Screening Disinfectants For A Vibrio Parahaemolyticus And High-throughput Study Of Its Application Parameters

As a product that can effectively kill and inhibit pathogens,disinfectants are responsible for the important mission of preventing and treating diseases in aquaculture.They have participated in the entire aquaculture process.However,in practice,disinfectants are often misused or improperly used,resulting in the inability to To achieve the desired disinfection effect,therefore,assessing the application parameters of the disinfectant has important practical significance for guiding production.In this paper,Vibrio parahaemolyticus,which causes acute hepatopancreatic necrosis disease （AHPND）in shrimp,was used as an object of study.Innovative high-throughput screening techniques were used to sterilize various species commonly used in production.The agent screening research has greatly improved the speed and accuracy of the research,and through in vitro antibacterial experiments,safe concentration experiments,regression verification experiments,etc.,collated analysis data to obtain the following results:1 Three viable counting methods,including MTT assay,ATP bioluminescence assay,and high-throughput growth curve assay,for the live Vibrio parahaemolyticus were compared in a high-throughput manner on 96-well culture plates.The standard curves and linear ranges of these three methods were determined.The results showed that the directly detected index of MTT assay is OD₅₅₅,which is the absorption at 555nm of DMSO-dissolved Formazan produced from MTT.The liner standard curve for viable V.parahaemolyticus between logarithmic viable counts[Log（C）]and the logarithmic OD₅₅₅[Log(OD₅₅₅)]was Log（C）=1.0478Log(OD₅₅₅)+8.0575,with a correlation index R=0.9968,and the linear detection range is 7.8×10⁶².5×10⁸ cfu/mL.The directly detected index of ATP bioluminescence assay is the relative luminescence unit（RLU）,which is produced by ATP of viable cells.The liner standard curve between Log（C）and Log（RUL）was Log（C）=0.9593Log（RLU）+0.9934,with a correlation index R=0.9990,and the linear detection range is 1×10⁴³×10⁸ cfu/mL.The directly detected index of high-throughput growth curve method is the spinodal time（Ts）,which is the time reaching at reflection point on the growth curve.The liner standard curve between Log（C）and Ts was Log（C）=-0.8838Ts+9.0834,with a correlation index R=–0.9976,and the linear detection range is 1×10⁰¹×10⁷ cfu/mL.These three methods were used to measure ten broths of V.parahaemolyticus and comparison with the plate counting method,respectively.The results showed both of the ATP bioluminescence assay and the high-throughput growth curve method had satisfying accuracy,but the MTT assay had lower accuracy.The high-throughput growth curve has a wildest liner range and is most suitable for high throughput measurement.2 Disinfection test of Vibrio parahaemolyticus by 16 kinds of disinfectants showed that the best disinfection effect was polyhexamethylene guanidine hydrochloride,followed by hydrogen peroxide;the effect of chlorine preparation was better than that of iodine preparation.The disinfection effect is good;the disinfection effect of the compound preparation is better than the single preparation.The multi-functional disinfectants for water diversion,potassium persulfate,quicklime,and several Chinese herbal disinfectants have poor disinfection effects,and they cannot completely kill Vibrio parahaemolyticus at the test concentration.Through the experiment of disinfectant action time,it was found that most of the disinfectants could kill 99%when they were sterilized for 2hours.After more than 2 hours,the disinfecting effect gradually remained stable;polyhexamethylene guanidine hydrochloride It is also the fastest disinfectant for disinfection,and the time for disinfecting povidone-iodine with the worst disinfection effect is the slowest.Different types of disinfectants are affected by different degrees of disinfecting time,and the disinfection effect of compound preparations is always better than that of a single preparation..By comparing the disinfection effect of disinfectants at different temperatures,the disinfecting effect of various disinfectants at low temperature is obviously lower than that at high temperature,and at low temperatures,the disinfecting effects of various disinfectants are less different;by comparing six disinfectants The killing rate can be found that the better the disinfectant is,the less affected the temperature is.3 The acute toxicity study of polyheptamethylene guanidine hydrochloride,combined with Rickett,glutaraldehyde,and povidone-iodine disinfectant on stage5 of nauplii of L.vannamei showed that:The 24 h LC₅₀ of the larvae were 1.07mg/L,0.20 mg/L,0.43 mg/L,and 2.27 mg/L,respectively;the safety concentrations were 0.11 mg/L,0.02 mg/L,0.04 mg/L,and 0.23 mg,respectively./L.It can be seen that the best safety of the four disinfectants on the nauplii of Penaeus vannamei is povidone-iodine,and the worst safety is the joint Rik.The safety evaluation test in this paper is supplemented by the lack of acute toxicity of polyhexamethylene guanidine hydrochloride,Rickett’s,and glutaraldehyde’s three disinfectants to the nauplii of Penaeus vannamei as a disinfectant in practice.The application in production provides a theoretical basis.