Filtropur s 0.2 μm filter

HEK293T cells at 30% confluency were transfected overnight in DMEM/10% FBS without P/S using jetPEI Transfection Reagent (#101‐40N, Polyplus‐Transfection SA). For each 10 cm dish, 5 μg DNA was incubated with 1.75 μg pMD2.G and 3.25 μg pCMV‐dR8.91 (#12259 Addgene). On the next day, the medium was replaced by DMEM/10%FBS/P/S. Cells were cultured for at least 48 h to allow production of the virus. The supernatant was filtered through a Filtropur S 0.2‐μm filter (#83.1826.001, Sarstedt) and stored at −80°C.

A sufficient volume to obtain an OD₆₀₀ = 0.5 after centrifugation, resuspension, and dilution was harvested from chemostat or batch cultures. This volume was centrifuged at 3000 ×g for 15 minutes, and the pellet was dissolved in 5 mL peptone water (0.1% peptone, 8.5% NaCl). This solution was diluted 1 : 39 in a total volume of 20 mL of inactivation media to obtain an E. coli concentration of 10⁶-10⁷ CFU per mL. Inactivation medium consisted of BHI broth (Brain Heart Infusion, Oxoid) supplemented with 10.3% NaCl and 150 mM lactic acid (DL-lactic acid, 90%, VWR, Sweden) filter-sterilized (Filtropur S 0.2 μm filter, Sarstedt, Germany) and adjusted to a pH of 4.6 with 1 M NaOH (Merck, Darmstadt, Germany). Survival at 20°C or 27°C was measured in triplicate eflasks. These inactivation temperatures are in the range commonly used for fermentation of cold-smoked sausages in Sweden [19 (link)]. At different time intervals the number of bacteria was quantified by plating decimal dilutions of the inactivation medium on TSA plates (Oxoid) and in some experiments also on violet red bile glucose agar (VRBGA, Oxoid) plates and incubating at 37°C. Colony forming units were counted after 24 hours.