Rhodamine 6G efflux assay

Rhodamine 6G efflux Assay:

1. In preparation for the assay, a single colony of each isolate was suspended in 5 ml Synthetic Dextrose (SD), (6.7 g Bacto Yeast Nitrogen Base without amino acids and 20 g glucose/litre) medium with 2% glucose and incubated at 37°C with shaking at 180 rpm. 
2. Next day, 0.1 OD of cells were exponentially grown (6-7 h) in SD medium with 2% glucose at 37°C with shaking at 180 rpm. 
3. 10⁸ exponentially growing cells from each isolate were then washed three times with 1× phosphate-buffered saline (PBS; pH 7.4), starved for 2 h in 1× PBS containing 5mM deoxy-glucose, and incubated at 37°C.
4. 5 μl of a 10 mM stock of Rhodamine 6G (R6G) was added to 5 ml of a starved cell suspension to a final R6G concentration of 10 μM. The 10mM stock solution of R6G was prepared in 100% ethanol
5. The cells were then incubated at 37^oC for 30 mins.
6. The cells were then pelleted and washed once with 1X PBS.
7. The washed cells were resuspended in 1× PBS plus 2% glucose to initiate efflux.
8. 200μl samples were then collected at 0-, 30-, and 60-mins intervals.
9. The collected samples were pelleted, and the supernatants were pipetted into black microtiter 96-well plate.
10. The fluorescence of the supernatants was measured at excitation and emission wavelengths of 525 nm and 555 nm respectively. 

Category