CN102914528B - A method for detecting fluorine content in water by using luminescent bacteria - Google Patents
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Abstract
一种利用发光细菌检测水体中氟含量的方法，它涉及一种检测水体中氟含量的方法，本发明是为解决现有的检测水体中氟含量的方法存在的对设施要求高、操作复杂、成本昂贵的技术问题，该检测方法过程如下：一、配置LB液体培养基；二、培养OP50-GFP大肠杆菌菌株菌液至平台期；三、在含菌液的新鲜LB液体培养基中加入不同含量的氟进行培养；四、通过荧光强度表征细菌生长状况；五、建立菌液发光强度和氟含量的关系曲线和回归方程；六、在含菌液的新鲜LB液体培养基中加入待测水样，检测其荧光强度，计算水样中的氟含量，本发明具有用时少，成本低，准确度、感度高，直观便捷，操作简单等优点，因此广泛应用于环境水质监测领域。
A method for detecting fluorine content in water by using luminescent bacteria, which relates to a method for detecting fluorine content in water. Expensive technical problems, the detection method process is as follows: 1. Configure LB liquid medium; 2. Cultivate the OP50-GFP E. coli strain bacterial liquid to the plateau stage; 3. Add different 4. Characterize the growth status of bacteria by fluorescence intensity; 5. Establish the relationship curve and regression equation between the luminous intensity of the bacterial solution and the fluorine content; 6. Add the water to be tested in the fresh LB liquid medium containing the bacterial solution sample, detect its fluorescence intensity, and calculate the fluorine content in the water sample. The present invention has the advantages of less time-consuming, low cost, high accuracy and sensitivity, intuitive and convenient operation, and simple operation. Therefore, it is widely used in the field of environmental water quality monitoring.
Description
技术领域technical field
本发明涉及一种检测水体中氟含量的方法。The invention relates to a method for detecting fluorine content in water.
背景技术Background technique
氟污染主要来源于主要来源于铝的冶炼、磷矿石加工、磷肥生产、钢铁冶炼和煤炭燃烧过程的排放物，以及电镀、金属加工等工业的含氟废水的排放。含氟烟尘沉降或受降水淋洗，会使土壤、地表水和地下水受污染。氟可以抑制脂肪酶、骨质磷酸酶和尿素酶等酶的活性，引起物质代谢紊乱。氟还可使甲状旁腺代偿性增生，干扰骨的钙磷代谢。骨骼氟中毒表现为骨硬化，韧带、关节囊钙化，椎管及椎间孔变窄后，可压迫脊髓神经根而导致麻痹、瘫痪。氟还可抑制内分泌作用，对生殖腺、肾上腺和胰腺产生不良影响。高浓度氟污染可刺激皮肤和粘膜，引起皮肤灼伤、皮炎、呼吸道炎症。Fluorine pollution mainly comes from emissions from aluminum smelting, phosphate rock processing, phosphate fertilizer production, iron and steel smelting, and coal combustion processes, as well as fluorine-containing wastewater from electroplating, metal processing and other industries. Fluorine-containing dust settles or is washed by precipitation, which will pollute soil, surface water and groundwater. Fluorine can inhibit the activity of enzymes such as lipase, bone phosphatase and urease, and cause substance metabolism disorders. Fluoride can also cause compensatory hyperplasia of parathyroid glands and interfere with bone calcium and phosphorus metabolism. Skeletal fluorosis manifests as osteosclerosis, calcification of ligaments and joint capsules, and narrowing of the spinal canal and intervertebral foramen, which can compress the spinal nerve roots and cause paralysis and paralysis. Fluorine can also inhibit endocrine effects and have adverse effects on the gonads, adrenal glands and pancreas. High-concentration fluorine pollution can irritate the skin and mucous membranes, causing skin burns, dermatitis, and respiratory inflammation.
我国国标规定的氟含量环境监测方法主要为类氟试剂比色法、茜素磺酸锆目视比色法、离子选择性电极法以及离子色谱法。这些化学分析和仪器检测一般都需要量药品或者昂贵设备，前处理步骤繁琐，操作复杂，而且往往由于缺乏特异标样而无法获得精确数值。化学分析中包括过硫酸铵分光光度法、高碘酸银钾分光光度法和甲醛肟分光光度法等测定氟的方法，这些方法都需要大量药品，步骤繁杂，工作量大，而且误差也较大。大肠杆菌（Escherichia coli）作为水质卫生学指标，在环境水质监测中起着非常重要的指示作用，其实验方法规范，生长培养操作简易，存在多种突变体菌株，可通过有绿色荧光蛋白胨GFP表达的OP50-GFP大肠杆菌菌株菌液指示水体污染程度并量化表征，并通过培养基扩散的变化，评价其生存条件的优劣，因此广泛应用于环境水质监测领域。The environmental monitoring methods of fluorine content stipulated in my country's national standard mainly include fluorine-like reagent colorimetry, alizarin zirconium sulfonate visual colorimetry, ion selective electrode method and ion chromatography. These chemical analyzes and instrumental tests generally require large amounts of drugs or expensive equipment, and the pretreatment steps are cumbersome and complicated, and it is often impossible to obtain accurate values due to the lack of specific standards. Chemical analysis includes ammonium persulfate spectrophotometry, silver potassium periodate spectrophotometry and formaldehyde oxime spectrophotometry and other methods for the determination of fluorine. These methods require a large amount of drugs, the steps are complicated, the workload is large, and the error is also large. . Escherichia coli (Escherichia coli), as an indicator of water quality hygiene, plays a very important role in the monitoring of environmental water quality. Its experimental method is standardized, its growth and culture are easy to operate, and there are many mutant strains, which can be expressed by green fluorescent peptone GFP The OP50-GFP Escherichia coli strain bacteria liquid indicates the degree of water pollution and quantitatively characterizes it, and evaluates the quality of its living conditions through the change of medium diffusion, so it is widely used in the field of environmental water quality monitoring.
发明内容Contents of the invention
本发明是为解决现有的检测水体中氟含量的方法存在的对设施要求高、操作复杂、成本昂贵的技术问题，而提供一种利用发光细菌检测水体中氟含量的方法。The present invention provides a method for detecting fluorine content in water by using luminescent bacteria to solve the technical problems of high requirements for facilities, complicated operation and high cost existing in the existing method for detecting fluorine content in water.
本发明的一种利用发光细菌检测水体中氟含量的方法按以下步骤进行：A method of utilizing luminescent bacteria to detect fluorine content in water according to the present invention is carried out in the following steps:
一、配置LB液体培养基；1. Configure LB liquid medium;
二、将菌液浓度为106~109cuf/mL的OP50-GFP大肠杆菌菌株菌液与步骤一的LB液体培养基按体积比为（1~2）：1000混合，在温度为34~40℃的条件下，培养9~11h，至平台期，得到平台期OP50-GFP大肠杆菌菌株菌液；2. Mix the OP50-GFP Escherichia coli strain with a bacterial concentration of 10 6 ~10 9 cuf/mL and the LB liquid medium in step 1 at a volume ratio of (1~2):1000, at a temperature of 34~ Under the condition of 40°C, cultivate for 9-11 hours until the plateau stage, and obtain the plateau stage OP50-GFP E. coli strain bacterial liquid;
三、将步骤二得到的平台期OP50-GFP大肠杆菌菌株菌液与步骤一的LB液体培养基按体积比为1：（90~110）混合，得到检测水体中氟含量的培养菌液；3. Mix the plateau OP50-GFP E. coli strain bacterial liquid obtained in step 2 with the LB liquid medium in step 1 at a volume ratio of 1: (90-110) to obtain a culture liquid for detecting fluorine content in water;
四、将不同氟含量的水样与步骤三得到的检测水体中氟含量的培养菌液按体积比为（1~2）：1000分别混合，在温度为34~40℃的条件下，震荡培养4~6h，得到不同浓度的含氟菌液；然后取每个浓度的含氟菌液，在荧光显微镜下进行荧光强度检测，利用图像软件显微分析系统量化荧光强度以表征细菌生长状况，建立菌液荧光强度与氟含量的关系曲线和回归方程y=-8.6575x+994.36；其中x为含氟水样的氟浓度，y为含氟菌液的荧光强度；不同氟含量的水样中氟浓度分别为0.01mg/L、0.05mg/L、0.1mg/L、0.5mg/L、1mg/L、5mg/L、10mg/L、20mg/L和50mg/L；4. Mix the water samples with different fluorine content with the culture solution obtained in step 3 to detect the fluorine content in the water body according to the volume ratio (1~2): 1000, respectively, and culture with shaking at a temperature of 34~40°C 4~6h, get different concentrations of fluorine-containing bacteria liquid; then take each concentration of fluorine-containing bacteria liquid, detect the fluorescence intensity under the fluorescence microscope, use the image software microanalysis system to quantify the fluorescence intensity to characterize the growth status of the bacteria, and establish The relationship curve and regression equation y=-8.6575x+994.36 between the fluorescence intensity of the bacteria solution and the fluorine content; where x is the fluorine concentration of the fluoride-containing water sample, and y is the fluorescence intensity of the fluoride-containing bacteria solution; the fluorine in water samples with different fluorine content Concentrations are 0.01mg/L, 0.05mg/L, 0.1mg/L, 0.5mg/L, 1mg/L, 5mg/L, 10mg/L, 20mg/L and 50mg/L;
五、取待测水样，加入到步骤三得到的检测水体中氟含量的培养菌液中，在温度为34~40℃的条件下，震荡培养4~6h，测量菌液荧光强度，利用步骤四获得的回归方程计算待测水样中的氟含量；其中待测水样的与步骤三得到的检测水体中氟含量的培养菌液的体积比（1~2）：1000。5. Take the water sample to be tested and add it to the culture bacteria solution obtained in step 3 to detect the fluorine content in the water body. Under the condition of temperature of 34-40°C, shake and cultivate for 4-6 hours, measure the fluorescence intensity of the bacteria solution, and use the steps The obtained regression equation calculates the fluorine content in the water sample to be tested; the volume ratio (1~2) of the water sample to be tested to the culture solution obtained in step 3 to detect the fluorine content in the water body: 1000.
本发明有益效果Beneficial effect of the present invention
本发明的一种利用发光细菌检测水体中氟含量的方法的优点在于，在较短时间内，通过细菌生长检测含氟水体的浓度并评价其毒性，与化学检测方法和仪器分析等传统技术相比成本低、步骤少、操作简单。本发明的一种利用发光细菌检测水体中氟含量的方法，利用发光细菌荧光敏感度强于传统的OD值的优点，通过荧光显微镜和图像软件显微分析系统量化荧光强度以表征细菌生长状况，然后建立菌液荧光强度与氟含量的关系曲线和回归方程，利用回归方程可快速计算出待测水样中氟的含量，与传统的琼脂扩散法比较，本发明的利用发光细菌检测水体中氟含量的方法更快捷和准确，且实验操作方便，精确度高，不需要特殊的高精分析仪器和检测设备，可应用于环境水质监测领域。The advantage of the method of using luminescent bacteria to detect fluorine content in water body of the present invention is that in a relatively short period of time, the concentration of fluorine-containing water body can be detected through bacterial growth and its toxicity can be evaluated, which is comparable to traditional techniques such as chemical detection methods and instrumental analysis. Low cost, few steps and simple operation. A method of using luminescent bacteria to detect fluorine content in water according to the present invention utilizes the advantage that the fluorescence sensitivity of luminescent bacteria is stronger than the traditional OD value, and quantifies the fluorescence intensity through a fluorescence microscope and an image software microanalysis system to characterize the growth status of bacteria. Then establish the relationship curve and regression equation between the fluorescence intensity of the bacteria liquid and the fluorine content, and use the regression equation to quickly calculate the fluorine content in the water sample to be tested. Compared with the traditional agar diffusion method, the present invention uses luminescent bacteria to detect fluorine in water The content method is faster and more accurate, and the experimental operation is convenient, the accuracy is high, and no special high-precision analytical instruments and testing equipment are required, and it can be applied to the field of environmental water quality monitoring.
附图说明Description of drawings
图1是实施例1中菌液荧光强度与氟含量的关系曲线图。Fig. 1 is the graph of the relationship between the fluorescence intensity of the bacterial solution and the fluorine content in Example 1.
具体实施方式Detailed ways
本发明的技术方案不局限于以下具体实施方式，还包括各具体实施方式间的任意组合。The technical solution of the present invention is not limited to the following specific embodiments, but also includes any combination of the specific embodiments.
具体实施方式一：本实施方式的一种利用发光细菌检测水体中氟含量的方法按以下步骤进行：Embodiment 1: A method of using luminescent bacteria to detect fluorine content in water according to this embodiment is carried out in the following steps:
一、配置LB液体培养基；1. Configure LB liquid medium;
二、将菌液浓度为106~109cuf/mL的OP50-GFP大肠杆菌菌株菌液与步骤一的LB液体培养基按体积比为（1~2）：1000混合，在温度为34~40℃的条件下，培养9~11h，至平台期，得到平台期OP50-GFP大肠杆菌菌株菌液；2. Mix the OP50-GFP Escherichia coli strain with a bacterial concentration of 10 6 ~10 9 cuf/mL and the LB liquid medium in step 1 at a volume ratio of (1~2):1000, at a temperature of 34~ Under the condition of 40°C, cultivate for 9-11 hours until the plateau stage, and obtain the plateau stage OP50-GFP E. coli strain bacterial liquid;
三、将步骤二得到的平台期OP50-GFP大肠杆菌菌株菌液与步骤一的LB液体培养基按体积比为1：（90~110）混合，得到检测水体中氟含量的培养菌液；3. Mix the plateau OP50-GFP E. coli strain bacterial liquid obtained in step 2 with the LB liquid medium in step 1 at a volume ratio of 1: (90-110) to obtain a culture liquid for detecting fluorine content in water;
四、将不同氟含量的水样与步骤三得到的检测水体中氟含量的培养菌液按体积比为（1~2）：1000分别混合，在温度为34~40℃的条件下，震荡培养4~6h，得到不同浓度的含氟菌液；然后取每个浓度的含氟菌液，在荧光显微镜下进行荧光强度检测，利用图像软件显微分析系统量化荧光强度以表征细菌生长状况，建立菌液荧光强度与氟含量的关系曲线和回归方程y=-8.6575x+994.36；其中x为含氟水样的氟浓度，y为含氟菌液的荧光强度；不同氟含量的水样中氟浓度分别为0.01mg/L、0.05mg/L、0.1mg/L、0.5mg/L、1mg/L、5mg/L、10mg/L、20mg/L和50mg/L；4. Mix the water samples with different fluorine content with the culture solution obtained in step 3 to detect the fluorine content in the water body according to the volume ratio (1~2): 1000, respectively, and culture with shaking at a temperature of 34~40°C 4~6h, get different concentrations of fluorine-containing bacteria liquid; then take each concentration of fluorine-containing bacteria liquid, detect the fluorescence intensity under the fluorescence microscope, use the image software microanalysis system to quantify the fluorescence intensity to characterize the growth status of the bacteria, and establish The relationship curve and regression equation y=-8.6575x+994.36 between the fluorescence intensity of the bacteria solution and the fluorine content; where x is the fluorine concentration of the fluoride-containing water sample, and y is the fluorescence intensity of the fluoride-containing bacteria solution; the fluorine in water samples with different fluorine content Concentrations are 0.01mg/L, 0.05mg/L, 0.1mg/L, 0.5mg/L, 1mg/L, 5mg/L, 10mg/L, 20mg/L and 50mg/L;
五、取待测水样，加入到步骤三得到的检测水体中氟含量的培养菌液中，在温度为34~40℃的条件下，震荡培养4~6h，测量菌液荧光强度，利用步骤四获得的回归方程计算待测水样中的氟含量；其中待测水样的与步骤三得到的检测水体中氟含量的培养菌液的体积比（1~2）：1000。5. Take the water sample to be tested and add it to the culture bacteria solution obtained in step 3 to detect the fluorine content in the water body. Under the condition of temperature of 34-40°C, shake and cultivate for 4-6 hours, measure the fluorescence intensity of the bacteria solution, and use the steps The obtained regression equation calculates the fluorine content in the water sample to be tested; the volume ratio (1~2) of the water sample to be tested to the culture solution obtained in step 3 to detect the fluorine content in the water body: 1000.
本实施方式的一种利用发光细菌检测水体中氟含量的方法的优点在于，在较短时间内，通过细菌生长检测含氟水体的浓度并评价其毒性，与化学检测方法和仪器分析等传统技术相比成本低、步骤少、操作简单。本实施方式的一种利用发光细菌检测水体中氟含量的方法，利用发光细菌荧光敏感度强于传统的OD值的优点，通过荧光显微镜和图像软件显微分析系统量化荧光强度以表征细菌生长状况，然后建立菌液荧光强度与氟含量的关系曲线和回归方程，利用回归方程可快速计算出水样中氟的含量，与传统的琼脂扩散法比较，本实施方式的利用发光细菌检测水体中氟含量的方法更快捷和准确，且实验操作方便，精确度高，不需要特殊的高精分析仪器和检测设备，可应用于环境水质监测领域。The advantage of the method of using luminescent bacteria to detect fluorine content in water in this embodiment is that in a relatively short period of time, the concentration of fluorine-containing water can be detected through bacterial growth and its toxicity can be evaluated, which is different from traditional techniques such as chemical detection methods and instrumental analysis. Compared with low cost, few steps and simple operation. A method of using luminescent bacteria to detect fluorine content in water in this embodiment uses the advantage that the fluorescence sensitivity of luminescent bacteria is stronger than the traditional OD value, and quantifies the fluorescence intensity through a fluorescence microscope and image software microanalysis system to characterize the growth status of bacteria , and then establish the relationship curve and regression equation between the fluorescence intensity of the bacterial solution and the fluorine content, and use the regression equation to quickly calculate the fluorine content in the water sample. The content method is faster and more accurate, and the experimental operation is convenient, the accuracy is high, and no special high-precision analytical instruments and testing equipment are required, and it can be applied to the field of environmental water quality monitoring.
具体实施方式二：本实施方式与具体实施方式一不同的是：步骤二中OP50-GFP大肠杆菌菌株菌液与步骤一的LB液体培养基的体积比为1.5：1000，其它步骤与参数与具体实施方式一相同。Specific embodiment two: the difference between this embodiment and specific embodiment one is that the volume ratio of the OP50-GFP E. coli strain bacterium liquid in step two to the LB liquid culture medium in step one is 1.5:1000, other steps and parameters and specific Embodiment 1 is the same.
具体实施方式三：本实施方式与具体实施方式一或二不同的是：步骤二中培养温度为37℃，培养时间为10h，其它步骤与参数与具体实施方式一或二相同。Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is that in Step 2, the culture temperature is 37° C., and the culture time is 10 h. Other steps and parameters are the same as Embodiment 1 or 2.
具体实施方式四：本实施方式与具体实施方式一至三之一的是：步骤三中步骤二得到的平台期OP50-GFP大肠杆菌菌株菌液与LB液体培养基的体积比为1：100，其它步骤与参数与具体实施方式一至三之一相同。Embodiment 4: This embodiment and one of Embodiments 1 to 3 are: the volume ratio of the plateau OP50-GFP Escherichia coli strain bacterium liquid obtained in step 2 in step 3 to the LB liquid medium is 1:100, and other The steps and parameters are the same as those in the specific embodiments 1 to 3.
具体实施方式五：本实施方式与具体实施方式一至四之一的是：步骤四中不同氟含量的水样中的水为蒸馏水，其它步骤与参数与具体实施方式一至四之一相同。Embodiment 5: This embodiment is different from Embodiment 1 to Embodiment 4 in that the water in the water samples with different fluorine contents in step 4 is distilled water, and other steps and parameters are the same as Embodiment 1 to Embodiment 4.
具体实施方式六：本实施方式与具体实施方式一至五之一不同的是：步骤四中不同氟含量的水样与步骤三得到的检测水体中氟含量的培养菌液的体积比为1.5：1000，其它步骤与参数与具体实施方式一或五之一相同。Embodiment 6: The difference between this embodiment and one of Embodiments 1 to 5 is that the volume ratio of the water samples with different fluorine content in step 4 to the culture solution obtained in step 3 to detect the fluorine content in the water body is 1.5:1000 , the other steps and parameters are the same as those in Embodiment 1 or 5.
具体实施方式七：本实施方式与具体实施方式一至六之一的是：步骤四和步骤五中振荡培养的温度条件均为37℃，时间为5h，其它步骤与参数与具体实施方式一至六之一相同。Embodiment 7: This embodiment and one of Embodiments 1 to 6 are: the temperature conditions of shaking culture in Step 4 and Step 5 are both 37° C., and the time is 5 hours. Other steps and parameters are the same as Embodiments 1 to 6. One is the same.
用以下试验验证本发明的有益效果：Verify beneficial effect of the present invention with following test:
实施例1、一种利用发光细菌检测水体中氟含量的方法按以下步骤进行：Embodiment 1. A method for utilizing luminescent bacteria to detect fluorine content in water is carried out in the following steps:
一、配置LB液体培养基：称取1g酵母膏，2g蛋白胨和2g NaCl，加入到1000mL蒸馏水中，在温度为120℃条件下，加热20min，进行灭菌，得到LB液体培养基；1. Prepare LB liquid medium: Weigh 1g yeast extract, 2g peptone and 2g NaCl, add to 1000mL distilled water, heat at 120°C for 20min, and sterilize to obtain LB liquid medium;
二、取2mL菌液浓度为109cuf/mL的OP50-GFP大肠杆菌菌株菌液，加入到步骤一的LB液体培养基中，在温度为37℃的条件下，培养10h，至平台期，得到平台期OP50-GFP大肠杆菌菌株菌液；其中OP50-GFP大肠杆菌菌株购买自美国NIH资助的国际线虫种质中心（CGC）；2. Take 2 mL of OP50-GFP E. coli strain bacterial liquid with a bacterial liquid concentration of 10 9 cuf/mL, add it to the LB liquid medium in step 1, and cultivate it for 10 hours at a temperature of 37°C until it reaches the plateau stage. Obtain the plateau phase OP50-GFP E. coli strain liquid; the OP50-GFP E. coli strain was purchased from the International Nematode Germplasm Center (CGC) funded by the NIH in the United States;
三、将步骤二得到的平台期OP50-GFP大肠杆菌菌株菌液与步骤一的LB液体培养基按体积比为1：100混合，得到检测水体中氟含量的培养菌液；3. Mix the plateau OP50-GFP Escherichia coli strain bacterium liquid obtained in step 2 with the LB liquid culture medium in step 1 in a volume ratio of 1:100 to obtain a culture bacterium liquid for detecting fluorine content in water;
四、将不同氟含量的水样与步骤三得到的检测水体中氟含量的培养菌液按体积比为1.5：1000分别混合，在温度为37℃的条件下，震荡培养5h，得到不同浓度的含氟菌液；然后取0.1mL每个浓度的含氟菌液，分别滴于载玻片上，用盖玻片平铺封盖，在型号为Axio ObserverA1的zeiss荧光显微镜下进行荧光强度检测，利用AxioVision LE图像软件显微分析系统量化荧光强度以表征细菌生长状况，建立菌液荧光强度与氟含量的关系曲线如图1所示、回归方程：y=-8.6575x+994.36（x为含氟水样的氟浓度，y为含氟菌液的荧光强度）；其中不同氟含量的水样中水为蒸馏水，水样中氟浓度分别为0.01mg/L、0.05mg/L、0.1mg/L、0.5mg/L、1mg/L、5mg/L,、10mg/L、20mg/L和50mg/L，设置标准参考生活饮用水水质标准和污水排放浓度；4. Mix the water samples with different fluorine content with the culture solution obtained in step 3 to detect the fluorine content in the water body at a volume ratio of 1.5:1000, and culture them with shaking for 5 hours at a temperature of 37°C to obtain different concentrations of Fluoride-containing bacteria solution; then take 0.1mL of each concentration of fluoride-containing bacteria solution, drop them on glass slides respectively, cover them with a cover glass, and detect the fluorescence intensity under a zeiss fluorescence microscope model Axio ObserverA1, using AxioVision The LE image software microanalysis system quantifies the fluorescence intensity to characterize the growth of bacteria, and establishes the relationship curve between the fluorescence intensity of the bacterial liquid and the fluorine content, as shown in Figure 1. The regression equation: y=-8.6575x+994.36 (x is the fluorine-containing water sample fluorine concentration, y is the fluorescence intensity of the fluorine-containing bacteria solution); the water in the water samples with different fluorine contents is distilled water, and the fluorine concentrations in the water samples are 0.01mg/L, 0.05mg/L, 0.1mg/L, 0.5 mg/L, 1mg/L, 5mg/L, 10mg/L, 20mg/L and 50mg/L, setting standards refer to drinking water quality standards and sewage discharge concentration;
五、取已知氟浓度为0.05mg/L的待测水样，将其加入到步骤三得到的检测水体中氟含量的培养菌液中，在温度为37℃的条件下，震荡培养5h，测量菌液荧光强度，利用步骤四得到的回归方程计算待测水样中的氟含量；其中待测水样与步骤三得到的检测水体中氟含量的培养菌液按体积比为1.5：1000。5. Take the water sample to be tested with a known fluorine concentration of 0.05 mg/L, add it to the culture solution obtained in step 3 to detect the fluorine content in the water body, and culture it with shaking for 5 hours at a temperature of 37°C. Measure the fluorescence intensity of the bacteria solution, and use the regression equation obtained in step 4 to calculate the fluorine content in the water sample to be tested; the water sample to be tested and the culture solution obtained in step 3 to detect the fluorine content in the water body have a volume ratio of 1.5:1000.
通过本实施例的方法，计算出待测水样的氟含量为0.048mg/L，与实际值相差无几，准确度高。Through the method of this embodiment, the fluorine content of the water sample to be tested is calculated to be 0.048 mg/L, which is almost the same as the actual value, and the accuracy is high.
本实施例的一种利用发光细菌检测水体中氟含量的方法的优点在于，在较短时间内，通过细菌生长检测含氟水体的浓度并评价其毒性，与化学检测方法和仪器分析等传统技术相比成本低、步骤少、操作简单。本实施例的一种利用发光细菌检测水体中氟含量的方法，利用发光细菌荧光敏感度强于传统的OD值的优点，通过荧光显微镜和图像软件显微分析系统量化荧光强度以表征细菌生长状况，然后建立菌液荧光强度与氟含量的关系曲线和回归方程，利用回归方程可快速计算出水样中氟的含量，与传统的琼脂扩散法比较，本实施例的利用发光细菌检测水体中氟含量的方法更快捷和准确，且实验操作方便，精确度高，不需要特殊的高精分析仪器和检测设备，可应用于环境水质监测领域。The advantage of the method of using luminescent bacteria to detect fluorine content in water in this embodiment is that in a relatively short period of time, the concentration of fluorine-containing water can be detected through bacterial growth and its toxicity can be evaluated, which is different from traditional techniques such as chemical detection methods and instrumental analysis. Compared with low cost, few steps and simple operation. A method of using luminescent bacteria to detect fluorine content in water in this embodiment utilizes the advantage that the fluorescence sensitivity of luminescent bacteria is stronger than the traditional OD value, and quantifies the fluorescence intensity through a fluorescence microscope and image software microanalysis system to characterize the growth status of bacteria , and then establish the relationship curve and regression equation between the fluorescence intensity of the bacterial solution and the fluorine content, and use the regression equation to quickly calculate the fluorine content in the water sample. The content method is faster and more accurate, and the experimental operation is convenient, the accuracy is high, and no special high-precision analytical instruments and testing equipment are required, and it can be applied to the field of environmental water quality monitoring.
实施例2、一种利用发光细菌检测水体中氟含量的方法按以下步骤进行：本实施例与实施例1不同的是：步骤七中待测水样浓度为0.1mg/L，其他过程及参数与实施例1相同。Embodiment 2, a method of utilizing luminescent bacteria to detect fluorine content in water is carried out according to the following steps: the difference between this embodiment and embodiment 1 is that the concentration of the water sample to be tested in step 7 is 0.1mg/L, and other processes and parameters Same as Example 1.
利用实施例1的回归方程，计算出待测水样的氟含量为0.116mg/L，与实际值相差无几，准确度高。Using the regression equation in Example 1, the calculated fluorine content of the water sample to be tested is 0.116 mg/L, which is almost the same as the actual value and has high accuracy.
实施例3、一种利用发光细菌检测水体中氟含量的方法按以下步骤进行：本实施例与实施例1不同的是：步骤七中待测水样浓度为0.5mg/L，其他过程及参数与实施例1相同。Embodiment 3, a method of utilizing luminescent bacteria to detect fluorine content in water is carried out according to the following steps: the difference between this embodiment and embodiment 1 is that the concentration of the water sample to be tested in step 7 is 0.5mg/L, and other processes and parameters Same as Example 1.
利用实施例1的回归方程，计算出待测水样的氟含量为0.485mg/L，与实际值相差无几，准确度高。Using the regression equation in Example 1, the calculated fluorine content of the water sample to be tested is 0.485 mg/L, which is almost the same as the actual value and has high accuracy.
实施例4、一种利用发光细菌检测水体中氟含量的方法按以下步骤进行：本实施例与实施例1不同的是：步骤七中待测水样浓度为1mg/L，其他过程及参数与实施例1相同。Embodiment 4, a method for utilizing luminescent bacteria to detect fluorine content in water is carried out according to the following steps: the difference between this embodiment and embodiment 1 is that the concentration of the water sample to be tested in step 7 is 1mg/L, and other processes and parameters are the same as Example 1 is the same.
利用实施例1的回归方程，计算出待测水样的氟含量为1.212mg/L，与实际值相差无几，准确度高。Using the regression equation in Example 1, the calculated fluorine content of the water sample to be tested is 1.212 mg/L, which is almost the same as the actual value and has high accuracy.
实施例5、一种利用发光细菌检测水体中氟含量的方法按以下步骤进行：本实施例与实施例1不同的是：步骤七中待测水样浓度为5mg/L，其他过程及参数与试验一相同。Embodiment 5, a method for utilizing luminescent bacteria to detect fluorine content in water is carried out according to the following steps: the difference between this embodiment and embodiment 1 is that the concentration of the water sample to be tested in step 7 is 5 mg/L, and other processes and parameters are the same as Test one is the same.
利用实施例1的回归方程，计算出待测水样的氟含量为5.626mg/L，与实际值相差无几，准确度高。Using the regression equation in Example 1, the calculated fluorine content of the water sample to be tested is 5.626 mg/L, which is almost the same as the actual value and has high accuracy.
实施例6、一种利用发光细菌检测水体中氟含量的方法按以下步骤进行：本实施例与实施例1不同的是：步骤七中待测水样浓度为10mg/L，其他过程及参数与实施例1相同。Embodiment 6, a method for utilizing luminescent bacteria to detect fluorine content in water is carried out according to the following steps: the difference between this embodiment and embodiment 1 is that the concentration of the water sample to be tested in step 7 is 10 mg/L, and other processes and parameters are the same as Example 1 is the same.
利用实施例1的回归方程，计算出待测水样的氟含量为11.302mg/L，与实际值相差无几，准确度高。Using the regression equation in Example 1, the fluorine content of the water sample to be tested was calculated to be 11.302 mg/L, which was almost the same as the actual value, and the accuracy was high.
实施例7、一种利用发光细菌检测水体中氟含量的方法按以下步骤进行：本实施例与实施例1不同的是：步骤七中待测水样浓度为20mg/L，其他过程及参数与实施例1相同。Embodiment 7, a method of utilizing luminescent bacteria to detect fluorine content in water is carried out according to the following steps: the difference between this embodiment and embodiment 1 is that the concentration of the water sample to be tested in step 7 is 20 mg/L, and other processes and parameters are the same as Example 1 is the same.
利用实施例1的回归方程，计算出待测水样的氟含量为21.446mg/L，与实际值相差无几，准确度高。Using the regression equation in Example 1, the fluorine content of the water sample to be tested was calculated to be 21.446 mg/L, which was almost the same as the actual value, and the accuracy was high.
实施例8、一种利用发光细菌检测水体中氟含量的方法按以下步骤进行：本实施例与实施例1不同的是：步骤七中待测水样浓度为50mg/L，其他过程及参数与实施例1相同。Embodiment 8, a method for utilizing luminescent bacteria to detect fluorine content in water is carried out according to the following steps: the difference between this embodiment and embodiment 1 is that the concentration of the water sample to be tested in step 7 is 50 mg/L, and other processes and parameters are the same as Example 1 is the same.
利用实施例1的回归方程，计算出待测水样的氟含量为52.181mg/L，与实际值相差无几，准确度高。Using the regression equation in Example 1, the calculated fluorine content of the water sample to be tested is 52.181 mg/L, which is almost the same as the actual value and has high accuracy.
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|---|---|---|---|---|
| CN1629621A (en) * | 2004-09-03 | 2005-06-22 | 集美大学 | Detection method for rapid determination of Escherichia coli |
Non-Patent Citations (3)
| Title |
|---|
| NADH荧光法快速检测细菌总数;王晶等;《微生物学通报》;20090520;第36卷(第05期);773-779 * |
| 发酵液中色氨酸含量高通量快速测定;于金龙等;《氨基酸和生物资源》;20081231;第30卷(第01期);70-71 * |
| 荧光分析法测定头孢氨苄和头孢拉定的含量;赵岚宁等;《中国抗生素杂志》;19960831;第21卷(第04期);267-269 * |
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