Culturing Conditions

Once you’ve selected your host, it is time to figure out the best way to grow it in a controlled setting. Ensuring the growth of a pure culture may involve delicate sterile technique, or the use of selective media. An easy way to minimise contamination risk is to also minimise the number of resources available in your petri dish or culturing tube.

If you’ve selected a sufficiently versatile host, you can achieve significantly different results simply by varying the culturing conditions. This may be important to know should you wish to maximise the efficiency of a downstream process such as protein expression.

This resource is not meant to be a complete work, but rather a guide of what to look for should your selected host not appear on the list. Temperature, pH, Media, Agitation & Atmosphere should be all you need to worry about for single-celled cell cultures. More complex processes, such as mammalian cell culture, may require many additional signaling peptides and resources in order to properly mimic a natural environment.

This list will grow in time, as I learn to work with new species - Alex

1. Escherichia coli (K12 Derived)

  • Mesophile that grows best at 37°C

    • Will happily grow at colder temperatures, between 4-30°C, but will be killed by freezing or temperatures exceeding 40°C

    • When attempting to induce protein production, it may be best to grow the cells at 18°C

  • Grows best around Neutral pH (7-8), but can survive between pH: 4.5-9.7

    • This is why you can skip pH balancing of your LB media, despite it being recommended by many guides.

  • Grows well on most types of media, but best on LB or SOC/SOB (richer broths with sugars may increase contamination risk however).

    • Grows equally well in liquid or on solid medium (agar added)

    • Can be grown on antibiotic medium if an appropriate resistance-containing plasmid is inserted.

  • Shaking a liquid suspension to improve oxygenation will significantly boost the doubling rate.

  • Atmospheric air is perfectly fine, no need to supplement growth with any CO2 or Methane.

2. Saccharomyces cerevisiae

3. Pichia pastoris

  • Methylotrophic Yeast - uses methanol as a source of carbon and energy

  • Optimal temperature 32.3 degrees C

  • Optimal glucose concentration 2.1%

  • Yeast extract 1.1%,

  • Optimal pH 5.4

  • Shaking a liquid suspension to improve oxygenation will significantly boost the doubling rate.

  • Atmospheric air is perfectly fine, no need to supplement growth with any CO2 or methane.

  • https://onlinelibrary.wiley.com/doi/full/10.1002/jcp.29583

  • https://pubmed.ncbi.nlm.nih.gov/16825788/

And so on…

This should give you a good idea of what to search for when deciding on the best way to culture a unique organism. Google widely and compare differing results for accuracy. Be sure to experiment with culturing conditions a little yourself as well. There’s always more to learn about how to keep life alive!