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VOL.101，206
23-25).Hyperchlorination(sodiumandcalciumhypochlo- rite，chlorinedioxide)，thermaleradication(superheatingand flushing)andcoper/silverionizationarewidelyusedinhos- pitalorpublicwatersystems.However，仕lesemeasuresare notalwayseficientbecauseofbiofilmsandthejrheteroge- neousfeatures.Nopreviousstudies(1.1， 26，27ronmo叩ho- logicalchangesofL.pneumophilabycultivationtempera- tureclarified出equantitativerelationshipbetweencultiva- tiontempera旬 reandlengthofthestrain.Thispapershows thelengthoftbrestrainsofL.pneumophila(atypestrain ATCC33152，twoclinicalisolates;Okinawa02-01and Nagasaki80~045) attemperaturerangingfrom30.0oCto 47.0oCatintervalsofO.50Cdeterminedbynewlydeveloped precisetempera印regradientincubator.Viabilityofbacteria atparticularterpera加rewasalsodetermined.Thosedata obtainedfromasetingwithprecisetempera印 reregulating modulewouldprovideinsightsforintroducingaguideline forregulatinghotwatertemperatureinaplumbingpipesof hotwatersuplysystems.
MATERIALSANDMETHODS
l>esignand.asemblyofthetemperaturegradientincub.ator ap par.atus (TGI) A bacterial incubator with an ac curate tem- peraturegradientcontrolwasfabricatedfo1'thisstudy(Figs.1and 2).Tenaluminumblocks(80x80x25mm)withcylindricalcham- bers(17mmdiameterand85mmdepth)holdingaglastesttube forincubatingL.pneumophilawereplacedsidebysideinarowto give10diferentculturetemperatures.Heatsourceswereaplied tothea1uminumblocksbytwoconstanttemperaturebathcircu- lators(ThermoNESLABRTE-7;ThermoElectronCorporation， Newington，NH，USA)set前 副 首erenttemperaturesusingwater
jacketsp1acedatbothendsoftherowofaluminumb10cks.The waterjacketsweremadeofbras，andcontainedapectinatedbras shettoprovideahigherheattransferrate.Eachaluminumblock wasseparatedbyasi1iconshetthata10wedaconstantstepedtem- peraturegradienttobeatained.Themoduleofaluminumb10cks withwaterjacketswaswrapedwithfiberglasfabrictominimize heat108.Temperaturesoutsideandinsideatest加 bewal1were measuredbystandardthenocouples(Chromel-Alume1sheathtype， 1mmdiameter，THERl¥征CTMB-KSI0/316レ10;YamatoIndustry， To旬。)andastandardthermometer(no.20907;JapanMeasuring Ins位um~nt IndustrialIncorporatedCompany，Tokyo)，respectively， andcompared，Temperaturesmeasuredby吐lestandardthenocou戸 plesweremonitoredbyadigitalmultimeter(Keithleymode1201 TCSCAN，mode1201;KeithleyInstruments，Cleveland，OH，USA) 飢 drecordedinapersonalcomputer.
TEMPERATUREONGROWTHOFLEGIONELABIOFILM 479
Bacterialstrainsandinoculation ThrestrainsofL.pneu- tenidenticalglastubescontainingbrothsampleswereinstaledin
mophikzserogroup1wereemployed;atypestrainandtwoclinical
isolates.AtypestrainofL.pneumophilaserogroup13152was
obtainedfromtheAmedcanTypeofCultureColection(ATC).
Twoclinica1isolatesofL.pneumophilaserogroup1，Okinawa
02-01(iso1atedin202，malepatient)andNagasaki80-045were
isolated企ompatientwithLegionaires'Disease(iso1atedin1980， phtlabiofilmadheredtotheglastesttubewalandtotheslid.e.
malepatient).Thethrestrainshadbenpreviouslyfrozenat-86PC invialscontaining10%skimmilk(BectonDickinsonandCompany， FranklinLakes，NJ，USA).Thevialswerethawedatromtem戸 pera旬 re，and the contents were streaked onto buf fered charcoa1- ye出 texむactagar(KantoKagaku，Tokyo)suplementedwith0.1% α-ketoglutarate(Sigma圃 Aldrich，S1.Louis，MO，USA)(BCYE-α). Theplateswerein
glasplate.Theslidegl出 splatewasreadilyremovedfromthetest 加 bewithoutlo08eningthebiofilm，Thee町ectofcultivationtemw pera旬 reonbacterial1engthwasverifiedonBCYE-αagar.Th~ vials werethawedatro omtempera加 re，andthecontentswerestreaked ontoBCYE-αagar.Theinoculatedagarwascutintosmalcubes (lOxlOx5mm)，andtheag訂 cubes.werethenplacedintoaglas tubeandincubatedfor48hataspecifiedtemperature.Theagar cubeonwhichL.pn.eumophilacolonieswereformedwaswithp d1'awnfromtheglastesttubeforanalysis.Thelengthofthre
FIG. 1. Photographoftemperaturegradientincubator(TGI).
1.Constant
t~mperature
/ 4 .Per:;onalcomput
2.PegisImu)!tmetfilr
!主選
1.Cpn:;tanttemperature bathclrculatorforco.plng
bathcircula!orforheating
口
FIG.2. Schematicdiagramoftheexperimentalaparatus.1，Con~ stanttemperaturebathcirculator(ThermoelectroncorporationTheno NESLABRTE-7);2，digitalmultimeter(KeithJeymodel201TC8CAN， model20MuItimeter);3，thermocouples(Clomel-Amumel1m.m sheath);4，personalcomputer(F吋itsuFMV-D5);5，aluminumbloc.ks (each80mmsquareand25mmthick，10pieces);6，brasblocks(each 80mmcubes，twopieces);7，sliconshets(each80mmsquareano 0.5mmthick，1pieces);8，glastesttubes(14mminternaldiameter and105mmlong，10pieces).
Theculturewasthendilutedwithsteriledisti1ledwatertoanoptト caldensityat60nm(OD60;Bio-RadLaboratories，'Hercules，CA， USA)ofaproximately0.6.Tostarttheexperiments，a50μ1ali" quotofsuspensionswastransferedintoatestglastubecontain~ ing4.5mlof仕eshBCYE-αbroth，Thefinalconcentrationiothe testtubereachedaproximately5x108cfuml-I• Tomeasureth.e lengthofL.pneumophilabiofilmgrownontheglaswalinbroth.;
thetemperaturegradientincubatoroperatingat10diferenttem~ pera加 ressimu1taneously.Ster1ieslideglasplates，13mmwideand 76mmlong，wereplacedineachtesttube.Foranalysisby8çan~ ningelectronmicroscopy(Hitachi，Tokyo)，asterilecoverglas (10mmsquare)wasusedinsteadofaslideglas，TheL.pneumor
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