# Antioxidant, Antibacterial, and Cosmeceutical Potential of Four Common Edible Mushrooms
## 2 Materials and Methods
### Antibacterial Activity  
Clinical isolates were collected from patients hospitalized in the Hospital Center of Tras-os-Montes and Alto Douro (CHTMAD), Portugal. Ethical approval was granted by the Ethics Committee of CHTMAD. These strains belong to MJH and MJMC collections and are stored at \(-70\)\({}^{\circ}\)C in aliquots of brain-heart infusion medium with 15% (\(v/v\)) glycerol. The antimicrobial activity of the mushroom extracts was assessed using Gram-positive and Gram-negative bacteria obtained from wound exudates. All the strains were identified by morphological and biochemical tests (Gram staining, morphological identification of colonies, conventional biochemical identification methods, and MicroScan WalkAway identification panels), followed by Kirby-Bauer antibiotic sensitivity assays with different antibiotics. _Escherichia coli_ (_E. coli_; ATCC 25922) and _Staphylococcus aureus_ (_S. aureus_; ATCC 25923) strains were obtained from the American Type Culture Collection.  
Minimum inhibitory concentration (MIC). The MIC was evaluated using the microdilution assay, as reported by [PERSON] et al. [29], with minor changes. Initially, each extract was dissolved in 50% dimethyl sulfoxide (DMSO, Sigma Aldrich) to reach a final concentration of 220 mg/mL. Then, 50 \(\upmu\)L of the extract solution in 50% DMSO at 220 mg/mL was added to 450 \(\upmu\)L of Mueller-Hinton Broth (MHB) medium (Oxoid, Basingstoke, United Kingdom), making a solution with a final concentration of 22 mg/mL at 5% DMSO. Subsequently, 190 \(\upmu\)L of this extract solution was added to the first well of each column of the 96-well microplate (Orange Scientific, Braine-I'Alleud, Belgium) and submitted to successive dilutions over the remaining wells containing 90 \(\upmu\)L of MHB medium, starting by withdrawing a volume of 100 \(\upmu\)L from the well containing the highest concentration and adding it to the well just below and thus repeating this procedure sequentially. Afterwards, 100 \(\upmu\)L of inoculum (1.5 \(\times\) 10\({}^{8}\) CFU/mL) was diluted in 9.9 mL of MHB medium. Then, 10 \(\upmu\)L of this solution was added to all the wells, achieving a final volume of 100 \(\upmu\)L in each well and concentrations ranging from 20 to 0.156 mg/mL. A negative control was prepared with MHB 5% DMSO. The positive control used was gentamicin (Applied Chemical Laboratories, Basel, Switzerland). The microplates were left at 38 \({}^{\circ}\)C and incubated for 24 h in a heated incubator (MIR-162-FE, Panasonic, Japan). The MICs of each extract were determined by adding 2,3,5-triphenyltetrazolzolum chloride (TTC, Merck; 0.2 mg/mL, 40 \(\upmu\)L) to each well and incubating the microplates at 38 \({}^{\circ}\)C for 2 h. In the presence of viable bacteria, the colorless TTC dye is reduced to formazan (red color). The lowest extract concentration that prevented the color change, i.e., that shows complete inhibition of microbial growth, is defined as the MIC [30].  
Minimum bactericidal concentration (MBC). The assessment of MBC, the lowest concentration of mushroom extract that kills 99.9% of the bacterial populations tested, was carried out as previously described [21]. Briefly, the content from each well without changes in color determined from the previous assay was plated on Mueller-Hinton Agar (MHA) medium (Oxoid, Basingstoke, United Kingdom) and left at 38 \({}^{\circ}\)C for 24 h. After this subculturing, the MBC was defined considering the lowest concentration that yielded no growth.