CN117106572A - 基于群体感应菌株原位评估消毒装置及方法 - Google Patents
基于群体感应菌株原位评估消毒装置及方法 Download PDFInfo
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Abstract
本发明提供了一种基于群体感应菌株原位评估消毒装置及方法,涉及微生物实验技术领域,具体而言,所述方法包括微流控反应器,所述微流控反应器内部对应芯片位置设有两个相同的独立培养空间,每个所述培养空间两端还连接有流入通道和流出通道,所述流入通道适于不同群体感应能力的菌液、培养基或消毒剂通过所述流入通道进入所述培养空间内,以培养生物膜或评估消毒情况。本发明可通过微流控反应器中的独立培养空间实现不同群体感应能力的菌液形成生物膜的观察和对比,以及对消毒刺激下不同群体感应细菌生物膜的生长进行详细研究,以全面了解其应激响应机制。
Description
技术领域
本发明涉及微生物实验技术领域,具体而言,涉及一种基于群体感应菌株原位评估消毒装置及方法。
背景技术
消毒是饮用水安全和污水处理回用的重要保障技术,污水处理厂和自来水厂设有专门的消毒程序来对目标水体进行消毒,可阻断水中病原微生物的繁殖和传播,对预防水传播疾病至关重要。耐药细菌的扩散以及耐药生物膜的形成已成为全球范围内影响公共健康的重要问题。许多细菌都以生物膜作为默认生长方式,在供水系统、医疗系统和环境自然水体中都普遍存在。然而,在大多情况下,生物膜的生长对人类健康和环境系统的可持续发展带来了严重的影响,给环境、工业和人类健康带来不利的影响,细菌与污水中悬浮物结合易形成聚集体,其具有更强的耐药性及抗消毒能力。群体感应系统是细菌通过分泌信号分子感知周围环境中细胞的数量,是影响细菌生长和代谢的重要调节系统。细菌的群体感应现象是影响消毒效果的重要因素之一,因此,水系统中对具有不同群体感应能力生物膜的生长抑制和去除尤为重要。然而,在废水处理过程中不同消毒技术是否会影响不同群体感应菌株生物膜的形成,目前尚不清楚。因此,需要对消毒刺激下不同群体感应细菌生物被膜的生长进行详细研究,以全面了解其应激响应机制。
发明内容
本发明解决的问题是解决现有技术中无法详细获知消毒刺激下不同群体感应细菌生物被膜的生长状况及应激响应机制情况。
为解决上述问题,本发明提供一种基于群体感应菌株原位评估消毒装置,包括微流控反应器,所述微流控反应器内部对应芯片位置设有两个相同的独立培养空间,每个所述培养空间两端还连接有流入通道和流出通道,所述流入通道适于不同群体感应能力的菌液、培养基或消毒剂通过所述流入通道进入所述培养空间内,以培养生物膜或评估消毒情况。
进一步地,还包括相连接的注射器和微量注射泵,所述注射器的端部与所述流入通道的一端相连接,所述微量注射泵适于控制所述注射器注入所述流入通道的注射量和注射时间。
进一步地,所述微流控反应器内部的芯片数量包括1-4个。
进一步地,所述流入通道和所述流出通道均为塑料软管;所述注射器的端部设有钢针,所述钢针与所述流入通道的一端相连接。
进一步地,所述微流控反应器由聚二甲基硅氧烷和玻璃键合而成,所述玻璃为结果基底。
进一步地,还包括收集容器,所述收集容器与所述流出通道的流出端相连接,适于收集所述培养空间通过所述流出通道流出的溶液。
本发明所述的基于群体感应菌株原位评估消毒装置相对于现有技术的优势在于,本发明可通过微流控反应器中的独立培养空间实现不同群体感应能力的菌液形成生物膜的观察和对比,以及对消毒刺激下不同群体感应细菌生物膜的生长进行详细研究,以全面了解其应激响应机制。
为解决上述问题,本发明还提供一种基于群体感应菌株原位评估消毒方法,基于所述的基于群体感应菌株原位评估消毒装置,包括如下步骤:
步骤S1:对基于群体感应菌株原位评估消毒装置进行紫外灭菌;
步骤S2:配置培养基和消毒剂,并配置不同群体感应能力的菌液,用不同注射器分别吸取所述培养基、所述消毒剂和所述菌液;
步骤S3:打开微量注射泵开关,分别设置相应的流速,通过控制所述注射器将培养基和所述菌液分别注入不同的培养空间,再将所述消毒剂分别注入所述培养空间,使生物膜在微流控反应器的所述培养空间中生长或灭活;
步骤S4:培养不同实验时间后,取出微流控反应器,置于激光共聚焦显微镜下染色观察。
进一步地,步骤S2中的所述菌液包括铜绿假单胞菌、革兰氏阳性菌和产氢菌中的一种,所述菌液均含有自带荧光蛋白。
进一步地,步骤S3中,所述消毒剂包括臭氧或含氯的培养基。
进一步地,步骤S2、步骤S3和步骤S4的操作适于在室温条件下进行。
本发明所述的基于群体感应菌株原位评估消毒方法相对于现有技术的优势在于,本发明所述的方法可适用于多种不同群体感应能力菌株的检测,并快速获得检测数据,也可原位评估不同消毒技术效率,也可探讨包括次氯酸钠和臭氧消毒在内的多种消毒剂对具有不同群体感应能力菌株的灭活效果,以此从群体感应的角度揭示信号分子对消毒的响应变化,为污水中条件致病菌的去除提供指导。
附图说明
图1是本发明实施例中基于群体感应菌株原位评估消毒装置的结构示意图;
图2是本发明具体实施方式的生物膜染色镜检示意图;
图3是本发明具体实施方式的四种具有不同群体感应能力生物膜的厚度变化散点图一;
图4是本发明具体实施方式的四种具有不同群体感应能力生物膜的厚度变化散点图二;
图5是本发明具体实施方式的四种具有不同群体感应能力生物膜的厚度变化散点图三。
附图标记:
1-微量注射泵;2-注射器;3-钢针;4-流入通道;5-微流控反应器;6-流出通道;7-收集容器。
具体实施方式
为使本发明的上述目的、特征和优点能够更为明显易懂,下面结合附图对本发明的具体实施例做详细的说明。
如图1所示,本发明实施例提供一种基于群体感应菌株原位评估消毒装置,包括微流控反应器5,所述微流控反应器5内部对应芯片位置设有两个相同的独立培养空间,每个所述培养空间两端还连接有流入通道4和流出通道6,所述流入通道4适于不同群体感应能力的菌液、培养基或消毒剂通过所述流入通道4进入所述培养空间内,以培养生物膜或评估消毒情况。
本发明实施例可通过微流控反应器5中的独立培养空间实现不同群体感应能力的菌液形成生物膜的观察和对比,以及对消毒刺激下不同群体感应细菌生物膜的生长进行详细研究,以全面了解其应激响应机制。
在一些优选的实施例中,还包括相连接的注射器2和微量注射泵1,所述注射器2的端部与所述流入通道4的一端相连接,所述微量注射泵1适于控制所述注射器2注入所述流入通道4的注射量和注射时间。由此,精确控制注射流速,提高精确率。
在一些优选的实施例中,所述微流控反应器5内部的芯片数量包括1-4个。
在一些优选的实施例中,所述流入通道4和所述流出通道6均为塑料软管;所述注射器2的端部设有钢针3,所述钢针3与所述流入通道4的一端相连接。
在一些优选的实施例中,所述微流控反应器5由聚二甲基硅氧烷和玻璃键合而成,所述玻璃为结果基底。由此,结构简单,易于清洗及实验观察。
在一些优选的实施例中,还包括收集容器7,所述收集容器7与所述流出通道6的流出端相连接,适于收集所述培养空间通过所述流出通道6流出的溶液。
本发明实施例还提供一种基于群体感应菌株原位评估消毒方法,基于所述的基于群体感应菌株原位评估消毒装置,包括如下步骤:
步骤S1:对基于群体感应菌株原位评估消毒装置进行紫外灭菌;
步骤S2:配置培养基和消毒剂,并配置不同群体感应能力的菌液,用不同注射器2分别吸取所述培养基、所述消毒剂和所述菌液;
步骤S3:打开微量注射泵1开关,分别设置相应的流速,通过控制所述注射器2将培养基和所述菌液分别注入不同的培养空间,再将所述消毒剂分别注入所述培养空间,使生物膜在微流控反应器5的所述培养空间中生长或灭活;
步骤S4:培养不同实验时间后,取出微流控反应器5,置于激光共聚焦显微镜下染色观察。
本发明实施例所述的方法可适用于多种不同群体感应能力菌株的检测,并快速获得检测数据,也可原位评估不同消毒技术效率,也可探讨包括次氯酸钠和臭氧消毒在内的多种消毒剂对具有不同群体感应能力菌株的灭活效果,以此从群体感应的角度揭示信号分子对消毒的响应变化,为污水中条件致病菌的去除提供指导。
在一些优选的实施例中,步骤S2中的所述菌液包括铜绿假单胞菌、革兰氏阳性菌和产氢菌中的一种,所述菌液均含有自带荧光蛋白。由此,适于后期收集观察数据。
在一些优选的实施例中,步骤S3中,所述消毒剂包括臭氧或含氯的培养基。由此,消毒剂易得,实验效果好。
在一些优选的实施例中,步骤S2、步骤S3和步骤S4的操作适于在室温条件下进行。
具体实施方式1
一种基于群体感应菌株原位评估消毒装置,包括微量注射泵1、注射器2、钢针3、流入通道4、微流控反应器5、流出通道6、收集容器7。注射器2、流入通道4和流出通道6均为一次性塑料制品,微流控反应器5为PDMS与玻璃键合制成。微流控反应器5最多可由4块芯片组成,每块芯片设置有2个独立培养空降,每个培养空间的长度为20mm、宽度为2mm、深度为0.2mm,芯片内部用于模拟微生物形成生物膜,收集容器7用于收集渗出培养基及菌液,微量注射泵1通过控制注射器2实现对溶液流速进行控制。
具体实施方式2
本具体实施方式基于具体实施方式1提供的基于群体感应菌株原位评估消毒装置,对携带绿色荧光蛋白的铜绿假单胞菌野生型菌株及突变体1、突变体2及突变体3进行次氯酸钠及臭氧模拟消毒,原位评估不同消毒技术效率的方法,具体步骤如下:
步骤S1:将基于群体感应菌株原位评估消毒装置的所有组件于超净工作台中紫外灭菌20min;用装有无水乙醇的注射器2连接原位评估消毒装置清洗整个通路,再在超净工作台中用装有无菌水的注射器2冲洗通路三次,设置微量注射泵1参数为200μl/min;
步骤S2:配置培养基及消毒剂:培养基配置方法:酵母粉5g、胰蛋白胨10g、NaCl10g,调节pH至7.2-7.4后定容到1L,接着将配制好的培养基用真空抽滤泵过膜后分装至若干个100mL锥形瓶中高压灭菌(121℃,20min),冷却至室温后保存备用;消毒剂配置方法:取一定体积的次氯酸钠溶液使培养基中氯浓度达到2mg/L;打开臭氧发生器,臭氧浓度为2mg/L,使臭氧连着橡胶管进入液体培养基中制备成曝有臭氧气体的水溶液;
制备菌液:按1%的接种量分别加入从4度冰箱中取出的铜绿假单胞菌野生型菌株及3种信号分子突变体菌液,放置37度摇床中培养至对数期活化后,用无菌注射器2吸出后置于微量注射泵1上方;
步骤S3:其中一个培养空间作为空白对照,只通入LB培养基,通入流速为200μl/min;其他培养空间分别通入野生型菌株及3种不同突变体悬浮液,微量注射泵1设置参数为60μl/min,静置2h,在芯片部位形成生物膜;最后,持续一段时间通入LB培养基,通入流速为200μl/min,使生物膜能够继续生长。与此同时,实验组培养空间依次通入含有次氯酸钠的培养基及曝有臭氧的培养基分别进行消毒实验;
步骤S4:分别取出培养2、24、36、48h的生物膜微流控反应器5,包括对照组及实验组,用浓度为3μM的PI染料室温避光孵育15min,无菌水漂洗后,立即置于激光共聚焦显微镜(CLSM)下观察并采集图像,监测各个芯片上生物膜形态结构及厚度。实验随机选取三个独立视野,进行10X、20X、40X下的图像采集,利用Zeiss Zen软件分析荧光强度。
检测观察,CLSM下呈现红色荧光代表生物膜中的死菌,绿色荧光代表生物膜中的活菌。如图2所示,培养48h后,发现信号分子缺失型菌株(突变体1及3)在消毒之后生物膜中死菌含量较高,信号分子过度分泌型菌株(突变体2)在消毒之后生物膜中活菌含量较高。通过3D层扫,监测菌体生物膜厚度变化,根据Z-Y俯视图,使用distance测定生物膜厚度随时间的变化,如图3所示,培养时间为2h、24h、36h、48h后,分别置于CLSM下观察。随培养时间的延长,野生型菌株的生物膜平均厚度依次为29.37μm、32.50μm、46.88μm、50.60μm;突变体1的生物膜平均厚度随时间变化依次为17.54μm、30.34μm、45.15μm、45.37μm;突变体2的生物膜平均厚度随时间变化依次为27.78μm、32.07μm、40.80μm、51.30μm;突变体3的生物膜平均厚度随时间变化依次为26.28μm、30.37μm、40.75μm、45.85μm。经过次氯酸钠消毒之后,四种菌体的生物膜形成都受到抑制,如图4所示;经过臭氧消毒之后,臭氧对生物膜形成的抑制作用最强,如图5所示。总而言之,经过48h的实时监测,可以得出以下两方面结论:首先,从生物膜厚度方面来看,对于铜绿假单胞菌菌株,信号分子突变体2能够形成较高的平均生物膜厚度,突变体3在48h之后能够形成的平均生物膜厚度最低,在生物膜形成初始阶段,信号分子突变体1形成的生物膜厚度最低。说明信号分子的分泌促进生物膜的形成,对消毒剂的破坏起到一定的保护作用。
虽然本发明公开披露如上,但本发明公开的保护范围并非仅限于此。本领域技术人员在不脱离本发明公开的精神和范围的前提下,可进行各种变更与修改,这些变更与修改均将落入本发明的保护范围。
Claims (10)
1.一种基于群体感应菌株原位评估消毒装置,其特征在于,包括微流控反应器,所述微流控反应器内部对应芯片位置设有两个相同的独立培养空间,每个所述培养空间两端还连接有流入通道和流出通道,所述流入通道适于不同群体感应能力的菌液、培养基或消毒剂通过所述流入通道进入所述培养空间内,以培养生物膜或评估消毒情况。
2.根据权利要求1所述的基于群体感应菌株原位评估消毒装置,其特征在于,还包括相连接的注射器和微量注射泵,所述注射器的端部与所述流入通道的一端相连接,所述微量注射泵适于控制所述注射器注入所述流入通道的注射量和注射时间。
3.根据权利要求1所述的基于群体感应菌株原位评估消毒装置,其特征在于,所述微流控反应器内部的芯片数量包括1-4个。
4.根据权利要求2所述的基于群体感应菌株原位评估消毒装置,其特征在于,所述流入通道和所述流出通道均为塑料软管;所述注射器的端部设有钢针,所述钢针与所述流入通道的一端相连接。
5.根据权利要求1所述的基于群体感应菌株原位评估消毒装置,其特征在于,所述微流控反应器由聚二甲基硅氧烷和玻璃键合而成,所述玻璃为结果基底。
6.根据权利要求1所述的基于群体感应菌株原位评估消毒装置,其特征在于,还包括收集容器,所述收集容器与所述流出通道的流出端相连接,适于收集所述培养空间通过所述流出通道流出的溶液。
7.一种基于群体感应菌株原位评估消毒方法,其特征在于,基于权利要求2至6任一项所述的基于群体感应菌株原位评估消毒装置,包括如下步骤:
步骤S1:对基于群体感应菌株原位评估消毒装置进行紫外灭菌;
步骤S2:配置培养基和消毒剂,并配置不同群体感应能力的菌液,用不同注射器分别吸取所述培养基、所述消毒剂和所述菌液;
步骤S3:打开微量注射泵开关,分别设置相应的流速,通过控制所述注射器将培养基和所述菌液分别注入不同的培养空间,再将所述消毒剂分别注入所述培养空间,使生物膜在微流控反应器的所述培养空间中生长或灭活;
步骤S4:培养不同实验时间后,取出微流控反应器,置于激光共聚焦显微镜下染色观察。
8.根据权利要求7所述的基于群体感应菌株原位评估消毒方法,其特征在于,步骤S2中的所述菌液包括铜绿假单胞菌、革兰氏阳性菌和产氢菌中的一种,所述菌液均含有自带荧光蛋白。
9.根据权利要求7所述的基于群体感应菌株原位评估消毒方法,其特征在于,步骤S3中,所述消毒剂包括臭氧或含氯的培养基。
10.根据权利要求7所述的基于群体感应菌株原位评估消毒方法,其特征在于,步骤S2、步骤S3和步骤S4的操作适于在室温条件下进行。
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