Muller hinton agar

Five lots of manufactured product were used over the course of the trial. A representative sample of the product from each lot was collected directly from a commercial supplement tub. A sample of the lyophilized base probiotic mix that was used for supplement manufacturing was also collected. Samples were placed in a −80 °C freezer until further processing.
Samples from all lots were cultured aerobically on phenylethyl alcohol agar (Oxoid Limited, Nepean, ON, Canada), as well as anaerobically on Muller Hinton agar (Sigma Aldrich, Oakville, ON, Canada). One (1.0) g of sample was serially diluted in 9 mL of sterile PBS and plated in triplicate from a concentration of 10⁻¹–10⁻⁹. Aerobic plates were cultured for 24 h at 37 °C, and anaerobic plates were cultured in a DG250 Whitley anaerobic chamber (Microbiology International, Frederick, MD, USA) at 37 °C for 48 h. Plates with <300 colonies were counted, and the average between all 3 plates was used to determine viable bacteria. Visual assessment of morphology was used to differentiate bacteria, and representative colonies were sub-cultured and sent to Animal Health Laboratory (University of Guelph, Guelph, ON, Canada) for MALDI-TOF identification.

The Prunus armeniaca gum (PAG) was collected from tree trunk from Malakwal, Punjab, Pakistan in August 2021. The collected gum along with plant parts was taxonomically identified at Department of Botany University of Sargodha by taxonomist Dr. Amin Ullah Shah (Associate Professor). A voucher specimen (No.UOS-PA-21-18) was deposited in the herbarium, University of Sargodha, Sargodha for further reference. All the procedures of collection and identification of plant material were in accordance with the guidelines of National Herbarium of Pakistan, Flora of Pakistan, and International Plant Name Index. All other reagents and chemicals used were commercially available and of analytical grade. Chemical used in experimental work include ethyl alcohol, sodium alginate: Mw 216, 2, 2- diphenyl-1-picrylhydrazyl (DPPH), Muller Hinton agar were obtained from Sigma-Aldrich and potassium dihydrogen phosphate was procured from Merck. All other chemicals and reagents used are commercially available and analytical grade.

Mueller-Hinton Broth (MHB), Muller-Hinton Agar (MHA), streptomycin, ciprofloxacin, ampicillin, tetracycline, metronidazole, myricetin, podophyllotoxin, dimethyl sulfoxide (DMSO ≥ 99.5%), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (≥ 98%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Potato dextrose agar (PDA), methanol, dichloromethane, HPLC-grade acetonitrile, and ethyl acetate were purchased from VWR (Fontenay-sous-Bois, France). Formic acid (FA) was purchased from Merck (Darmstadt, Germany). Water was purified by 0.22 µM membrane filtration and deionization by using a Barnstead Nanopure system from Thermo Scientific (Waltham, MA, USA) or a Milli-Q Plus system (Millipore, Billerica, MA, USA), while the DNeasy UltraClean Microbial kit was purchased from Qiagen (Hilden, Germany) and the GFX PCR DNA and Gel Band Purification Kit were purchased from GE Healthcare (Chicago, IL, USA).

A total of ninety (n = 90) composite specimens were tested for their antibacterial activity with an agar diffusion test. A standardized bacterial suspension (200 µL) was uniformly spread onto petri plates containing Muller–Hinton agar (Sigma, St. Louis, MO, USA). Using a sterile cotton swab, the agar plates were streaked by spreading inoculum over the entire agar surface evenly while the plate was held at approximately 60°. With the help of sterile metal tweezers, the specimen discs were placed by firmly pressing down onto the inoculated agar plate’s surface with a 2 cm distance from each other. Four discs were placed on each plate, including two experimental RBDC discs, a positive control, and a negative control disc. For the positive control, 0.2% chlorhexidine gluconate mouthwash (Protect, Karachi, Pakistan) was poured on filter paper discs with dimensions matching the RBDC discs. The concentration applied was 0.075 ppm. The plates were then placed in a CO₂ incubator for 48 h set at 37 °C and 5% CO₂. After incubation, the diameter of the inhibition zone around each specimen was measured using vernier calipers in two perpendicular locations [2 (link)].

Micro-curcumin (MC), dichloromethane, Nutrient agar, Muller Hinton agar, glycerol, phosphate-buffered saline, white paraffin, petroleum jelly, ketamine, Soframycin ointment and Ciprofloxacin, purchased from Sigma Chemicals Co. (USA). Experimental research on used plants complied with relevant international guidelines and legislation.

A. pullulans 201253 was obtained from the American
Type Culture Collection and
was maintained at 4 °C on Sabouraud dextrose agar (SDA) and subcultured
regularly every 2 weeks. For long-term storage, cultures were maintained
at −80 °C in a 15% glycerol solution. For inoculum preparation, A. pullulans was grown at 28 °C for 48 h in a medium
shown in Table S1. Silver nitrate (AgNO₃, ≥ 99.0%) and gold(III) chloride trihydrate (HAuCl₄·3H₂O, ≥ 99.9%) were purchased from
Sigma-Aldrich. Luria–Bertani broth, Muller–Hinton agar,
and other chemicals used in the antimicrobial analyses (Table S1) were purchased from Sigma-Aldrich.
Mica was supplied from Agar Scientific. Twenty-five micrometer thick
poly(ethylene-alt-tetrafluoroethylene) (ETFE) film
(Tefzel 100LZ) was kindly donated by the Paul Scherrer Institute,
PSI, Switzerland. Poly(styrene sulfonic acid)-grafted ETFE film (ETFE-g-PSSA, degree of grafting: 54%) was synthesized in a previous
study of ours.^{19 (link)}