Orion 9156 bnwp ph probe

At appropriate time points, 5 mL of culture was withdrawn from the experiment cultures for measurement of optical density and pH. Specifically, samples were placed in a quartz cuvette of pathlength 10 mm (volume: 3.5 mL), and optical density measured using a UV-Visible spectrophotometer (Shimadzu Biospec Mini) at 600 nm. Samples were diluted with deionized water if the optical density exceeded 1. On the other hand, pH of samples were measured with an Orion 9156 BNWP pH probe outfitted to a Mettler Toledo Delta 320 pH meter. A formulated medium with yeast extract and a high capacity phosphate buffer system was formulated to enable the high cell density aerobic cultivation of E. coli DH5α at 37 o C and 230 rpm rotational shaking in 250 mL shake flasks (Table 1). Specifically, K2HPO4 and KH2PO4 comprise the phosphate buffer system, while yeast extract provided a secondary source of carbon and nitrogen for further biomass formation.

After 15 hours of incubation, 2.5 mL of culture broth was withdrawn and diluted with 37.5 mL of non-sterile wash buffer in a 50 mL polypropylene centrifuge tube and mixed vigorously by hand and vortex mixer. This content was centrifuged at 3300 x g for 10 minutes at 25 o C. Following centrifugation, wash buffer was carefully decanted off without disturbing the cell pellet. 40 mL of wash buffer was added to the centrifuge tube and the contents resuspended by vigorous mixing by hand and vortex mixer. The centrifugation and washing steps were repeated a total of three times prior to use of 40 mL of deionized water for resuspending the cell pellet through vigorous shaking by hand and vortex mixer. Nitric acid and sodium hydroxide was added to the samples for pH adjustment with pH measured by an Orion 9156 BNWP pH probe outfitted to a Mettler Toledo Delta 320 pH meter.