Hello,
I am trying to establish a growth curve for E.coli k-12 strain. I picked a colony from a fresh overnight plate and inoculated in 20 ml of fresh TSB in a 50 ml flask for 18 hours at 37 degrees Celsius at 220 rpm. Then I transferred 100 micro-liters of this culture into 250 ml of fresh TSB in a 500 ml flask at 37 degrees at 220 rpm. I took 5-ml aliquots immediately at time 0 and then every hour for up to 8 hours. I took optical density measurements of the aliquots and also did plate counts to get cfu/ml data. My calculated generation time was about 1 hour which seems to be a bit slow for E.coli and I was expecting to only get a lag phase of about a couple of hours. Does the outcome I obtained reasonable for E.coli? My plot has the typical shape of E.coli growth curve but the time I obtained for generation time and lag phase seem off to me.
I think 100 ul of an overnight culture for a 250ul is too little. Did you measured the starting OD just after adding the 100ul? Which OD values did you get?
Hello Miguel Fernández-Niño it was 100 ul of overnight culture transferred to 250 mL of fresh TSB in a 500 mL flask. My OD Values averaged for 5 readings from T0-T8 were the following:
T0: 0.309 ; T1 0.512 ; T2 0.345 ; T3 0.432; T4 0.584; T5 0.424; T6 0.57; T7 0.976; T8 0.919
From each 5 mL aliquot of each time point I transferred 200 ul into a 96 well plate and performed the OD measurements at 600 nm using a perkin elmer victor 1420 3v plate reader.
I think my OD values are less reliable since they increase and decrease for some reason that I need to figure out, but attached is my plot.
Jaqueline,
I just made some calculations and according to your description and I wonder what was the initial concentration. I mean the concentration of your overnight culture?
Miguel Fernández-Niño ,
In the overnight culture I prepared in the shaker incubator at 37 degrees Celsius at 220 rpm for 18 hours I calculated to have about 3.676 × 10^8 cfu/ml .
Hi. I an just trying to guess what was your overnight initial od. So, you took a volume of 0.1 mL from that overnight culture, right? The final volume was 250 ml and the final concentration was 0.309 right? When I do the calculations I found a discrepancy. I will double check you overnight od. I think you have too little
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