Growth curve

[Linden]

Hey guys!

I would like to know if an E. coli, growth curve can be created in two steps. So on the first day I would start at OD 0.1 and measure it for 8 hours, and on the second day, I would start the culture at the OD it stopped the day before and measure for another 8 hours. Is it ok to do it?

I really appreciate your insight!

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[Sebastian Cocioba]

If its the same strain and conditions it should be repeatable. Then again spectrophotometers in general can vary their signal depending on the cuvette used to do the reading. A long time ago people used to match or pair cuvettes made of glass of similar optical characteristics but that practice is rarely seen as manufacturing standards have gotten so tight that within reason one can have some confidence in optical similarity. That being said I've noticed differences between two plastic cuvettes from the same box. I'm actually doing some research on growth curves and am hoping to publish by winter in PLoS and will be introducing a new device that should help the whole growth curve data acquisition logistics issue. Through the studies I've conducted, I've noticed very repeatable curves using similar techniques but initial inoculation and state of said inoculum may vary the rate quite a lot. These are relative rates so it should not be metered from an absolute starting point if you are doing curves with intermission-like situations like yours. When in doubt, do a few repeats as well as trying various start and stop points and see if you can stitch the data together with reasonable error.

My biggest concern with your design is that the state at which the cells are diluted or seeded into new media can affect the growth characteristic. Old cells are slower to come out of lag phase (personal experimental experience) and optical density, unless calibrated to cells/mL or dry weight, does not always reflect cell count...biomass is biomass living or dead and anything that life pulls out of solution stays out of solution until solubilized thus creating turbidity. Why not just dose a fresh media flask with more and more cells and record the effects to see if you can shorten your experiment. You can also set a flask to shake overnight in such a way that when you return in the morning, they will be at the optical density desired. This will require trial and error but if u get the timing down, you can record the composite curve across two days and stitch them together to form one longer one. Just be sure to replicate the conditions of the first flask in the second to avoid discrepancies especially in inoculation volume....or wait until late fall for my paper and use my device ;) ::shameless plug::

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[Linden]

Hey Sebastian!

Great tips, I will try that! When your paper is published post it here, if possible. I am looking forward to read it ;).

Thanks a lot.

[John Griessen]

On 08/15/2015 09:37 PM, Linden wrote:
> I would start at OD 0.1 and measure it for 8 hours, and on the second day, I would start the culture at the OD it stopped the day
> before and measure for another 8 hours.

How do you get it to "start the culture at the OD it stopped the day before"? By temperature drop to stop growth?
Seems like you'd then be measuring how slowly they wake up, and some of how quickly you heat them up.
If taking data all along, I'd not count on the first hour to have a logarithmic response like all the other hours
until they exhaust nutrients and increase waste enough to slow.

[Bryan Jones]

I don't think you will get very good results by starting a second day culture at the OD that you left off at. Like Sebastian mentioned, the condition of the cells matter. Say you stopped on day 1 during log phase at an OD of 0.8, and then on day 2 diluted a stationary phase culture to OD of 0.8. These cultures are not going to be the same. Probably the biggest factor is that a stationary phase culture will take some time (lag phase) to switch into growth mode. But also the nutrient content may be different (i.e. a culture that has been growing in and using the media for 8 hours vs one that was just put in the fresh media).