loaded in the wells and incubated for 24 h at 37 °C.The negative control and positive standard were tested by sterile water and tetracycline (200 μg/mL), respectively.The resulting clear zone around the wells was scored for the antibacterial activity of the samples.diameter of 6 mm were put on the solid MHA plates and loaded with 50 μL of AgNPs measured as they reflect the samples' a The optimization parameters to synthesize AgNPs-Cur were monitored with spectrophotometer UV-Vis (200-800 nm).The optimum conditions were carried out by varying the turmeric extracts, AgNO3 concentration, and the incubation time.The synthesis of AgNPs-Cur was occurred owing to the redox reaction between Ag + ions and the OH groups of the turmeric.The formation of AgNPs-Cur was proven by the appearing surface plasmon resonance (SPR) band in the wavelengths 400-500 nm [2].
The optimization of aqueous turmeric extracts in the synthesis of AgNPs-Cur was performed first.It was revealed in the Fig. 1 that the red peak assigned to 3 g of turmeric extracts had the absorbance value of around 1. 1 g of turmeric extracts led to a lower SPR peak and increasing the turmeric extracts to 5 and 7 g caused the peaks broader.A broad SPR peak was indicative of a large size of AgNPs-Cur [5].Therefore, 3 g of turmeric extracts was chosen as the optimum condition for the synthesis of AgNPs-Cur.
The influence of AgNO3 concentration was determined next.Fig. 2 showed that increasing concentration led to a reduced absorbance.0.005 M had the highest and the broadest SPR peak.This phenomenon explained that under this condition the silver nitrate produced more Ag + to be reduced to Ag 0 , so the AgNPs-Cur were formed in a larger amount than other concentrations.Hence, 0.005 M was considered as the best condition to prepare AgNPs-Cur.
After optimizing the turmeric extracts and AgNO3 concentration, the effect of incubation time was studied.It was observed from Fig. 3, the color of the AgNPs-Cur solution became darker and there was an increase in absorbance intensity when the incubation time was increased from 5 to 30 min.It showed that 30 min of reaction time could produce more AgNPs-Cur, so it was the optimum time to synthesize AgNPs-Cur.
All the optimum conditions were applied in the next synthesis process.The SPR peak of the AgNPs-Cur appeared at 424 nm with the absorbance value of 1.73 and the solution color was reddish brown (Fig. 4A).Further, the solution was stored in the fridge for 5 months.The stability of AgNPs-Cur could be studied from the change in the absorbance intensity and the peak [11].It showed that there was an increase in absorbance and the peak became broader after storage time.The increasing absorbance was occurred due to the on-going reducing process of Ag + to Ag 0 .The broad peak indicated that the particle size of AgNPs-Cur became larger.These phenomenon showed that AgNPs-Cur were agglomerated (Fig. 4B).It explained that the AgNPs-Cur solution was relatively stable for 5 months.
The formation of AgNPs-Cur happened owing to the redox reaction between silver ions and the hydroxyl groups of curcumin.FTIR analysis revealed the wavenumber shifts of turmeric extracts after AgNPs-Cur synthesis.Table 1 below enlisted the wavenumbers of each functional groups in turmeric.The peak appearing at 1665 cm - 1 (AgNPs-Cur) was attributed to ketone C=O stretching.The existence of carbonyl groups evidenced that Ag + underwent a reducing process to Ag 0 by the OH groups, and simultaneously they were self-oxidized into carbonyls.

C=H streching aromatic
The morphology and the size of AgNPs-Cur were examined using TEM.The formed AgNPs-Cur had spherical shape with the average size of 22.12 ± 0.60 nm (Fig. 5).It was observed that the particle size was relatively small which corresponded with the statement that a narrow SPR peak indicated a small size of particle [12].
The antibacterial properties of AgNPs-Cur were tested on Gram-positive bacteria (B.subtilis and S. aureus) and Gram-negative bacteria (E. coli and P. aeruginosa).The assay was performed using two different methods, i.e. well diffusion and disc diffusion.Both methods used sterile water and tetracycline as the negative control and positive standard, respectively.The resulting clear zones surrounding the wells and discs were measured as the antibacterial activity.
The results of inhibition zone in the well diffusion method were shown in Table 2, meanwhile the measurements of the clear zone diameter were displayed in Fig. 6 and Table 3.It was observed that turmeric extracts (Cur) did not exhibit any bacterial growth inhibition since the diameters of the clear zones were the same as the negative controls.AgNO3 solution had the potential to inhibit the bacterial growth of B. subtilis, S. aureus, and P. aeruginosa as there were clear zones around the wells with the diameters of 9.5 ± 0 mm; 7 ± 0 mm; and 9.75 ± 0.75 mm, respectively.The Gram-positive bacteria (B.subtilis and S. aureus) and the Gram-negative (P.aeruginosa) were inhibited effectively by AgNPs-Cur, while the Gram-negative E. coli was not susceptible.This might be caused by the power of the silver ions to penetrate into the cell membrane of the E. coli.The cell membrane of Gram-negative bacteria was built by hydrophilic groups and acted as a barrier against hydrophilic dyes, antibiotics, and detergents [14].Another study reported that the inactive antibacterial activity could be owing to the low concentrations or the presence of lipopolysaccharide in the surface membrane of Gram-negative bacteria, which had a role as a permeability barrier and restricted diffusion of active compounds [15].
In accordance with the results, the well diffusion method exhibited better bactericidal properties than the disc diffusion method did.Previous study stated that this might occur because in the well diffusion method, AgNPs-Cur were loaded directly into the wells, meanwhile in the disc diffusion method, the discs were impregnated first by the AgNPs-Cur and placed on the MHA plates later [14].It was also reported that the discs were prepared from paper filter Whatman which was composed of cellulose.There were many free hydroxyl groups consisting in each glucose monomer that covered the hydrophilic surface of the discs.Hence, the cationic silver ions were assumed to adsorb to the disc surface, not diffuse into the agar plates.Also, the discs might undergo diffusion through capillarity, where the AgNPs-Cur could be evaporated before placing the discs on the agar.Furthermore, a precipitation of water-insoluble matter in the discs would prevent any diffusion of the antibacterial agents into the agar [16].It could be concluded that the well diffusion method yielded greater diameter of inhibition zone than the disc diffusion method.