# Biochars as a Solution for Silver Removal and Antimicrobial Activity in Aqueous Systems
## 2 Materials and Methods
### The In Vitro Antimicrobial Effect of Biochars Adsorbing Silver Ions  
_Microorganisms:_ Pure cultures of four strains of _Escherichia coli_ were tested: 1 reference, ATCC 8739, and 3 clinical isolates. The clinical strains used were from the Microbial Strain Collection of the Microbiological Laboratory, Faculty of Veterinary Medicine, University of Forestry, Sofia, Bulgaria, which belongs to the Global Register of Scientific Collections. Suspensions with a density of 2 by McFarland were prepared in sterile saline and brought to a concentration of 10\({}^{7}\) cells/mL.  
_Nutrient media:_ Nutrient agar and the selective and differentiating Endo agar for Gram-negative aerobic and facultatively anaerobic bacteria (BUL BIO--National Centre for Infectious and Parasitic Diseases--NCIPD--Sofia, Bulgaria) were used.  
_Antimicrobial tests:_ The antibacterial effect was investigated using the classic agar-gel diffusion method of Bauer-Kirby, according to [PERSON] and [PERSON] [35; 36; 37], designed for rapidly growing microbes under aerobic conditions on nutrient agar with a pH of 7.2-7.4 and a layer thickness of 4 mm. The 10% suspension of biochar loaded with silver in saline solution, homogenized at Vortex model Heidolph, 100-2500 rpm, was administered by the instillation of 0.1 mL into wells with a diameter of 9 mm. The control antibiotic thiamphenicol was tested by instilling into wells 0.1 mL of a solution in sterile saline at standard final concentrations of 30 \(\upmu\)g per well. Inoculation of the bacterial suspensions at a dose of 2.10\({}^{6}\) cells/mL was followed by cultivation at 35-37 \({}^{\circ}\)C for 18-24 h.  
The results were recorded by measuring the diameters of the inhibitory zones in millimetres, rounded to the nearest whole millimetres. This measurement included the diameter of the well and was performed using a transparent ruler on the outside bottom of the Petri dishes.  
The measured zones of inhibition were interpreted according to the Bauer-Kirby three-level categorization system. The sensitivity of the tested microorganisms to the examined product was determined, as in the case of non-antibiotic agents, as sulfonamides, namely: resistant (R)--for areas with diameters <12 mm; intermediate (I)--for areas within 13-16 mm; and sensitive (S)-->17 mm. For thiamphenicol, the corresponding limits were R < 12 mm; I--13-17 mm; and S-->18 mm [35; 37].  
_Suspension method:_ Suspensions of the tested plant material with silver were made at a ratio of 1:10 in a sterile physiological saline solution. To 9 mL solutions of the studied material, 1 mL of a suspension of each studied strain with a concentration of 10\({}^{7}\) cells/mL was added. At such a tenfold dilution, a bacterial cell density of 10\({}^{6}\) cells/mL was achieved. Cultures were made on nutrient agar and Endo agar at different time intervals (0 min, 30 min, and 60 min) of exposure to the studied product with silver. Cultures were also made directly from the tested bacterial strains, with a concentration of 10\({}^{6}\) cells/mL without treatment serving as controls. They were cultured at 37 \({}^{\circ}\)C for 24 h. Colonies that developed were then counted and compared to those in untreated controls.  
The broad-spectrum antibiotic thiamphenicol (Nikovet--Sofia) was used as a positive control. Sterile saline served as a negative control.  
The statistical analysis of the results was carried out using the classic Student-Fisher method.