Page 220 - konishipaper

480 KONISHIETAL.
strainsgrownattemperaturesranging企om30.0oCto47.0oCwas mea.suredandevaluated.Fortheanalysisofcelmultiplicationat diferenttemperatures，threstrainswereculturedatatemperature ranging企om41.0oCto45.0oCfor72h.Twoseparateexperiments withtemperaturewindowsof41.0oCto43.0oCand43.0oCto 45.0oCwereperfoロnedwiththeTGIapara旬 s古oastoachievea serialstep-wiseculturetempera加reranging企om41.0oCto45.0oC atintervalsofO.2OC.EvenonecolonyofL.pneumophilaresulted inapositivefindingforcelmultip1ication.Theexperimentswere repeated2to3times.
Scaningele.ctronmicroscopy Forscaningelectronmi- croscopy，thebiofilmgrownonacoverglaswasfixedwith5% glutaraldehydeandstainedwith1%osmiumtetroxideandwashed in0.1mMphosphatebufer拭 pH6.9for2handthendehydrated throughanalcoholseries.Samplesweremountedon20mmscan- ningelectronmicroscopyspecimenstubswithhigh-conductivity silverpaint.Specimenswerecoatedwitha20nmlayerofgoldina vacumcoatingunitand10wereexaminedinaHitachiscaning electronmicroscopyoperatedat10kVaceleratingvoltage.
1.BIOSCI.BIOENG).
Bacterialstainingandmeasurementofbacterialength Biofilmsgrownonglasslidesinbrothandcoloniesonagarcubes wereharvestedwithaplatinumlopandplacedinto2mlvolumes ofsteriledistiledwaterinavial.ThevialwasmixedinaVortex mixer.Weverifiedthatthelengthofbacteriawasnota百ectedby mixing(datanotshown).Thesamplewasstainedconventionaly onaslideglasbytheGimenez(MutoPureChemicals，To匂0) methodandobservedwithamicroscope(NikonTE20;Nikon， Tokyo)orwithaCCDcamera(SonyDXC-390，Sony，Tokyo)and recordedinapersonalcomputer.AfterGimenezstaining，photo- graphsincludingbacterialimagesweretakenandrecordedina personalcomputerandprocesedwithimageprocesingso食ware (AdobePhotoshop).Aproximately20wel-isolatedbacterial imageswererandomlychosenfromphotosandthelengthofbac- teriawasmeasuredwithScionImagesoftware(Beta4.02forWin- dowsXP;ScionCorporationFrederick，Matγland，MD，USA)by tracingtheimageonthecomputerdisplaywithamouse/pende- Vlce.
RESULTS
StatusofcuIturetemperaturesgeneratedbythetem-
peraturegradientincubator(TGI)aparatus A step-
mumandminimumtempera印 reswererecordedintheper
目
sonalcomputer. InfluenceofculturetemperatureonthelengthofL.
pneumophila T1restrainswereculturedinbrothand onagarat10diferenttempera旬 resfor48hintheTGIap- para旬 s.Asnosignificantdiferencebetwentwoclinical isolateswasobserved，theresultswereonlyshciwnfora 句'pestrainATCC3152andOkinawa02-01.Theresults forATCCtypestrain33152andaclinicalisolate;Okinawa 02-01strainincubatedonagarandonbrothareshownin Fig.3.TheaveragelengthofthestrainATCC33152ex- ceded50μmwhenculturedattemperaturesrangingfrom
50
pedtempera旬 regradientwith10diferenttempera印 resin 0
o 38.50CforOkinawa02心01andat43.0CforATCC3152.
therangeof30.0oC(testtubeno.1)to47.0oCin2.0C(test
tubeno.10)stepswasachievedwhentheconstanttempera-
旬 re bath circulators were set at 30.0oC and 50.0oC in the
TGIapara印 s.Eachtesttubereachedastabletempera-
印 rewithin2handwasabletomaintainthistempera旬 re
t1roughouttheexperimentwithatemperaturefluctuationof turesrangingfrom43.10Cto44.10C.AlnineATCC33152 土0.10C.Thetempera同 reineachtesttubeandthemaxi-
o 39.00Cto4.0C.Thebacteriahadthelongestaverage
tures higher 由 an 4 4.10 C (Fig. 4a). Dead cel ls were deter- minedbyplatemethodonwhichthestraindidnotforma colonyat370Canylonger.A1136Okinawa02-01cultures testedwereabletomultiplywhenculturedonBCYE-αagar attempera加 resrangmg企om41.0oC to43.1oC.Fi丘e enout of22(68.2%)culturesmultipliedwhenculturedatthetem- pera加 reranging合om43.1oCto4.1oC.Eightoutofnine (8.9%)Okinawa02-01culturesdidnotmultiplywhen culturedatthetemperaturehigherthan4.1OC(Fig.4b). TheresultsofNagasakihadnomeaningfuldiferenceto Okinawa.
GrowthofL.pneumophilabiofilmonglaswalsatdif- ferenttemperatures TheclinicalisolatestrainOkinawa
ω
吉コー
40
Legiolelapnumophila
ATC3152 E30
ωLn詰f 20 0 o
。
-一一一一一
一 - "，山!...，!，~~~ι立法必立与ムー~
a
b
FIG. 3. Variationoftheaver.agedlengthofL.pneumophilawith cu1tivationtemperatureforATCC3152(a)andOkinawa02-01(b).
iーE~ 10 O
250 ω220
30
Okinawa02-01
t旧ーJ3'
150
工-‘
o
」iちωE~ 10 0
怜
Legiolelapnumophila
30 35 40 45 CultivationtemperatureO(C)
betwenthebacteriallengthformedonagarorinbroth mediawerealsoshown.Theresultsarepresentedasmean:tSD(n>10).
lerigthofaproximately115μmwhencu1turedat42.50C.
TheaveragelengthoftheclinicalisolateOkinawa02-01
The
di宜erenc~e
exceded50μmwhencultured瓜 tempera旬 resranging企om
41.00Cto43.0oC，withthegreate~t averagelengthofap- 0
proximately70μmat42.5C.Figure3showsthediferent bacteriallengthsformedonagarorinbrothmediaofthe Okinawa02-01c1inicalisolateandtheATCC33152strain. TheaveragelengthofthestraInsATCC33152andOkinawa 02-01cu1turedintesttubeswaslongerthanwhencultured onagarattemperaturesrangingfrom35.0oCto43.0oC.A significantdiferenceofbacteriallengthwasrecognizedat
Influenceofculturetemperatureoncelmultiplication ofL.pneumophila Al136ATCC3152culturestested wereabletomultiplywhenculturedonBCYE-αagarat
0 temperaturesrangingfrom41.0oCto43.1C.Thirtenout
of22(59.1%)culturesmu1tipliedwhenculturedattempera- cultureswerenotabletomultiplywhenculturedattempera-