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将绿色荧光蛋白标记的嗜水气单胞菌 (Ah4332 GFP)置模拟越冬水体 ( 8— 1 0℃ )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DVC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 (Viablebutnonculturable ,VBNC)状态。复苏试验表明 ,升高温度、添加鱼血清或新鲜培养的Ah4332 GFP细菌上清及通过兔肠管结扎 ,均使VBNC状态的Ah4332 GFP得到复苏。荧光显微镜和电镜观察处于可培养和VBNC状态的Ah4332 GFP,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。
Green fluorescent protein-labeled Aeromonas hydrophila (Ah4332 GFP) was used to simulate the survival of the overwintering water (8- 10 ℃), and the hunger survival test was carried out. The change of the number of bacteria in the water was examined by three counting methods. The plate count (PC) dropped to 0 after 41 days, while the total bacterial count (DC) was similar to the live viable count (DVC) except that the DVC count was 1-2 orders of magnitude lower than the total number of bacteria. This shows that the bacteria have become viable (Viablebutnonculturable, VBNC). Resuscitation experiments showed that Ah4332 GFP in VBNC status was resuscitated at elevated temperature, supplemented with fish serum or freshly cultured Ah4332 GFP bacterial supernatants and ligation by rabbit intestine. Fluorescence microscopy and electron microscopy of Ah4332 GFP in cultureable and VBNC state showed that the bacterial cells in the latter had a significantly smaller volume than the former, and the morphology changed from rod to sphere, but the cell membrane and cell wall were intact, not bacterial L type.