This project was funded by the National Institutes of Health under GM018568. This protocol was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Coordinated Infrastructure Network (NNCI), which is supported by the National Science Foundation under NSF award no. 1541959. CNS is part of Harvard University. Many thanks are due to Thomas Norman and Nathan Lord for their work in conceiving and fabricating the master template used for the devices shown here and in building the original version of the apparatus. We also thank Johan Paulsson for his valuable collaborative advice and thank members of his lab for their advice and continued improvements to bacterial microfluidic apparatuses.

1. PDMS Device Casting
<ol>
	<li>In a dust-free environment, mix PDMS polymer and curing agent at a 10:1 ratio and degas under vacuum for at least 10 min.</li>
	<li>Place a silicon master (or an epoxy or polyurethane replica thereof) on a piece of unbroken aluminum foil in a polystyrene Petri plate. Pour the degassed PDMS mixture over the master to a depth of approximately 5 mm. Degas the Petri plate for at least 10 min. Use a gentle stream of air to break surface bubbles. 
	NOTE: The dimensions of the two-tiered ...

<ol>
	<li>Moffitt, J. R., Lee, J. B., Cluzel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single-cell+chemostat:+an+agarose-based,+microfluidic+device+for+high-throughput,+single-cell+studies+of+bacteria+and+bacterial+communities.">The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities.</a> Lab Chip. 12 (8), 1487-1494 (2012).</li><li>Young, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+single-cell+gene+expression+dynamics+in+bacteria+using+fluorescence+time-lapse+microscopy.">Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy.</a> Nat Protoc. 7 (1), 80-88 (2011).</li><li>Dusny, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+bias+of+microcultivation+environments+on+single-cell+physiology.">Technical bias of microcultivation environments on single-cell physiology.</a> Lab Chip. 15 (8), 1822-1834 (2015).</li><li>Wang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+growth+of+Escherichia+coli.">Robust growth of Escherichia coli.</a> Curr Biol. 20 (12), 1099-1103 (2010).</li><li>Norman, T. M., Lord, N. D., Paulsson, J., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+and+modularity+in+cell-fate+decision+making.">Memory and modularity in cell-fate decision making.</a> Nature. 503 (7477), 481-486 (2013).</li><li>Perestrelo, A. R., Aguas, A. C., Rainer, A., Forte, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Organ/Body-on-a-Chip+Devices+at+the+Convergence+of+Biology+and+Microengineering.">Microfluidic Organ/Body-on-a-Chip Devices at the Convergence of Biology and Microengineering.</a> Sensors. 15 (12), 31142-31170 (2015).</li><li>Johari, S., Nock, V., Alkaisi, M. M., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+analysis+of+C.+elegans+muscular+forces+and+locomotion+patterns+in+microstructured+environments.">On-chip analysis of C. elegans muscular forces and locomotion patterns in microstructured environments.</a> Lab Chip. 13 (9), 1699-1707 (2013).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+between+distant+biofilms+and+emergence+of+nutrient+time-sharing.">Coupling between distant biofilms and emergence of nutrient time-sharing.</a> Science. 356 (6338), 638-642 (2017).</li><li>Choi, C. 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B., Jun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Physiology.">Single-Cell Physiology.</a> Annu Rev Biophys. 44, 123-142 (2015).</li><li>Karimi, A., Karig, D., Kumar, A., Ardekani, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+of+physical+mechanisms+and+biofilm+processes:+review+of+microfluidic+methods.">Interplay of physical mechanisms and biofilm processes: review of microfluidic methods.</a> Lab Chip. 15 (1), 23-42 (2015).</li><li>Yu, F. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+microfluidic+tracking+of+coccoid+cyanobacterial+cells+reveals+robust+control+of+division+timing.">Long-term microfluidic tracking of coccoid cyanobacterial cells reveals robust control of division timing.</a> BMC Biol. 15 (1), 11 (2017).</li><li>Campos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+constant+size+extension+drives+bacterial+cell+size+homeostasis.">A constant size extension drives bacterial cell size homeostasis.</a> Cell. 159 (6), 1433-1446 (2014).</li><li>Taheri-Araghi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-size+control+and+homeostasis+in+bacteria.">Cell-size control and homeostasis in bacteria.</a> Curr Biol. 25 (3), 385-391 (2015).</li><li>Siryaporn, A., Kim, M. K., Shen, Y., Stone, H. A., Gitai, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization,+competition,+and+dispersal+of+pathogens+in+fluid+flow+networks.">Colonization, competition, and dispersal of pathogens in fluid flow networks.</a> Curr Biol. 25 (9), 1201-1207 (2015).</li><li>Connell, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+prokaryotic+social+behaviors+with+bacterial+"lobster+traps".">Probing prokaryotic social behaviors with bacterial "lobster traps".</a> MBio. 1 (4), (2010).</li><li>Long, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+bacterial+adaptation+dynamics+at+the+single-cell+level+using+a+microfluidic+chemostat+and+time-lapse+fluorescence+microscopy.">Measuring bacterial adaptation dynamics at the single-cell level using a microfluidic chemostat and time-lapse fluorescence microscopy.</a> Analyst. 139 (20), 5254-5262 (2014).</li><li>Okumus, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+slowing-down+of+cytoplasmic+diffusion+allows+in+vivo+counting+of+proteins+in+individual+cells.">Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells.</a> Nat Commun. 7, 11641 (2016).</li><li>Teng, S. W., Mukherji, S., Moffitt, J. R., de Buyl, S., O'Shea, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+circadian+oscillations+in+growing+cyanobacteria+require+transcriptional+feedback.">Robust circadian oscillations in growing cyanobacteria require transcriptional feedback.</a> Science. 340 (6133), 737-740 (2013).</li><li>Baker, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable,+Pneumatically+Actuated+Microfluidic+Device+with+an+Integrated+Nanochannel+Array+To+Track+Development+of+Individual+Bacteria.">Programmable, Pneumatically Actuated Microfluidic Device with an Integrated Nanochannel Array To Track Development of Individual Bacteria.</a> Anal Chem. 88 (17), 8476-8483 (2016).</li><li>Russell, J. R., Cabeen, M. T., Wiggins, P. A., Paulsson, J., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+in+a+phosphorelay+drives+stochastic+entry+into+sporulation+in+Bacillus+subtilis.">Noise in a phosphorelay drives stochastic entry into sporulation in Bacillus subtilis.</a> EMBO J. 36 (19), 2856-2869 (2017).</li><li>Cabeen, M. T., Russell, J. R., Paulsson, J., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+microfluidic+platform+to+uncover+basic+features+of+energy+and+environmental+stress+responses+in+individual+cells+of+Bacillus+subtilis.">Use of a microfluidic platform to uncover basic features of energy and environmental stress responses in individual cells of Bacillus subtilis.</a> PLoS Genet. 13 (7), e1006901 (2017).</li></ol>

This microfluidics protocol is flexible in that many of the steps may be modified to optimize its use with a particular species or strain or for a specific purpose. Indeed, in this protocol we have made modifications to the original &#34;mother machine&#34; concept4 to optimize its use with B. subtilis. Often, the trenches in which the cells are confined constitute a single-layer feature, whereas in this protocol we use two-layer cell trenches, with a shallow channel surrounding the cells...

One of the most striking features of life on Earth is its great resilience and variety. A central goal of molecular biology is to understand the logic by which cells use genes and proteins to maximize their growth and fitness under a wide variety of environmental conditions. To achieve this goal, scientists must be able to confidently observe how individual cells grow, divide, and express their genes under a given set of conditions, noting how cells respond to subsequent changes in their environment. However, traditional analytical methods in microbiology have technical limitations that affect the types of questions that can be addressed. For example, bulk culture-based analyses have been very useful over the years, yet they offer only population-level data that can mask meaningful cell-to-cell variations or the behaviors of smaller sub-populations of cells in the total population. Single-cell analyses of living bacteria based on light microscopy reveal single-cell behavior but are also technically limited. Bacteria are typically immobilized on agarose pads containing growth medium, but cell growth and division crowds the microscopic view and depletes the available nutrients after just a few cell cycles, substantially limiting the observation time1,2. Moreover, the local depletion of nutrients and the concomitant buildup of metabolic byproducts due to cell growth are constantly changing the local cell growth environment in ways that are difficult to measure or predict. Such environmental changes using agarose pads pose a challenge to studies of steady-state behaviors or of cellular responses to specific changes in growth conditions3.
Microfluidic technology, in which a liquid medium is continuously flowed through microfabricated devices, offers a solution to classic experimental limitations. A microfluidic device can keep individual cells in position for live-cell microscopy while the flow of growth medium constantly provides cells with fresh nutrients and washes away metabolic byproducts and excess cells, thereby creating a highly uniform growth environment. Under constant growth conditions, cell behaviors can be observed in isolation from the influence of environmental factors, permitting an unimpeded view of the internal logic of cells. As the fluid flow prevents the microfluidic device from becoming crowded with cells, observation of single cell lineages for tens or hundreds of generations becomes possible4,5. Such long observation times permit the detection of otherwise undetectable long-term or rare cell behaviors. Finally, the composition of the medium that flows through the device can be altered at will, allowing cells to be observed as they respond to the onset of a stress or to the introduction or removal of a particular compound.
Microfluidics has already enjoyed a number of important applications. For instance, it has been used in tissue-, organ-, or body-on-a-chip devices, in which multiple human cell types are co-cultured to simulate an in vivo condition6; for the study of nematode movement in microstructured environments7; to examine interactions among bacterial biofilms (e.g., 8); and for the encapsulation and manipulation of tiny volumes of cells or chemicals (e.g., 9). Microfluidic devices have also become increasingly popular in the field of microbiology (for excellent reviews, see 10 and 11), especially as their physical and flow properties are well-matched to natural microbial niches12. For instance, microfluidics has been recently employed by microbiologists for such purposes as precisely measuring cell growth and division13,14,15, analyzing pathogen movement16, monitoring quorum sensing17 and physiological transitions18, and for protein counting19, among many other examples. The method presented here is specifically designed for the analysis of single bacterial cell lineages rather than combinations of strains or species. The microfluidic device demonstrated here utilizes one variation of the &#34;mother machine&#34; design4, in which cells are grown single-file within a microfluidic trench with one closed end and one open end; cell growth and division pushes progeny cells up and out of the open end into the fluid flow. Our analyses typically focus only on the &#34;mother&#34; cell that is confined at the closed end of the trench. We consider this method as an advancement over previous light microscopy-based single-cell analytical techniques, such as cell immobilization on agarose pads. While B. subtilis is used as a model here, the method is also applicable to other bacterial species (Escherichia coli is another common model; some species with different cell sizes or morphologies may require the fabrication of new devices with different dimensions). The use of fluorescent reporters to mark cells and to visualize changes in gene expression requires the use of genetically tractable species; however, analyses of cell growth and morphology are possible even without fluorescent markers.
The present protocol excludes the process of fabricating the silicon master using photolithography, which has been extensively described elsewhere5; masters can also be easily outsourced from microfabrication facilities. It includes the molding of a PDMS device from a reinforced silicon master; bonding the device to a piece of cover glass; assembling the microfluidic inlet and outlet plumbing, including pinch valves to permit medium switching; passivating the device, preparing bacterial cells, and loading the device with bacterial cells; attaching the plumbing to the device and equilibrating the cells; and loading the device onto a fluorescence microscope for imaging. Because many different image acquisition and processing software tools can be used to visualize and analyze different data of interest4,5, example images are shown, but image-capture methods are not included in this protocol.

<table border="0" cellpadding="0" cellspacing="0" width="580">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:71px;">Dow Corning</td>
			<td style="width:119px;">(240)4019862</td>
			<td style="width:167px;">This is referred to as PDMS in the protocol. There are 2 components that are mixed at a 10:1 ratio</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">0.75-mm biopsy punch</td>
			<td style="width:71px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">504529</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">22 x 40 mm No. 1.5 cover glass</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48393 172</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Plasma Etch PE-50</td>
			<td style="width:71px;">Plasma Etch Inc.</td>
			<td style="width:119px;">PE-50</td>
			<td>Instrument used to bond PDMS to glass using oxygen plasma treatment</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Tygon flexible tubing ID 0.02&#34;, OD 0.06&#34;</td>
			<td style="width:71px;">Saint-Gobain PPL Corp.</td>
			<td>AAD04103</td>
			<td>For the main part of the tubing, e.g. attached to the needles</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:223px;">Silicone Tubing, 0.04&#34; ID, 0.085&#34; OD</td>
			<td style="width:71px;">HelixMark Standard Silicone Tubing</td>
			<td style="width:119px;">60-795-05</td>
			<td>For pinch valves and Y-junction connection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Bovine Serum Albumin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906-100G</td>
			<td>Used as passivation agent</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">ART Gel Pipet Tips (P200)</td>
			<td style="width:71px;">Molecular BioProducts</td>
			<td align="right" style="width:119px;">2155</td>
			<td>For loading the device with medium and cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Acrodisc 32-mm syringe filter with 5-&#956;m Supor membrane</td>
			<td style="width:71px;">Pall Life Sciences</td>
			<td align="right" style="width:119px;">4560</td>
			<td>For filtering cultures before device loading</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">21-ga blunt needles, 1&#34;</td>
			<td style="width:71px;">McMaster-Carr</td>
			<td style="width:119px;">75165A681</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Y connector with 200-series barbs, 1/16&#34; ID tubing, polypropylene</td>
			<td style="width:71px;">Nordson Medical</td>
			<td style="width:119px;">Y210-6005</td>
			<td>The company is AKA Value Plastics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Eclipse Ti-E inverted microscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>This unit has been discontinued by the manufacturer and is replaced by the Ti 2 E.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">LB Lennox</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">L3022</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Microfuge 18</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td align="right">367160</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Bacillus subtilis strain NCIB3610</td>
			<td style="width:71px;">Bacillus Genetic Stock Center</td>
			<td style="width:119px;">3A1</td>
			<td>wild-type parental strain modified for use in the experiments shown in this protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Gravity convection oven</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">414005-106</td>
			<td>for curing PDMS and baking assembled devices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Scotch-Weld Epoxy Kit</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;">2216 B/A</td>
			<td>may be used to bond a silicon wafer to an aluminum backing plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Scotch Magic tape, 3/4&#34; width</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;"></td>
			<td>to clean dust from PDMS devices and to place over features to increase visibility (denoted &#34;office tape&#34; or &#34;adhesive tape&#34; in protocol)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Stereomicroscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">SMZ800N</td>
			<td>listed here as an example; nearly any stereomicroscope will do</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Isopropyl alcohol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">W292907</td>
			<td>To clean cover glass</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Syring pump, 6 channel</td>
			<td style="width:71px;">New Era Pump Systems Inc.</td>
			<td style="width:119px;">NE-1600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Kimwipes</td>
			<td style="width:71px;">Kimberly-Clark</td>
			<td align="right" style="width:119px;">34120</td>
			<td>a brand of dust-free wipes</td>
		</tr>
	</tbody>
</table>

single-cell microfluidic analysis of bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Successful initial cell loading, as assessed by phase-contrast microscopy before attaching the microfluidic plumbing to the device, would be considered as having all or nearly all of the microfluidic side channels containing one or more bacterial cells (Figure 3A). Optimal loading would show several cells in each channel, but channels will nonetheless fill with cells due to cell growth during the equilibration period (Figure 3B)....

Watch this Scientific Journal Video about Single-cell Microfluidic Analysis of Bacillus subtilis at JoVE.com

Single-cell Microfluidic Analysis of Bacillus subtilis

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

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

The overall goal of this microfluidic experimental set-up is to enable the longterm observation of individual bacterial cells under constant, highly controlled environmental conditions. This method can help answer key questions in the bacterial physiology field, such as what are the longterm response patterns of individual cells to stress or how does a heterogeneous phenotype across a population vary with time. The main advantage of this technique is that we can observe individual cell lineages for tens or even hundreds of generations, under constant, uniform and highly controlled environmental conditions.After preparing PDMS polymer according to the text protocol, place a silicone master on a piece of unbroken aluminum foil in a polystyrene Petri dish. Pour the degassed PDMS mixture over the master to a depth of approximately five millimeters. Then degas the dish for at least 10 minutes.Cure the PDMS in an oven at 60 degrees Celsius for at least one hour and cool it to room temperature. Then remove the aluminum foil containing the master and cured PDMS from the plate. Carefully score it and peel off the aluminum foil from the back of the master.Then carefully peel the PDMS from the master. As a wafer, typically include several microfluidic devices with slightly different dimensions. Use a clean, sharp scalpel to cut a single device to the appropriate size.Place a piece of translucent office tape over the patterned side of the device to enhance the visibility of the patterned features. The inlet and outlet ports are identified as circular areas at opposite ends of the visible fluidic channels. Flip the PDMS device with the pattern side down and use a 0.75 millimeter biopsy punch to punch through the device to the marked dots.Then discard the punchings. A tweezer may be required to remove the punchings from the device. Insert the cleaned and dried cover glass and PDMS device into an oxygen plasma cleaner and plasma treat the device with the settings shown here.Immediately after the plasma treatment finishes, remove the device and cover glass from the plasma cleaner. Invert the PDMS and place it on the center of the cover glass so that the pattern side contacts the glass. Ensure that the feeding channels are aligned with the long access of the cover glass.Press down very gently to ensure that the PDMS seals against the glass. Bake the assembled device in an oven at 60 degrees Celsius for at least one hour. After baking, manually verify that the PDMS is bonded to the glass by gently pushing on one corner of the device.After cutting polymer tubing according to the text protocol, assemble Y-junctions for pinch valves, if using them, by cutting and attaching two-centimeter lengths of flexible silicone tubing to the top barbs of the Y and one-centimeter lengths of silicone tubing to the bottom barb of the Y.Next, cut 10-centimeter lengths of polymer tubing then connect them at one end to the switch junction by inserting them into the one-centimeter silicone tubing segments on the bottom barb of the Y.Connect the lengths of tubing at the other end to 21-gauge needles that have been removed from their plastic syringe adapters and bent approximately 90 degrees about nine millimeters from the needle end. Connect 21-gauge blunt needles to one end of the tubing segments that will run from the syringes to the switch apparatus by inserting the needles into the tubing. Insert the other ends of each length of tubing into the silicone tubing segments on the inlet barbs of the Y-junctions.After filling syringes with bacterial growth medium, supplemented with BSA, according to the text protocol, connect the loaded syringes to the prepared 21-gauge needles, attach to the inlet tubing and load the syringes into the syringe pumps. Purge air from the inlet plumbing by running the syringe pumps at a high flow rate and orienting the syringe pumps vertically, tapping the syringes to bring air bubbles to the top. Once air has been purged from the syringe, position the syringe pump horizontally and progressively tap or flick the polymer lines from the syringes, up through the switch apparatus to dislodge and purge any air bubbles.Repeat this process for the second bank of syringes if switching media. After the air bubbles are purged, set the phase-two syringe pump to 1.5 microliters per minute then pause the flow. Place small binder clips onto the segments of flexible tubing on the branch of the Y-junctions corresponding to the second medium phase and continue to run the phase-one pump to purge the second medium downstream of the Y-junction.To load the PDMS device, begin by gently placing empty, thin gel-loading micropipette tips into the outlet holes of the microfluidic device. Using thin gel-loading tips with the P200 micropipette, passivate the device by injecting medium containing one milligram per milliliter BSA into the inlet holes, observing the tips and the outlet holes to monitor filling of the microfluidic channels. Incubate the device at room temperature for approximately five minutes.Next, using similar tips, load each channel with the re-suspended cells using the tips in the outlet channel to monitor the progress of the cells through the device. Gently remove the gel-loading tips from the outlet holes, working one at a time to prevent damage to the device. Centrifuge the device in a benchtop microcentrifuge in an appropriate rotor adapter at approximately 6, 000 times g for 10 minutes.Verify successful loading under the microscope but without affixing the device to the slide holder stage insert. Carefully mount the loaded device onto a stage insert by taping the cover glass on either side of the PDMS to the bottom of the stage insert. Invert the stage insert device assembly so that the PDMS is facing up and place it on a soft surface such as a dust-free wipe.Adjust the flow rate of the phase-one syringe pump to 35 microliters per minute. Then working with one lane at a time, insert the inlet needle and then the outlet needle that runs to the waste beaker. After visually inspecting the device for leaks, examine the outlet tubing for the appearance of excess cells that appear as a turbid stripe in the medium meniscus, which will slowly move towards the waste beaker.Once the device has run for approximately 15 minutes, set the phase-one syringe pump to 1.5 microliters per minute and pause the flow. Bring the entire pump and device apparatus to an inverted fluorescent microscope and restart the phase-one medium flow at 1.5 microliters per minute. Carefully mount the stage insert with the device on to the microscope, using tape as necessary to route the inlet and outlet tubing.Locate the appropriate positions on the device for imaging and begin imaging as desired. Note that the cells typically require a few hours to read steady-state, exponential phase growth, and the onset of image acquisition may be delayed as desired so that imaging begins after the equilibration period. To switch medium between successive rounds of imaging, pause the flow of the phase-one syringe pump, carefully unclip the binder clips from the phase-two silicone tubing, moving each one to the silicone tubing on the phase-one branch of the Y-junction, then unpause the flow of the phase-two syringe pump and continue imaging.As assessed by phase contrast microscopy, before attaching the microfluidic plumbing to the device, initial cell loading would be considered successful if all or nearly all of the microfluidic side channels contained one or more bacterial cells. Once the device is initially loaded, it is equilibrated at a relatively high flow rate. As shown here, microscopic inspection of an equilibrated device should reveal a few cells confined in single file in most of the side channels.Once an equilibrated device is placed on the microscope under flow at 1.5 microliters per minute at 37 degrees Celsius, the cells will resume uniform and constant exponential phase growth over the course of approximately two hours. Cells that have resumed exponential phase growth can be reliably imaged using bright-field or fluorescence microscopy for at least 24 hours. Introducing a medium switch expands the range of experiments that can be conducted but also increases the frequency of catastrophic cell death events through the presence of air bubbles or cell clusters at the inlet that become dislodged and pass through the feeding channel.Once mastered, this technique can be done in approximately five hours, if it is performed properly. By using this procedure, with different fluorescent reporters, strains and environmental conditions, a wide variety of physiological phenomena can be examined, including very rare or transient events.

single-cell-microfluidic-analysis-of-bacillus-subtilis

Research

JoVE Journal

Biology

11.8K vistas.  Oklahoma State University. The overall goal of this microfluidic experimental set-up is to enable the longterm observation of individual bacterial cells under constant, highly controlled environmental conditions. This method can help answer key questions in the bacterial physiology field, such as what are the longterm response patterns of individual cells to stress or how does a heterogeneous phenotype across a population vary with time. The main advantage of this technique is that we can observe individual cell lineag...