Aim: Assess the impact of toluene on a bacteria population
3 replicates for each toluene concentration were realized in order to improve the results statistics
Every toluene manipulations were performed under a chemical hood and following the safety protocol available here . Bacterial culture containing toluene were only openned under the chemical hood. Spectrometer tank as well as 96 wells plate were covered with parafilm before proceeded to OD or luciferase activity measurement.
Toluene preparation: stock solutions
1g/L: Dilution of absolute liquid toluene (Cm= 867g/L) to obtain a final toluene concentration at 1g/L: Add 11,53µL of toluene into 10mL ethanol (calculations done from toluene's molecular weight) 100µg/L: Dilute 1µL 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 (100mL) containing chloramphenicol. Mix and separate the bacterial culture into several falcon tubes (10mL in each tube) and add toluene to reach the desired toluene concentration.
C0 Control
C1
C2
Bacterial culture (mL)
10
10
10
Toluene from stock solution 100µg/L (µL)
0
0,5
50
Obtain concentration (ng/L)
0
5
500
C3
Bacterial culture (mL)
10
Toluene from stock solution 1g/L (µL)
50
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 8 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 500µL of culture and dilute in 500µL LB medium used for the culture (dilution 1/10)
Make the blank
Measure OD
Multiply OD of samples with dilution factor (2 times here)
