CN113684239A - 一种用发光细菌对阻垢剂类物质毒性进行检测的方法 - Google Patents
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Abstract
本发明公开了一种用发光细菌对阻垢剂类物质毒性进行检测的方法，主要包括三个步骤：发光细菌复苏，毒性实验和数据处理分析。本发明充分考虑海水淡化工程和石油开采工程回注水处理过程中，阻垢剂类物质应用的扩大和发展，提供了一种利用发光细菌对阻垢剂类物质进行急性毒性检测的方法。
Description
技术领域
本发明属于环境毒理学技术领域，具体涉及一种用发光细菌对阻垢剂类物质毒性进行检测的方法。
背景技术
发光细菌是一类在正常的生理条件下能够发射可见光的细菌，属革兰氏阴性菌，兼性好氧化能自养型。其发光机理为特异性荧光酶(LE)、还原性黄素单核苷酸(FMNH2)、八碳以上长链脂肪醛(RCHO)、氧分子(O2)所参与的复杂反应。当受到外界环境的干扰时，发光细菌的正常生理反应受阻，其发光过程也会受到影响，发光强度改变，并且反应较为灵敏，因此，发光细菌常用于毒性物质的检测。自1672年BOYLE观察到发光细菌所发出的光易被化学物质抑制，引起科学界对细菌发光效应的大量研究起，发光细菌逐渐被用于各种环境污染物监测和食品单一及综合毒性评价。使用发光细菌对毒性物质进行检测，具有快速、灵敏、重现性好等优点，并且将发光细菌制成冻干粉进行保存更是增添了该实验的便捷性。目前，常用于环境毒理学领域进行急性生物毒性检测的发光细菌包括费氏弧菌、明亮发光杆菌和青海弧菌。
阻垢剂是一类能阻止结垢的化学药剂，其原理是通过破坏结垢过程，阻断晶体的形成和发育，从而起到阻碍结垢发生的作用。常见的阻垢剂可以分为磷酸类阻垢剂、聚羧酸类阻垢剂、磺酸类阻垢剂与绿色阻垢剂四类。随着油田开采工程、海水淡化工程和其他工业循环系统的发展，阻垢剂在油田开采回注水处理、海水淡化水处理和工业冷却水循环系统中的应用也在进一步扩大。当含有阻垢剂的海水排放入海，势必会对海水水质环境产生扰动，甚至会影响水生生物的正常生命活动，进而对生物多样性和海水生态系统的平衡造成影响。
因此，研究阻垢剂类物质的环境效应和生物毒性是十分必要，且具有实践意义的。目前，有关阻垢剂类物质的生物毒性和生态效应的研究还有待进一步深入，关于其生物毒性部分的研究相对较少。
发明内容
针对目前阻垢剂类物质生态毒性效应方面研究不足的现象，本发明提供了一种用发光细菌对阻垢剂类物质毒性进行检测的方法，该方法可操作性强，使用的发光细菌可以直接通过冻干粉复苏获得，简便易行，为目前阻垢剂类物质生物毒性领域提供了研究参考。
本发明所述的一种用发光细菌对阻垢剂类物质毒性进行检测的方法，包括以下步骤：
1.发光细菌复苏：在超净工作台取适量复苏液加入发光细菌冻干粉(具体用量根据发光量和仪器要求确定)中，轻轻震荡摇匀，复苏等待10min。
2.取复苏后发光菌液，用2％NACl溶液按照1：40的体积比稀释。
3.根据实验所需，用2％NACl溶液作为溶剂，配置10mg/L的ZnSO4·7H2O溶液作为阳性质控溶液。
4.根据实验所需，用2％NACl溶液作为溶剂，配置阻垢剂类物质溶液，共设置5个浓度梯度和一个空白对照组，每个组设置3个平行。
5.以96孔板第一行为阴性指控，第二行为阳性质控，其余行用于其他实验组，各孔加入样品180μL和菌液20μL，上机对初始发光强度进行测定。
6. 15min后，再次测试各孔发光强度。
7.计算各浓度阻垢剂类物质对发光细菌的发光抑制率并进行分析。
其特征在于，步骤五中，每个实验样品平行加样3-5次对发光强度进行测定，以避免偶然因素干扰实验结果。
其特征在于，向96孔板中加入菌液时应迅速，避免时间因素干扰实验结果。
本发明的优点：
1.优先选用生态毒理学领域具有代表性的生物，发光细菌
2.所述的毒性实验每个浓度组设立三个平行实验，并在后续测定时，对每个实验样品的发光强度测定多次，多次重复有利于消除偶然因素对实验结果的干扰，获得更可靠的实验结果。
附图说明
图1为所述的一种用发光细菌对阻垢剂类物质毒性进行检测的方法流程图。
具体实施方式
为了阐明本发明的技术方案，现结合附图及使用费氏弧菌测定磷酸类阻垢剂2-磷酸基丁烷-1，2，4-三羧酸(PBTCA)的毒性流程方式对本发明做进一步的详细介绍。
下列实施例中的方法，可以使本专业技术人员更全面的理解本发明，但不以任何方式限制本发明。
本实施例具体步骤如下。
1.配置2％NACl溶液2L，供后续实验使用。
2.在超净工作台取复苏液1mL放入装有1g费氏弧菌冻干粉的西林瓶中，轻轻震荡摇匀，复苏等待10min。
3.取复苏后发光菌液1mL，用2％NACl溶液稀释到40mL。
4.用2％NACl溶液作为溶剂，配置10mg/L的ZnSO4·7H2O溶液200mL作为阳性质控溶液。
5.用2％NACl溶液作为溶剂，分别配置0、40、80、160、320、640μL/L的PBTCA(50％inwater)溶液，每个浓度配置200mL。
6.以96孔板第一行为阴性指控，第二行为阳性质控，其余行用于其他实验组，各孔加入样品180μL和菌液20μL，每个样品平行加样五次，使用化学发光仪对初始发光强度进行测定。
7. 15min后，再次测试各孔发光强度。
8.计算各浓度阻垢剂类物质对发光细菌的发光抑制率并进行分析。
Claims (8)
1.一种用发光细菌对阻垢剂类物质毒性进行检测的方法，特征是包括如下步骤：
(1)发光细菌复苏：在超净工作台取复苏液加入发光细菌冻干粉中，轻轻震荡摇匀，复苏等待10min。
(2)毒性实验
①取复苏后发光菌液，用2％NACl溶液按照1∶40的体积比稀释。
②根据实验所需，配置阻垢剂类物质溶液和阳性质控溶液。大多数阻垢剂可以直接溶于水中，用2％NACl溶液作为溶剂，根据需要配置所需浓度的阻垢剂溶液，用于后续实验。
③浓度组的设定，根据实验需要设置5个浓度梯度和1个空白对照组，每个实验组设置3个平行。
④上机测定：以96孔板第一行为阴性指控，记录发光强度为C0；第二行为阳性质控，其余行用于其他五个实验组，测定发光强度并记为Ci。
⑤15min后，再次测试各孔发光强度。
(3)数据处理分析：依次计算各浓度阻垢剂类物质对发光细菌的发光抑制率IR并进行分析。
2.据权利要求1所述的一种用发光细菌对阻垢剂类物质毒性进行检测的方法，其特征在于，步骤(1)中的发光细菌冻干粉的取用量具体用量根据发光量和仪器要求确定，复苏液取用量根据发光细菌冻干粉用量决定。
3.据权利要求1所述的一种用发光细菌对阻垢剂类物质毒性进行检测的方法，其特征在于，步骤(2)中的阳性质控液一般可选用10mg/L的ZnSO4·7H2O溶液。
4.据权利要求1所述的一种用发光细菌对阻垢剂类物质毒性进行检测的方法，其特征在于，步骤(2)中配置的阳性质控液和各浓度组溶液，溶剂均为2％NACl溶液。
5.据权利要求1所述的一种用发光细菌对阻垢剂类物质毒性进行检测的方法，其特征在于，步骤(2)中进行上机测试时，可使用多功能酶标仪、化学发光仪等多种仪器。
6.据权利要求1所述的一种用发光细菌对阻垢剂类物质毒性进行检测的方法，其特征在于，步骤(2)中进行上机测试加样时，各组分别向96孔板对应各孔中加入样品180μL和菌液20μL。
7.据权利要求1所述的一种用发光细菌对阻垢剂类物质毒性进行检测的方法，其特征在于，步骤(3)中计算公式为IR＝(1-Ci/C0)×100％。
8.据权利要求1所述的一种用发光细菌对阻垢剂类物质毒性进行检测的方法，其特征在于，整个实验尽量在16-25℃的条件下进行。
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