Page 232 - konishipaper

for rnation of rnicro-colonies on the surface (2hours to 3days)， rnaturation of at tached bacteria into a diferentiated into exopolysacharide-encased (3days to 9days) and detachrnentanddispersalofplanktoniccels企ornbiofilrns(9daysto12days)(14)(19)(20).The eachexperirnentwasperfornedoveraperiodof8daysafter1daypre-incubation.We avoidedtheexperirnentsafter9daysduetofewcelclustersrnightbeobservedforthe dispersionofatachedbacteria企ornslideglases.Theaveragef10wvelocityinthechanel is0.64crn/sec.Atthisf10wvelocity，theReynolds(Re)nurnberswere108.TheRenurnbers forlarninarf10wconditionwasdeterninedinordertocornparisonwithotherstudies(21). Duringtheexperirnent，thesarnplesweretakentodeterninethebacterialgrowthandfind outcontarnination.Theacesportwasopenedandrernovedtheportionofthernediurn. Carewastakenthattheacesportwasopenforarninirnumlengthoftime.Inthisway，the survival and replication of planktonic and biofilm-asociated L. pneumophila were investigatedinliquidf10wbiofilmsimulator.
Comparisonsweremadeamongbiofilmformationsintheliquidf1ow，thoseinstatic liquidandthoseonagarplate(18). Theincubationperiodis8daysinliquidf1ow，3daysin thestaticliquidand1dayontheagarplateinordertoadjusttheincubationperiodtothe cycleofbiofilrnmaturationandcolonygrowthunderdiferentconditions(18). Confocallaserscanningmicroscopy(CLSM)
Attheendoftheexperirnent，fourslideglasplateswereswiftlyremovedfromthe f10wchanelwithoutloseningthebiofilms.Aftertheywereremoved，eachslidewas quicklybutgentlyimmersedthretimesinsterilewatertoremoveunatachedorlosely atachedcels.ThebiofilmswerelabeledusingtheLIVE/DEADBacLightTM Bacterial Viabilitystainingkit(InvitrogenL-13152)acordingtothemanufacturer'sinstructions. Brie臼y，biofilmswerelabeledwith1.67μMSYT09(agrenf1uorescentdyethatcancros intact membranes) and 1 0 μ M propidium iodide (a red f1uorescent dye that can only penetrate into cel ls that have lost mernbrane integrity). The biofilms were monitored through a 40x oil-irnmersion lens with a PASCAL LSM5 confocal laser scaning microscope(Zeis，Germany)andexaminedtocharacterizethebiofilmsize，andstructure under varying incubation conditions. Z-stack images were colected v
3.Results
TocharacterizetheabilityofL.pneumophilatoformbiofilmonglassurfacesinliquid f1ow，the thre圃 dimensional structure of8-days biofilrns was analyzed using CLSM. Representative biofilrn structures incubated at 370 C are presented in Fig.4. By day 8， biofilms of L. pneumophila began to form， where they formed microcolonies with a diarneter of20 to 120μm (Fig.4A). The most interesting observation was that the microcoloniesshowedareasofholowing(Fig.4B).A thre-dimensionalreconstruction imageoftheholowingmicrocolony(Fig.4B)isshowninFig.4C.Inordertoclariか the propertiesofbiofilrns，theblack/whitepicture，whichwasperformedimageprocesingby PhotoshopCS4TMandMoticImagesVersion2.2sSoftware，isshowninFig.4D.
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