Aim: Assess the impact of toluene on a bacteria population
3 replicates for each toluene concentration were realize in order to improve the results statistics
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 a colony in 20mL 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 .
Separate the initial bacterial culture into several falcon tubes (5mL in each tube) and add toluene to reach the desired toluene concentration.
C0 Control
C1
C2
LB (mL)
10
10
10
Toluene from stock solution 100µg/L (µL)
0
0,5
50
Obtain concentration (ng/L)
0
5
500
C3
LB (mL)
10
Toluene from stock solution 100mg/L (mL)
0,5
Obtain concentration (mg/L)
5
Incubate culture at 37°C and 250 rpm.
Regularly measure the OD with a spectrophotometer
OD measurement over time
OD measurment were realize every hours until 6 hours after toluene addition 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 100µL of culture and dilute in 900µL LB medium used for the culture (dilution 1/10)
Make the blank
Measure OD
Multiply OD of samples with dilution factor (10 times here)
