Triathler liquid scintillation counter

Rapid dilution-filtration assays were performed as described previously.^{26 (link)} MG1655 E. coli were grown to stationary phase as described above for the solute distribution measurements. An aliquot equivalent to 2 mL of OD600 = 2.0 was thrice washed and resuspended in 2 mL 10 mM HCl, pH 2, 150 mM KCl, 10 mM glucose and 1 mM glutamic acid. ³⁶Cl was added at a concentration where 100 uL of the suspension contained approximately 80,000 counts per minute. After incubating for 20 minutes, 100 uL of the suspension was diluted into 10 mL unlabeled 10 mM HCl, pH 2, 150 mM KCl, 10 mM glucose and 1 mM glutamic acid and the solution was immediately vacuum-filtered through a 2.5 cm 0.45 um Nitrocellulose membrane (Millipore cat. no. HAWP02500) using a Millipore XX2702550 vacuum manifold. The time between dilution and complete filtration was about 15 seconds. The filter was then resuspended in 10 mL scintillation fluid and the radioactivity was assayed in a Hidex Triathler liquid scintillation counter. Three replicates were performed. To determine the amount of ³⁶Cl from residual liquid retained on the filter, the above dilution-filtration procedure was performed on a 2 mL sample of buffer containing 80,000 cpm ³⁶Cl per 100 uL without E. coli.