The Optimal Study On Methods Of Detecting Environment Microorganism By Use Of Fluorescence In Situ Hybridization Assay

This study is the use of fluorescence in situ hybridization (fluorescent in situ hybridization, FISH) to detective the microorganisms in activated sludge and the microorganisms which isolated and culture from activated sludge by monochrome, synchronous two-color and duplicate two-color ways, through the tests to find out the best two-color FISH hybridization. After compare several hybrid major factors which can affect the test results, come to the following conclusions:1 The best sludge concentration fit for fluorescence in situ hybridization is 250mg/L- 350mg / L, For the hybridization conditions, the best temperature suitable for the PAO651 probe for hybridization is 46℃, the concentration of formamide for hybridization is 40%, hybridization time is 2.5 hours.2 When we use two kinds of probes, the result of duplicate multicolor is better than synchronous multicolor, that is the result of hybridization which used separately probe is better than the result of hybridization which use probe at the same time .3 In using duplicate two-color FISH, when the hybridization temperature and formamide concentration in the hybridization solution of the two probes are similar but the hybridization time is quite different, it can take a hybridization with one probe without washing then re-hybridization with another probe; and when the hybridization conditions of the two probes are very different, take a washing after the completion of a hybridization with one probe then re-hybridization with another probe.