Development Of The Methods For Rapid Detection Of Fungal Spores’ Viability By Flow Cytometry And Their Application In Chlorine-based Disinfection

Fungal contamination in drinking water has become a hot issue.The conventional method for counting fungal spores during water treatment and disinfection is heterotrophic plate count（HPC）.However,this method is time-consuming and labor-intensive,and has the difficulty of not counting living but uncultured cells.In addition,the plate method only characterized the culturability of fungal spores and could not judge the damage of other structures in the process of fungal spore damage.Flow cytometry（FCM）is a fast,simple and accurate method for counting cells.At present,FCM has been widely used in the enumeration of bacteria in water treatment and the evaluation of bacterial viability in specific disinfection processes.In this study,Aspergillus niger（A.niger）was taken as the test microorganism in the development of enumeration and viability method for fungal spores using FCM combined with several kinds of fluorescent dyes,and discuss the applicability of the method to other fungi,and the applications of FCM to traditional oxidant（Cl₂,NH₂Cl,Cl O₂）in the assessment of disinfection of the dominant fungi（A.niger,Trichoderma harzianum（T.harzianum）,Penicillium polonicum（P.polonicum））isolated from groundwater.The following conclusions are drawn:（1）The staining method for counting fungal spores by flow cytometry was as follows:fungal spores were ultrasonic treated at 495 W for 5 min,then 10μL SYBR Green I（SG）（100×）and 30 m M ethylene diamine tetraacetic acid（EDTA）were added to 500μL of treated spore suspension,and then incubated at 35°C for 20 min without light.The results of flow cytometry were linearly correlated with those of microscope and plate counting,which proved that flow cytometry was an accurate and feasible method.In addition,flow cytometry can be applied to the detection of other fungal spores,and can also be applied to the quantitative detection of fungal spores in actual water.（2）The method for detecting fungal spore membrane integrity based on flow cytometry was:500μL sample,10μL of SG/propidium iodide（SG/PI）（1:50）and 30 m M EDTA were added to 1.5 m L centrifuge tube,and then incubated at 35°C for 20 min without light.The flow cytometry method was used to detect the esterase activity of fungal spores by adding 500μL sample,0.1 M phosphate buffered solution（PBS）,10μM carboxyfluorescein diacetate（CFDA）and 50 m M EDTA into a 1.5ml centrifuge tube,and then incubated at 35°C for 10min without light.The method for detecting intracellular ROS of fungal spores based on flow cytometry was as follows:500μL sample and 2μg/m L dihydroethidium（DHE）were added into 1.5 m L centrifuge tube,and then incubated at 35°C for 20 min without light.In addition,these viability methods can be applied to the the membrane integrity detection,esterase activity detection and intracellular ROS level detection of other fungal spores.（3）After treatment with three disinfectants,the total number of cells quantified by flow cytometry of the spores of the three genera of fungal spores did not change,that is,there was little or no damage to the double-stranded DNA during disinfection.The rate constants of membrane integrity of the spores were all lower than the rate constants of their culturability,indicating that the cells first lost their culturability and then lost their membrane integrity in the process of inactivation.In the process of chloramine treatment,fungal spores would first decrease their culturability and esterase activity,then increase their oxidative stress response,and finally their membrane integrity would be damaged.Chloramine will first penetrate the cell,destroy the internal enzyme activity,and then damage the cell membrane.During the treatment of chlorine and chlorine dioxide,fungal spores would first decrease their culturability and increase their oxidative stress response,then the membrane integrity would be damaged,and finally the esterase activity would be decreased.Compared with chloramine,chlorine and chlorine dioxide first target the cell membrane of the spores,then destroy the enzyme activity.