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Preparation, maintenance and propagation of synchronous cultures of photoactive Chlamydomonas cells

Nature Protocols volume 20, pages 2125–2150 (2025)Cite this article

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Abstract

The systematic cultivation of species of photosynthetically active ‘green’ microorganisms in research labs started in the 1940s. Among these microorganisms, Chlamydomonas represents a genus of green biciliated microalgae, of which Chlamydomonas reinhardtii has become the main describing species. For decades C. reinhardtii has been used as an established model organism in biology, including research areas such as molecular biology of eukaryotes, photosynthesis, light receptors, cell metabolism, the dynamics of microtubule assembly and protein transport along cilia. More recently, the use of suspensions of light-responsive living microorganisms has seen a major expansion from the life sciences to the biophysics, statistical physics, fluid dynamics and bioengineering communities. Studies that substantially advance the state of the art in these research areas require the reliable preparation and maintenance of viable, monodisperse and synchronous cell cultures. Although some technical aspects are shared with standard procedures in cell biology and microbiology, Chlamydomonas and its relatives are photosensitive and, simultaneously, motile, meaning this microorganism requires tailored cultivation protocols that are specific to this species. Here we provide guidance on which Chlamydomonas wild-type and mutant strains are suitable for specific experiments and provide detailed step-by-step procedures to measure culture synchronicity, growth rate of the population, average cell size and motility features. The reliable preparation of cell cultures may facilitate future interdisciplinary research using living suspensions of photoactive microorganisms.

Key points

  • Short-term Chlamydomonas cultures, prepared in liquid, are used for experiments, whereas long-term cultures are prepared on agar for the preservation of strains.

  • The preparation of synchronous suspensions of cells facilitates the reproducibility of data obtained in disciplines such as biophysics, statistical physics and fluid dynamics, where motility and collective behavior of large populations of cells is dependent on the health and synchronicity of the culture.

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Fig. 1: Structure and reproductive cycles of a Chlamydomonas cell.
Fig. 2: Schematics of the experimental protocol for handling Chlamydomonas and other green microalgae.
Fig. 3: Incubation of agar and liquid cultures of Chlamydomonas strains.
Fig. 4: Manual cell counting to estimate the growth rate of Chlamydomonas cells in a suspension.
Fig. 5: Cell size distribution of a Chlamydomonas suspension.
Fig. 6: Evaluating the synchronicity of Chlamydomonas cells in a suspension.
Fig. 7: Assessment of the velocity distribution of Chlamydomonas cells in confinement.
Fig. 8: Hints of culture contamination and alterations of swimming motility of Chlamydomonas in confinement.

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Data availability

The source data supporting Figs. 4–8 can be retrieved via Zenodo at https://doi.org/10.5281/zenodo.11191785 (ref. 73).

Code availability

The code in MATLAB for cell detection, tracking and calculating the MSD is available via Zenodo at https://doi.org/10.5281/zenodo.13485141 (ref. 74) under Creative Commons Attribution v.4.0 International Public License and includes a user’s guide.

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Acknowledgements

The authors thank the Göttingen Algae Culture Collection (SAG) for providing SAG-labeled microalgal strains used in this work and T. Pröschold for insightful discussions. R.E.C. acknowledges generous financial support from the German Academic Exchange Service (DAAD).

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Author notes

  1. These authors contributed equally: Rodrigo E. Catalan, Alexandros A. Fragkopoulos.

Authors and Affiliations

  1. Experimental Physics V, University of Bayreuth, Bayreuth, Germany

    Rodrigo E. Catalan, Alexandros A. Fragkopoulos, Antoine Girot & Oliver Bäumchen

  2. Department of Experimental Phycology and SAG Culture Collection of Algae, Georg-August-University Göttingen, Göttingen, Germany

    Maike Lorenz

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All authors contributed to the development of the protocol. R.E.C., A.A.F. and O.B. led the data analysis. R.E.C. and A.A.F. wrote the first draft of the manuscript. All authors contributed to the discussions and the final version of the manuscript.

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Correspondence to Oliver Bäumchen.

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Key References

Kreis, C. T. et al. Nat. Phys. 14, 45–49 (2018): https://doi.org/10.1038/nphys4258

Cammann, J. et al. Proc. Natl Acad. Sci. USA 118, e2024752118 (2021): https://doi.org/10.1073/pnas.2024752118

Ostapenko, T. et al. Phys. Rev. Lett. 120, 068002 (2018): https://doi.org/10.1103/PhysRevLett.120.068002

Böddeker, T. J. et al. J. R. Soc. Interface 17, 20190580 (2020): https://doi.org/10.1098/rsif.2019.0580

Till, S. et al. Phys. Rev. Res 4, L042046 (2022): https://doi.org/10.1103/PhysRevResearch.4.L042046

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Catalan, R.E., Fragkopoulos, A.A., Girot, A. et al. Preparation, maintenance and propagation of synchronous cultures of photoactive Chlamydomonas cells. Nat Protoc 20, 2125–2150 (2025). https://doi.org/10.1038/s41596-024-01135-3

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