Measuring Bacterial Tobramycin Uptake

The bacterial uptake of tobramycin was measured by two independent methods. In the radioactivity assay, a stock solution of 15 μCi ³H-labeled tobramycin/ml was prepared in pure water and stored at –20°C. Referring to the method described in our earlier report for assaying ³H-labeled estrogen uptake in E. coli cells (79 (link)), exponential-phase E. coli cell cultures were concentrated 4-fold by centrifugation and resuspension, thoroughly mixed with ³H-labeled tobramycin at a final concentration of 0.015 μCi per 100 μl, and then subjected to the rapid freezing treatment. After thawing in an ice-water bath, the cells were centrifuged (16,000 rpm, 15 s), washed once with the PBS buffer, resuspended in a lysis buffer (0.2 M NaOH, 1% SDS), and incubated at 90°C for 30 min. The cell lysates were then cooled, centrifuged quickly (800 rpm, 10 s), and mixed with a 5-volume scintillation cocktail before subjected to radioactivity measurement on a PerkinElmer Tri-Carb3110TR liquid scintillation analyzer. The nonradioactivity assay was performed as recently described by us (41 (link)), such that tobramycin as taken up by E. coli cells was extracted by cell lysis and then dropped on LB agar dishes to inhibit bacterial cell growth.