NSI Technology Services Corporation and Environmental Research Laboratory, U.S. Environmental Protection Agency, Corvallis, Oregon 97333.
Appl Environ Microbiol. 1991 Jan;57(1):51-6. doi: 10.1128/aem.57.1.51-56.1991.
Determining the fate and survival of genetically engineered microorganisms released into the environment requires the development and application of accurate and practical methods of detection and enumeration. Several experiments were performed to examine quantitative recovery methods that are commonly used or that have potential applications. In these experiments, Erwinia herbicola and Enterobacter cloacae were applied in greenhouses to Blue Lake bush beans (Phaseolus vulgaris) and Cayuse oats (Avena sativa). Sampling indicated that the variance in bacterial counts among leaves increased over time and that this increase caused an overestimation of the mean population size by bulk leaf samples relative to single leaf samples. An increase in the number of leaves in a bulk sample, above a minimum number, did not significantly reduce the variance between samples. Experiments evaluating recovery methods demonstrated that recovery of bacteria from leaves was significantly better with stomacher blending, than with blending, sonication, or washing and that the recovery efficiency was constant over a range of sample inoculum densities. Delayed processing of leaf samples, by storage in a freezer, did not significantly lower survival and recovery of microorganisms when storage was short term and leaves were not stored in buffer. The drop plate technique for enumeration of bacteria did not significantly differ from the spread plate method. Results of these sampling, recovery, and enumeration experiments indicate a need for increased development and standardization of methods used by researchers as there are significant differences among, and also important limitations to, some of the methods used.
确定释放到环境中的基因工程微生物的命运和存活需要开发和应用准确实用的检测和计数方法。进行了几项实验来检查常用或具有潜在应用的定量回收方法。在这些实验中，将草生欧文氏菌和阴沟肠杆菌应用于温室中的蓝湖菜豆（菜豆）和凯尤塞燕麦（燕麦）。采样表明，叶片中细菌计数的方差随时间增加，并且这种增加导致相对于单个叶片样本，批量叶片样本对平均种群大小的估计过高。批量样本中叶片数量的增加（超过最小数量）不会显著降低样品之间的方差。评估回收方法的实验表明，与混合、超声或洗涤相比，用 stomacher 混合从叶片中回收细菌的效果明显更好，并且在样品接种密度范围内回收效率是恒定的。将叶片样品储存在冰箱中以延迟处理，在短期储存和叶片未储存在缓冲液中时，不会显著降低微生物的存活和回收。用于细菌计数的滴板技术与平板法没有显著差异。这些采样、回收和计数实验的结果表明，需要增加研究人员使用的方法的开发和标准化，因为其中一些方法之间存在显著差异，并且一些方法也存在重要限制。
