Optimization And Application Of Rapid Quantitative Detection Method For Bacterial Activity Based On Flow Cyometry

The flow cytometry(FCM)is capable of fast and accurate multi-parameter quantitative analysis and sorting on the cells based on the physical and chemical properties of the cells.it is widely used in the analysis of environmental microbial samples.With the improvement of analysis requirements,using FCM for quantitative testing has become a necessary means to study the number and activity of microorganisms.However,due to the wide variety of factors affecting the results of FCM determination,the results of different determination methods vary greatly and the comparability is poor.Therefore,it is of great significance for the application and popularization of FCM technology to clarify the characteristics and influencing factors of FCM quantitative detection and to establish the method of FCM quantitative detection,which is helpful to the standardization of the technology.In this study,the quantitative detection method of bacterial activity was developed by using FCM combined with fluorescent dyes to solve the problem of quantitative detection of bacteria in different states.Through the systematic analysis of various factors affecting the quantitative detection results of FCM,the adjustment of measurement parameters is optimized,and a rapid quantitative detection method for the analysis of bacterial activity of water environment samples is established.Using Escherichia coli NK5449 preparation in a different state of the tested strains,and the FCM was analyzed by using SYBR GreenⅠ dyes and PI dyes for bacterial staining.The effects of sample pretreatment processes such as centrifugal treatment,diluent and dyeing-dilution sequence on FCM determination results were investigated.Determine the optimal sample concentration,sample rate and the delimitation of the door(Gate).The effects of dye concentration,dyeing temperature and dyeing time on FCM test results were studied,and the single dyeing method using SYBR GreenⅠ and PI was determined respectively,as well as the best determination scheme using SY-PI double dyeing method.The established SY-PI double dyeing method is applied to the rapid quantitative detection of bacteria in water samples after ultraviolet disinfection and in urban surface waters.The results show that the centrifugal 10 min under the relative centrifugal force of223.6xg and the use of phosphoric acid buffer Salt solution(PBS)heavy suspension fungus can effectively remove the small particulate matter impurities in the fungus liquid,and have little effect on the bacterial activity.Both PBS and ultrapure water can be used as diluent for samples,and the dyeing-dilution sequence has no effect on FCM determination results.The optimum sample concentration of FCM is 105 CFU/m L,the sample rate is medium speed,And by setting the door on the FL1-FL3 two dimensional scatter plot to accurately distinguish the living bacteria from the dead bacteria.The SYBR Green I was dyed 100 times after dilution by two methyl sulfoxide to dye the bacterial samples,and the total number of dead and live bacteria could be accurately determined by FCM.If the activated fresh fungus solution is used as the sample,the determination results are in good correlation with the results of the plate counting method.The results show that the centrifugal 10 min under the relative centrifugal force of 223.6xg and the use of phosphoric acid buffer Salt solution(PBS)heavy suspension fungus can effectively remove the small particulate matter impurities in the fungus liquid,and have little effect on the bacterial activity.When the dilution of DMSO was 100 times,the SYBR Green I and PI dye were mixed according to the volume ratio of 100 μmol/L.when the final concentration of PI was 100 μmol/L,the number of living bacteria and dead bacteria in the sample could be quickly quantified by dyeing the sample at 35℃ for 10 min,.The relative standard deviation of multiple detection values is less than 10%,and the precision is good.The optimum concentration of SYBR dye is the same as that of single dyeing method,and the optimum concentration of PI dye is 100 μmol/L.The number of living bacteria determined by SY-PI double staining method is consistent with that of HPC,but the results are determined.The number of dead bacteria needs to be corrected by coefficient 2.The modified SY-PI double staining method can quickly and accurately detect the bacterial activity in the samples.Using SYBR-FCM can accurately detect bacteria that are in VBNC state after Ultraviolet disinfection,and combined with HPC method,the resurrection rate of bacteria can be calculated and analyzed.In addition,SY-PI double dyeing method combined with FCM can efficiently and accurately carry out quantitative detection of VBNC bacteria in various surface water bodies,and the accuracy and accuracy of the test results are very well.