Angle J S, Levin M A, Gagliardi J V, McIntosh M S, Glew J G
Department of Agronomy, University of Maryland, College Park, MD 20742.
Microb Releases. 1994 Jul;2(4):247-54.
Efficient methods for the recovery of genetically engineered organisms (GEM) added to soil are critical if the safety of potential releases is to be evaluated and the actual release is to be monitored. Pseudomonas aureofaciens strain 3732 RN-L11 (lacZY) was added to 10 g sieved soil microcosms and incubated for 5 and 28 days. Various diluents, shaking methods, and settling of soil were examined to determine the optimum method for recovery of the GEM from the soil. Of the diluents examined, 0.1% agar gave significantly lower numbers than distilled water, 1.0% sodium metaphosphate, 1% peptone, and phosphate-buffered water. After 5 days of incubation, shaking for 10 min with glass beads and shaking for 30 min without glass beads resulted in the highest recovery of the GEM from soil, while sonification resulted in the lowest recovery. After 28 days of incubation, sonification produced significantly lower numbers than any of the other treatments. The addition of 1% CaCl2 to enhance settling significantly increased recovery efficiency. Although the use of CaCl2 in distilled water and shaking for 10 min was an effective method for recovering P. aureofaciens from a Maryland soil, when the same extraction procedure was compared with a standard technique (dd H2O, shaking for 10 min) for eight divergent soils, neither extraction method was consistently better than the other. Statistical analysis of the data showed the need for log transformation of the raw data. Four microcosm and two plate replicates for each dilution provided the greatest ability to detect differences between treatment means while maximizing experimental efficiency.
如果要评估潜在释放的安全性并监测实际释放情况，那么从土壤中回收添加的基因工程生物（GEM）的有效方法至关重要。将金黄色假单胞菌菌株3732 RN-L11（lacZY）添加到10克经筛分的土壤微观世界中，并孵育5天和28天。研究了各种稀释剂、振荡方法和土壤沉降情况，以确定从土壤中回收GEM的最佳方法。在所研究的稀释剂中，0.1%琼脂培养出的数量明显低于蒸馏水、1.0%偏磷酸钠、1%蛋白胨和磷酸盐缓冲水培养出的数量。孵育5天后，用玻璃珠振荡10分钟和不使用玻璃珠振荡30分钟可使从土壤中回收的GEM数量最多，而超声处理回收的数量最少。孵育28天后，超声处理得到的数量明显低于其他任何处理方法。添加1%氯化钙以增强沉降显著提高了回收效率。虽然在蒸馏水中使用氯化钙并振荡10分钟是从马里兰州土壤中回收金黄色假单胞菌的有效方法，但当将相同的提取程序与八种不同土壤的标准技术（双蒸水，振荡10分钟）进行比较时，两种提取方法都并非始终优于另一种。对数据的统计分析表明需要对原始数据进行对数转换。每个稀释度设置四个微观世界重复和两个平板重复，在最大限度提高实验效率的同时，具有最大的检测处理均值差异的能力。
