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Eliminate the Growth Lag with Large E. coli Cultures

If you purify proteins expressed in E. coli, then you’re probably familiar with this scenario: you come in bright and early in the morning and inoculate your large flasks of media with the overnight culture, start shaking them at 37 °C, and now you wait. And watch. And wait some more. You can’t venture far, because sometimes the bugs take off right away and you might miss your window to induce protein expression, but some days it can take hours before the cultures show the first signs of life. And in a procedure like this, hours of delays at the beginning of the day means you are going to be late for dinner, even by grad student/postdoc standards. Here I’ll detail some approaches you can take to get large E. coli cultures up and growing quickly, not only shortening your day, but hopefully also taking some of the time variability out of the procedure.

What’s the hold up?

When we inoculate a flask of media with an overnight culture, we are dropping in a mixture of dead cells, stationary cells and dividing cells. The living cells have to then recognize the fact that things are looking up for them – the pH is nice and neutral (saturated overnight cultures have likely acidified their media), nutrients abound, oxygen is plentiful and the density is low. The dividing cells take off pretty quickly, while the stationary cells take a little time to ‘wake up’ and start dividing. Therefore, the ratio of these different cell types in your inoculating culture is the determining factor in how quickly your large cultures will get up and running.

Now some will look at this situation and say “just add more overnight culture, and you’ll add more dividing cells.” While true, you’ll also be adding more dead cells (which can complicate your OD readings) as well as acidified media, both of which can lead to poor or at least inconsistent protein expression. The better solution is to inoculate with a smaller volume of a culture that contains more dividing cells – ideally a culture that is still in log-growth phase. If your overnight culture was a flask of LB inoculated in the early evening and shaken at 37 °C, however, it probably left log-growth phase several hours before you climbed out of bed in the morning, so when you inoculate your large flasks the majority of the cells are snoozing in stationary phase.

Growing a “fresh” overnight

So how do you attain a fresh, log-phase starter culture in the morning without having to go into the lab to inoculate it at some crazy time of the night? There are two parameters that you can easily manipulate. The first is to simply use a media that has a larger log-phase window (relative to cell density) than the relatively simple LB media. There are many formulations, with Terrific Broth probably being the most popular, but the two components to look for are an extra energy source and a pH buffering system. Since sugars can complicate protein expression using the most popular Lac expression system, it is probably best to avoid using them as the energy source unless you’re really confident you know what you’re doing. Personally, I keep a sterile solution of 1M potassium phosphate (pH 7.0), 10% glycerol (the extra energy source) at my bench which I add to my lab’s pre-made LB at a 1:20 ratio. Even though this media isn’t as “terrific” as Terrific Broth, it still extends the log-phase window about 3-fold relative to straight LB, giving me a little more time to catch my bugs while they’re still happily growing.

(Using a buffered media doesn’t mean that you can’t still use regular old LB for the large cultures, although I might encourage you to test a richer, buffered media if your expression protocol requires you to induce protein expression overnight).

The second tip to finding a happily dividing starter culture in the morning is to slow the growth rate of the culture by lowering the incubation temperature. Simply growing the culture at 30 °C overnight can make a big difference, but for most strains I find that I get even more consistent results if I grow the overnight culture at room temperature (22-25 °C). (The exception is when I must use 3 or more antibiotics in the starter culture – for these slower growing strains I incubate at 30 °C overnight). Ideally the room temperature culture would be grown in a refrigerated shaking incubator, but I have also done this simply by autoclaving the flask with a stir-bar in it, then growing the culture on my bench with a stir-plate set at a reasonable speed. When using this method it’s unlikely that the culture will be saturated in the morning, but trust me – this is a good thing. Virtually all of these cells are happy campers, and will take off as soon as they’re moved to 37 °C.

One more thing…

There is one more trick to getting your large culture up to induction density fast. The night before your expression, put the flasks of sterilized media at 37 °C so the media will be nice and toasty warm when the starter culture is added to them. If your flasks are large (1 liter of media per flask) and at room temperature, it can take an hour or more for the media to warm to 37 °C in a warm-air incubator, and as I mentioned above the temperature will affect the growth rate of bacteria. This probably doesn’t matter if you are using a saturated, stationary-phase starter culture, but it causes a noticeable growth lag when you are using a log-phase starter culture.

Using these approaches, I dilute my starter culture 1 to 500 (2 mL per liter) into the large flasks with the appropriate antibiotics, and with good aeration the cell density is approaching an OD of 0.6 within 2 to 2.5 hours, quite reliably. (The first time you try this method, be very attentive and check the OD of the culture every 30 minutes or so). This should give you time for a four hour induction, cell pelleting and freezing with time to spare before dinner.

If you have other tips for getting your large E. coli cultures up and running quickly, let us know in the comments.


  1. Peter on November 29, 2018 at 10:02 am

    I’ve found that pelleting your starter culture, then resuspending in fresh media before inoculating the main culture can help. Also, grow starter cultures in the same media as the main cultures, as cells adapt to specific media.

  2. Esha Pandit on June 3, 2018 at 4:08 pm

    Should I add Glycerol and Potassium phosphate both at 1;20 ratio? Should this be added to the primary culture/starter culture or the large culture/secondary culture or both?

  3. Stephanie Dawes on May 17, 2017 at 11:54 pm

    I work part-time and so have had to come up with some time saving methods. I do a fresh transformation one day and then resuspend the entire plate of colonies next morning in approx 1 ml LB, and inoculate 1L of autoinduction terrific broth. I leave it at 37 for a few hours, and then transfer to 18 at lunch. No carry over of acidic media, and it seems the plated cells are happier than a similar stationary phase ON liquid culture. I haven’t noticed a lag phase after inoculation. Next morning I harvest cells. I have had 20g/L cell pellets from this procedure with great production of protein. I have even transformed on the Friday and left the plates on the bench till Monday. Still works. If I have a glycerol stock I also go through a plating first to get my inoculum.

    • Shravasti Misra on June 13, 2018 at 4:55 pm

      But you aren’t working with a single colony by doing this. Unless it doesn’t matter to you, but we mostly prefer working with a single colony so that any downstream problem can be traced back to a single colony

  4. TINI on November 30, 2015 at 6:04 am

    You have mentioned about autoclaving a bead in the flask along with media…can I use this procedure for making competent cells? The Inoue method requires growing the cultures at 18-23 degrees. We dont have facility for it..so can I keep in a magnetic stirrer in a room where the temp is maintained at 18-23 degrees? Can the bead hamper/damage the cells in anyway?

    • Dr Amanda Welch on November 30, 2015 at 11:30 am

      The magnetic stirrer won’t hurt your cells at all–as long as you autoclaved it along with the media (otherwise there might be contaminants). I did this for making competent cells all the time.

      • TINI on November 30, 2015 at 2:55 pm

        Okay.. thank you 🙂

  5. Jeremy Anderson on April 16, 2015 at 1:51 pm

    I recently employed the strategy of growing a large (500 mL) LB culture(s) at room temperature (~25 C) overnight, roughly 16 hours, at 250 rpm. I use the LB cultures for the production of double (2H/15N) or triple (2H/13C/15N) labeled NMR samples using the method employed in:

    Murray, Victoria, et al. “A Novel bacterial expression method with optimized parameters for very high yield production of triple-labeled proteins.” Protein NMR Techniques. Humana Press, 2012. 1-18.

    In the above method the authors stress to not use starter cultures that have grown to saturation but only those that have not grown beyond mid log phase. My results have been more reproducible using non-saturated starter cultures in regular non-labeled protein expression as well.

    As an aside, I choose to not use auto induction as I can produce more protein from a smaller volume due to the higher cell density achievable with the method cited above. This is important to me as D2O and 2H/13C glucose is expensive.

    • Nicola Evans on June 9, 2015 at 2:18 pm

      Thank you, this is really useful! I have just started working with NMR samples.

  6. kelvone on February 19, 2013 at 9:45 pm

    How do I know I’m in the mid-log phase? In other words, what OD600 reading should my starter culture be at before I seed a larger culture with it? 0.5 to 1.0? I grow my E.coli in either LB or TB broth.

    Thank you

    • Ria Sanyal on April 6, 2017 at 1:22 pm

      Precisely it should be around 0.6.
      You can use it till 0.8.

  7. Jode Plank on January 17, 2011 at 3:46 pm

    I’ve been playing with auto induction and have had some constructs that work great with it, but I’ve also had others that haven’t. In addition, if you work on a protein with a long and sordid history, many lab heads aren’t going to be crazy about changing the expression method. I think that auto induction will become the go-to method in the future, but there will always be some proteins that are best expressed “old school”.

  8. Jeff Hollins on January 17, 2011 at 1:43 pm

    Why not use auto induction media instead. That way all you need to do is innoculate then come in the next day and you have your protein. Its so much easier. Here is the reference:
    F. W. Studier, Protein
    expression and purification
    , 2005, 41, 207-34.

    DOI: 10.1016/j.pep.2005.01.016

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