High-throughput quantification of microbial birth and death dynamics using fluorescence microscopy



Microbes live in dynamic environments where nutrient concentrations fluctuate. Quantifying fitness in terms of birth rate and death rate in a wide range of environments is critical for understanding microbial evolution and ecology.


Here, using high-throughput time-lapse microscopy, we have quantified how Saccharomyces cerevisiae mutants incapable of synthesizing an essential metabolite (auxotrophs) grow or die in various concentrations of the required metabolite.We establish that cells normally expressing fluorescent proteins lose fluorescence upon death and that the total fluorescence in an imaging frame is proportional to the number of live cells even when cells form multiple layers. We validate our microscopy approach of measuring birth and death rates using flow cytometry, cell counting, and chemostat culturing.


For lysine-requiring cells, very low concentrations of lysine are not detectably consumed and do not support cell birth, but delay the onset of death phase and reduce the death rate compared to no lysine. In contrast, in low hypoxanthine, hypoxanthine-requiring cells can produce new cells, yet also die faster than in the absence of hypoxanthine. For both strains, birth rates under various metabolite concentrations are better described by the sigmoidal-shaped Moser model than the well-known Monod model, while death rates can vary with metabolite concentration and time.


Our work reveals how time-lapse microscopy can be used to discover non-intuitive microbial birth and death dynamics and to quantify growth rates in many environments.


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We thank Jose Pineda for performing the experiment in Supplementary Figure S3 and Li Xie for consultation regarding models.

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Corresponding author

Correspondence to Wenying Shou.

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Author summary: How fast microbes give birth or die (fitness) is influenced by their genetic makeups (genotypes) and environment. Microbes can mutate to many different genotypes, and the environment can change in many different ways. Thus, it is important to measure fitness for many genotypes in many environments so that we can understand, for example, why one genotype outcompetes another genotype. Here, we have developed a high-throughput method to quantify the fitness of fluorescent cells using time-lapse microscopy. We applied this method to two S. cerevisiae (budding yeast) mutants that had lost their ability to synthetize an essential metabolite.We found that the mutants behaved differently from one another in response to metabolite limitation, and in some cases, behaved differently from our expectations. Our method will be useful for quantifying growth phenotypes of fluorescent microbes.

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Hart, S.F.M., Skelding, D., Waite, A.J. et al. High-throughput quantification of microbial birth and death dynamics using fluorescence microscopy. Quant Biol 7, 69–81 (2019). https://doi.org/10.1007/s40484-018-0160-7

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  • Saccharomyces cerevisiae
  • fluorescence microscopy
  • microbial growth
  • birth rate
  • death rate