Page 221 - konishipaper

M;y;己主時討議邦弘\~!.Ji:.....，'.
VOL.101，206
10寸 aATCC3152
E E Ea R-6 | B C Y E α 司 ar o
TEMPERATUREONGROWTHOFLEGIONELLABIOFILM 481
。 10-，41
42
] b ~ð
畳BB BGYE4agar
』o 6 E呈E 42 。
Cultivationtemperature(oC)
FIG.4. Theefectofcultivationtemperatureontheviabiliザ of1.
pneumophila，ATCC33152(a)andOkinawa02-01(b).
山
0 ・0
FIG.5. Thegrowthof1.pneumophiZα(clinicalisolate;Okinawa 02・01)onaverticalglassurfaceobservedat0h(a)，after25h(b) andafter48h(c).
02ー01wasincubatedinBCYE-αbrothintheglastubeat 42.00C for72h.Figure5showsL.pneumophilabiofilm growthontheglaswal.lThefieldofviewofthephoto- graphs.coverstheglas加 bewalattheair-liquidinterface. Threpicturesshow0h(Fig.5a)，24h(Fig.5b)，48h(Fig. 5c)afterinoculation.Theleveloftheair-liquidinterfacein thebrothfel1duetoevaporationofthemedium.Wespecu- latedthatthesuspendedcelswouldaglomerateonthesur- faceofthebroth(Fig.5a).Thickbibfilmdevelopedonthe glaswalabovetheair-liquidinterfaceandgranularbio四 filmadheredtotheglaswal1belowtheair-liquidinterface at24h(Fig.5b).Af王er48hofincubation，thebiofilmabove theair-liquidinterfaceextendedupwardstoaheightof3 m mabovetheinterfa凶 (Fig.5c).Belowtheair-liquidinter- face，ascrenmeshpa抗emwasobservedwith0.2-0.5'm m pitch.Thebiofilmsloughedoftheglaswal1surfaceand sanktothebotlomofthetubeafter72hofincubation.
Figures6and7showthetypicalresultsofbiofilmformed ontheglaswalsurfaceat35.0oCand42.0oC，respectively. At35.00Cthebiofilmadherenttotheglaswal1surfacedid notgrowcloselytogetherorinlargeamounts.Thebiofilm wasgranulatedbutwasnoteasytodetach企omthewal;血
FIG.6. The1.pneumophilastrain(clinicalisolate;Okinawa02聞 01)wasculturedfor72hingIastube.(a)Typicalresultsofbiofilm formedontheglaswaIsurfaceat35.0oC.Scaningelectronmicro- graphswereobtainedfortheentireimage(b)，overtheair-liquidinter- face(c)，ne町 theair-liquidinterface(d)，undertheair-liquidinterface (e)，andmagnifiedimages(f，g).
otherwords，itwassticky.Wecouldnotobserveanydifer- encebetweenthethrestrainsat35.0oC.Threlocationsin thebiofilmweremagnified:abovetheair-liquidinterface (Fig.6c)，neartheair-liquidinterface(Fig.6d)，andbelow theair-liquidinterface(Fig.6e).Figures6fand6gshowa magnifiedimageofFig.6e(twodi庄erentlocations).The biofilmexhibitedatypicalshortrodmorphologythatdid notchangemuchwiththelocationinthebiofilm.Thecel was0.2-0.3μmindiameterand2-4μmlong.Abovetheair interface，theisolatedcelsrestedontheslideglaswalas showninFig.6c.Celsembeddedinashetofamorphous materialontheglassurfaceapearednearorbelowtheair- liquidinterfaceasshowninFig.6dand6e.Figure6fshows themagnifiedimageoftheshetofamorphousmateria.l Figure6gshowsthrestrandsconectingthecelstoone another.At42.0oCthebiofilmofL.pneumophiladeveloped aheterogeneousstructurewiththredistinctlayers.The lowerlayerwasgranulatedbiofilmaproximately0.2-0.5 m mindiameter，andlayaproximately2m mbelowtheair- liquidinterface.Themidlelayerwasathickbiofilmand 2-3m minheight.TheuperlayerconsistedofsurfacepeI・ l i c l e s 1 - 3 m m i n h e i g h .t A 1t h o u g h t h e t h i c k n e s s o f t h e t h r e e layerswasdiferentinalthrestrains，nosignificantdifer-