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 37oC 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.