Bactiter glo microbial viability assay

To determination the influence of REPHA-OS^® and ethanol on bacterial growth of planktonic cultures, the optical density (absorption) was measured at 600 nm (OD₆₀₀) with a plate reader (Tecan, Mennedorf, Switzerland). To show the effects on metabolic activity, the BacTiter-Glo™ Microbial Viability Assay (Promega, Mannheim, Germany) was used to quantify the amount of adenosine triphosphate (ATP). BacTiter-Glo™ reagent was prepared according to the manufacturer's instructions and then mixed 1:1 with the bacterial suspension. After 5 min incubation at room temperature, luminescence was measured again with a plate reader (Tecan, Mennedorf, Switzerland). All results were normalized to the full medium control (BHI/VitK).

ATP was quantified using the BacTiter-Glo microbial viability assay (Promega, Madison, WI). To quantify bacterial ATP, the microbial viability assay reagent was first prepared according to the manufacturer’s instructions. 100μl BacTiter-Glo assay reagent was added to 100μl of the test sample (of which ATP content is to be measured), and incubated for 15 min at room temperature, to release bacterial ATP and generate a bioluminescent signal. Bioluminescent light, measured as relative light units (RLU), was recorded by a GloMax Integrated Luminescence System (Promega, Madison, WI), with a 1-s integration time. Background RLU values, determined using sterile MHB, were then subtracted from the RLU values obtained at 0h and at 24h.