The Optimal Study On Experimental Conditions Of Fluorescence In Situ Hybridization Assay For Detecting Microorganism In Reactor

Fluorescent in situ hybridization (FISH) is used to investigate the target species such as phosphate accumulating bacteria, nitrifying bacteria in the reactor.The experimental steps and better conditions of detection are set up, the groping experiment of anammox bacteria is done by FISH.By comparing some important factors that affect the hybridization tests, following results are shown:⒈Optimized pretreatment and hybridization conditions of detecting phosphate accumulating bacteria are as follows.⑴The sample must be washed twice by 1×PBS.⑵FISH conditions for detecting phosphate accumulating bacteria in the reactor are: temperature is 46℃, hybridization time is 2h, sodium chloride concentration in the eluate is 70mmol/L.⒉Optimized pretreatment and hybridization conditions of detecting nitrifying bacteria on slide of the attached biofilm are as follows.⑴No sample washing before fixing.⑵FISH conditions for detecting nitrifying bacteria on slide of the attached biofilm are as follows.①Nitrobacter: temperature is 46℃, hybridization time is 5h, sodium chloride concentration in the eluate is 55 mmol/L.②Nitrosomonas: temperature is 46℃, hybridization time is 3h, sodium chloride concentration in the eluate is 30～40 mmol/L.⒊Initial optimal conditions of anammox are as follows.⑴After sliced, it must be dehydrated by ethanol of 50%,80%,100% in order to get a better result.⑵The ratio of glutin and potassium sulfate dodecahydrate in liquid adhesion is:10:1.⑶The ratio of liquid paraffin and sludge when embedding is:1:10.