阿萨希毛孢子菌生物膜形成研究

Study of biofilm formation by Trichosporon asahii

目的 观察阿萨希毛孢子菌（T.asahii）生物膜形态特征、活性分布.方法 用倒置显微镜和扫描电镜观察T.asahii生物膜的形态特征,2,3-二（2-甲氧基-4-硝基-5-磺苯基）-5[（苯胺基）羧基]-2氢-氢氧化四氮唑（XTT）法和活菌计数法测定生物膜活性作定量分析,双荧光染色结合激光共聚焦显微镜观察生物膜不同层面活菌分布、活性及生物膜厚度.结果 T.asahii在聚苯乙烯表面形成生物膜,该膜扫描电镜下呈复杂的三维立体结构,包括孢子、假菌丝和真菌丝.随时间延长,其活性不断升高,XTT法和活菌计数结果一致（r=0.94,P＜0.01）,激光共聚焦显微镜下显示T.asahii生物膜不同层面的活性曲线坡度缓和,无明显规律.生物膜的厚度为14.3～31 μm.结论 体外形成的T.asahii生物膜比其浮游状态下具有更加复杂的结构,生物膜不同层面菌活性无明显差异.

Objective To observe morphological characteristics and activity distribution of T. asahii biofilm. Methods The morphological characteristics of T. asahii biofilm were observed under an inverted microscope and scanning electron microscope, and activity was measured and quantitatively analyzed by 2,3-bis （2-methoxy-4-nitro-5-sulfophenyl）-5-[（phenylamino） carbonyl]-2H-tetrazo-lium hydroxide （XTT） assay and viable count, respectively. Spatial distribution of dead/vital cells, activity and thickness of biofilm at different layers were assessed under a confocal laser scanning microscope （CLSM） following double staining with FDA/PI. Results T. asahii formed a biofilm in vitro on the surface of polystyrene materials. Under a scanning microscope, the biofilm displayed a complex three-dimensional structure which composed of spores, pseudohy-pha and true hypha. As time prolonged, the activity and quantity of biofilm increased. The results of XTT assay were correlated with those of viable count （r = 0.94, P 〈 0.01）. The activity was of no obvious difference between different layers of the biofilm. The thickness of biofilm varied from 14.3 μm to 31 μm. Conclusions The structure of T. asahii biofilm in vitro is more complex than that of planktonic T. asahii. The activity is of no significant difference between different layers of T. asahii biofilm.