## 1 Introduction
#### 2.1.7 Antibacterial Bioassays  
Test microorganisms  
The antibacterial efficacy of both leaf and stem AgNP samples was evaluated against five distinct strains of bacteria. These strains included Gram-positive bacteria: _Bacillus subtilis_ (ATCC 6633), methicillin-resistant _Staphylococcus aureus_ (ATCC 43300), and _Staphylococcus aureus_ (ATCC 25923), in addition to Gram-negative bacteria: _Pseudomonas aeruginosa_ (ATCC 25783), and _Escherichia coli_ (ATCC 35218). The aforementioned bacterial strains were provided by Professor [PERSON] from the School of Life Sciences (Microbiology Department) at UKZN, and were preserved in a 75% glycerol solution at \(-80\)\({}^{\circ}\)C for experimental use.  
Preparation of sample  
Synthesized nanoparticles were obtained from methanolic, aqueous-fresh, and powdered leaf and stem extracts. These extracts were further dissolved in a 10% dimethyl sulfoxide (DMSO) solution at different concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 mg/mL. Samples were then transferred into Eppendorf tubes (mL) and stored at 4 \({}^{\circ}\)C in preparation for further experimental procedures.  
Preparation of culture media and bacterial cultures  
Mueller-Hinton agar (MHA, 38 g) (Biolab, Merck, South Africa) was dissolved in 1 L of distilled water, agitated for 15 min, microwave heated for 10 min, and then autoclaved (Model HL-340, Durban, South Africa) for a duration of 1 h at 121 \({}^{\circ}\)C. The medium was aliquoted into Petri dishes (sterile) (90 mm) and cooled at room temperature (23 \({}^{\circ}\)C). Each bacterial strain, encompassing both Gram-positive and Gram-negative categories, was sub-cultured onto freshly prepared agar plates and incubated overnight (24 h) at 37 \({}^{\circ}\)C. Glass test tubes containing 15-20 mL of distilled water were similarly autoclaved at 121 \({}^{\circ}\)C for 1 h. Bacterial colonies from each agar plate were inoculated by transferring 5 \(\upmu\)p-\(\mu\)L-1 of the respective strain into the prepared glass test tubes containing 15 mL of sterile distilled water, corresponding to the 0.5 McFarland scale. The absorbance of each bacterial culture was subsequently measured, adjusted, and diluted to achieve an appropriate viable cell count utilizing a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA).  
Freshly cultured bacterial strains were uniformly distributed over the MHA plates using an L-shaped metal spreader (hockey stick). The disk diffusion method was employed to evaluate the antibacterial properties of the synthesized AgNPs. Approximately 20 \(\upmu\)L of AgNPs, prepared using the methanolic and aqueous-fresh and powdered extracts, were carefully pipetted onto sterile filter paper discs (Whatman(r) no. 1 filter paper) with a diameter of 6 mm. Sterile discs impregnated with AgNPs at various concentrations were allowed to dry overnight before being positioned onto Petri plates inoculated with bacterial cultures. The Petri plates were subsequently sealed and incubated for 24 h at 37 \({}^{\circ}\)C. The presence of clear zones of inhibition surrounding the filter paper was recorded as indicative of positive antibacterial results, and the diameters of these zones were measured and documented within 18-24 h post-incubation to ascertain whether the synthesized AgNPs exhibited antibacterial activity. The clear zones of inhibition were quantified and tabulated. Filter paper discs containing streptomycin and gentamycin served as positive controls, while 10% DMSO was utilized as a negative control. The analyses were conducted in triplicate, and the data were presented as mean \(\pm\) standard deviation.