Warning: in order to get proper result analysis, it is better to perform 3 replicates for each toluene concentration

Toluene preparation: stock solutions

100mg/L: Dilution of absolute liquid toluene (Cm= 867g/L) to obtain a final toluene concentration at 100mg/L: Add 11,53uL of toluene into 100mL Ethanol (calculations done from toluene's molecular weight)
100µg/L: Dilute 10µL of the first stock solution into 10mL of Ethanol

Inoculation

From 1 UFC: Resuspend colony in 20 mL of LB medium containing chloramphenicol at 25µg/mL but free of toluene and grow over night.
When turbidity is high, inoculate the appropriate volume of bacteria culture to liquid LB medium containing chloramphenicol to reach 0,1 OD in the flask (100mL). Incubate culture at 37°C and 250 rpm .
When the OD reach 0.1, separate the initial bacterial culture to several falcon tubes (5mL in each tube) and add toluene to reach the desired toluene concentration.

	C0 Control	C1	C2		C3
LB (mL)	10	10	10	LB (mL)	10
Toluene from stock solution 100µg/L (µL)	0	0,5	50	Toluene from stock solution 100mg/L (mL)	0,5
Obtain concentration (ng/L)	0	5	500	Obtain concentration (mg/L)	5

Incubate culture at 37°C and 250 rpm. Regularly measure the OD with a spectrophotometer

OD measurement over time (at t=0, t=30min, t=1h, t=2h…)

NB: The blank must be the clean liquid LB medium
NB : Samples must be diluted if OD is too high (>1)
  For each culture:

Pipette 100ul of culture and dilute in 900uL LB medium used for the culture (dilution 1/10)

Make the blank

Measure OD

Multiply OD of samples with dilution factor (10 times here)