Page 230 - konishipaper

Legionelaea1'eplacedsidebysideinarowtogive10dife1'entculturetempe1'atures.The inf1uenceoftempe1'atureonthemo1'phologicalchangesofLegionelapneumophilabiofilm wasinvestigatedinbrothandonaga1' attempe1'atu1'esbetwen30.0to47.0oC unde1' the staticcondition.Itwas1'evealedthattempe1'aturewasakeyfacto1'whichafectsthebiofilm fo1'mationonglassu1'faceunde1'thestaticcultu1'es.Howeve，'1 inpublicwate1'ingsystems，it isimpo1'tanttoconside1' liquidf10wwithinthesystem， fo1' liquidf10winf1uencesthe procesesofbacte1'ialatachmentanddetachmentanddevelopmentwithinthebiofilm.The velocityofliquidf10wwhichdi1'ectlyafectsthesurfaceplaysanimpo1'tantpa1'tinthe g1'owthofbiofilms.
Ino1'de1' todesignandope1'atewate1'ingsystemsthatdelive1' thebacte1'ia-frequality wate，'1 weshouldunde1'standhowbiofilmsdevelopunde1' liquidf10wconditions， what problemstheycancause，andhowtheycanbecont1'oled.Thisstudydemonstratesthe developmentofamonospeciesmodelbiofilmfo1' L.pneumophila， bacte1'iawhicha1'e knowntofo1'm biofilmsinthenaturalenvi1'onment，thoughthismodeldoesnot1'ef1ectthe complexityofthesesilelifestyleofL.pneumophilafoundinthenaturalenvi1'onmentWe desc1'ibethedevelopmentofthef10wtypebiofilmsimulato1' inorde1' toanalyzebiofilm fo1'mationunde1'liquidf10wconditions.Bacterialbiofilmformationsa1'einf1uencedmo1'eby tempe1'aturechangesthanf10wconditions.Wewouldliketo1'evealsomediferencesof bacterialbiofilmformationsinliquidf1ow，thoseinstaticliquidandthoseonaga1'plate.
2.MaterialsandMethods
Bacterialstrain，inoculation Thest1'ainsofL.pneumophilaserogroup1we1'eemployedforthisexperimen.tA type
st1'ain of L. pneumophila se1'ogroup 1 33152 was obtained f1'o m the A m e1'ican Type of Cultu1'eColection(ATCC).Thest1'ainhadbenp1'eviously-・白ozenat・860C invials containing10%skimmilk.Thevialswerethawedat1'oomtempe1'ature，andthecontents we1'est1'eakedontobuferedcha1'coal-yeastextractaga1' suplementedwith0.1%α
0
α).Theaga1' plateswereincubatedfo1' 72hat37C.G1'owthwas
ha1'vested ・白 o m plates with a platinum lo op and placed into 合 esh BCYE-a medium containingsuplementsof1% L-cysteineand1% felTicpyrophosphateandincubated shakinginf1askovemightat370C.T1'ansfe1' thecultu1'einto50mlcentr汀ugetubeand centrifugeat1501'pmfo1' 10minatRT.Supematantwas1'emovedandthepeletcelswe1'e resuspended in 10ml sterile distil led water. The culture was then diluted with sterile distiledwatertomeasureanopticaldensityat660nm(OD660). Liquidflowchambersystem
Ino1'de1' tomonito1' thebiofilmfo1'mationofL.pneumophilaunde1' dynamicf10w conditions，aliquidf10wchambersystemwassetupasshowninFig.3(A).The4removable clearglasslides(26mmwideand76mmlong)we1'emountedinthef10wchaneltogiveL.
pneumophilatheoportunitytodevelopbiofilmsontheslidesasshowninFig.3(B).The testsectionofbiofilmsamplingdevice(thef10wchanel)hasalengthof304mmanda r e c t a n g u l a r c r o s s s e c t i o n o f 2 6 m m w i d e a n d 1 0 m m h e i g h t. T h e c u l t u r e m e d i u m w a s ci1'culatedthroughsilicontubes(I/D4.8mm)betwentwobotles(NalgeNuncIntemational K.K.， 2126・ ・20 0 0) by means of a peristaltic pump (Masterf1ex Inc.， 7524-40， 7 7201-60). Twoglasf10wbreaks(BioSurfaceTechnologiesCorp.，FC50)wereusedonboththeinlet andtheoutletsidesofthebiofilmsamplingdevicetoinsureasteadyfedrate.Acylinder bub ble trap (BioSurface Technologies Corp.，FC33) with air release cock captu1'es air bublesreleasedfromthef10wingculturemedium. SinceLegionelarequi1'eoxygenfor thei1'survival，toensuretheaerobicconditionsduringtheexpe1'iment，airwaspumpedinto thesystemthroughtheairinletline.Smalairpumpswereusedtopumpairthroughsilicon tube to a 0.2μm polycarbonate membrane filter (Nip pon Genetics Co. Ltd.，GM4210)
欄 -ketogluta1'ate(BCYE
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