Abstract
ABSTRACTBacterial growth is classically assessed by measuring the increases in optical density of pure cultures in shaken liquid media. Measuring growth using optical density has severe limitations when studying multistrain interactions, as it is not possible to measure the growth of individual strains within mixed cultures. Here, we demonstrated that constitutively expressed fluorescent proteins can be used to track the growth of individual strains in different liquid media. Fluorescence measurements were highly correlated with optical density measurements and cell counts. This allowed us to assess bacterial growth not only in pure cultures but also in mixed bacterial cultures and determine the impact of a competitor on a focal strain, thereby assessing relative fitness. Furthermore, we were able to track the growth of two different strains simultaneously by using fluorescent proteins with differential excitation and emission wavelengths. Bacterial densities measured by fluorescence yielded more consistent data between technical replicates than optical density measurements. Our setup employs fluorescence microplate readers that allow high throughput and replication.IMPORTANCE We expand on an important limitation of the concept of measuring bacterial growth, which is classically limited to one strain at a time. By adopting our approach, it is possible to measure the growth of several bacterial strains simultaneously with high temporal resolution and in a high-throughput manner. This is important to investigate bacterial interactions, such as competition and facilitation.
Highlights
Bacterial growth is classically assessed by measuring the increases in optical density of pure cultures in shaken liquid media
We performed our experiments in 96-well microtiter plates and microtiter plate readers, which allowed time series monitoring of fluorescence signals and cell growth under different growth conditions with a high degree of reproducibility. By combining these measurements with a series of controls, we demonstrated that our system allowed us to determine the strength of interaction and relative fitness of bacterial strains in competition
To highlight the advantage of fluorescence intensity measurements over optical density measurements, we followed the growth of Pantoea eucalypti strain 299R labeled with a constitutively expressed red fluorescent protein in five replicated cultures using optical density and fluorescence intensity in nutrient broth (NB) (Fig. 1A)
Summary
Bacterial growth is classically assessed by measuring the increases in optical density of pure cultures in shaken liquid media. We demonstrated that constitutively expressed fluorescent proteins can be used to track the growth of individual strains in different liquid media. Fluorescence measurements were highly correlated with optical density measurements and cell counts. This allowed us to assess bacterial growth in pure cultures and in mixed bacterial cultures and determine the impact of a competitor on a focal strain, thereby assessing relative fitness. We were able to track the growth of two different strains simultaneously by using fluorescent proteins with differential excitation and emission wavelengths. Bacterial densities measured by fluorescence yielded more consistent data between technical replicates than optical density measurements. Our setup employs fluorescence microplate readers that allow high throughput and replication
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
