# Honey as a Bioindicator: Pollution's Effects on Its Quality in Mining vs. Protected Sites
## 2 Materials and Methods
### 2.2.1. Phyxico-Chemical Analyses
#### 2.2.8 Microbiological Analysis  
To assess the antibacterial activity of the honey, 7 bacterial strains (Gram-negative and Gram-positive bacteria) were used: _Escherichia coli_ (ATCC 25922), _Salmonella enteritidis_ (ATCC 13076), _Pseudomonas aeruginosa_ (ATCC 27853), _Staphylococcus aureus_ (ATCC 25923), _Staphylococcus epidermidis_ (ATCC 14990), _Bacillus cereus_ (ATCC 11788), and _Listeria monocytogenes_ (ATCC 19115).  
Several colonies from each bacterial strain were dispersed in 10 mL of nutrient broth (Mikrobiologie Labor-Technik, Arad, Romania) and incubated for 18 h at 37 \({}^{\circ}\)C. The turbidity was adjusted to a 0.5 McFarland standard and was measured using a McFarland Densitometer (Mettler Toledo, Columbus, OH, USA), corresponding approximately to a homogeneous suspension of 1.5 \(\times\) 10\({}^{8}\) CFU (colony forming units)/mL.  
The antifungal activity of the honey was tested using 7 fungal strains: _Candida albicans_ (ATCC 10239), _Aspergillus niger_ (derived from ATCC 16888), _Aspergillus flavus_ (ATCC 9643), _Penicillium chrysogenum_ (ATCC 10106), _Rhizopus stolonifer_ (ATCC 14037), _Fusarium oxysporum_ (ATCC 48112), and _Alternaria alternata_ (TX 8025).  
For the fungal cultures, colonies from each fungal strain were dispersed in 10 mL of nutrient broth, incubated for 72 h at 25 \({}^{\circ}\)C, and adjusted to a 0.5 McFarland standard.  
All strains used in this study were sourced from Thermo Fisher Scientific, Inc. (Waltham, MA, USA) and MicroBioLogics, Inc. (St. Cloud, MN, USA).  
For evaluating the antimicrobial activity, the well diffusion method was used, following the procedures recommended by the CLSI [43]. The inhibition zones diameters (IZDs) were measured and served as a semi-quantitative indicator of antimicrobial effectiveness. Mueller-Hinton agar (Merck KGaA, Darmstadt, Germany) was used for culturing the bacterial strains, while Sabouraud 4% dextrose agar (Merck KGaA) was used for the fungal strains. The surfaces of the Petri dishes were inoculated by flooding them with 1 mL of culture. Using a sterile stainless-steel tube, 6 mm diameter circles were created by pressing into the culture medium on the Petri dishes. Each hole was then filled with 150 \(\upmu\)L of undiluted and liquefied honey. The Petri dishes were incubated with their lids up for 18 h at 37 \({}^{\circ}\)C for bacterial growth and for 5 days at 25 \({}^{\circ}\)C for fungal growth. Discs with 5 \(\upmu\)g ciprofloxacin (Bio-Rad, Hercules, Marnes-la-Coquette, France) were used as positive controls for bacterial growth, and discs with 1 \(\upmu\)g voriconazole (Bio-Rad Laboratories, Marnes-la-Coquette, France) were used as positive controls for fungal growth. An artificial honey sample was prepared by dissolving 40.5 g fructose, 33.5 g glucose, 7.5 g maltose, and 1.5 g sucrose in 17 mL sterile deionized water. This solution contained the proportions of the four predominant sugars in the natural honey samples and was used as the sugar control.  
The antimicrobial activity was evaluated by measuring the diameter of the inhibition zones (in mm) using a DIN 862 ABS digital caliper (Fuzhou Conic Industrial Co., Ltd., Fuzhou, China) [44].