Name: single-cell microfluidic analysis of bacillus subtilis
Uploaded: 2018-01-26T14:30:00
Description: Watch this Scientific Journal Video about Single-cell Microfluidic Analysis of Bacillus subtilis at JoVE.com

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>
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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. 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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 ...

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Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon ...