Studies On The Flow Cytometry Method To Detect Microbial Indexes In Raw Milk

Objective: Raw milk is rich in nutrition, in which microorganism grows and propagates veryfast. Traditional detection methods are labourious, which bring inconveniences for enterprise'sroutine manufactures and economic loss. So a method which can detect the microorganism in ashort time is required. The objective of this study is to make research on the detection on the totalbacteria and E. coli.Methods: In this study, pretreatment method which was used to remove the big granule'sdisturbance to the flow cytometry method was established; PI, fluorescence antibody ab30522 wereused to dye on the total bacteria, E. coli, and the dyeing condition was optimized; Themicroorganism detection procedure was established on the flow cytometry's EXPO32 ADCoperation system; the total bacteria and E. coli was deteced by FCM, and the results were comparedwith Standard Plate Count method for total bacterial and MPN method for E. coli.Results: The result is as following:1. Raw milk pretreatment method: Raw milk sample was filtrated by 300 mesh nylonmembrane. We used 5μl of proteinase K(20mg/ml) and 1μl of Triton X-100 to treat 200 ml of rawmilk samples. Treated raw milk samples were incubated at 50℃for 15 min, after which 800 ml ofPBS was added and mixed by EP tubes. Then Treated raw milk samples were heated up 100℃for5 min, Samples were then centrifuged at 14000g for 5 min and lipids (top layer) and digestedproteins of the milk were drawn off with a micropipette. The deposition was bacteria.Remark: while detecting total bacterial of raw milk, the time parameter of heating disposalwas 5 min;, and 10 min for detecting E. coli.2. The process FCM for detection of total bacteria in raw milk: Raw milk was dispose bypretreatment method firstly, then the deposition was resuspended in 190 ml of 0.1M PBS(pH=7.2).We used 10μl of PI (20μg/ml) to stain bacteria for 5min in the dark at room temperature. Thesample was diluted at appropriate multiple. We use EXPO32 ADC operation system to analyse thetreated sample.3. The process FCM for detection of E. coli in raw milk: Raw milk was dispose by pretreatmentmethod firstly, then treated raw milk sample was stained by 100μl fluorescence antibody ab30522for 20min, which was diluted 1:100 in 0.1M PBS(pH=7.2). The sample was diluted at a appropriatemultiple. We use EXPO32 ADC operation system to analyse the treated sample.4. The detecting time of FCM for detection of total bacteria in raw milk was 40 min; Thedetecting time of FCM for detection of E. coli in raw milk was 60 min.5. The lowest limitation of FCM was 1 counts/ml; The highest limitation of FCM was 2.4×10~7/ml. So when the detecting value approach 2.4×10~7/ml, we should dilute the sample at aappropriate multiple6. Comparing with FCM and Standard Plate Count method in detecting total bacteria of rawmilk, the result showed that strong linear correlations (r=0.9360, P＜0.01)between FCM andStandard Plate Count method; Comparing with FCM and MPN method in detecting E. coli of rawmilk, the result showed that strong linear correlations (r=0.96, P＜0.01)between FCM and MPNmethod, and regress equation was that y=1.2443x-1.4718。Conclusion: It could be shortened detecting time greatly by using FCM, that was capable ofachieving the request of detecting raw milk. The result showed that strong linear correlationsbetween FCM and conventional method.