DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology
The physiological heterogeneity of cells within a microbial population imparts resilience to stresses such as antimicrobial treatments and nutrient limitation. This resilience is partially due to a subpopulation of cells that can survive such stresses and regenerate the community. Microfluidic appro...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-09-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2019.02112/full |
| _version_ | 1818516820560183296 |
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| author | Shawna L. Pratt Shawna L. Pratt Geoffrey K. Zath Geoffrey K. Zath Tatsuya Akiyama Tatsuya Akiyama Kerry S. Williamson Kerry S. Williamson Michael J. Franklin Michael J. Franklin Connie B. Chang Connie B. Chang |
| author_facet | Shawna L. Pratt Shawna L. Pratt Geoffrey K. Zath Geoffrey K. Zath Tatsuya Akiyama Tatsuya Akiyama Kerry S. Williamson Kerry S. Williamson Michael J. Franklin Michael J. Franklin Connie B. Chang Connie B. Chang |
| author_sort | Shawna L. Pratt |
| collection | DOAJ |
| description | The physiological heterogeneity of cells within a microbial population imparts resilience to stresses such as antimicrobial treatments and nutrient limitation. This resilience is partially due to a subpopulation of cells that can survive such stresses and regenerate the community. Microfluidic approaches now provide a means to study microbial physiology and bacterial heterogeneity at the single cell level, improving our ability to isolate and examine these subpopulations. Drop-based microfluidics provides a high-throughput approach to study individual cell physiology within bacterial populations. Using this approach, single cells are isolated from the population and encapsulated in growth medium dispersed in oil using a 15 μm diameter drop making microfluidic device. The drops are arranged as a packed monolayer inside a polydimethylsiloxane (PDMS) microfluidic device. Growth of thousands of individual cells in identical microenvironments can then be imaged using confocal laser scanning microscopy (CLSM). A challenge for this approach has been the maintenance of drop stability during extended time-lapse imaging. In particular, the drops do not maintain their volume over time during incubation in PDMS devices, due to fluid transport into the porous PDMS surroundings. Here, we present a strategy for PDMS device preparation that stabilizes drop position and volume within a drop array on a microfluidic chip for over 20 h. The stability of water-in-oil drops is maintained by soaking the device in a reservoir containing both water and oil in thermodynamic equilibrium. This ensures that phase equilibrium of the drop emulsion fluids within the porous PDMS material is maintained during drop incubation and imaging. We demonstrate the utility of this approach, which we label DropSOAC (DropStabilization On AChip), for time-lapse studies of bacterial growth. We characterize growth of Pseudomonas aeruginosa and its Δhpf mutant derivative during resuscitation and growth following starvation. We demonstrate that growth rate and lag time heterogeneity of hundreds of individual bacterial cells can be determined starting from single isolated cells. The results show that the DropSOAC capsule provides a high-throughput approach toward studies of microbial physiology at the single cell level, and can be used to characterize physiological differences of cells from within a larger population. |
| first_indexed | 2024-12-11T00:47:36Z |
| format | Article |
| id | doaj.art-b1ef86dc12f945b7820d19903b9a9d04 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-11T00:47:36Z |
| publishDate | 2019-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-b1ef86dc12f945b7820d19903b9a9d042022-12-22T01:26:43ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2019-09-011010.3389/fmicb.2019.02112460473DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial PhysiologyShawna L. Pratt0Shawna L. Pratt1Geoffrey K. Zath2Geoffrey K. Zath3Tatsuya Akiyama4Tatsuya Akiyama5Kerry S. Williamson6Kerry S. Williamson7Michael J. Franklin8Michael J. Franklin9Connie B. Chang10Connie B. Chang11Center for Biofilm Engineering, Montana State University, Bozeman, MT, United StatesDepartment of Chemical and Biological Engineering, Montana State University, Bozeman, MT, United StatesCenter for Biofilm Engineering, Montana State University, Bozeman, MT, United StatesDepartment of Chemical and Biological Engineering, Montana State University, Bozeman, MT, United StatesCenter for Biofilm Engineering, Montana State University, Bozeman, MT, United StatesDepartment of Microbiology and Immunology, Montana State University, Bozeman, MT, United StatesCenter for Biofilm Engineering, Montana State University, Bozeman, MT, United StatesDepartment of Microbiology and Immunology, Montana State University, Bozeman, MT, United StatesCenter for Biofilm Engineering, Montana State University, Bozeman, MT, United StatesDepartment of Microbiology and Immunology, Montana State University, Bozeman, MT, United StatesCenter for Biofilm Engineering, Montana State University, Bozeman, MT, United StatesDepartment of Chemical and Biological Engineering, Montana State University, Bozeman, MT, United StatesThe physiological heterogeneity of cells within a microbial population imparts resilience to stresses such as antimicrobial treatments and nutrient limitation. This resilience is partially due to a subpopulation of cells that can survive such stresses and regenerate the community. Microfluidic approaches now provide a means to study microbial physiology and bacterial heterogeneity at the single cell level, improving our ability to isolate and examine these subpopulations. Drop-based microfluidics provides a high-throughput approach to study individual cell physiology within bacterial populations. Using this approach, single cells are isolated from the population and encapsulated in growth medium dispersed in oil using a 15 μm diameter drop making microfluidic device. The drops are arranged as a packed monolayer inside a polydimethylsiloxane (PDMS) microfluidic device. Growth of thousands of individual cells in identical microenvironments can then be imaged using confocal laser scanning microscopy (CLSM). A challenge for this approach has been the maintenance of drop stability during extended time-lapse imaging. In particular, the drops do not maintain their volume over time during incubation in PDMS devices, due to fluid transport into the porous PDMS surroundings. Here, we present a strategy for PDMS device preparation that stabilizes drop position and volume within a drop array on a microfluidic chip for over 20 h. The stability of water-in-oil drops is maintained by soaking the device in a reservoir containing both water and oil in thermodynamic equilibrium. This ensures that phase equilibrium of the drop emulsion fluids within the porous PDMS material is maintained during drop incubation and imaging. We demonstrate the utility of this approach, which we label DropSOAC (DropStabilization On AChip), for time-lapse studies of bacterial growth. We characterize growth of Pseudomonas aeruginosa and its Δhpf mutant derivative during resuscitation and growth following starvation. We demonstrate that growth rate and lag time heterogeneity of hundreds of individual bacterial cells can be determined starting from single isolated cells. The results show that the DropSOAC capsule provides a high-throughput approach toward studies of microbial physiology at the single cell level, and can be used to characterize physiological differences of cells from within a larger population.https://www.frontiersin.org/article/10.3389/fmicb.2019.02112/fulldrop-based microfluidicssingle cellgrowth ratelag timetime-lapse imagingbiofilm |
| spellingShingle | Shawna L. Pratt Shawna L. Pratt Geoffrey K. Zath Geoffrey K. Zath Tatsuya Akiyama Tatsuya Akiyama Kerry S. Williamson Kerry S. Williamson Michael J. Franklin Michael J. Franklin Connie B. Chang Connie B. Chang DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology Frontiers in Microbiology drop-based microfluidics single cell growth rate lag time time-lapse imaging biofilm |
| title | DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology |
| title_full | DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology |
| title_fullStr | DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology |
| title_full_unstemmed | DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology |
| title_short | DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology |
| title_sort | dropsoac stabilizing microfluidic drops for time lapse quantification of single cell bacterial physiology |
| topic | drop-based microfluidics single cell growth rate lag time time-lapse imaging biofilm |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2019.02112/full |
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