Detection and estimation of toluene degrading bacteria in samples by using molecular biology techniques

• Main Authors: Li-Chia Huang, 黃禮佳
• Other Authors: Wei-Liang Chao
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/75721929008710139789

碩士 === 東吳大學 === 微生物學系 === 87 === Tradtionally,it is very diffucult to rapidly and accurately estimate the numbers of specific bacteria in the environmental samples. Several methods have been proposed by various researchers, but they all have their limitations such as, can nto detect viable but nonculturable cells.With the develpoment in molecular biology,DNA probes have been used extensively in detecting the presence of specific bacteria in samples.In the present study, a fluorescent pseudomonad(isolate F5) which can utilize toluene as carbon and energy source was used as target organism. Attempts to isolate TOL plasmid-like extrachromosomal DNA were a failure.Therefore, the polymerase chain reaction approach was taken,base on its ability to degrade toluene, we have designed a pair specific primer,and the reaction, a 560-base DNA fragment in the target bacteria''s genome was amplified.Sequence analysis indicated that there is a 99.46% similarity between this 560-base fragment and todC1 gene of the tod operon. It is to be used as a functional probe in our future studies. When a biodegrader was introduced into the environment and failed to do its work,frequently it is through the activities of various biotic components. Therefore, the ability to analyze the changes in the composition of microbial community has become crucial. In this study, we employed Reverse Sample Genome Probing developed by Voordouw,using a mathematical formula to try to quantify the introduced bacteria. Using 100ng of lamda phage DNA as internal standard, we calculate the k(lamda)/kx value for four different common soil bacteria. When using the equation proposed by Voordouw,the k(lamda)/kx values for fluorescent Pseudomonas isolate F5, Acinetobacter calcoaceticus, Alcaligens faecalis, Enterobacter cloacae, and Rhizobium fredii are 0.82, 33.86, 1.16, 7.48, and 6.43 respectively. When using these values to calculate the actual amounts of individual DNA in the probe preparation, results indicated that if specific DNA concentration in the probe preparation was too high, a significant difference to the actual added amount of DNA were observed. Therefore, a modification was done in the way of calculation of k(lamda)/kx value. When using the modified method, the k(lamda)/kx values for fluorescent Pseudomaonas isolate F5, A. calcoaceticus, A. faecalis, E. cloacae, and R. fredii are 1.17,21.63,1.48,12.11, and 3.25 respectively. The validities validities of these number were examined by converting the DNA concentration, detected by RSGP method, to cell number and compared them with the actual added cell number. The result indicated that at high cell number ( For example, more than 74000 CFU for isolate F5 ), the estimation provided by Voordouw method tends to be lower than the actual number, while the modified method provide a better estimation.