METHODOLOGY Open Access Microbe observation and ......Figure 1 Scheme of microbe observation and...

Gao et al. Microbiome 2013, 1:4http://www.microbiomejournal.com/content/1/1/4

METHODOLOGY Open Access

Microbe observation and cultivation array (MOCA)for cultivating and analyzing environmentalmicrobiotaWeimin Gao, Dena Navarroli, Jared Naimark, Weiwen Zhang, Shih-hui Chao* and Deirdre R Meldrum

Abstract

Background: The use of culture-independent nucleic acid techniques, such as ribosomal RNA gene cloning libraryanalysis, has unveiled the tremendous microbial diversity that exists in natural environments. In sharp contrast tothis great achievement is the current difficulty in cultivating the majority of bacterial species or phylotypes revealedby molecular approaches. Although recent new technologies such as metagenomics and metatranscriptomics canprovide more functionality information about the microbial communities, it is still important to develop thecapacity to isolate and cultivate individual microbial species or strains in order to gain a better understanding ofmicrobial physiology and to apply isolates for various biotechnological applications.

Results: We have developed a new system to cultivate bacteria in an array of droplets. The key component of thesystem is the microbe observation and cultivation array (MOCA), which consists of a Petri dish that contains anarray of droplets as cultivation chambers. MOCA exploits the dominance of surface tension in small amounts ofliquid to spontaneously trap cells in well-defined droplets on hydrophilic patterns. During cultivation, the growth ofthe bacterial cells across the droplet array can be monitored using an automated microscope, which can produce areal-time record of the growth. When bacterial cells grow to a visible microcolony level in the system, they can betransferred using a micropipette for further cultivation or analysis.

Conclusions: MOCA is a flexible system that is easy to set up, and provides the sensitivity to monitor growth ofsingle bacterial cells. It is a cost-efficient technical platform for bioassay screening and for cultivation and isolationof bacteria from natural environments.

Keywords: Microbe observation and cultivation array (MOCA), Cultivation, Growth, Microbiota

BackgroundDuring the past over two decades, the use of culture-independent nucleic acid techniques, represented byribosomal RNA gene cloning library analysis, hasunveiled the tremendous microbial diversity that existsin natural environments [1]. In sharp contrast to thisgreat achievement is the current inability to cultivate themajority of bacterial species or phylotypes revealed bymolecular approaches. One of the major difficulties formicrobial ecology is that conventional cultivation meth-ods provide access to only a very small fraction of themicrobial diversity and more than 99% of naturally

* Correspondence: [email protected] State University, Tempe, AZ 85287, USA

© 2013 Gao et al.; licensee BioMed Central LtdCommons Attribution License (http://creativecreproduction in any medium, provided the or

occurring microbes are considered ‘unculturable’ onstandard culture media [2]. Although recent new tech-nologies such as metagenomics and metatranscriptomicscan provide more functionality information about micro-bial communities [2,3], it is still important to developthe capacity to isolate and cultivate individual microbialspecies or strains in order to gain better understandingof microbial physiology and to apply isolates for variousbiotechnological applications [4].A new view is emerging among microbial ecologists

that the majority of so-called ‘unculturable’ microbialspecies simply have not been cultured yet. In line withthis view, more research is necessary to enhance theability to culture microbes, in order to reduce the de-pendence on indirect and cumbersome metagenomicapproaches [5,6]. Recent developments in improving

. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited.

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Gao et al. Microbiome 2013, 1:4 of 8http://www.microbiomejournal.com/content/1/1/4

traditional cultivation techniques have shown that someconventionally unculturable species can in fact be grownas pure cultures. Tamaki et al. reported that the use ofgellan gum instead of agar as the solidifying agent couldgreatly improve the cultivability of novel microbes onsolid media [7,8]. Kaeberlein et al. designed a diffusionchamber to grow previously uncultivated pure isolates ofmarine origin [9], and the same strategy was also suc-cessfully used in cultivation of groundwater microorgan-isms [10]. Stevenson et al. achieved similar results withsoil microbes by fine-tuning the oxygen concentrationand nutrient levels [11]. Knowledge obtained throughmetatranscriptome analysis has also been used in direc-ted cultivation of bacteria [12]. These examples shownthat many microbial species can be cultured as long asthe environments are optimized for growth.Recently, some non-traditional cell-isolation technolo-

gies have been introduced to isolate targeted cells for pureculture cultivation. For example, Huber et al. used opticaltweezers to track and isolate an extremophilic archaeonfrom a microbial community in a terrestrial hydrothermalvent field [13]. This method of single-cell manipulationprovides a new way to grow pure microbial cultures fromsingle mother cells, but it has the disadvantage that theidentification and manipulation of the bacteria is anextremely labor-intensive process. By contrast, a high-throughput isolation method has been presented usingencapsulated single bacteria in droplets of gel [14], result-ing in the successfully cultivation of pure cultures frommarine microorganisms. Oligonucleotide probes wereused to identify the species after isolation.One of the remaining hurdles for bacterial cultivation

is that fine-tuning the growth condition for any specificspecies is a daunting effort, especially with the widelydiverse microbiota in the ocean and soil environments.Consequently, a high-throughput platform is urgentlyneeded to perform trials of different cultivation condi-tions in parallel. Previously, several high-throughputmicrotiter-plate-based cultivation platforms have beendeveloped for marine and aquatic water column bacteria,and these have contributed greatly to the successfulcultivation of previously uncultivated bacteria [15,16].Most recently, a chip-based version of a Petri dish anddiffusion chamber has been developed to addressthe same purpose [17,18]. Microfluidic ‘lab-on-a-chip’(LOC) devices have been used for co-cultivation of vari-ous bacterial strains and species [19,20]. These devicesare complicated in structure, utility, and fabrication, andare therefore less useful for general microbiologists.In this study, we developed a parallel cultivation set-up

that incorporates streamlined processes and is compatiblewith downstream genomic analysis. It spontaneously iso-lates environmental bacteria into miniature incubationchambers from a mixed microbial community. The key

component of the system is the microbe observation andcultivation array (MOCA), which uses a Petri dish thatcontains an array of droplets with an oil covering as culti-vation chambers. During cultivation, the growth of bac-teria across the droplet array can be monitored using anautomated microscope, which can produce a real-timegrowth record. Compared with conventional cultivationmethods, MOCA provides streamlined preparation, paral-lel cultivation, and real-time observation, and unlike otherchip-based platforms [16-19], MOCA does not requirecomplicated engineering techniques or equipment for fab-rication. We have found that bacterial growth across thedroplet array has a high level of uniformity when the ini-tial cell density is more than 10 cells/μl, and thus MOCAprovides a novel platform for bioassay screening. Whenthe cell occupancy in droplets is at the single-cell level,real-time image recording can be used to monitor thegrowth and morphological development of microcoloniesderived from single bacterial cells. The droplet culturedeveloped from a single bacterial cell can be transferredusing a micropipette [21] for bulk cultivation or furthermolecular analysis.

MethodsMicrobe observation and cultivation arrayThe MOCA Petri dish is produced using a techniqueknown as microscale plasma activated templating(μPLAT) [22,23]. The μPLAT method uses an inexpen-sive plasma-treatment process to create hydrophilic pat-terns on Petri dishes (60 mm diameter; Falcon; BectonDiskinson Labware, Franklin Lakes, NJ, USA); the airplasma makes the originally hydrophobic polystyrenesurfaces of the Petri dishes hydrophilic.Briefly, a μPLAT stencil is made from polydimethylsilox-

ane (PDMS) film, an inexpensive, soft polymer. The sten-cil has an array of 4 × 6 holes, each 3.18 mm in diameter,which is adhered to the surface of a Petri dish, (Figure 1a).The assembly is placed in a plasma cleaner (HarrickPlasma, Ithaca, NY, USA), and exposed to air plasma for1 minute (Figure 1b). The stencil is removed after plasmatreatment. Because only the polystyrene surface under thecircular openings is exposed to the plasma, these areas be-come hydrophilic on the hydrophobic background of theplate (Figure 1c). The patterned MOCA Petri dish is thensurface-sterilized under UV light for 15 minutes in a PCRcabinet (PCR Workstation; Thermo Fisher Scientific,Hudson, NH, USA). The bacteria and medium mixture isloaded using a pipette into the prepared Petri dish, produ-cing a 4 × 6 array of 1-μl droplets. The borders of theliquid are confined at the hydrophilic/hydrophobic inter-face, forming discrete droplets defined by the stencilpattern. To prevent evaporation and contamination fromthe ambient environment, 5 ml mineral oil (M5904; Sigma

Figure 1 Scheme of microbe observation and cultivation array (MOCA) droplet preparation. (a) A stencil with an array of holes is adheredto a Petri dish, which is then (b) exposed to air plasma to generate an array of hydrophilic disks. (c) The stencil is then removed, and the mediumcontaining the bacteria is added to the hydrophilic disks, after which (d) the droplet array is covered with mineral oil.


Chemical Co., St Louis, MO, USA) is loaded to cover thedroplet array (Figure 1d).

Loading of bacteria in medium and culturing usingmicrobe observation and cultivation arrayLuria broth (Difco; Becton, Dickinson and Company,Sparks, MD, USA) was used for both bulk cell anddroplet cultivation of the gram-negative bacteriumEscherichia coli DH5α and the gram-positive bacteriumBacillus subtilis 168. Marine broth (Difco‘ Becton,Dickinson and Company) was used for droplet cultiva-tion of marine bacteria from sea water sampled from thedeep sea of the northwest Pacific Ocean [24]. Cells werecounted using a counting chamber (Hausser ScientificPartnership, Horsham, PA, USA) under a microscope toestimate the bacterial density for both pure cultures andsampled sea water. Four cell concentrations (103, 102,10, and 1 cell(s)/μl) were used in the experiments. Celloccupancy in the droplets is a Poisson random variable[23]; when 1 cell/μl medium is loaded into the 1 μl dro-plets, the average cell occupancy is one cell per droplet.For each individual hydrophilic MOCA spots, 1 μl

bacteria-medium mixture was pipetted onto it. The dro-plets were then covered with 5 ml of mineral oil and thelid placed on the Petri dish. The dishes were kept in the

dark at room temperature, and growth was monitoredby direct microscopy.

Automated time-series observationA MOCA dish can be monitored on an automated op-tical microscope to observe the growth of the loadedbacteria. The MOCA Petri dish was placed on the auto-mated microscope (Eclipse TiE, Nikon Instrument Inc.,Japan) with a 4× objective, so the field of view coveredan entire droplet (Figure 2). The microscope stage wasprogrammed to travel through all the droplets and to ac-quire bright-field images of all droplets every 30 minutesfor 72 hours. The microscope illumination was turnedon only during image acquisition to avoid heating of thedish. After the experiments, the time-lapse micrographsof each droplet were used to estimate bacterial growthusing measurement of light transmission; the micro-graphs appeared darker as bacteria grew. Although anautomated microscope was used in this study, other op-tical platforms (for example, conventional microscopesor even the naked eye when the droplets are saturatedwith microbes) can also be used for estimation.

Phylogeny analysis of isolated bacteria from single cellsThe cultivated isolates developed from single bacterialcells of interest can be transferred using a micropipette

Figure 2 Demonstration of automated time-series observation by optical microscopy of bacterial droplet cultivation using microbeobservation and cultivation array (MOCA).


from the MOCA chip to other containers for archival anddownstream phylogenetic analyses using 16S rRNA se-quencing. In this study, bacteria were removed from thedroplets using a micropipette and transferred to a 1.5 mlmicrocentrifuge tube containing 100 μl sterilized water.After being spun in a centrifuge at 12000 g for 1 minute,the supernatant was decanted to remove possible carry-over of the mineral oil, then the bacterial cells were re-suspended in 5 μl sterilized water and transferred to a0.2 ml PCR tube. The PCR tubes were then put in a PCRthermal cycler (model 9700; Applied Biosystems Inc.,Foster City, CA, USA) and heated at 95°C for 5 minutes,followed by chilling on ice to induce cell lysis. Using heat-treated bacterial cells as the template, conventional PCRwas carried out to amplify the highly variable V6 region ofthe small subunit (SSU) rRNA gene. The PCR primersequences are shown in Table 1 [25]. The 20 μl PCRvolume contained: 10 μl 2 × PCR Master Mix (FermentasLife Science Inc., Glenburnie, MD, USA), 2 μl of each pri-mer (4 μmol/l), 2 μl of DNA template and 4 μl of double-distilled water. The conditions for PCR amplification wereas follows: 94°C for 5 minutes; 35 cycles of 94°C for 15 sec-onds, 56°C for 20 seconds, and 72°C for 45 seconds; witha final extension step of 72°C for 10 minutes and soak at4°C. The expected PCR products were recovered with agel and extracted (Qiaquick Gel DNA Extraction Kit; Qia-gen Inc., Valencia, CA, USA). The purified PCR productswere used as templates for DNA sequencing, using a com-mercial kit (BigDye Terminator Cycle Sequencing Kit, ver-sion 3.1; Applied Biosystems Inc.) and a PCR analyzer

Table 1 Primers used for PCR

Primer name Sequence (5′→3′)

U968 AACGCGAAGAACCTTAC-

L1401 CGGTGTGTACAAGACCC

(model 3700; Applied Biosystems Inc.) using the samePCR primers as before. The resultant DNA sequenceswere edited manually using Sequence Scanner Software(version 2.0; Applied Biosystems Inc.). Sequences werecompared against those in GenBank through the NationalCenter for Biotechnology Information (NCBI) portal andBLAST software (version 2.2.10) [26], and the phylogenyaffiliation of each isolates were then inferred.

Results and discussionBacterial growth in dropletsBecause MOCA droplet settings create a different physicalenvironment from that of bulk cultures, this may influ-ence many of the growth factors required by the bacteria[27,28]. Thus, given that the total volume for cultivation isa microliter, bacterial growth within the droplets may havedifferent properties from that in regular cultures. To testif the droplets could inhibit bacterial growth, we initiallycultivated E. coli and B. subtilis cells within droplets witha cover of mineral oil and assessed their growth in realtime. Using the method described above, a 4 × 6 array of1 μl droplets for each species were generated for the fourcell concentrations (103, 102, 10, 1 cell(s)/μl), using sixdroplets for each cell concentration. Cell occupancy in thedroplets is a Poisson random variable [23]: when loading1 cell/μl medium into droplets, around 37% of dropletsare empty, and 37%, 18%, 6%, and 1.5% of the droplets areoccupied by one, two, three, and four cells, respectively.Therefore, the lowest cell concentration in all presentedcultivation experiments yielded single-digit bacterial cellsin droplets.Figure 3 shows the growth of these two bacteria, where

each curve represents the growth in an individual droplet.We measured light transmission through the acquiredmicrographs as an indication of cell density; the lowerthe transmission, the larger the cell population. The

Figure 3 Real-time growth record of bacteria in microbe observation and cultivation array (MOCA). (a,c) Growth curves of (a) Escherichiacoli and (c) Bacillus subtilis with different initial cell concentrations (the lower the transmission, the larger the cell population). Each curverepresents the growth in an individual droplet. (b,d) Threshold growth time distribution versus (b) E. coli and (d) B. subtilis cell concentrations.The threshold growth time was defined as the time when the normalized transmission intensity of a droplet intersected 0.99 (Figure 3a and 3c,red dotted line).


transmitted light intensity was normalized with respect toits initial value to reduce the light variation between dro-plets., therefore, all growth curves started from 1. The col-ors of the curves in the figure indicate initial cellconcentrations. The curves of the individual cell concen-tration were clustered before reaching the stationaryphase, with a few outliers appearing for droplets withlow initial cell concentrations. We defined the thresholdgrowth time as the time when the normalized transmis-sion intensity of a droplet intersected 0.99 (Figure 3, reddotted lines). The box plots (Figure 3b,d) show the thresh-old growth time, which was mostly linear to the individualcell concentrations. Because of random cell occupancyand cellular heterogeneity, the variation was large whenbacterial number within the droplets was in single figures.The growth curves of both E. coli (Figure 3a) and

B. subtilis (Figure 3c) shared similar features. The curvesof individual cell concentration were clustered beforegrowth reached the stationary phase. One distinct featureof the B. subtilis growth is that the transmission intensitybecame brighter after the population peaked. The time-lapse micrographs showed that the number of cells did

not significantly change, but the size of cells became smal-ler, suggesting that the B. subtilis cells started to sporeafter the nutrition was depleted [29]. Only two of the mostdiluted droplets contained B. subtilis cells that were ableto grow into colonies in this experiment. Similar to theE. coli cultivation result, the threshold growth time wasmostly linear to individual cell concentrations (Figure 3d),whereas the lowest concentration had a large variationbecause only two droplets resulted in growth.These results show that, when the initial cell density is

more than 10 cells/μl, bacterial growth across the drop-let array is strongly uniform and reproducible. For drop-lets with single-cell occupancy, the platform can be usedto monitor the growth heterogeneity at the single-cellresolution.

Morphological development of microcolonies withindropletsWe found that the droplet setting permitted the growthof both E. coli and B. subtilis. In addition, we also foundthat the bacterial growth pattern within a droplet wasdifferent from that of a regular liquid medium. This was


especially evident when cultivating single-digit numbersof bacterial cells within a droplet. Using the time-seriesmicrographs of the 24 droplets with E. coli growthrecorded at 0, 8, 16, and 24 hours (Figure 4), we foundthat each single E. coli cell could develop its own micro-colony, and these did not merge with each other untilthe solution became cloudy. Similar results were seenfor B. subtilis (data not shown). In this respect, thegrowth pattern of both E. coli and B. subtilis within adroplet is more similar to that on a solid agar plate thanin a liquid medium. This may due to the use of the oildroplet cultivation platform, in which the physical envir-onment is different from that of routine liquid cultiva-tion. For instance, oil coverage may create a microoxicenvironment, which may inhibit or reduce the move-ment of bacteria [30].

Environmental bacteria cultivation and isolation using amicrobe observation and cultivation arrayWe further used MOCA to cultivate and isolate environ-mental marine microbes, which were sampled from theHydrate Ridge site (80 km off the Oregon coast and 5 mabove the 780-m deep seafloor) during a research cruisebetween 22 July and 5 August 2008 [24]. To avoid loadingmultiple cells from each droplet, the cell concentration

Figure 4 Proliferation of Escherichia coli in droplets. The time-series mithe droplets are E. coli colonies. For all four panels, the nominal cell numberespectively.

was diluted to 0.25 cells per droplet with marine brothmedium. Based on Poisson statistics, the possibility ofmulticell occupancy with this concentration is around 2%and that of single-cell occupancy is 20%.Using the automated time-series observation system,

we could identify which droplets had bacterial growthand how many microcolonies had been developed withinthese droplets. Droplets showing single microcolonygrowth were identified and the cultivated pure microbesrecovered directly. For verification of purity, the reco-vered cultures were subjected to further purification ontraditional Petri dishes.In one experiment, thirty-six droplets were generated,

and eight of these were successfully cultivated during a2-day culture period. Time-lapse micrographs showedthat they were all derived from a single bacterium (datanot shown). The profiles of the growth curves showedsimilar trends to those of the previous results of thesingle-digit bacterial cell growth in a droplet (Figure 3),but the variation was larger, perhaps due to species dif-ferences or to the different time requirement for exitfrom dormancy status (Figure 5). Some fast-growingdroplets (for example, those in droplets 8 and 9) startedgrowing within the first 8 hours, whereas others (for ex-ample, droplet 21) started as late as the 30th hour. The

crographs of 24 droplets at 0, 8, 16, and 24 hours. The dark clusters inrs of the five rows are 104, 103, 102, 10, and 1 cell(s) per droplet,

Figure 5 Growth curve of marine bacterial isolates within the droplets. Each curve represents the growth in an individual droplet. Thenumber in the legend is the droplet number associated with Table 2.


threshold growth times of these droplets are listed inTable 2.Single colonies were identified in these droplets and the

transferred cells were subjected to colony PCR. Aftersequencing the obtained PCR products of 400 bp, whichrepresent the highly variable V6 region of the SSU rRNAgene, a phylogeny analysis was conducted based on NCBIdatabase searches. Sequences were deposited in GenBankunder accession numbers JQ178347 to JQ178354. Theclosest species/genus with which each isolated strain wasidentified are listed in Table 2. Of these identified cul-tures, the majority (six out of eight cultures) belonged toPseudoalteromonas spp. (E value = 0), with the remainingtwo identified as Shewanella sp. (E value = 0) andColwellia piezophila (E value = 0), respectively. Thephylogeny affiliation of recovered isolates is consistentwith the source of the microbial community [31-33].Because MOCA droplets create a different physical en-

vironment from that of bulk cultures, , this may influ-ence many of the growth factors required by bacteria.For example, the use of an oil cover reduces gas ex-change with the ambient environment, which may favor

Table 2 Cultivated bacteria, their phylogeny affiliation and co

Droplet number GenBank accession number Closest match in

1 JQ178347 Pseudoalteromon






21 JQ178353 Shewanella halifa

33 JQ178354 Colwellia piezoph

the growth of microaerobic bacteria, but not strict aer-obic bacteria. In addition, putting MOCA into an anaer-obic chamber and loading with a bacteria-media mixtureunder anaerobic conditions can allow an anaerobic en-vironment to be created. We will explore the potentialof this technique and its limitations in cultivating novelenvironmental bacteria in future studies.

ConclusionsWe have developed a new system to cultivate bacteria ina droplet array. Our results show that the set-up ofdroplet does not inhibit the growth of bacteria, as wefound that both E. coli and B. subtilis cells could prolif-erate uniformly across the MOCA. We also found thatthe bacterial growth pattern within a droplet is moresimilar to that on a solid than in a liquid medium. Givenits flexibility, ease of set-up, and sensitivity to growth ofa single bacterial cell, we believe that this droplet plat-form could provide a cost-efficient way to cultivate andisolate novel bacteria from different environments, andhad the potential for use in bioassay screening.

rresponding threshold growth times

GenBank (percentage identity) Threshold growth time, hours

as elyakovii (99%) 10.51

as tetraodonis (99%) 11.02





xensis (98%) 31.13

ila (98%) 27.94


AbbreviationsMOCA: Microbe observation and cultivation array; DNA: Deoxyribonucleicacid; rRNA: Ribosomal ribonucleic acid; SSU rRNA: Small subunit ribosomalRNA; PCR: Polymerase chain reaction; μPLAT: Microscale plasma activatedtemplating; PDMS: Polydimethylsiloxane.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsWG conceived of the study, participated in its design, carried out thephylogenetic studies, and was involved in the interpretation of all data. DNand JN carried out the preparation, cultivation processes, and phylogeneticstudies. WZ conceived of the study, participated in its design, and wasinvolved in the interpretation of all data. SC conceived of the study,participated in its design, and analyzed the results. DM conceived of thestudy. All authors read and approved the final manuscript and were involvedin the interpretation of data.

AcknowledgementsWe thank the crew of the R/V Thompson TN221 Research Cruise with chiefscientists Dr John Delaney and Dr Deborah Kelley of the University ofWashington, and we thank Dr Cody Youngbull at Arizona State University(ASU) for the help with the water sampling, and the staff in the ASU DNAlaboratory for providing DNA sequencing service. We also thank ASU for thesupport of this research. DN and JN appreciate the high-school summerinternship program of the Biodesign Institute at ASU.

Received: 30 March 2012 Accepted: 23 May 2012Published: 9 January 2013

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doi:10.1186/2049-2618-1-4Cite this article as: Gao et al.: Microbe observation and cultivation array(MOCA) for cultivating and analyzing environmental microbiota.Microbiome 2013 1:4.

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