绿色荧光蛋白标记的嗜水气单胞菌在越冬水体的饥饿存活及复苏
STARVATION-SURVIVAL AND RESUSCITATION OF GFP-MARKED Ah4332 IN THE OVERWINTERING WATER
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摘要: 将绿色荧光蛋白标记的嗜水气单胞菌(Ah4332GFP)置模拟越冬水体(8-10℃)内,进行饥饿存活试验,用三种计数方法检查水体中的细菌数量变化 . 在41d后平板计数法(PC)降至0,而细菌总数法(DC)和活菌直接计数法(DVC)结果相似,只是D VC计数结果低于细菌总数1-2个数量级.显示细菌已经变成活的非可培养(Viable but nonculturable,VBNC)状态 .复苏试验表明,升高温度、添加鱼血清或新鲜培养的Ah4332GFP细菌上清及通过兔肠管结扎,均使VBNC状态的Ah4332GFP得到复苏.荧光显微镜和电镜观察处于可培养和VBNC状态的Ah4332GFP,后者的细菌细胞比前者体积明显缩小,形态由杆状变成了球形,但细胞膜和细胞壁是完整的,不是细菌L型.Abstract: Starvation survival of GFP marked Ah4332 strain had been detected at 8～10℃ in the overwintering water in laboratory. The changes of bacterial amount were monitored by three counting ways.After 41 days the plate counts)PC)of Ah4332GFP dropped to zero. In all cases,the total number of Ah4332GFP remained the same level with direct viable count(DVC)or direct count(DC),but the DVC was about one or two log lower than the DC.It suggested that the Ah4332GFP had entered a dormant,viable but nonculturable(VBNC)state. The resuscitation of VBNC Ah4332GFP strain was tested in different conditions.Under increasing the temperature,culturable Ah4332GFP strain was recovered from VBNC state by two methods of either adding some substances e.g.fish serum,supernatant from late logarithmic phase Ah4332GFP cultures or injecting VBNC Ah4332GFP into the rabbit ligated ileal loop. By using electron microscopy and vertical fluorescent microscopy,the culturable and VBNC Ah4332GFP were observed.Comparing with the culturable cells of Ah4332GFP ,the VBNC bacteria processed a significant reduction in cell size and a morphological change from bacilli to cocci,meanwhile the cell membrane and cell wall were intact.It demonstrated that the VBNC state of Ah4332GFP was not the L-form bacterium.
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