1991 Volume 37 Issue 1 Pages 85-92
A sensitive method using selective plating media to detect a genetically engineered microorganism (GEM) released into the environment was developed. In experiments, a strain of Pseudomonas sp. capable of assimilating monofluoroacetate (FA) as a sole carbon source was chosen as the GEM model. Two sorts of Japanese paddy soil were used as a microcosm. In a trial when serial 10-fold soil dilutions were spread on minimal FA agar plates and incubated, native oligotrophic microorganisms (105-106CFU/g of dry weight of soil) grew on the plates. So, it was difficult to pick out a few populations of target bacteria from such a high number of background microbial communities. A further examination showed that those indigenous microorganisms colonized even on plates containing only water and agar. For this reason, we tested silica-gel instead of agar as an agent to solidify the selective media. On minimal FA silica-gel plates, the number of indigenous oligotrophic colonies was greatly decreased. When a definite number of target Pseudomonas cells was mixed with the soil dilutions and cultured on minimal FA silica-gel plates, only the target colonies were appeared. We applied the most-probable-number technique to the plate-counting method to attain as low as 4CFU/g of dry weight of soil as the statistical limit of detecting FA-assimilating bacteria. This selective plating method is suitable for environmental monitoring of GEMs which assimilate FA aerobically, as it is on highly sensitive, specific and feasible.
