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Fig.4BiofilmformationbyL.pnewηophilaunderliquidflowwithBCYE司 α mediumat370C.(A)LSMSapearanceofarepresentativebiofilmsection formed in a glas slide of 8・day-old L. pneumophila biofilm; (B) high magnification LSMS micrograph of (A);.(C) a thre-dimensional reconstructionof(B);(D)identicalblack/whitepictureof(B)
Table 1showsthenumberofanalyzedcolonies，equivalentouterdiameter(Dod)， at tached circular diameter (Dat)，emptines s (Em p) ofthe at tached cel l clusters on each ofthe four glas s slides in continues-flow chamber incubated at 370 C. The equivalent outer diameterofanon-circularcolonyisequaltoadiameterofacircularcolonythatexhibits identicalarea.Theatachedcirculardiameterisequaltoadiameterofacircularcolonythat atachedareaofbacteriaexhibitsidentica.lTheemptines(holownes)ofcolonyisdefined astheratioofbiofilmnon-atachedareastoatachedareas，
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mp n 2
ム /od TheglasslidesareidentifiedasP1toP4inthedirectionofupstreamtodown-stream
inthecontinues-flowchamber.InFig.5，thebarsindicatetheaveragesfortheanalyzed colonies，andtheeITorbarsindicatestandarddeviations.OnP1neartheinletoftheflow chamber，nobiofilmcolonieswereobserved.L.pneumophilaformedmicrocolonieswithan equivalentouterdiameterof87土 32μmonP2，138士 81μmonP3and35士 14μmonP4. Theatachedcirculardiameterwas62土 23μmonP2，81土 25μmonP3and27土 9μmon P4.Theemptinesofcolonywas43士4%onP2，43土 18%onP3and35土 16%onP4.In statisticalsignificancetesting，thePvalueistheprobabilityofobtainingateststatisticat leastasextremeastheonethatwasactualyobserved，withavaluerangingfI"omzeroto one. The smal ler the P value is，the more significant the test result is statistical ly. The statisticalysignificantdiferenceswereobservedbetweenP2andP4(P<0.01)，betweenP3 andP4(P<0.01)inanequivalentouterdiameterandatachedcirculardiameter.However， therewasnosignificantdiferenceswereobservedforemptines(P>0.1).
Figure6showsCLMStwo-dimensionalapearanceofarepresentativebiofilmsection formed in a glas s slide of ιday-old L. pneumophila biofilm under the liquid flow in
000
αmedium(A)at37C，(B)40C and(C)42C.Table2showsthepropertiesofL.
開
pneumophilabiofilmmicrocoloniesat37，40，and420Conglasslidessurfacesinoculated inliquidflowcondition.Datafromthreindependentexperimentswereaveragedoverfour
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