Anyone who has worked with microorganisms, be it bacteria or yeast, is familiar with subculturing – the act of transferring some cells from a previous culture to a fresh growth medium. You do it either to reset the growth phase of your culture or to increase the biomass for downstream experiments.

But there’s more to subculturing than just scraping cells from any old petri dish or dumping a liquid culture of the indeterminate date of origin into fresh medium.  Especially if you want your experiments to work and be reproducible.

Using Stock Culture as a Starting Point

Let’s start at the beginning: you have a long-term stock culture with a determined genotype and phenotype stored at – 70ºC. Using it to start an everyday culture is passage number one. It’s not a good idea to do this often, as it increases the chances of decreased stock culture viability due to partial defrosting/refreezing. You can also contaminate the stock. And finally, defrosted culture is in a state of cold shock, and it takes time for it to recover to normal physiological state.

It is a good idea to check genotype of the culture at this stage, especially if the stock is made by somebody else. You wouldn’t believe how many times cultures are stored where the contents don’t match the label on the vial, and weeks of experiments go down the drain (not literally – dispose of your microbes responsibly).

Further Passaging

Important note: The inoculum size varies, but when your culture starts growing, it’s worth noting when it does and how long a division takes. Just count the cells at regular intervals and calculate the division time.

A freshly grown to saturation liquid culture or newly formed colonies are the best starting point for subculturing. They are in late logarithmic or early stationary phase containing a maximum density of live cells with active or just paused metabolism. Putting them into fresh medium gets their metabolism going after a brief pause. Obviously, if you change the medium during passaging, it’ll take cells longer to adjust and re-tune their metabolism (1, 2).

Now you have the second passage culture. Should you keep going indefinitely, never going back to the stock? It’s not a good idea for several reasons:

  • In liquid culture, you might get contamination and not notice it. With every passage, the probability of contamination increases, and you will not always be able to notice it, especially in a liquid medium.
  • Because of the founder effect, you might accidentally select a fast-growing mutant that will outcompete the parent strain so that the strain will have a different phenotype. The probability of this also increases with passaging.

As a rule, microorganisms grow slower on plates, so it’s better to keep “passage culture one” on a plate and use several colonies to inoculate liquid medium. Using liquid culture one is not optimal beside the steadily reducing number of viable cells, as many microorganisms accumulate secondary mutations in stationary phase (3, 4).

Take Home Messages:

  • Always check your culture before passage making sure that it’s not contaminated. Start by making sure that colonies are uniform and look as normal.
  • As a rule, do not exceed 4 – 5 passages.
  • Do not use cultures in late stationary/death phase as they accumulate mutations and take much longer to enter log-phase.
  • Preferably use colonies from a plate.
  • If using a liquid starter culture, dilute at least 50 times so not to transfer toxic metabolites with old medium. For slow-growing microorganisms, concentrate and wash cells before using it for subculturing.

Happy subculturing.


  1. Sezonov G, Joseleau-Petit D, and D’Ari (2007). Escherichia coli Physiology in Luria-Bertani Broth. Bacteriol. V.189:8746-8749
  2. Bergman L.W. (2001) Chapter 2. Growth and Maintenance of Yeast. Methods in Molecular Biology, Vol. 177, Two-Hybrid Systems: Methods and Protocols Edited by: P. N. MacDonald. Humana Press Inc., Totowa, NJ
  3. Kivisaar M (2010) Mechanisms of stationary-phase mutagenesis in bacteria: mutational processes in pseudomonads. FEMS Microbiol Lett. Nov;312(1):1-14. doi: 10.1111/j.1574-6968.2010.02027.x.
  4. Robleto EA1, Yasbin R, Ross C, Pedraza-Reyes M. (2007) Stationary phase mutagenesis in B. subtilis: a paradigm to study genetic diversity programs in cells under stress. Crit Rev Biochem Mol Biol. Sep-Oct;42(5):327-39.

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