In overnight bacterial culture, why do we put bacteria in a small flask to culture before moving it to a larger flask? Why not just put the sample in a large flask to begin with? This is in reference to growing e. coli to produce proteins in a 125mL flask overnight first, then over a liter after that. For recombinant protein expression.

  • $\begingroup$ Not sure, but it may have to do with equalizing/standardizing the size of the initial innoculum for the large culture. Normally you want to harvest the large flask while it is still growing in log phase or otherwise appropriate condition. If your initial innoculum varies widely in number of cells, time to harvest of the large flask can also vary substantially. It might also have to do with maintaining selection for a plasmid, as partially permissive culture conditions under many generations can lead to selection for faster growth/loss/deletion of plasmid. $\endgroup$
    – Armand
    Commented Jun 13, 2021 at 3:43
  • $\begingroup$ @Val Can you please give a specific example of a protocol you're working with? With what you've written right now, there's too much unknown to be able to answer your question clearly. $\endgroup$
    – jakebeal
    Commented Jun 13, 2021 at 11:58

1 Answer 1


While this is old and therefore unlikely to be marked as answered, it can be easily answered.

The comments have it correct; it is to standardize the inoculum so that you get your culture to the correct density after a set time of incubation. It also helps eliminate problems associated with not selecting the correct colony and ending up with nothing growing.

In culture of E. coli, assuming ideal growth conditions, the cells reach a maximum density of greater than 109 colony forming units per milliliter1.

For your protein expression culture ideally you want the culture growing vigorously as this is the point where the most proteins are being produced and you want the culture to be at a high density too, so that you maximize the amount of protein produced. Typically this is done by inoculating using your overnight culture at 0.5-1% of final medium volume (e.g. 5-10 ml overnight culture into 1 litre protein production medium) and then growing to mid-log phase growth at optical densities (600 nm; OD600) between 0.6 and 0.9. See flow chart at Figure 2 from this publication2.

You can, of course, inoculate with a much lower volume or even from a colony picked off a plate, however, this results in a very long incubation time before you reach the log-phase growth you are targeting, often extending the incubation time from a couple of hours to 8 or more, which is not ideal for a working day and you'll spend much of your day monitoring the culture for growth hoping to not overshoot the point wanted. The difference in incubation time between 0.6 and 0.9 can be as little as about 30-40 min, so you don't want to miss this after having incubated for 8 hours - better to do it with the short incubation.

Growth in a large volume from single or even a few colonies also leaves you prone to time wasting in the form of selecting colonies that don't contain your target protein; they've lost the plasmid somehow. A quick overnight culture allows you to be sure that you have something growing before inoculation.


  1. Tuttle AR, Trahan ND, Son MS. Growth and Maintenance of Escherichia coli Laboratory Strains. Curr Protoc. 2021 Jan;1(1):e20. doi: 10.1002/cpz1.20. Erratum in: Curr Protoc. 2022 Aug;2(8):e552. Erratum in: Curr Protoc. 2022 Aug;2(8):e551. PMID: 33484484; PMCID: PMC8006063.

  2. Francis DM, Page R. Strategies to optimize protein expression in E. coli. Curr Protoc Protein Sci. 2010 Aug;Chapter 5(1):5.24.1-5.24.29. doi: 10.1002/0471140864.ps0524s61. PMID: 20814932; PMCID: PMC7162232.


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