Abstract: Objective To establish a closed continuous circulatory incubation system to prepare Escherichia coli(E coli) biofilm for investigating the effect of different incubation time on biofilm formation.Methods The bacteria within biofilm was assessed by counting adherent bacteria and the biofilm was assessed by scanning electron microscopy on the 5th,7th,and 10th day of the cutlure.Results On the 5th,7th,and 10th day,the viable bacterial counts(lg CFU/cm²) within the biofilm adhering to the internal surface of Teflon tube were 4.98?0.47,4.82?0.46,and 4.43?0.52,respectively.The viable bacterial counts were significantly different among the three groups(P<0.05).Bacterial colony count on the 10th day within the biofilm was significantly less than that on the 5th and 7th day(P<0.05).There was no difference between the bacterial colony count on the 5th and 7th day(P>0.05).The observation with scanning electron microscope showed that a large number of bacteria closely adhered to the Teflon tube'internal surface with extracellular polymeric substance(EPS) crossing network-like shape after continuous perfusion for 5 days.EPS adhered to single cell forming microcolony after continuous perfusion for 7 days.The stable mature biofilm formed on the internal surface of Teflon tube after continuous perfusion for 10 days.Conclusion Biofilm formation of E coli on the internal surface of Teflon tube is closely related to incubation time.With the increase of incubation time,the bacteria within biofilm gradually decreased and a mature and stable biofilm formed.