Team:Ionis Paris/Protocol 12
Warning: in order to get proper result analysis, it is better to perform 3 replicates for each toluene concentration 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 water (calculations done from toluene's molecular weight) From 1 UFC: Incubate culture at 37°C and 250 rpm.
Regularly measure the OD with a spectrophotometer NB: The blank must be the clean liquid LB medium 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)
Protocol 12: Cell Survival assay
Aim: Assess the impact of toluene on a bacteria population
Toluene preparation: stock solutions
100µg/L: Dilute 10µL of the first stock solution into 10mL of water
Inoculation
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 (µL)
0,5
Obtain concentration (ng/L)
0
5
500
Obtain concentration (mg/L)
5
OD measurement over time (at t=0, t=30min, t=1h, t=2h…)
NB : Samples must be diluted if OD is too high (>1)
For each culture:
