中国农业科学 ›› 2021, Vol. 54 ›› Issue (13): 2906-2915.doi: 10.3864/j.issn.0578-1752.2021.13.018

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

酸性硫酸钙对临床常见致病微生物的杀灭能力

付霞丽(),郑紫方,马志倩,徐乐乐,李志伟,李洋,肖书奇,李爽()   

  1. 西北农林科技大学动物医学院,陕西杨凌712100
  • 收稿日期:2020-06-01 修回日期:2020-12-30 出版日期:2021-07-01 发布日期:2021-07-12
  • 通讯作者: 李爽
  • 作者简介:付霞丽,Tel:18702910446;E-mail: 1579459296@qq.com
  • 基金资助:
    国家重点研发计划项目(2017YFD0500605);陕西省重点研发计划项目(2019NY-076);陕西高校青年创新团队项目;西北农林科技大学试验示范站科技创新与成果转化项目(TGZX2020-24)

The Ability of Acidic Calcium Sulfate to Kill Common Clinical Pathogenic Microorganisms

FU XiaLi(),ZHENG ZiFang,MA ZhiQian,XU LeLe,LI ZhiWei,LI Yang,XIAO ShuQi,LI Shuang()   

  1. College of Veterinary Medicine, Northwest Agricultural and Forestry University, Yangling 712100, Shaanxi
  • Received:2020-06-01 Revised:2020-12-30 Online:2021-07-01 Published:2021-07-12
  • Contact: Shuang LI

摘要:

【目的】研究消毒剂酸性硫酸钙(ACS)对微生物的杀灭效果,为用消毒剂防控人畜疫病提供基础数据和理论依据。【方法】将ACS与中和剂(3%卵磷脂、5%吐温-80的磷酸盐缓冲液)共孵育一段时间后,再分别加入大肠杆菌、金黄色葡萄球菌、沙门氏菌和猪繁殖与呼吸综合征病毒(PRRSV)共培养一段时间,将与细菌的混合物涂布于营养琼脂平板,置于37℃生化培养箱培养18—24 h;将与病毒的混合物接种于细胞板,在37℃细胞培养箱内培养一定时间,评价中和剂的中和效果。其对照设置和评判标准如下:消毒剂+菌悬液或病毒悬液(第1组),(消毒剂+菌悬液或病毒悬液)+中和剂(第2组),(中和剂+消毒剂)+菌悬液或病毒悬液(第3组),(无菌硬水+菌悬液或病毒悬液)+中和剂(第4组),(无菌硬水+菌悬液或病毒悬液)+PBS(第5组),无菌硬水+PBS(第6组);细菌杀灭试验中和剂效果评价标准:第1组有极少量细菌生长或无菌生长,第2组有菌生长且明显少于第3、4、5组但多于第1组,第3、4、5组细菌数接近且与阳性对照组细菌数接近,第6组无菌生长,3次重复试验均符合上述条件,结果一致,判定所选中和剂及浓度合适;病毒灭活试验中和剂效果评价标准:第1组有极少量病毒生长或无病毒生长,第2组有病毒生长且明显少于第3、4、5组但多于第1组,第3、4、5组病毒生长与原接种量相近,第6组细胞正常生长,3次重复试验结果一致,判定所选中和剂及浓度合适。利用悬液定量杀菌法和测定病毒滴度的方法评价ACS对上述细菌和病毒的消灭效果,将ACS 稀释200、300、400、500、600、700倍分别与上述细菌或病毒作用不同时间后加入中和剂进行中和,然后将细菌混合物涂布于营养琼脂平板,通过计算菌落数评价ACS杀灭细菌的效果,测定病毒混合物中病毒滴度评价ACS对病毒的杀灭效果。【结果】所选用的中和剂能够有效地中和ACS 200倍稀释液对细菌和病毒的残留作用,且该中和剂对细菌、病毒和细胞无毒性。当ACS与细菌作用0 h后立即被中和,最大稀释300倍对大肠杆菌的灭菌率为100%,最大稀释600倍对金黄色葡萄球菌的灭菌率为100%,最大稀释700倍时对沙门氏菌的灭菌率为100%。当ACS 稀释至700倍时,分别与上述细菌作用1d或6d后被中和,灭菌率均为100%;此外,ACS稀释200倍时,与PRRSV作用60 min后被中和,未检测出病毒滴度。【结论】ACS稀释700倍与大肠杆菌、金黄色葡萄球菌、沙门氏菌作用1 d或1 d以上均能产生较好的杀灭效果;ACS稀释200倍与PRRSV作用60 min能完全杀灭病毒。这可为养殖场选用消毒剂防控疫病提供参考。

关键词: 酸性硫酸钙, 微生物, 中和剂, 杀灭效果

Abstract:

【Objective】The purpose was to study the effect of disinfectant acid calcium sulfate (ACS) on the killing of microorganisms, and to provide basic data and theoretical basis for disinfectant prevention and control of human and animal diseases.【Method】After incubating ACS with 3% lecithin and 5% Tween-80 as phosphate buffer neutralizer for a period of time, Escherichia coli, Staphylococcus aureus, Salmonellaand porcine reproductive and respiratory syndrome virus (PRRSV) were added respectively for a period of time, We spread the culture mixture with bacteria on nutrient agar plates and cultured at 37℃ biochemical incubator for 18-24 h, the mixture with virus was inoculated on the cell plates and incubate in 37℃ cell incubator for a certain period of time to evaluate the neutralizing effect of the neutralizer. The comparison settings and evaluation criteria were as follows: disinfectant mixed with bacterial suspension or virus suspension (group 1), the mixture of disinfectant and bacterial suspension or virus suspension was mixed with neutralizer (group 2), the mixture of neutralizer and disinfectant was mixed with bacterial suspension or virus suspension (group 3), the mixture of sterile hard water and bacterial suspension or virus suspension was mixed with neutralizer (group 4), the mixture of sterile hard water and bacterial suspension or virus suspension was mixed with PBS (group 5), sterile hard water mixed with PBS (group 6). The evaluation criteria of the neutralizer effect in the bacteria killing test were as follows: group 1 had a very small amount of bacterial growth or aseptic growth; group 2 had bacterial growth, which was significantly less than that of groups 3, 4, and 5, but more than the group 1; the number of bacteria in group 3, 4,5 was close to that of the positive control group, no bacterial growth in group 6. All three repeated tests met the above conditions, and the results were consistent, it was determined that the selected neutralizer and concentration were appropriate. The evaluation criteria of the neutralizer effect in the virus inactivation test were as follows: group 1 had very little virus growth or no virus growth; group 2 had virus growth and was significantly less than that of groups 3, 4, and 5, but more than group 1. The growth of viruses in groups 3, 4, and 5 was similar to the original inoculation; the cells in group 6 grew normally. The results of the three repeated tests were consistent, and it was determined that the selected neutralizer and concentration were appropriate. We used the suspension quantitative sterilization method and the method of determining the virus titer to evaluate the elimination effect of ACS on the above-mentioned bacteria and viruses. We diluted the ACS 200, 300, 400, 500, 600, and 700 times to interact with the above-mentioned bacteria or virus for different time and added neutralizer for neutralization, then spread the bacterial mixture on nutrient agar plates to evaluate the killing effect of ACS by calculating the number of colonies, and determined the virus titer in the virus mixture to evaluate the killing effect of ACS on the virus.【Result】 The selected neutralizer can effectively neutralize the residual effects of ACS 200-fold dilution on bacteria and viruses, and the neutralizer was non-toxic to bacteria, viruses and cells. When ACS interacted with the bacteria for 0 h, it was neutralized immediately. The sterilization rate ofEscherichia coli at the maximum dilution of 300 times was 100%, the sterilization rate of Staphylococcus aureusat the maximum dilution of 600 times was 100%, and when the maximum dilution was 700 times, the sterilization rate of Salmonella was 100%. When ACS was diluted to 700 times, it was neutralized one day or six days after treatment with the above bacteria, and the sterilization rate was 100%. In addition, when ACS was diluted to 200 times, it was neutralized 60 minutes after treatment with PRRSV, and no virus titer was detected.【Conclusion】 When ACS diluted 700 times withEscherichia coli,Staphylococcus aureus and Salmonella for one day or more, it can produce better killing effect; when ACS diluted 200 times with PRRSV for 60 minutes, it can completely kill the virus, which will provide strong data support for the selection of disinfectants in the farms and provide reference for the prevention and control of epidemic diseases.

Key words: acid calcium sulfate, microbial, neutralizing agent, killing effect