Monitoring microbial population dynamics at low densities

Abstract

We propose a new and simple method for the measurement of microbial concentrations in highly diluted cultures. This method is based on an analysis of the intensity fluctuations of light scattered by microbial cells under laser illumination. Two possible measurement strategies are identified and compared using simulations and measurements of the concentration of gold nanoparticles. Based on this comparison, we show that the concentration of Escherichia coli and Saccharomyces cerevisiae cultures can be easily measured in situ across a concentration range that spans five orders of magnitude. The lowest measurable concentration is three orders of magnitude (1000×) smaller than in current optical density measurements. We show further that this method can also be used to measure the concentration of fluorescent microbial cells. In practice, this new method is well suited to monitor the dynamics of population growth at early colonization of a liquid culture medium. The dynamic data thus obtained are particularly relevant for microbial ecology studies.

Quantitative high-throughput population dynamics in continuous-culture by automated microscopy

“…Most abundance measurements are made by sampling and plating 3 or hemacytometry 5 ; but sampling methods are labor-intensive, subject to uncontrolled bias and have poor temporal resolution. More sophisticated methods to measure abundances include optical density measurements 2 , flow cytometry 7 , digital holography 8 9 and light scattering measurements 10 . Automated optical density measurements are high-throughput and cost-effective but are indirect measures of abundance that can vary with cell size or shape, and have a small dynamic range.…”

“…Flow cytometry solves these problems, but is challenging to automate and requires costly lasers and sensitive light detection hardware. Another technique for measuring abundances in microbial populations based on fluctuations in the intensity of scattered light 10 was recently demonstrated; however, this approach does not image single cells directly, requires precise alignment of optical components and is sensitive to changes in cell morphology. Digital holography has also been used to make long term measurements of abundance dynamics in complex microbial communities 8 9 .…”

“…These devices measure abundance dynamics in bacterial communities through automated single-cell imaging. Our instrument provides complementary capabilities to light scattering 10 , holography 8 9 and flow cytometry. The advantages of our instrument are that single-cell fluorescence imaging does not require morphologically distinct species, is immune to artifacts from light scattering by detritus or aggregates and requires little precise optical alignment, allowing stable acquisition for many weeks.…”

Mass and Density Measurements of Live and Dead Gram-Negative and Gram-Positive Bacterial Populations

“…Julou et al present an imaging method based on fluctuations of light scattered by particles or cells passing through a laser beam to determine low microbial concentrations in the range of 10 3 to 10 8 cells per ml (13). Elfwing et al developed a flow chamber microscopy system to monitor growth and division of individual Listeria innocua cells and E. coli K-12 (14)(15)(16).…”

Evolutionary rescue of sphericalmreBdeletion mutants of the rod-shape bacteriumPseudomonas fluorescensSBW25

“…A duct was cut 156 through the center of the pad to allow for oxygen diffusion into the gel. Temperature 157 was maintained at 30°C using a custom-made temperature controller 35 . ( Bacteria were 158 imaged on a custom built microscope using a 100X/NA 1.4 objective lens (Apo-ph3, 159…”