I need to grow my E.coli host strain to late log phase in LB broth, however I cannot find an approximate time that the cells would hit late log phase.
I appreciate that it is approximate, and I will be taking OD readings, however I need a narrower time window.
Some sources say 8 hours, others 12-16 hours, more say 18-20 and another 24 hours.
Any advice?
ADD: I'll be using 20 mL LB and an inoculation of less than a mL (1 colony), at 37 degrees with at least 250 rpm shaking.
It depends on both your inoculum size and the final culture volume.
A fairly reproducible procedure is this:
(1) streak plate from -80C glycerol stock.
(2) pick single colonies and inoculate liquid LB (2-3 mL), overnight (10-12 h).
(3) Take a small inoculum (say, 20 uL), and inoculate larger volume of LB (say, 2 mL), in multiple tubes (for a prelim kinetics study)
(4) in about 2.5 to 3 h's time, you should get cells progressing through the log phase (until late log and stationary phases)
(5) introduce a theoretical lag phase here (say 30 min), before first sampling. For this point, cells are presumed to experience little growth.
(6) Monitor the OD by taking a sample in 30 min (or less) interval.
(7) Obtain a growth curve for OD = 0.1, 0.2, 0.4, 0.8, 1.2, 1.5, until saturation point (remember to resuspend well, because cells settle and this distorts OD)
(8) Next time, repeat the same inoculation from step (3) (either maintaining the same culture scale, or upscale it to get approximate results).
Doing a kinetics study would be quite easy (and worthwhile) if you have a Nanodrop for sampling.
It depends on both your inoculum size and the final culture volume.
A fairly reproducible procedure is this:
(1) streak plate from -80C glycerol stock.
(2) pick single colonies and inoculate liquid LB (2-3 mL), overnight (10-12 h).
(3) Take a small inoculum (say, 20 uL), and inoculate larger volume of LB (say, 2 mL), in multiple tubes (for a prelim kinetics study)
(4) in about 2.5 to 3 h's time, you should get cells progressing through the log phase (until late log and stationary phases)
(5) introduce a theoretical lag phase here (say 30 min), before first sampling. For this point, cells are presumed to experience little growth.
(6) Monitor the OD by taking a sample in 30 min (or less) interval.
(7) Obtain a growth curve for OD = 0.1, 0.2, 0.4, 0.8, 1.2, 1.5, until saturation point (remember to resuspend well, because cells settle and this distorts OD)
(8) Next time, repeat the same inoculation from step (3) (either maintaining the same culture scale, or upscale it to get approximate results).
Doing a kinetics study would be quite easy (and worthwhile) if you have a Nanodrop for sampling.
Depends on the flask, volume of LB and the state of seed inoculum. I used to make 20ml LB in 100ml flask. With 2ml of overnight seed culture it takes 12 hours to reach late log phase (200rpm shaking at 37oC).
hey.....the bacterial growth depends on 3 factors...agitation..aeration and finally the inoculum used. If this things are good then it takes not more than 5-6 hours.
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