Table Of ContentGaoetal.Microbiome2013,1:4
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METHODOLOGY Open Access
Microbe observation and cultivation array (MOCA)
for cultivating and analyzing environmental
microbiota
Weimin Gao, Dena Navarroli, Jared Naimark, Weiwen Zhang, Shih-hui Chao* and Deirdre R Meldrum
Abstract
Background: The use ofculture-independent nucleic acid techniques, such as ribosomalRNA gene cloninglibrary
analysis, has unveiled thetremendous microbialdiversity that exists innatural environments.Insharp contrast to
this great achievementis the current difficulty in cultivating themajorityof bacterial species or phylotypes revealed
bymolecular approaches. Although recent new technologies such as metagenomics and metatranscriptomics can
provide more functionality information about themicrobial communities, it is still important to develop the
capacity to isolate and cultivateindividual microbial species or strains in order to gain a better understanding of
microbial physiology and to apply isolatesfor various biotechnological applications.
Results: We have developed a new system to cultivate bacteria in an array of droplets. The key component of the
system is the microbe observation and cultivation array (MOCA), which consists ofa Petri dish that contains an
array of droplets as cultivation chambers. MOCAexploitsthe dominance of surface tension insmall amountsof
liquid to spontaneously trap cells inwell-defined droplets on hydrophilic patterns. During cultivation, thegrowth of
thebacterialcells across the droplet array can be monitored using anautomated microscope, which can produce a
real-time record of the growth. When bacterial cells grow to a visible microcolony level inthesystem,they can be
transferred using a micropipette for further cultivationor analysis.
Conclusions: MOCA is a flexible system that is easy to set up, and provides thesensitivity to monitor growth of
single bacterial cells. It is a cost-efficient technical platform for bioassay screening and for cultivationand isolation
ofbacteria from natural environments.
Keywords: Microbe observation and cultivationarray (MOCA), Cultivation, Growth, Microbiota
Background occurring microbes are considered ‘unculturable’ on
During the past over two decades, the use of culture- standard culture media [2]. Although recent new tech-
independent nucleic acid techniques, represented by nologies such as metagenomics and metatranscriptomics
ribosomal RNA gene cloning library analysis, has canprovidemorefunctionalityinformationaboutmicro-
unveiled the tremendous microbial diversity that exists bial communities [2,3], it is still important to develop
in natural environments [1]. In sharp contrast to this the capacity to isolate and cultivate individual microbial
great achievement is the current inability to cultivate the species or strains in order to gain better understanding
majority of bacterial species or phylotypes revealed by of microbial physiology and to apply isolates for various
molecular approaches. One of the major difficulties for biotechnologicalapplications [4].
microbial ecology is that conventional cultivation meth- A new view is emerging among microbial ecologists
ods provide access to only a very small fraction of the that the majority of so-called ‘unculturable’ microbial
microbial diversity and more than 99% of naturally species simply have not been cultured yet. In line with
this view, more research is necessary to enhance the
ability to culture microbes, in order to reduce the de-
*Correspondence:[email protected]
pendence on indirect and cumbersome metagenomic
ArizonaStateUniversity,Tempe,AZ85287,USA
approaches [5,6]. Recent developments in improving
©2013Gaoetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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traditional cultivation techniques have shown that some component of the system is the microbe observation and
conventionally unculturable species can in fact be grown cultivation array (MOCA), which uses a Petri dish that
as pure cultures. Tamaki et al. reported that the use of containsanarrayofdropletswithanoilcoveringasculti-
gellan gum instead of agar as the solidifying agent could vation chambers. During cultivation, the growth of bac-
greatly improve the cultivability of novel microbes on teria across the droplet array can be monitored using an
solid media [7,8]. Kaeberlein et al. designed a diffusion automated microscope, which can produce a real-time
chamber to grow previously uncultivated pure isolates of growth record. Compared with conventional cultivation
marine origin [9], and the same strategy was also suc- methods,MOCAprovidesstreamlinedpreparation,paral-
cessfully used in cultivation of groundwater microorgan- lelcultivation,andreal-timeobservation,andunlikeother
isms [10]. Stevenson et al. achieved similar results with chip-based platforms [16-19], MOCA does not require
soil microbes by fine-tuning the oxygen concentration complicatedengineeringtechniquesorequipmentforfab-
and nutrient levels [11]. Knowledge obtained through rication. We have found that bacterial growth across the
metatranscriptome analysis has also been used in direc- droplet array has a high level of uniformity when the ini-
ted cultivation of bacteria [12]. These examples shown tial cell density is more than 10 cells/μl, and thus MOCA
that many microbial species can be cultured as long as provides a novel platform for bioassay screening. When
theenvironmentsareoptimizedforgrowth. the cell occupancy in droplets is at the single-cell level,
Recently, some non-traditional cell-isolation technolo- real-time image recording can be used to monitor the
gieshavebeenintroducedtoisolatetargetedcellsforpure growth and morphological development of microcolonies
culture cultivation.Forexample,Huberet al.usedoptical derived from single bacterial cells. The droplet culture
tweezers to track and isolate an extremophilic archaeon developed from a single bacterial cell can be transferred
fromamicrobialcommunityinaterrestrialhydrothermal using a micropipette [21] for bulk cultivation or further
vent field [13]. This method of single-cell manipulation molecularanalysis.
provides a new way to grow puremicrobial cultures from
single mother cells, but it has the disadvantage that the
identification and manipulation of the bacteria is an Methods
extremely labor-intensive process. By contrast, a high- Microbeobservationandcultivationarray
throughput isolation method has been presented using The MOCA Petri dish is produced using a technique
encapsulated single bacteriain droplets of gel [14],result- known as microscale plasma activated templating
ing in the successfully cultivation of pure cultures from (μPLAT) [22,23]. The μPLAT method uses an inexpen-
marine microorganisms. Oligonucleotide probes were sive plasma-treatment process to create hydrophilic pat-
usedtoidentifythespeciesafterisolation. terns on Petri dishes (60 mm diameter; Falcon; Becton
One of the remaining hurdles for bacterial cultivation Diskinson Labware, Franklin Lakes, NJ, USA); the air
is that fine-tuning the growth condition for any specific plasma makes the originally hydrophobic polystyrene
species is a daunting effort, especially with the widely surfacesofthePetri disheshydrophilic.
diverse microbiota in the ocean and soil environments. Briefly,aμPLATstencilismadefrompolydimethylsilox-
Consequently, a high-throughput platform is urgently ane (PDMS) film, an inexpensive, soft polymer. The sten-
needed to perform trials of different cultivation condi- cil hasanarrayof 4× 6holes, each3.18mmindiameter,
tions in parallel. Previously, several high-throughput whichisadheredtothesurfaceofaPetridish,(Figure1a).
microtiter-plate-based cultivation platforms have been The assembly is placed in a plasma cleaner (Harrick
developedformarineandaquaticwatercolumnbacteria, Plasma, Ithaca, NY, USA), and exposed to air plasma for
and these have contributed greatly to the successful 1 minute (Figure 1b).The stencil is removed after plasma
cultivation of previously uncultivated bacteria [15,16]. treatment.Becauseonlythepolystyrenesurfaceunderthe
Most recently, a chip-based version of a Petri dish and circularopeningsisexposedtotheplasma,theseareasbe-
diffusion chamber has been developed to address come hydrophilic on the hydrophobic background of the
the same purpose [17,18]. Microfluidic ‘lab-on-a-chip’ plate (Figure 1c).Thepatterned MOCA Petri dish is then
(LOC) devices have been used for co-cultivation of vari- surface-sterilizedunder UV lightfor15minutesin aPCR
ous bacterial strains and species [19,20]. These devices cabinet (PCR Workstation; Thermo Fisher Scientific,
are complicated in structure, utility, and fabrication, and Hudson, NH, USA). The bacteria and medium mixture is
aretherefore lessusefulfor general microbiologists. loadedusingapipetteintothepreparedPetridish,produ-
In this study, we developed a parallel cultivation set-up cing a 4 × 6 array of 1-μl droplets. The borders of the
thatincorporatesstreamlinedprocessesand iscompatible liquid are confined at the hydrophilic/hydrophobic inter-
with downstream genomic analysis. It spontaneously iso- face, forming discrete droplets defined by the stencil
lates environmental bacteria into miniature incubation pattern. To prevent evaporation and contamination from
chambers from a mixed microbial community. The key theambientenvironment,5mlmineraloil(M5904;Sigma
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Figure1Schemeofmicrobeobservationandcultivationarray(MOCA)dropletpreparation.(a)Astencilwithanarrayofholesisadhered
toaPetridish,whichisthen(b)exposedtoairplasmatogenerateanarrayofhydrophilicdisks.(c)Thestencilisthenremoved,andthemedium
containingthebacteriaisaddedtothehydrophilicdisks,afterwhich(d)thedropletarrayiscoveredwithmineraloil.
Chemical Co., St Louis, MO, USA) is loaded to cover the dark at room temperature, and growth was monitored
dropletarray(Figure1d). bydirectmicroscopy.
Automatedtime-seriesobservation
Loadingofbacteriainmediumandculturingusing A MOCA dish can be monitored on an automated op-
microbeobservationandcultivationarray tical microscope to observe the growth of the loaded
Luria broth (Difco; Becton, Dickinson and Company, bacteria. The MOCA Petri dish was placed on the auto-
Sparks, MD, USA) was used for both bulk cell and mated microscope (Eclipse TiE, Nikon Instrument Inc.,
droplet cultivation of the gram-negative bacterium Japan) with a 4× objective, so the field of view covered
Escherichia coli DH5α and the gram-positive bacterium an entire droplet (Figure 2). The microscope stage was
Bacillus subtilis 168. Marine broth (Difco‘ Becton, programmedtotravelthrough allthedropletsandtoac-
Dickinson and Company) was used for droplet cultiva- quire bright-field images of all droplets every 30 minutes
tion of marine bacteria from sea water sampled from the for 72 hours. The microscope illumination was turned
deep sea of the northwest Pacific Ocean [24]. Cells were on only during image acquisition to avoid heating of the
counted using a counting chamber (Hausser Scientific dish. After the experiments, the time-lapse micrographs
Partnership, Horsham, PA, USA) under a microscope to of each droplet were used to estimate bacterial growth
estimate the bacterial density for both pure cultures and using measurement of light transmission; the micro-
sampled sea water. Four cell concentrations (103, 102, graphs appeared darker as bacteria grew. Although an
10, and 1 cell(s)/μl) were used in the experiments. Cell automated microscope was used in this study, other op-
occupancy in the droplets is a Poisson random variable tical platforms (for example, conventional microscopes
[23]; when 1 cell/μl medium is loaded into the 1 μl dro- or even the naked eye when the droplets are saturated
plets,the average celloccupancyisone cellper droplet. with microbes) canalso beusedfor estimation.
For each individual hydrophilic MOCA spots, 1 μl
bacteria-medium mixture was pipetted onto it. The dro- Phylogenyanalysisofisolatedbacteriafromsinglecells
plets were then covered with 5 ml of mineral oil and the The cultivated isolates developed from single bacterial
lid placed on the Petri dish. The dishes were kept in the cells of interest can be transferred using a micropipette
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Figure2Demonstrationofautomatedtime-seriesobservationbyopticalmicroscopyofbacterialdropletcultivationusingmicrobe
observationandcultivationarray(MOCA).
fromtheMOCAchiptoothercontainersforarchivaland (model 3700; Applied Biosystems Inc.) using the same
downstream phylogenetic analyses using 16S rRNA se- PCR primers as before. The resultant DNA sequences
quencing. In this study, bacteria were removed from the were edited manually using Sequence Scanner Software
droplets using a micropipette and transferred to a 1.5 ml (version 2.0; Applied Biosystems Inc.). Sequences were
microcentrifuge tube containing 100 μl sterilized water. comparedagainstthoseinGenBankthroughtheNational
After being spun in a centrifuge at 12000g for 1 minute, Center for Biotechnology Information (NCBI) portal and
the supernatant was decanted to remove possible carry- BLASTsoftware (version 2.2.10) [26], and the phylogeny
over of the mineral oil, then the bacterial cells were re- affiliationofeachisolatesweretheninferred.
suspended in 5 μl sterilized water and transferred to a
0.2 ml PCR tube. The PCR tubes werethen put in a PCR Results and discussion
thermal cycler (model 9700; Applied Biosystems Inc., Bacterialgrowthindroplets
Foster City, CA, USA) and heated at 95°C for 5 minutes, BecauseMOCAdropletsettingscreateadifferentphysical
followedbychillingonicetoinducecelllysis.Usingheat- environment from that of bulk cultures, this may influ-
treated bacterial cells as the template, conventional PCR ence many of the growth factors required by the bacteria
wascarriedouttoamplifythehighlyvariableV6regionof [27,28].Thus,giventhatthetotalvolumeforcultivationis
the small subunit (SSU) rRNA gene. The PCR primer amicroliter,bacterialgrowthwithinthedropletsmayhave
sequences are shown in Table 1 [25]. The 20 μl PCR different properties from that in regular cultures. To test
volume contained: 10 μl 2×PCR Master Mix (Fermentas if the droplets could inhibit bacterial growth, we initially
LifeScienceInc.,Glenburnie,MD,USA),2μlofeachpri- cultivated E. coli and B. subtilis cells within droplets with
mer(4μmol/l),2μlofDNAtemplateand4μlofdouble- a cover of mineral oil and assessed their growth in real
distilledwater.TheconditionsforPCRamplificationwere time. Using the method described above, a 4 × 6 array of
asfollows:94°Cfor5minutes;35cyclesof94°Cfor15sec- 1 μl droplets for each species were generated for the four
onds, 56°C for 20 seconds, and 72°C for 45 seconds; with cell concentrations (103, 102, 10, 1 cell(s)/μl), using six
a final extension step of 72°C for 10 minutes and soak at dropletsforeachcellconcentration.Celloccupancyinthe
4°C. The expected PCR products were recovered with a droplets is a Poisson random variable [23]: when loading
gelandextracted(QiaquickGelDNAExtractionKit;Qia- 1 cell/μl medium into droplets, around 37% of droplets
gen Inc.,Valencia, CA, USA). The purified PCR products areempty,and37%,18%,6%,and1.5%ofthedropletsare
wereusedastemplatesforDNAsequencing,usingacom- occupied by one, two, three, and four cells, respectively.
mercialkit(BigDyeTerminatorCycleSequencingKit,ver- Therefore, the lowest cell concentration in all presented
sion 3.1; Applied Biosystems Inc.) and a PCR analyzer cultivation experiments yielded single-digit bacterial cells
indroplets.
Table1PrimersusedforPCR Figure 3 shows the growth of these two bacteria, where
eachcurverepresentsthegrowthinanindividualdroplet.
Primername Sequence(5′→3′)
We measured light transmission through the acquired
U968 AACGCGAAGAACCTTAC-
micrographs as an indication of cell density; the lower
L1401 CGGTGTGTACAAGACCC
the transmission, the larger the cell population. The
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Figure3Real-timegrowthrecordofbacteriainmicrobeobservationandcultivationarray(MOCA).(a,c)Growthcurvesof(a)Escherichia
coliand(c)Bacillussubtiliswithdifferentinitialcellconcentrations(thelowerthetransmission,thelargerthecellpopulation).Eachcurve
representsthegrowthinanindividualdroplet.(b,d)Thresholdgrowthtimedistributionversus(b)E.coliand(d)B.subtiliscellconcentrations.
Thethresholdgrowthtimewasdefinedasthetimewhenthenormalizedtransmissionintensityofadropletintersected0.99(Figure3aand3c,
reddottedline).
transmittedlightintensitywasnormalizedwithrespectto notsignificantlychange,butthesizeofcellsbecamesmal-
its initial value to reduce the light variation between dro- ler, suggesting that the B. subtilis cells started to spore
plets.,therefore,allgrowthcurvesstartedfrom1.Thecol- afterthenutritionwasdepleted[29].Onlytwoofthemost
ors of the curves in the figure indicate initial cell diluted droplets contained B. subtilis cells that were able
concentrations. The curves of the individual cell concen- to grow into colonies in this experiment. Similar to the
tration were clustered before reaching the stationary E. coli cultivation result, the threshold growth time was
phase, with a few outliers appearing for droplets with mostlylineartoindividualcellconcentrations(Figure3d),
low initial cell concentrations. We defined the threshold whereas the lowest concentration had a large variation
growth time as the time when the normalized transmis- becauseonlytwodropletsresultedingrowth.
sion intensity of a droplet intersected 0.99 (Figure 3, red These results show that, when the initial cell density is
dottedlines).Theboxplots(Figure3b,d)showthethresh- more than 10 cells/μl, bacterial growth across the drop-
oldgrowthtime,whichwasmostlylineartotheindividual let array is strongly uniform and reproducible. For drop-
cell concentrations. Because of random cell occupancy lets with single-cell occupancy, the platform can be used
and cellular heterogeneity, the variation was large when to monitor the growth heterogeneity at the single-cell
bacterialnumberwithinthedropletswasinsinglefigures. resolution.
The growth curves of both E. coli (Figure 3a) and
B. subtilis (Figure 3c) shared similar features. The curves Morphologicaldevelopmentofmicrocolonieswithin
of individual cell concentration were clustered before droplets
growth reached the stationary phase. One distinct feature We found that the droplet setting permitted the growth
of the B. subtilis growth is that the transmission intensity of both E. coli and B. subtilis. In addition, we also found
became brighter after the population peaked. The time- that the bacterial growth pattern within a droplet was
lapse micrographs showed that the number of cells did different from that of a regular liquid medium. This was
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especially evident when cultivating single-digit numbers was diluted to 0.25 cells per droplet with marine broth
of bacterial cells within a droplet. Using the time-series medium. Based on Poisson statistics, the possibility of
micrographs of the 24 droplets with E. coli growth multicell occupancy with this concentration is around 2%
recorded at 0, 8, 16, and 24 hours (Figure 4), we found andthatofsingle-celloccupancyis20%.
that each single E. coli cell could develop its own micro- Using the automated time-series observation system,
colony, and these did not merge with each other until we could identify which droplets had bacterial growth
the solution became cloudy. Similar results were seen andhow many microcolonies hadbeendevelopedwithin
for B. subtilis (data not shown). In this respect, the these droplets. Droplets showing single microcolony
growth pattern of both E. coli and B. subtilis within a growth were identified and the cultivated pure microbes
droplet is more similar to that on a solid agar plate than recovered directly. For verification of purity, the reco-
in a liquid medium. This may due to the use of the oil vered cultures were subjected to further purification on
droplet cultivation platform, in which the physical envir- traditionalPetri dishes.
onment is different from that of routine liquid cultiva- In one experiment, thirty-six droplets were generated,
tion. For instance, oil coverage may create a microoxic and eight of these were successfully cultivated during a
environment, which may inhibit or reduce the move- 2-day culture period. Time-lapse micrographs showed
mentof bacteria[30]. that they were all derived from a single bacterium (data
not shown). The profiles of the growth curves showed
Environmentalbacteriacultivationandisolationusinga similar trends to those of the previous results of the
microbeobservationandcultivationarray single-digit bacterial cell growth in a droplet (Figure 3),
We further used MOCA to cultivate and isolate environ- but the variation was larger, perhaps due to species dif-
mental marine microbes, which were sampled from the ferences or to the different time requirement for exit
Hydrate Ridge site (80 km off the Oregon coast and 5 m from dormancy status (Figure 5). Some fast-growing
above the 780-m deep seafloor) during a research cruise droplets (for example, those in droplets 8 and 9) started
between22Julyand5August2008[24].Toavoidloading growing within the first 8 hours, whereas others (for ex-
multiple cells from each droplet, the cell concentration ample, droplet 21) started as late as the 30th hour. The
Figure4ProliferationofEscherichiacoliindroplets.Thetime-seriesmicrographsof24dropletsat0,8,16,and24hours.Thedarkclustersin
thedropletsareE.colicolonies.Forallfourpanels,thenominalcellnumbersofthefiverowsare104,103,102,10,and1cell(s)perdroplet,
respectively.
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Figure5Growthcurveofmarinebacterialisolateswithinthedroplets.Eachcurverepresentsthegrowthinanindividualdroplet.The
numberinthelegendisthedropletnumberassociatedwithTable2.
threshold growth times of these droplets are listed in the growth of microaerobic bacteria, but not strict aer-
Table2. obic bacteria. In addition, putting MOCA into an anaer-
Single colonieswereidentifiedinthesedropletsandthe obic chamber and loading with a bacteria-media mixture
transferred cells were subjected to colony PCR. After under anaerobic conditions can allow an anaerobic en-
sequencing the obtained PCR products of 400 bp, which vironment to be created. We will explore the potential
represent the highly variableV6 region of the SSU rRNA of this technique and its limitations in cultivating novel
gene, aphylogeny analysis wasconducted basedonNCBI environmental bacteriainfuturestudies.
database searches. Sequences were deposited in GenBank
under accession numbers JQ178347 to JQ178354. The
closest species/genus with which each isolated strain was Conclusions
identified are listed in Table 2. Of these identified cul- We have developed a new system to cultivate bacteria in
tures, the majority (six out of eight cultures) belonged to a droplet array. Our results show that the set-up of
Pseudoalteromonasspp.(Evalue=0),with the remaining droplet does not inhibit the growth of bacteria, as we
two identified as Shewanella sp. (E value=0) and found that both E. coli and B. subtilis cells could prolif-
Colwellia piezophila (E value=0), respectively. The erate uniformly across the MOCA. We also found that
phylogeny affiliation of recovered isolates is consistent the bacterial growth pattern within a droplet is more
withthesourceofthemicrobialcommunity[31-33]. similar to that on a solid than in a liquid medium. Given
BecauseMOCAdropletscreateadifferentphysicalen- its flexibility, ease of set-up, and sensitivity to growth of
vironment from that of bulk cultures, , this may influ- a single bacterial cell, we believe that this droplet plat-
ence many of the growth factors required by bacteria. form could provide a cost-efficient way to cultivate and
For example, the use of an oil cover reduces gas ex- isolate novel bacteria from different environments, and
change with the ambient environment, which may favor hadthepotentialfor useinbioassayscreening.
Table2Cultivatedbacteria,theirphylogenyaffiliationandcorrespondingthresholdgrowthtimes
Dropletnumber GenBankaccessionnumber ClosestmatchinGenBank(percentageidentity) Thresholdgrowthtime,hours
1 JQ178347 Pseudoalteromonaselyakovii(99%) 10.51
2 JQ178348 Pseudoalteromonastetraodonis(99%) 11.02
3 JQ178349 Pseudoalteromonastetraodonis(99%) 11.16
8 JQ178350 Pseudoalteromonastetraodonis(96%) 9.43
9 JQ178351 Pseudoalteromonastetraodonis(98%) 9.48
14 JQ178352 Pseudoalteromonastetraodonis(99%) 15.37
21 JQ178353 Shewanellahalifaxensis(98%) 31.13
33 JQ178354 Colwelliapiezophila(98%) 27.94
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Abbreviations 15. BrunsA,CypionkaH,OvermannJ:CyclicAMPandacylhomoserine
MOCA:Microbeobservationandcultivationarray;DNA:Deoxyribonucleic lactonesincreasethecultivationefficiencyofheterotrophicbacteria
acid;rRNA:Ribosomalribonucleicacid;SSUrRNA:Smallsubunitribosomal fromthecentralBalticSea.ApplEnvironMicrobiol2002,68:3978–3987.
RNA;PCR:Polymerasechainreaction;μPLAT:Microscaleplasmaactivated 16. ConnonSA,GiovannoniSJ:High-throughputmethodsforculturing
templating;PDMS:Polydimethylsiloxane. microorganismsinvery-low-nutrientmediayielddiversenewmarine
isolates.ApplEnvironMicrobiol2002,68:3878–3885.
Competinginterests 17. InghamCJ,SprenkelsA,BomerJ,MolenaarD,vandenBergA,etal:The
Theauthorsdeclarethattheyhavenocompetinginterests. micro-Petridish,amillion-wellgrowthchipforthecultureandhigh-
throughputscreeningofmicroorganisms.ProcNatlAcadSciUSA2007,
Authors’contributions 104:18217–18222.
18. NicholsD,CahoonN,TrakhtenbergEM,PhamL,MehtaA,etal:Useof
WGconceivedofthestudy,participatedinitsdesign,carriedoutthe
Ichipforhigh-throughputinsitucultivationof“uncultivable”microbial
phylogeneticstudies,andwasinvolvedintheinterpretationofalldata.DN
andJNcarriedoutthepreparation,cultivationprocesses,andphylogenetic species.ApplEnvironMicrobiol2010,76:2445–2450.
19. KimHJ,BoedickerJQ,ChoiJW,IsmagilovRF:Definedspatialstructure
studies.WZconceivedofthestudy,participatedinitsdesign,andwas
stabilizesasyntheticmultispeciesbacterialcommunity.ProcNatlAcadSci
involvedintheinterpretationofalldata.SCconceivedofthestudy,
USA2008,105:18188–18193.
participatedinitsdesign,andanalyzedtheresults.DMconceivedofthe
study.Allauthorsreadandapprovedthefinalmanuscriptandwereinvolved 20. ParkJ,KernerA,BurnsMA,LinXN:Microdroplet-enabledhighlyparallel
co-cultivationofmicrobialcommunities.PLoSOne2011,6:e17019.
intheinterpretationofdata.
21. AnisYH,HollMR,MeldrumDR:Automatedvision-basedselectionand
placementofsinglecellsinmicrowellarrayformats.IEEETransAutomSci
Acknowledgements Eng2010,7(3):598–606.
WethankthecrewoftheR/VThompsonTN221ResearchCruisewithchief
22. ChaoS-H,CarlsonR,MeldrumDR:Rapidfabricationofmicrochannels
scientistsDrJohnDelaneyandDrDeborahKelleyoftheUniversityof usingmicroscaleplasmaactivatedtemplating(μPLAT)generatedwater
Washington,andwethankDrCodyYoungbullatArizonaStateUniversity molds.LabChip2007,7:641–643.
(ASU)forthehelpwiththewatersampling,andthestaffintheASUDNA
23. LinL-I,ChaoS-H,MeldrumDR:Practical,microfabrication-freedevicefor
laboratoryforprovidingDNAsequencingservice.WealsothankASUforthe
single-cellisolation.PLoSOne2009,4:e6710.
supportofthisresearch.DNandJNappreciatethehigh-schoolsummer
24. GaoW,ShiX,WuJ,JinY,ZhangW,MeldrumDR:Phylogeneticandgene
internshipprogramoftheBiodesignInstituteatASU.
expressionanalysisofcyanobacteriaanddiatomsinthetwilightwaters
ofthetemperatenortheastpacificocean.MicrobEcol2011,62:765–775.
Received:30March2012Accepted:23May2012
25. GaborEM,deVriesEJ,JanssenDB:Efficientrecoveryofenvironmental
Published:9January2013
DNAforexpressioncloningbyindirectextractionmethods.FEMS
MicrobiolEcol2003,44:153–163.
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