Name: A Continuous Culture Millifluidic Device for the Study of Escherichia coli under Low and Fluctuating Nutrient Conditions (Video) | JoVE Journal
Uploaded: 2026-07-07T13:05:00.000+00:00
Duration: 6 min 22 s
Description: Learn how to study microbial responses to low and fluctuating nutrient conditions using the millifluidic continuous culture device with Escherichia coli. This method enables precise control of nanomolar nutrient environments.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We gratefully acknowledge funding from a Gordon and Betty Moore Foundation Symbiosis in Aquatic Systems Initiative Investigator Award (GBMF9197; https://doi.org/10.37807/GBMF9197), the Simons Foundation through the Principles of Microbial Ecosystems (PriME) collaboration (grant 542395FY22), and Swiss National Science Foundation grant 205321_207488 to R.S. K.S.L. acknowledges support from the Korea Basic Science Institute (National Research Facilities and Equipment Center; Grant No. RS2025-00554860) and the National Research Foundation of Korea (NRF; Grant No. RS2025-23523643), both funded by the Korea Government (MSIT), and from the 2025 Research Fund (1.260002.01) of UNIST.

<ol><li>Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Marine+microbes+see+a+sea+of+gradients%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Marine microbes see a sea of gradients.</a> Science. 338 (6107), 628-633 (2012).</li>
<li>Vorholt, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+life+in+the+phyllosphere%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">Microbial life in the phyllosphere.</a> Nat Rev Microbiol. 10 (12), 828-840 (2012).</li>
<li>Or, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physical+constraints+affecting+bacterial+habitats+and+activity+in+unsaturated+porous+media%3A+a+review%5BTitle%5D)+AND+%22Adv+Water+Resour%22%5BJournal%5D)">Physical constraints affecting bacterial habitats and activity in unsaturated porous media: a review.</a> Adv Water Resour. 30 (6–7), 1505-1527 (2007).</li>
<li>Nguyen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+fluctuations+and+their+effects+on+microbial+communities%2C+populations+and+individuals%5BTitle%5D)+AND+%22FEMS+Microbiol+Rev%22%5BJournal%5D)">Environmental fluctuations and their effects on microbial communities, populations and individuals.</a> FEMS Microbiol Rev. 45 (1), (2021).</li>
<li>Moore, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Processes+and+patterns+of+oceanic+nutrient+limitation%5BTitle%5D)+AND+%22Nat+Geosci%22%5BJournal%5D)">Processes and patterns of oceanic nutrient limitation.</a> Nat Geosci. 6 (9), 701-710 (2013).</li>
<li>Doranga, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nutrition+of+Escherichia+coli+within+the+intestinal+microbiome%5BTitle%5D)+AND+%22EcoSal+Plus%22%5BJournal%5D)">Nutrition of Escherichia coli within the intestinal microbiome.</a> EcoSal Plus. 12 (1), eesp-0006(2024).</li>
<li>Rodríguez-Verdugo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+rate+of+environmental+fluctuations+shapes+ecological+dynamics+in+a+two-species+microbial+system%5BTitle%5D)+AND+%22Ecol+Lett%22%5BJournal%5D)">The rate of environmental fluctuations shapes ecological dynamics in a two-species microbial system.</a> Ecol Lett. 22 (5), 838-846 (2019).</li>
<li>Thaiss, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transkingdom+control+of+microbiota+diurnal+oscillations+promotes+metabolic+homeostasis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis.</a> Cell. 159 (3), 514-529 (2014).</li>
<li>Kearney, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orthogonal+dietary+niche+enables+reversible+engraftment+of+a+gut+bacterial+commensal%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Orthogonal dietary niche enables reversible engraftment of a gut bacterial commensal.</a> Cell Rep. 24 (7), 1842-1851 (2018).</li>
<li>Elena, S. F., Lenski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evolution+experiments+with+microorganisms%3A+the+dynamics+and+genetic+bases+of+adaptation%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Evolution experiments with microorganisms: the dynamics and genetic bases of adaptation.</a> Nat Rev Genet. 4 (6), 457-469 (2003).</li>
<li>Beaumont, H. J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+evolution+of+bet+hedging%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Experimental evolution of bet hedging.</a> Nature. 462 (7269), 90-93 (2009).</li>
<li>Schaechter, M., Maaloe, O., Kjeldgaard, N. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dependency+on+medium+and+temperature+of+cell+size+and+chemical+composition+during+balanced+growth+of+Salmonella+typhimurium%5BTitle%5D)+AND+%22J+Gen+Microbiol%22%5BJournal%5D)">Dependency on medium and temperature of cell size and chemical composition during balanced growth of Salmonella typhimurium.</a> J Gen Microbiol. 19 (3), 592-606 (1958).</li>
<li>Ballesta, J. P. G., Schaechter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+shift-down+and+growth+inhibition+on+phospholipid+metabolism+of+Escherichia+coli%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Effect of shift-down and growth inhibition on phospholipid metabolism of Escherichia coli.</a> J Bacteriol. 107 (1), 251-258 (1971).</li>
<li>Nguyen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+distinct+growth+physiology+enhances+bacterial+growth+under+rapid+nutrient+fluctuations%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">A distinct growth physiology enhances bacterial growth under rapid nutrient fluctuations.</a> Nat Commun. 12 (1), (2021).</li>
<li>Novick, A., Szilard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Description+of+the+chemostat%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Description of the chemostat.</a> Science. 112 (2920), 715-716 (1950).</li>
<li>Egli, T., Lendenmann, U., Snozzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetics+of+microbial+growth+with+mixtures+of+carbon+sources%5BTitle%5D)+AND+%22Antonie+Van+Leeuwenhoek%22%5BJournal%5D)">Kinetics of microbial growth with mixtures of carbon sources.</a> Antonie Van Leeuwenhoek. 63 (3-4), 289-298 (1993).</li>
<li>Lendenmann, U., Snozzi, M., Egli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetics+of+the+simultaneous+utilization+of+sugar+mixtures+by+Escherichia+coli+in+continuous+culture%5BTitle%5D)+AND+%22Appl+Environ+Microbiol%22%5BJournal%5D)">Kinetics of the simultaneous utilization of sugar mixtures by Escherichia coli in continuous culture.</a> Appl Environ Microbiol. 62 (5), 1493-1499 (1996).</li>
<li>Tappe, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cultivation+of+nitrifying+bacteria+in+the+retentostat%2C+a+simple+fermenter+with+internal+biomass+retention%5BTitle%5D)+AND+%22FEMS+Microbiol+Ecol%22%5BJournal%5D)">Cultivation of nitrifying bacteria in the retentostat, a simple fermenter with internal biomass retention.</a> FEMS Microbiol Ecol. 19 (1), 47-52 (1996).</li>
<li>Neidhardt, F. C., Bloch, P. L., Smith, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culture+medium+for+enterobacteria%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Culture medium for enterobacteria.</a> J Bacteriol. 119 (3), 736-747 (1974).</li>
<li>Controlling a solenoid valve with Arduino. , Available from: 
 <a target="_blank" href="https://bc-robotics.com/tutorials/controlling-a-solenoid-valve-with-arduino/">https://bc-robotics.com/tutorials/controlling-a-solenoid-valve-with-arduino/</a> (2015).</li>
<li>Egli, T. The Ecological and Physiological Significance of the Growth of Heterotrophic Microorganisms with Mixtures of Substrates. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aEgli%2C+T+%22Advances+in+Microbial+Ecology%22">Advances in Microbial Ecology</a>. , Plenum Press. New York. (1995).</li>
<li>Koch, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oligotrophs+versus+copiotrophs%5BTitle%5D)+AND+%22BioEssays%22%5BJournal%5D)">Oligotrophs versus copiotrophs.</a> BioEssays. 23 (7), 657-661 (2001).</li>
<li>Muenster, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concentrations+and+fluxes+of+organic+carbon+substrates+in+the+aquatic+environment%5BTitle%5D)+AND+%22Antonie+Van+Leeuwenhoek%22%5BJournal%5D)">Concentrations and fluxes of organic carbon substrates in the aquatic environment.</a> Antonie Van Leeuwenhoek. 63 (3-4), 243-274 (1993).</li>
<li>Murugan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roadmap+on+biology+in+time+varying+environments%5BTitle%5D)+AND+%22Phys+Biol%22%5BJournal%5D)">Roadmap on biology in time varying environments.</a> Phys Biol. 18 (4), (2021).</li>
<li>Cox, C. D., Bavi, N., Martinac, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+mechanosensors%5BTitle%5D)+AND+%22Annu+Rev+Physiol%22%5BJournal%5D)">Bacterial mechanosensors.</a> Annu Rev Physiol. 80 (1), 71-93 (2018).</li>
</ol>

The authors declare no conflict of interest.

Bacterial culturing methods that capture the nutrient complexity of natural environments are essential for advancing the study of microbial physiology beyond simplified laboratory conditions. Natural ecosystems contain mixtures of nutrients spanning a wide concentration range, which often do not support maximal growth rates1,3,4,21,22,<sup class="xr...

Bacteria in natural habitats encounter a nutrient landscape that often varies in both composition and concentration and can fluctuate across a broad range of timescales1,2,3,4. In environments such as the ocean5, soil3, and gut6, microbial growth is frequently constrained by one or more essential nutrients, and nutrient availability can change due to physical, chemical, and biological processes4. These nutrient mixtures and their fluctuations drive microbial strategies for survival and growth, shaping responses across different levels of biological organization4.
Microbial adaptations to nutrient dynamics occur at the community, population, and single-cell levels. At the community level, fluctuations can promote species coexistence7, coordinate community composition and metabolic activity with temporal variation8, and create opportunities for new species to colonize9. At the population level, changing nutrient conditions can accelerate evolution by selecting for genetic changes10 or induce phenotypic variability, including bet-hedging strategies11, that improve survival under unpredictable conditions. At the single-cell level, bacteria adjust their physiology in response to the nutrient environment12,13. One recently reported example is a specific physiology that confers bacteria a growth advantage under fluctuating conditions14. Because bacteria frequently encounter nutrient limitation, studying how they respond to low and variable nutrient conditions—both as individual cells and within populations or communities— is essential for understanding their behavior in their natural environments.
Chemostats are well-suited for studying growth under limitation by a single nutrient, but are difficult to use when the goal is to control the concentrations of multiple nutrients in low-concentration mixtures. Chemostats have been valuable tools for studying bacterial adaptation to nutrient limitation by providing a well-controlled, steady environment. However, their operation inherently restricts them to conditions in which growth is limited by a single essential nutrient element. In a chemostat, the dilution rate is adjusted so that cellular growth balances the supply of one limiting nutrient, thereby fixing the growth rate rather than the concentrations of individual nutrients15. This framework does not readily generalize to environments containing mixtures of substitutable nutrients—such as multiple carbon sources—because the dilution rate controls only the overall growth rate, not the concentrations of each component in the medium16,17. As a result, the concentrations of individual nutrients and their co-utilization patterns emerge from microbial utilization rather than being externally prescribed16,17.
Additionally, while nutrient fluctuations can be induced in chemostats, rapid fluctuations or single shifts on the scale of minutes are difficult to achieve due to the typically large culture volumes. Microfluidic devices have emerged as powerful tools for creating precise low-concentration and fluctuating nutrient environments14. When paired with video microscopy, they enable measurement of single-cell growth. Although microfluidic systems permit quantification of the expression of selected proteins using fluorescent reporters, unlike chemostats, typical microfluidic devices are too small to produce the biomass needed for omic analyses, for example, to study the proteome of a bacterial population. To bridge this gap, we developed the millifluidic continuous culture device (MCCD).
The MCCD enables bacterial growth under controlled nutrient conditions, including constant low-nutrient conditions with minimal or no depletion, as well as fluctuating nutrient conditions, using a continuous flow culture system capable of generating sufficient biomass for downstream analyses. The MCCD uses a 0.45 µm PVDF filter as the culture vessel, in which medium is continuously replenished by a peristaltic pump delivering fresh medium from a reservoir (Figure 1A). The porous membrane of the filter retains bacteria while allowing a prescribed flow of nutrients, which prevents nutrient depletion even at low nutrient concentrations (hundreds of nanomolar to micromolar range). Because biomass is retained while fresh medium continuously flows through the device, the MCCD shares similarities with retentostats18, in which cells are retained while medium is renewed. However, the MCCD differs from classical retentostats in its architecture and mode of nutrient delivery. The retentostat typically operates as a stirred bulk reactor, where cells are suspended in a well-mixed culture volume. Under such conditions, nutrient availability depends on the balance between mixing, diffusion, and cellular uptake, and local depletion or gradients can arise, particularly at low nutrient concentrations and high cell densities. In contrast, in the MCCD, cells assemble directly on the membrane inside the small-volume Sterivex cartridge, forming layers only a few cells thick that are continuously exposed to flowing medium. No active mixing is imposed within the cartridge; instead, medium is homogenized upstream and delivered by continuous flow. Nutrients are supplied by convective transport through the thin cell layers, and because the diffusion distance is small and the flow rate is high, local nutrient gradients are minimized. As a result, the nutrient conditions experienced by the population are primarily determined by the composition of the incoming medium, enabling more consistent and well-defined low-nutrient conditions than can be ensured in stirred retention systems.
<img alt="Automated fluid control system using peristaltic pump, solenoid valves, Arduino setup for filtration." class="xfigimg" src="/files/ftp_upload/70652/70652fig1.jpg" data-altupdatedat="1785816036000" data-alt="Figure 1"> 
Figure 1: The millifluidic continuous culture device (MCCD) for bacterial culture under low-concentration or fluctuating nutrient conditions. (A) Schematic of the MCCD for constant nutrient conditions. A peristaltic pump drives the flow of fresh medium from a reservoir through a 0.45 µm PVDF filter containing bacterial cells. The porous membrane in the filter retains the bacteria while allowing medium to pass through. Continuous medium flow maintains constant nutrient conditions inside the filter, even at very low nutrient concentrations. (B) Schematic of the MCCD for fluctuating nutrient conditions. A software-controlled three-way solenoid valve upstream of the peristaltic pump alternates medium inflow between two reservoirs (e.g., high-nutrient concentration, CHigh, and low-nutrient concentration, CLow). The software controls the valve's plunger position to determine the active inflow source. (C) Fluidic assembly for constant nutrient conditions. A cap with three silicone tubes that traverse it seals the bottle containing fresh medium. One tube connects to a vent filter for aeration, while the other two carry medium to the filters. Barb-to-Male Luer fittings connect these tubes to the peristaltic tubing, and connect each peristaltic tube to a one-way check valve, serving as the entry point to the filter. The filter outlet is also fitted with a one-way check valve. The image shows two samples, each contained in one filter. (D) Fluidic assembly for fluctuating nutrient conditions. The metal platform houses the microcontroller and electronic components controlling two solenoid valves via custom MATLAB software. Threaded-to-Barb fittings connect both the inlets and outlets of the solenoid valves to the tubing. Each valve has two inlets for the two alternate media, here a high-nutrient concentration medium, CHigh, red-labeled tubes, and a low-nutrient concentration medium, CLow, blue-labeled tubes. The media are contained within capped bottles with a vent filter, as in (C). The outlets from the solenoid valves connect to the filters via peristaltic tubing, as in the steady-state assembly (C). The image shows two samples, each contained in one filter. <a href="https://www.jove.com/files/ftp_upload/70652/70652fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Additionally, the incorporation of a software-controlled, three-way solenoid valve upstream of the Sterivex filter enables the user to switch the medium flow between two media contained in different reservoirs (Figure 1B). As a result, the device can be used to study the response of bacteria to either a stable environment at a constant (including low concentration) nutrient condition or to nutrient fluctuations, with fluctuation timescales of several minutes or longer.
This protocol outlines the assembly and operation of the MCCD for studying bacteria under both low-nutrient and fluctuating-nutrient conditions. As an example, this protocol provides a step-by-step guide to conducting a growth experiment using Escherichia coli NCM3722 in two operating modes of the MCCD: constant low-nutrient conditions and fluctuating nutrient conditions. The MCCD setup can be readily adapted to other nutrient conditions and microbial species, making it a versatile platform for studying bacterial physiology under nutrient environments typical of those encountered in nature.

<table><tbody><tr><td>1K Ohm Resistor</td><td>BC Robotics</td><td>ELC-021</td><td>Power Rating: 1/6 W</td></tr><tr><td>1N4001 Diode</td><td>BC Robotics</td><td>ELC-001</td><td>DC Reverse Voltage: 50 V; Average Rectified Current: 1 A</td></tr><tr><td>3-stop platinum-cured silicone peristaltic tubing</td><td>Innofluid</td><td>SE-TUB-SIL-SSS-2.4*0.8</td><td>Inner diameter 0.8 mm; outer diameter 2.4 mm; compatible with peristaltic pump used</td></tr><tr><td>3-way Solenoid valves</td><td>Emerson Electric Co.&amp;nbsp;</td><td>0055A301L0T00F3</td><td>12 V DC, 1 W</td></tr><tr><td>Aluminum foil</td><td></td><td></td><td>Suitable for autoclaving</td></tr><tr><td>Arduino Uno Rev4</td><td>Arduino</td><td>SKU&amp;nbsp;ABX00173</td><td></td></tr><tr><td>Autoclave</td><td>Systec</td><td>D-200</td><td>Or different company</td></tr><tr><td>Cap closure with three ports &amp;ndash; for 1 and 2 L bottles</td><td>Nalgene</td><td>2162-0531</td><td>Or different company (make sure they fit the selected bottles)</td></tr><tr><td>Cap closure with three ports &amp;ndash; for 4 L bottle</td><td>Nalgene</td><td>2162-0830</td><td>Or different company (make sure they fit the selected bottles)</td></tr><tr><td>Cell density meter CO8000</td><td>WPA biowave</td><td></td><td>OD meter</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804R</td><td>Centrifuge that fits 50 mL Falcon tubes</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424R</td><td>Centrifuge that fits 1.5 and 2 mL eppendorf tubes</td></tr><tr><td>Conical centrifuge tube - 50 mL</td><td>Fischer Scientific</td><td>10788561</td><td>Falcon tube</td></tr><tr><td>Culture tubes (14 mL, sterile)</td><td>Greiner bio-one</td><td></td><td>Culture tubes</td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>493511</td><td>Dilute to 70%</td></tr><tr><td>Flow cytometer</td><td>Beckman</td><td>C09756</td><td>CYTOFlex</td></tr><tr><td>Glutaraldehyde 25%</td><td>Sigma Aldrich</td><td>G5882</td><td>Or different company</td></tr><tr><td>Graduated cylinder - 500 mL</td><td>Nalgene</td><td>3662-0500&amp;nbsp;</td><td>Or different company</td></tr><tr><td>Incubator box</td><td></td><td></td><td>Custom-built incubator box &amp;ndash; contact corresponding authors for construction details</td></tr><tr><td>LabV1 Intelligent Low Flow Rate Peristaltic Pump</td><td>Innofluid</td><td>SE-LABV1-AMC12(10)</td><td>Available at Darwin Microfluidics</td></tr><tr><td>Male Luer-to-barb fitting</td><td>Aldrich</td><td>Z277142</td><td>Male Luer-to-barb adapter compatible with 0.8 mm ID tubing (see Figure 1C)</td></tr><tr><td>Male Luer-to-threaded fitting</td><td>Aldrich</td><td>Z277142</td><td>Used to connect the solenoid valve to the &quot;inlet tubing&quot; (see Figure 1D)</td></tr><tr><td>Matlab</td><td>Mathworks</td><td>R2022b or later</td><td>Software to run the custom-made software for the solenoid valves</td></tr><tr><td>MOPS EZ Rich Defined Medium Kit</td><td>Teknova</td><td>M2105</td><td>MOPS medium for Enterobacteria</td></tr><tr><td>One-way Luer check valve</td><td>Masterflex</td><td>30505-92</td><td>Prevents backflow into medium reservoirs</td></tr><tr><td>Pipe cutter - 0-37 mm</td><td>KS Tools</td><td>2222051</td><td>Or different company</td></tr><tr><td>Pipette set</td><td>Fisher Scientific</td><td>05-403-151</td><td>Or different company</td></tr><tr><td>Platinum-Cured Silicone Tubing - 1/16 in</td><td>Nalgene</td><td>8060-0020</td><td>Inner diameter 1/16 in (1.6 mm); used for fluctuating-nutrient experiments</td></tr><tr><td>Platinum-Cured Silicone Tubing - 1/8 in</td><td>Nalgene</td><td>8060-0030</td><td>Inner diameter 1/8 in (3.2 mm); used for fluctuating-nutrient experiments</td></tr><tr><td>Polypropylene copolymer bottle - 1 L&amp;nbsp;</td><td>Nalgene</td><td>2126-1000</td><td>Or different company</td></tr><tr><td>Polypropylene copolymer bottle - 2 L</td><td>Nalgene</td><td>2126-2000</td><td>Or different company</td></tr><tr><td>Polypropylene copolymer bottle - 4 L</td><td>Nalgene</td><td>2126-4000</td><td>Or different company</td></tr><tr><td>Serological pipette - 5 mL</td><td>Corning</td><td>CLS4487</td><td>Or different company</td></tr><tr><td>Solderless Breadboard</td><td>BC Robotics</td><td>PROTO-001</td><td></td></tr><tr><td>Sterivex-HV filter - 0.45 &amp;micro;m PVDF</td><td>Millipore</td><td>SVHV01015</td><td></td></tr><tr><td>SYBR Green I Nucleic Acid Gel Stain</td><td>Invitrogen</td><td>S7563</td><td>Nucleic acid stain for cell counting in flow cytometer</td></tr><tr><td>TIP120 Darlington Transistor</td><td>BC Robotics</td><td>TRN-005</td><td></td></tr><tr><td>Tubes - 1.5 mL</td><td>Eppendorf</td><td>3810X</td><td>Tubes for centrifuging cell cultures and storing pellets</td></tr><tr><td>Vent filter</td><td>Millex</td><td>SLFG05000</td><td>0.2 &amp;micro;m hydrophobic vent filter</td></tr><tr><td>Waste receptacle</td><td></td><td></td><td>Volume capacity should be at least the total media volume to be used in the experiment</td></tr></tbody></table>

a continuous culture millifluidic device for the study of escherichia coli under low and fluctuating nutrient conditions

A Continuous Culture Millifluidic Device for the Study of Escherichia coli under Low and Fluctuating Nutrient Conditions

Microbial habitats in nature are often characterized by low concentrations of mixed nutrients, spatial heterogeneity, and temporal fluctuations. However, traditional laboratory culturing methods fail to replicate these conditions. Batch cultures cannot sustain growth in low-nutrient environments, while chemostats maintain steady-state growth with a single limiting nutrient but are challenging to implement when the goal is to maintain defined low concentrations of nutrient mixtures or to introduce rapid fluctuations. Microfluidic systems generate dynamic environments but yield insufficient biomass for population-level omic analyses. To address these limitations, we introduce the millifluidic continuous culture device (MCCD), a versatile platform for studying microbial responses to stable and fluctuating nutrient conditions. The MCCD houses bacterial populations inside a Sterivex filter (0.45 &#181;m polyvinylidene fluoride [PVDF] porous filtering membrane), where a continuous flow of media sustains stable culture conditions while preventing nutrient depletion. A three-way solenoid valve system, controlled via custom Matlab software, enables precise, minute-scale nutrient fluctuations. This protocol provides a step-by-step guide to operating the MCCD in two modes: (1) constant low-nutrient conditions and (2) fluctuating-nutrient conditions. Using this system, Escherichia coli grew exponentially in a mixture of amino acids and nucleobases present at tens to hundreds of nanomolar concentrations, reaching cell concentrations on the order of 109 cells/mL. By recreating key features of natural microbial habitats, the MCCD enables the study of bacterial growth and physiology under controlled yet ecologically relevant conditions in E. coli and other microbial species.

In this study, we used the MCCD to quantify the growth rate of E. coli NCM3722 under constant low-nutrient conditions at three RDM concentrations. These concentrations were derived from the rich-defined medium (RDM) formula described by Neidhardt19, with the amino acid and nucleobase supplements diluted to 0.5%, 0.25%, and 0.01% of the original formulation, respectively.
Replicate growth experiments were performed in each medium to quantify growth rates. For th...

We present a protocol for the millifluidic continuous culture device (MCCD), which allows the cultivation of bacterial populations under constant low-nutrient conditions (down to hundreds of nanomolar concentrations) or temporal nutrient fluctuations on a minute timescale.

Microbial habitats in nature are often characterized by low concentrations of mixed nutrients, spatial heterogeneity, and temporal fluctuations. However, ...

My research focuses on examining how bacteria adapt physiologically to low and fluctuating nutrient conditions typical of natural environments. Conventional cultivation systems poorly replicate low dynamic nutrient conditions, whereas our system enables cultivation under these environmental relevant conditions. Prepare the overnight culture the day before the experiment.Prepare a pre-culture by adding six microliters of the overnight culture to a tube containing six milliliters of full-rich defined media, or RDM. Divide the mixture equally into two 15-milliliter culture tubes and label them as A and B.Incubate both tubes at 37 degrees Celsius, with shaking at 200 RPM. Monitor the optical density at 600 nanometers using tube B, and do not proceed until the optical density reaches approximately 0.5.During incubation, prepare the Low RDM medium, and allocate the total volume across bottles for different time points. Then seal each bottle with caps fitted with three tubes. Connect the third-widest silicone tube to a 0.2-micrometer vent filter for sterile air exchange.Attach one end of each peristaltic tube fitted with male Luer lock connectors to the smaller-diameter silicone tubes. Leave the aluminum foil cover on the free male Luer lock fitting at the other end of the peristaltic tubes until the experiment begins to prevent contamination. Place the bottles and peristaltic pump into an incubator set to 37 degrees Celsius.Secure all peristaltic tubing into the channels of the same pump head, ensuring identical flow rates across all filters. Dilute the pre-culture at a ratio of one to 10 by adding 600 microliters of culture into a tube containing 5.4 milliliters of Low RDM. Using a one-milliliter pipette, introduce one milliliter of diluted culture into each of six 0.45-micrometer polyvinylidene fluoride, or PVDF filters.After rinsing one-way check valves with autoclaved ultra-pure water, attach them to each end of the filters. Remove the aluminum foil covers from tubing fittings. Connect each filter to the peristaltic tubing fittings.Set the flow rate to two milliliters per minute on the peristaltic pump. Place a waste receptacle below the filters to collect outflow, and start the flow. After two hours, unplug one of the filters connected to the one liter bottle, unscrew the one-way check valves, and place the filter into a 50-milliliter centrifuge tube with the inlet facing downward.Centrifuge at 2, 000 G for two minutes. Using a sterilized pipe cutter, apply gentle pressure at the outlet end of the plastic casing to open the filter. Release the inner cylinder and discard the membrane-containing section.Rinse the inner walls of the filter casing with the collected culture medium. Measure the total collected volume using a five-milliliter serological pipette. Take three 100-microliter aliquots from the sample and fix them with glutaraldehyde to a final concentration of 1.5%Unplug the corresponding filters at subsequent time points and repeat the collection procedure.Label the collected samples according to their respective time points. Label the samples with the fluorescent DNA dye, and count the cells using flow cytometry. Over time, the log 10 transformed cell counts followed linear trajectories, indicating exponential growth in all conditions.The growth rates were approximately 0.88 per hour for the 0.5%RDM condition, 0.64 per hour for the 0.25%condition, and 0.18 per hour for the 0.01%condition. The cell counts reached up to one billion cells per sample, demonstrating that the millifluidic continuous culture device, or MCCD, supports both exponential growth, and high biomass yield at nanomolar to micromolar nutrient concentrations. Cells grown in 0.5%RDM had a greater mean cell length than cells grown in 0.01%RDM.The main challenge during low nutrient cultivation is maintaining adequate cell densities to prevent clogging, pressure buildup, and nutrient depletion. Following this procedure, cells can be harvested for other downstream analysis, including proteomics, metabolomics, and other omics approaches. Future studies can investigate microbial adaptation to nutrient limitation and fluctuations by integrating this protocol with a quantitative omic analysis.

a-continuous-culture-millifluidic-device-for-study-escherichia-coli

Research

JoVE Journal

Biology

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

A Microfluidic Device for Studying Multiple Distinct Strains

Source: Pioli, R., et al., Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly. J. Vis. Exp. (2021)This video demonstrates the method of capturing single fluorescently tagged Escherichia coli (E. coli) cells in microfluidic traps using a carbon-free suspension and controlled flow, followed by nutrient addition to trigger growth and fluorescence imaging of microcolonies.

Microfluidic Patterning and Fluorescence-Based Tracking of Single-Cell Bacterial Growth

Source: Jian, X. et. al., Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC). J. Vis. Exp. (2022)This video demonstrates the use of a microfluidic droplet-based platform for real-time monitoring of bacterial growth. By generating uniform droplets containing bacterial suspensions and guiding them through a detection pathway, the system enables continuous optical measurement of cell density to construct a growth curve.

Real-Time Monitoring of Bacterial Growth Using a Microfluidic Microdroplet Culture System

Source: Cabeen, M. T., et al. Single-cell Microfluidic Analysis of Bacillus subtilis. J. Vis. Exp. (2018)The video demonstrates the use of a microfluidic device to observe bacterial growth at the single-cell level. It showcases how non-motile bacterial cells, confined within micro-scale trenches, are continuously nourished and imaged under a fluorescence microscope. The setup enables real-time visualization of cell division and lineage tracking across multiple generations.

Tracking Bacterial Growth at Single-Cell Resolution in a Microfluidic System

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

Single-cell Microfluidic Analysis of Bacillus subtilis

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This protocol presents a method for fabricating a double-layer microfluidic chip and analyzing microbial growth under rapid oxygen oscillations. The device's performance was validated through oxygen sensing in fluid channels and microbial cultivation.

Creating Rapid Oxygen Oscillations in Microbial Single-cell Growth Analysis using a Microfluidic Double-layer Device

Learn how to study microbial responses to low and fluctuating nutrient conditions using the millifluidic continuous culture device with Escherichia coli. This method enables precise control of nanomolar nutrient environments.

A Continuous Culture Millifluidic Device for the Study of Escherichia coli under Low and Fluctuating Nutrient Conditions

Explore More Videos