Abstract: ObjectiveTo conduct real-time observation of living biofilm formation and determine the thickness of the Pseudomonas aeruginosa biofilm.MethodsAfter green fluorescent prote in(GFP)expression plasmid was transfected to Pseudomonas aeruginosa,the horizontal section scanning of living biofilm was done in real-time with confocal laser scanning micro scope(CLSM).The thickness of living biofilm was determined according to Zaxial distance.ResultsThe biofilm formation of Pseudomonas aeruginosa was observed in three stages,which were the adherence phase from 0 to 6 hours,the assembly phase from 6 to 24 hours,and the maturation phase from 24 to 72 hours.The thickness of the biofilm at 6,24,48, and 72 hr were 6.1±2.8,29.2±2.3,61.4±1.4,and 61.8±1.1μm,respectively.Statistical analysis showed that the thickness of the biofilm at 6 hr was less than that of at 24 hr(P<0.01)and the thickness of the biofilm at 24 hr was less than that of at 48 hr(P<0.01).There was no difference in the thickness of the biofilm between the time of 48 hr and 72 hr(P>0.05).ConclusionCLSM can be used to observe the biofilm formation of Pseudomonas aeruginosa in real-time and to determine its thickness.