| Objective:The interest in environmentally friendly,non-toxic and degradable yet potent biocides has never been so high.Vaporized hydrogen peroxide(VHP)is intensely concerned because of its properties of highly effective,safe and no-residual.VHP decontamination systems have been widely applied in hospitals,biological laboratories,the pharmaceutical industries food industries and other health care environments to prevent and control microbial contamination.Evaluation of disinfection efficacy is of great significance in ensuring the disinfection efficacy,reducing the risk of microbial contamination and controlling the spread of disease-causing microorganisms.The bactericidal rate,the percentage of killed cells in total cells,is the key index to evaluate the efficacy of disinfection process.There are many methods that can distinguish live and dead cells in biological indicators(BIs),including the plate counting,polymerase chain reaction(PCR)assay,fluorescence microscopy and flow cytometry.The standard pate counting method is the most frequently used method for its low cost and low request for the equipment,but it does not meet the requirements of instant detection in practice owing to its disadvantages in time consuming,cumbersome manual operation and low efficiency and accuracy.The PCR-based assay is mature in technology,highly sensitive and specific.However,the general testing instrument on the market cost highly,cannot use in the disinfecting scene because of its strict demands of the work environment.Development of fluorescence technology,such as fluorescence microscopy and flow cytometry make the faster detection possible by discriminating live cells from dead cells with fluorescent dyes.Nevertheless,general specking,fluorescence microscopy is used to observe the labeled cells,but the analysis process is tedious.Fluorescence microscopy has a narrow field of vision,small depth of field and requires repeated focusing.Flow cytometry is a low-flux single-cell analysis method,having the detection limit of approximately 10~3 cells/ml.Since on-site evaluation of disinfection efficacy requires accurate and efficient quantification of minimal residual microorganisms after disinfection treatment,it is of practical significance to develop a sensitive detection system that can automatically quantify live and dead cells and calculate the bactericidal rate.In this study,a rapid and accurate detection strategy based on single-cell fluorescence imaging technology,which can accurately identify and quantitatively analyze the live and dead bacteria,is proposed to evaluate the efficacy of VHP surface disinfection process.Contents:The research contents of this paper are as follows:(1)Research on sample pretreatment methods.Base on the investigation and study,we mainly introduce the realization of double fluorescent staining of live and dead bacteria and the comparative study of bacterial enrichment methods.By comparing the staining results of different fluorescent dyes on bacteria,two fluorescent dyes with excellent fluorescence properties and high spectral separation were selected to reliably distinguish live and dead bacteria in minutes.In terms of bacterial enrichment,we compared the adsorption efficiencies of different bare magnetic beads on bacteria.Moreover,we discussed the enrichment efficiency of membrane filtration method and magnetic bead adsorption method and analyzed the advantages and disadvantages of the two methods in practical application.On the basis of summarizing the pre-treatment process,the research had also preliminarily designed a reagent packaged detection card,which was of great significance for the integration of the detection processes.(2)Design of the optical path of the detection system and optimization design of signal processing algorithm.The optical path mainly includes the two simultaneous scanning lasers,the beam expansion and shaping part of the lasers,the design of the two fluorescence detection channels and the selection of the main components.A Lab VIEW program was designed to control the deflection of the two-axis galvanometer and collect and store signals.The data processing algorithm involved was programmed in Mat Lab to extract the images feature points for calculation of labeled cells and reconstruct the scan images.Finally,the results were directly visualized by a scan image and a scatter plot with the percentage of dead cells in total cells.(3)Performance analysis of the dual-channel laser scanning cytometry.First,fluorescent microspheres were used as the detected objects.By comparing with the results from fluorescence microscope,the design of the optical path of the scanning detection system was inspected and calibrated.After completing the preliminary test of the scanning system,the stained bacteria were used as the detected objects to measure the accuracy,reliability and detection range of the system.(4)Practical application in the evaluation of VHP disinfection efficacy.Method:According to the actual requirements of the evaluation of disinfection efficacy and based on extensive literature research about the current technology of live and dead bacteria detection,a rapid detection assay based on single-cell fluorescence imaging technology was proposed to evaluate the disinfection efficacy.The research included research on sample pre-treatment method,design of the dual-channel laser scanning cytometer,performance analysis and its application in evaluating disinfection efficacy.The first two parts of the work were carried out independently and then combined.After the independent problems were solved,the sample processing and detection section were organically integrated.And then,the performance of the system was tested by multiple experiments.In the sample pretreatment section,literature research combined with experimental verification was used to solve the technical difficulties of sample pretreatment.The development of the dual-channel laser scanning cytometer mainly adopted research methods of the design of the optical system,primarily development of the laser scanning system and the optimization design of the processing algorithm.Then the fluorescent microspheres and stained bacteria were used as the detected objects to verify the performance of the detection system.Finally,the VHP-exposed BIs were used as the test object,and the actual performance of this method in the evaluation of disinfection efficacy was tested by comparing with the plate counting method.Results:In this study,a microbial detection assay based on single-cell fluorescence imaging technology was proposed to identify and quantify live and dead bacteria.A dual-channel laser scanning cytometer was designed and the experimental prototype was produced,correspondingly.Compared with the fluorescence microscope,the system proposed had high resolution,strong sensitivity and good consistency.In the evaluation of VHP disinfection efficacy,the proposed approach was accurate and effective,with an average detection efficiency of 98.47±1.55%,compared with the standard plate counting method.Moreover,excellent correlation between the concentrations of B.subtilis var niger(ATCC 9372)measured by the proposed detection system and by the plate counting method was obtained(R~2=0.9971),indication that this approach had advantages in detection of trace microorganisms.Conclusion:Based on the importance of the evaluation of disinfection efficacy,the advantages and disadvantages of the commonly used detection methods were discussed through extensive investigation.The research on the evaluation method of disinfection efficacy based on single-cell fluorescence imaging technology was proposed.Based on the characteristics of the stained bacteria,a dual-channel laser scanning system was designed.Combined with two-color fluorescence staining technology,it could reliably identify and quantify live and dead cells in bacterial samples.The proposed method can not only be used for rapid evaluation of disinfection efficacy,but also had practical significance and brilliant prospects in many fields requiring microbial counting and identification. |
PDF Full Text Request