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Airpump
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Peristarpump Samplingport
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Outlet rl
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1:↑ SideView
Fig.3 (A)Liquidf10wchambersystem(CFCS)，(B)Flowchanel
conectedtothebotle. A checkvalvewasusedtostopbackf10wandprotectLegionela containingmediumspiloutofthesystembyasuddenbackf10wsurgethatmightocur. Theairpresureinsidethebotlewassodevisedtopushthemediumintotheotherbotle throughsilicontubeandthef10wadjustvalve.Then，theairpasedthrougha0.2μm membranefiltertoremoveLegionelaandwasexhaustedtotheatmosphere.Thesystem wasrinsedexcesivelywithsterilewaterandautoclavedbeforeuseandwasasembledin anincubator.Thef10wchanel，twobotles，andotheraparatuswereasembledcarefuly toavoidcontamination.
Flowchamberexperiments
A c.a.200μ1aliquotofL.pneumophilasuspensionswastransferedintoacentrifuge
tubecontaining50mloffreshBCYE-αmediumcontainingsuplementsof1% L-cysteine and1% fericpyrophosphate.Priertothebiofilmexperiments，BCYE-αcontainingwithL pneumophilawasinstaledinthef10wchanelandincubatedovernightat370C forthe
initialcoloniesontheglasslidestogrow.Then，300mLofsterilizedBCYE-αmedium containingsuplementsof1% L-cysteineand1% fericpyrophosphatewasdilutedwiththe distil led water to make 1200mL medium. The final concentration in the system was adjustedtoaproximately1x106bacteriamL-1.Themediumwasdividedequalybetween thetwobotles.TheexperimentswereperformedbyrecirculatingBCYE-αcontainingwith L.pneumophilainthef10wchanel，wherebiofilmsacumulatedontotheslides.Theliquid f10wchambersystemwasincubatedbetween370Cto420Cineachindependentexperiment. Theroutineoperatingconditionsincludedaf10wrateof10mLlmin.andeachexperiment
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