Tryptic soy agar

M. lacticum D84 (EF204392), isolated from extended shelf-life (ESL) milk, and S. capitis subsp. capitis, isolated from an air decontamination step prior to packaging at a meat production facility, were selected for biofilm studies, as they are persistent food-related contaminants with less studied biofilm-related characteristics. The isolates were preserved in a 50% (v/v) glycerol stock at −80 °C. To obtain a stock culture, bacteria were sub-cultured overnight in tryptic soy broth (TSB; Carl Roth, Karlsruhe, Germany) at 37 °C and in 0.8% (v/v) skimmed milk broth (MB; Carl Roth, Karlsruhe, Germany) at 30 °C, for S. capitis and M. lacticum, respectively. Subsequently, stock cultures were then streaked onto either tryptic soy agar (TSA) or milk agar (MA) (Carl Roth, Karlsruhe, Germany) and incubated at 37 °C or 30 °C overnight and stored at 4 °C. Before each experiment, one colony was inoculated in 10 mL fresh TSB or MB, and the optical density 600 (OD) was standardized to 0.1 (corresponding to 6.7 ± 0.2 log₁₀ colony-forming units (CFUs) per mL⁻¹ and 8.5 ± 0.2 log₁₀ CFUs mL⁻¹ for S. capitis and M. lacticum, respectively) to obtain a working culture.

Two Gram-positive bacteria frequently found in the food industry, especially in the milk or meat industry were selected because of their strong biofilm forming ability [25 (link)]. Microbacterium lacticum (M. lacticum) D84 (EF 204392) was isolated from extended shelf-life (ESL) milk, and Staphylococcus capitis subsp. capitis (S. capitis) was isolated from an air decontamination step prior to packaging at a meat production facility. Bacterial stock and working cultures were maintained and prepared according to Zand et al. [25 (link)]. The isolates were preserved in a 50% (v/v) glycerol stock at −80 °C. To obtain a stock culture, bacteria were sub-cultured overnight in tryptic soy broth (TSB; Carl Roth, Karlsruhe, Germany) at 37 °C and in 0.8% (v/v) skimmed milk broth (MB; Carl Roth) at 30 °C, for S. capitis and M. lacticum, respectively. Subsequently, stock cultures were then streaked onto either tryptic soy agar (TSA) or milk agar (MA) (Carl Roth) and incubated at 37 °C or 30 °C overnight and stored at 4 °C. Before each experiment, one colony was inoculated in 10 mL fresh TSB or MB, and the optical density₆₀₀ (OD) was standardized to 0.1 to obtain a working culture.

Staphylococcus aureus 04-02981 (Nuebel et al., 2010 (link)) was grown at 37°C in Tryptic Soy Broth [TSB] (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) with constant agitation at 150 rpm or on Tryptic Soy Agar [TSA] (Carl Roth GmbH & Co. KG, Karlsruhe, Germany). Growth inhibition tests on agar surface were performed according to CLSI guidelines for disk diffusion test (Naas et al., 2006 (link)). For this assay, 0.25 cm² sheets of Ag and AGXX^® were used.
For generation of growth curves, bacteria were pre-cultured overnight, diluted in TSB to an optical density at 600 nm (OD₆₀₀) of 0.05 and incubated for further 8 h either in presence of AGXX^® or in the presence of silver (Ag), 24 cm² each in 30 mL medium to obtain a sheet surface to medium volume ratio (A: V) of 0.8. Cultures grown in the absence of a metal sheet served as controls. OD₆₀₀ of the cultures was measured using the Genesys 10S UV-Vis spectrophotometer (Thermo Scientific, China). Colony forming units (CFU) per mL were determined hourly from 0 to 8 h post inoculation. Growth experiments were performed in triplicate with independent biological replicates.