Glucose colorimetric assay

17-h stationary phase cultures grown in 0.5 g/L (nutrient and salt depletion) or 10 g/L NaCl LB (nutrient depletion) were prepared (as described above) in biological triplicate, reaching an optical density at 600 nm of 5 in both conditions. 1 mL of each culture was centrifuged at 13,400 rpm for 5 min using a microcentrifuge. Each pellet was subsequently resuspended in 1 mL 30 µM D-glucose and incubated for different time intervals. Following incubation, centrifugation was carried out as above but at 0 °C, to reduce any further glucose uptake by cells. The supernatant of each sample was collected while the pellet was discarded. Using a glucose assay kit (Sigma Aldrich, Montana, United States), 50 µL of each supernatant sample was added to 50 µL master reaction mix (46 µL glucose assay buffer, 2 µL glucose probe and 2 µL glucose enzyme mix) and incubated in the dark at room temperature for 15 min. Oxidisation of any glucose present in the sample occurred during incubation, thus generating a colorimetric product, the absorbance of which was measured at 570 nm using a CLARIOstar PLUS plate reader (BMG Labtech, UK). For each experimental repeat, glucose standards were obtained by performing a serial dilution between 0 and 10 µL of a 1 nmole/µL glucose standard solution added to 50 µl of the master reaction mix above, brought to 100 µL per well with glucose assay buffer as needed. Absorbances collected for glucose standard wells were then used as a standard curve by which sample absorbances were compared and interpolated.

To calculate the concentration of glucose present in each sample, background absorbance (assay blank of standard curve where 0 µl of glucose standard solution was present in the sample) was first subtracted and then the following equation was used:

Where S_a is the amount of glucose in the unknown sample (in nmole) from standard curve, S_v is the sample volume (µL) added into the well and C describes the concentration of glucose in the sample.