Research On Rapid Detection Method Of Three Pathogenic Bacteria In Food

Staphylococcus aureus, Bacillus cereus and Salmonella are considered the mostcommon cause of food-borne illness worldwide. The plate count method is acommonly accepted, reliable method for the detection of them, but is timeconsuming for needing a series of steps such as enrichment, separation andpurification, biochemical identification and serological test and cannot meet therequirements for achieving rapid analysis. Thus, in order to shorten the analyticaltime requirement, simplify testing procedures, and meet the necessity of rapiddetection of S. aureus, B. cereus and Salmonella for the modern food industry, wedeveloped a rapid detection method using staining and computer vision technology.Compared to the plate count method, which requires3to7d, this new detectionmethod offers great time savings. The total analysis time was2to5h. Our new,rapid detection method for microorganisms in foods has great potential for routinefood safety control and microbiological detection applications. In this paper, thepretreatment methods, staining methods and computer vision technologies for S.aureus, B. cereus and Salmonella were studied using the Food MicrobiologicalRapid Detection system (FMRDS) manufactured by our laboratory.The main research work and results are as follows:1. In this section, we studied a rapid detection method based on computer visionfor selective isolation and identification of S. aureus from foods. The researchincluded preparation of selective medium, selection of inhibitors and accelerators,optimizing of selective medium, staining of S. aureus, image denoising,segmentation, morphological operation treatment, characteristic parametersextraction and artificial neural network identification. The detection range for S.aureus was confirmed and this method was compared with the plate count method.The conclusions are as follows:Through single factor and response surface analysis experiments, the formula ofS. aureus selective medium was determined to be17.0g/L tryptone,3.0g/L phytone,68.0g/L NaCl,2.5g/L KH2PO4,2.5g/L glucose,5g/L sodium pyruvate,9.0g/Lglycine,60mg/L K2TeO3, and3.8g/L phenethyl alcohol.Rapid detection by the FMRDSSolid sample (25g) was placed in a sterile homogeneous bag containingphysiological saline (225mL) and flapped for1to2min with a flapping homogenizer to create a liquid bacterial sample (1:10). To minimize large particles interference andfacilitate subsequent enrichment process, medium speed quantitative filter paper wasused to remove large particles. The large particulate matter was removed and filterpaper was rinsed with physiological saline (100mL) in order to minimize bacterialloss.A0.1-mL aliquot of the S. aureus suspension was added into a centrifuge tube(1.5mL) containing S. aureus selective medium (0.9mL). It was then fermented at37C for4h, and an aliquot of the fermentation solution (0.1mL) was taken anddiluted to10mL with sterile distilled water.The10-mL dilution was concentrated using a syringe filter (Ф13/0.45μm).Using disposable syringe,50μL of air was drawn and pushed through the filter3times to mix. The concentrated fermentation solution (2μL) close to the filtermembrane was then transferred onto the slide and air-dried. Sterile distilled water wasdripped on the slide for rinsing, and excess moisture on the slide was absorbed withfilter paper. Finally, the slide was analyzed using the rapid detection system.The image identification program of the food microbiological rapid detectionsystem included the following steps: median filtering denoising, the global thresholdsegmentation, open operation smooth processing and edge extraction, using BPneural network model for image discriminant4-5-1.Compared to the Baird-Parker plate count method, this rapid detection methodoffers great time savings. The total analysis time was5h. Results using the rapiddetection method agree with those using the traditional Baird-Parker method with acorrelation coefficient of>0.99in the range of10-107cfu/mL.2. In this section, we researched the rapid detection method of B. cereus basedshort-term fermented. The research content including B. cereus nature, heattreatment, bacteria suspension preparation, microwave germination and short-termfermentation, bacteria liquid concentration and dyeing, image denoising,segmentation, morphological operation treatment, characteristic parametersextraction and artificial neural network identification. The rapid method wascompared with the plate count method. The conclusions are as follows:Sample was made into solution and heated it at65℃for30min to sterilize nontarget bacteria. Based on LB medium, the germination agent combination wasdetermined to be0.05%ZnSO4,0.1%MgCl,0.15%MnCl. The spore suspensionwas used microwave to germinate the spores for60s under high grade condition. Thegerminated suspension was fermented for2h and filtered with needle filter.Rapid detection by the FMRDSSolid sample (25g) was taken which was crumbled fully and put into conical flask which contained225mL physiological saline and shook up. Put conical flask inwater bath at65℃for30min, and cooled it to room temperature.The bacteria solution above (1mL) was absorbed with pipette and put into1.5mL centrifugal pipe and then centrifuged for10min at11000rpm. Thesupernatant (0.85mL) was discarded and the germination solution (0.85mL) wasmixed and shook up. The spore suspension was put in microwave oven andgerminated for60s under the condition of high-grade and then cooled to roomtemperature rapidly.0.1mL germinated suspension was drew and put into1.5mLcentrifugal pipe which contained0.9mL short-term fermented solution. Afterfermented for2h at150rpm at37℃,0.1mL fermented solution was drew and madegradient dilution in turn with0.9mL peptone solution.1mL was absorbed by a disposable syringe and inserted it in a needle filter(Ф13/0.45μm), the bacteria liquid was concentrated with the needle filter before thecentrifugal pipe was flushed and filtered twice with1.0mL peptone solution (0.5%).Then disposable syringe was drawn out and1mL air was indrawn into filter bottomand5μL bacteria liquid closed to filter membrane was drew. Finally, the bacterialiquid was put on the slide and air-dried. Dripped6-8μL dye liquor (X-Glu) on theslide and covered glass, kept at37℃for10-20min under conditions of avoidinglight, then swilled and put it in the rapid detection system to identify.The image identification program of the system included the following steps:median filtering denoising, RGB color space segmentation, open and close operationsmooth processing and morphological and color feature extraction, using BP neuralnetwork model for image discriminant8-6-1.Compared to plate count method, there was a good linear relationship comparedwith plate method. The total analysis time was5h. The range of detection was50-1×106cfu/mL.3. In this section, we researched the rapid detection method of Salmonella. Theresearch content including the preparation of immunogold nanoparticles andantibodies, Salmonella liquid concentration, dyeing, image denoising, segmentation,morphological operation treatment, characteristic parameters extraction and artificialneural network identification. The rapid method was compared with the plate countmethod. The conclusions are as follows:Determines the conditions of the immunogold nanoparticles and antibodies,heated the0.01%gold chloride acid aqueous solution, add reducing agent citrate,after continuous boiling, the size of15nm-25nm colloidal gold solution wascompounded. Adjusted pH to9.0, mixed second antibody, and then purified thesynthetic solution with high-speed centrifugation. Rapid detection by the FMRDSSolid sample (25g) was placed in a sterile homogeneous bag containing PBS-T(175mL) and flapped for1to2min with a flapping homogenizer to create a liquidbacterial sample. To minimize large particles interference and facilitate subsequentenrichment process, medium speed quantitative filter paper was used to remove largeparticles. Then use flush the filter paper with50mL PBS-T solution and collected.10mL was absorbed by a disposable syringe and inserted it in a needle filter,2μLbacteria liquid closed to filter membrane was drew and put on the slide and air-dried.Dripped75%ethanol and kept6-9min, flushed with PBS-T solution, mixed2μlSalmonella primary antibody and immunogold nanoparticles respectively, kept at37℃for30min. And then4μl growth solution is dropped into the specimen in twicewith a total reaction time4-8min. Then swilled and put it in the rapid detection systemto identify.The image identification program of the system included the following steps:spatial domain filtering method combined with gray level window changes denoising,iterative threshold method segmentation, open and close operation smoothprocessing and fill the hole corrosion method extraction, using BP neural networkmodel for image discriminant4-3-1.Compared to plate count method, there was a good linear relationship comparedwith plate method. The total analysis time was2h. The range of detection was50-1×106cfu/mL.4. Compared the nature, training, detection methods of Staphylococcus aureus,Bacillus cereus and Salmonella. Three methods have their principles andcharacteristics, they are all based on the processing of pretreatment, staining andimage recognition. The detection time of these methods are very short which onlycost2-5hours.