Hello!
I am trying to establish a growth curve of E. coli XL-2 Blue. I have tried several times changing the conditions. I achieved the most optimal growth at: 37 °C, shaking 300 rpm, 50 ml of LB medium in a 500 ml bottle. I started from a pre-culture at OD600=0.7 (1/100 dilution), I used the following protocol:
(1. day: inoculate 25 ml LB with a colony, once the OD600 is 0.7-0.8, stop the growth and put the bottle in a fridge
2. day: inoculate 50 ml LB with pre-culture)
Is a growth curve ok?
I did not correct OD600 when the measured OD is above 0.4. Is this necessary? Could you advise me in wich OD should I do dilutions, how to dilute (1:1 ?) and how often?
Thank you for help!!! :)
Maybe the fridge cold shocks the cells? Yes, I’d dilute the cells above a certain OD at a certain wavelength due to scattering at higher densities. OD 600 is good - at lower wavelengths ’noise’ increases. Optimisation can be faster if you have access to a multi-well plate instrument with built-in software for growth curves.
Article Precise, High-throughput Analysis of Bacterial Growth
. This is what I meant about access to a multi-well growth curve analyser.
Following your results, the growth rate is very low for an E. coli strain. The species is known to be a very fast grower with generation times ranging from 20 min to 30 min in nutrient broth, Luria Broth, and usual culture media.
As suggested by Mr. Jim Philip, maybe the preculture was stressed by cold temperature.
Now, you can check if your suspension is not contaminated by other strains.
You can restart with one colony as you did the first time, then use the obtained culture as a preculture. Don't put it in the fridge.
The next day, start with an OD around 0.1.
You can start your dilutions once you have an OD higher than 0.6 because there is no more linearity between the OD and the bacterial density. The dilutions will vary with time. The first ones for example would b v/v; then next 1v/2v... etc. The point is to have each time an OD lower than 0.6 and you will multiply by the dilution factor.
Regards,
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