Overnight (18 h) grown cultures at 20 °C were used to inoculate (1%) 12-well polystyrene microtiter plates containing 1 ml BHI,
BHI-Mn, or BHI-Mn-G and incubated at 20 °C for 48 h. Single and mixed species biofilms were washed once with PBS and exposed to 1 ml of 50 or 100 μg/ml benzalkonium chloride (Merck, Darmstadt, Germany) or peracetic acid (Sigma-Aldrich, Steinheim, Germany) up to 15 min at 20 °C. After exposure, the see more biofilms were washed once with PBS and resuspended in 1 ml PBS by pipetting rigorously. To verify that washing and resuspending in PBS inhibited further inactivation of the cells by residual benzalkonium chloride or peracetic
acid, a control experiment was performed in which the cells after standard treatments were incubated in 1 ml PBS for up to 1 h. No further inactivation during this incubation period was observed for both benzalkonium chloride and peracetic acid treatments. The cells were serial diluted in PBS and plated on BHI agar containing 2 μg/ml erythromycin and/or MRS agar containing 20 μg/ml GDC-0973 solubility dmso kanamycin. Plates were incubated for 3-5 days at 30 °C and colonies were enumerated. Overnight (18 h) grown cultures at 20 °C were used to inoculate (1%) 2 ml BHI, BHI-Mn, or BHI-Mn-G in 12 ml polystyrene tubes (Greiner Bio-One, Frickenhausen, Germany). Planktonic cultures were grown statically for 24 h at 20 °C and 1 ml culture was centrifuged (2 min at 5000 × g). The pellet was resuspended
in 1 ml of 20 or 50 μg/ml benzalkonium chloride or peracetic acid up to 15 min at 20 °C. Samples were serial diluted in PBS and plated on BHI agar or MRS agar and incubated for 3-5 days at 30 °C. /www.selleck.co.jp/products/MG132.html All disinfection treatments were performed in two independent biological replicates. To determine whether planktonic cells, single species biofilms, and mixed species biofilms showed differences in resistance against benzalkonium chloride and peracetic acid, the inactivation curves were fitted with the reparameterized Gompertz model (Zwietering et al., 1990) using the following equation: equation(1) log10Nt=log10N0+A⋅exp−expk⋅e−A⋅(ts−t)+1where A is the difference between the surviving population and the initial population (log10 cfu/well), k is the maximum specific inactivation rate (log10/min), and ts is the duration of the shoulder (min). The model was fitted in Microsoft Excel by minimizing the residual sum of squares using the Excel Solver add-in. Statistical significant differences between the average parameter estimates of the inactivation curves were identified using the Student’s t-test (p < 0.05). The L. monocytogenes strain EGD-e, which is one of the most widely studied L.