CN102914528A - Method for detecting fluorine content in water by utilizing luminous bacteria - Google Patents
Method for detecting fluorine content in water by utilizing luminous bacteria Download PDFInfo
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- CN102914528A CN102914528A CN2012104006351A CN201210400635A CN102914528A CN 102914528 A CN102914528 A CN 102914528A CN 2012104006351 A CN2012104006351 A CN 2012104006351A CN 201210400635 A CN201210400635 A CN 201210400635A CN 102914528 A CN102914528 A CN 102914528A
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Abstract
The invention provides a method for detecting fluorine content in water by utilizing luminous bacteria and relates to the method for detecting the fluorine content in water. The method provided by the invention is used for solving the technical problems of the present method for detecting the fluorine content in water that the demand on facilities is high, the operation is complex and the cost is low. The method comprises the following steps: 1) preparing an LB fluid medium; 2) cultivating OP50-GFP escherichia coli strain liquid to a platform stage; 3) culturing by adding the fluorine in different contents into the fresh LB fluid medium containing bacterium fluid; 4) representing the growing condition of bacteria by fluorescence intensity; 5) establishing a relation curve and a regression equation between the fluorescence intensity of the bacterium fluid and the fluorine content; and 6) adding a to-be-detected water sample into the fresh LB fluid medium containing the bacterium fluid, detecting the fluorescence intensity and calculating the fluorine content in the water sample. The method provided by the invention has the advantages of less time consumption, low cost, high accuracy, high sensitivity, directness, convenience, simpleness in operation, and the like, so that the method is widely applied to the field of environmental water quality monitoring.
Description
Technical field
The present invention relates to a kind of method that detects Fluoride Concentration In Waters.
Background technology
Fluoride pollution is mainly derived from the emission of smelting, rock phosphate in powder processing, production of phosphate fertilizer, smelting iron and steel and the coal burning process of aluminium, and the discharging of the fluoride waste of the industry such as plating, metal processing.Fluorine-containing flue dust sedimentation or the water wash of accepting a surrender can make soil, surface water and groundwater contaminated.Fluorine can suppress the activity of the enzymes such as lipase, sclerotin phosphatase and urease, causes that metabolism is disorderly.Fluorine also can make the parathyroid gland compensatory hypertrophy, disturbs the alcium and phosphor metabolization of bone.The bone fluorosis shows as osteosclerosis, ligament, capsular ligament calcification, after canalis spinalis and intervertebral foramen narrow down, can oppress spinal cord and nerve roots and cause the paralysis, the paralysis.Fluorine also can suppress the endocrine effect, and gonad, adrenal gland and pancreas are produced harmful effect.But high concentration fluoride pollution chafe and mucous membrane cause skin burn, dermatitis, respiratory inflammation.
The fluorine content method of environmental monitoring of China's GB regulation is mainly class fluorine reagent colourimetry, alizarin sulfonic acid zirconium visual colorimetry, ion-selective electrode method and the chromatography of ions.These chemical analyses and instrument detect generally all requirement medicine or expensive device, and pre-treatment step is loaded down with trivial details, complicated operation, and often can't obtain accurate numerical value owing to lack special standard specimen.Comprise the method for the mensuration fluorine such as ammonium persulfate spectrophotometric method, periodic acid silver potassium spectrophotometric method and formaldoxime spectrophotometric method in the chemical analysis, these methods all need a large amount of medicines, and step is numerous and diverse, and workload is large, and error is also larger.Escherichia coli (Escherichia coli) are learned index as hygienic quality, in environmental water quality monitoring, play very important indicative function, proved recipe method standard in fact, the grown cultures operation is simple and easy, there is various mutations body bacterial strain, can be by OP50-GFP coli strain bacterium liquid indication water pollution degree and the quantization signifying that has green fluorescent protein peptone GFP to express, and the variation by the nutrient culture media diffusion, estimate the quality of its life condition, therefore be widely used in the environmental water quality monitoring field.
Summary of the invention
The present invention requires the technical matters of height, complicated operation, cost costliness for what the method that solves existing detection Fluoride Concentration In Waters existed to facility, and a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters is provided.
A kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters of the present invention is carried out according to the following steps:
One, configuration LB fluid nutrient medium;
Two, be 10 with bacterial concentration
6~ 10
9The OP50-GFP coli strain bacterium liquid of cuf/mL is (1 ~ 2) by volume with the LB fluid nutrient medium of step 1: 1000 mix, be under 34 ~ 40 ℃ the condition in temperature, cultivate 9 ~ 11h, to plateau, obtain plateau OP50-GFP coli strain bacterium liquid;
Three, the plateau OP50-GFP coli strain bacterium liquid that step 2 is obtained is 1:(90 ~ 110 by volume with the LB fluid nutrient medium of step 1) mix, obtain detecting the cultivation bacterium liquid of Fluoride Concentration In Waters;
The cultivation bacterium liquid of the detection Fluoride Concentration In Waters that four, the water sample of different fluorine content and step 3 is obtained is (1 ~ 2) by volume: 1000 mix respectively, are under 34 ~ 40 ℃ the condition in temperature, and concussion cultivation 4 ~ 6h obtains the fluorine-containing bacterium liquid of variable concentrations; Then get the fluorine-containing bacterium liquid of each concentration, carrying out fluorescence intensity under fluorescent microscope detects, utilize image software microscopic analysis system quantifies fluorescence intensity to characterize the bacterial growth situation, set up relation curve and the regression equation y=-8.6575x+994.36 of bacterium liquid fluorescence intensity and fluorine content; Wherein x is the fluorine concentration of fluorine-containing water sample, and y is the fluorescence intensity of fluorine-containing bacterium liquid; Fluorine concentration is respectively 0.01mg/L, 0.05mg/L, 0.1mg/L, 0.5mg/L, 1mg/L, 5mg/L, 10mg/L, 20mg/L and 50mg/L in the water sample of different fluorine content;
Five, get water sample to be measured, joining in the cultivation bacterium liquid of the detection Fluoride Concentration In Waters that step 3 obtains, is that 4 ~ 6h is cultivated in concussion under 34 ~ 40 ℃ the condition in temperature, measure bacterium liquid fluorescence intensity, utilize the fluorine content in the regression equation calculation water sample to be measured that step 4 obtains; The volume ratio (1 ~ 2) of the cultivation bacterium liquid of the detection Fluoride Concentration In Waters that obtains with step 3 of water sample to be measured wherein: 1000.
Beneficial effect of the present invention
A kind of advantage of utilizing photobacteria to detect the method for Fluoride Concentration In Waters of the present invention is, within a short period of time, detect the concentration of fluorine-containing water body and estimate its toxicity by bacterial growth, compare with conventional arts such as instrumental analysis with the chemical detection method that cost is low, step is few, simple to operate.A kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters of the present invention, utilize photobacteria sensitive fluorescent degree to be better than the advantage of traditional OD value, by fluorescent microscope and image software microscopic analysis system quantifies fluorescence intensity to characterize the bacterial growth situation, then set up relation curve and the regression equation of bacterium liquid fluorescence intensity and fluorine content, utilize regression equation can calculate fast the content of fluorine in the water sample to be measured, compare with traditional agar diffusion method, the method of utilizing photobacteria to detect Fluoride Concentration In Waters of the present invention is faster and accurate, and convenient experimental operation, degree of accuracy is high, do not need special high-precision analytical instrument and checkout equipment, can be applicable to the environmental water quality monitoring field.
Description of drawings
Fig. 1 is the graph of relation of bacterium liquid fluorescence intensity and fluorine content among the embodiment 1.
Embodiment
Technical scheme of the present invention is not limited to following embodiment, also comprises the combination in any between each embodiment.
Embodiment one: a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters of present embodiment is carried out according to the following steps:
One, configuration LB fluid nutrient medium;
Two, be 10 with bacterial concentration
6~ 10
9The OP50-GFP coli strain bacterium liquid of cuf/mL is (1 ~ 2) by volume with the LB fluid nutrient medium of step 1: 1000 mix, be under 34 ~ 40 ℃ the condition in temperature, cultivate 9 ~ 11h, to plateau, obtain plateau OP50-GFP coli strain bacterium liquid;
Three, the plateau OP50-GFP coli strain bacterium liquid that step 2 is obtained is 1:(90 ~ 110 by volume with the LB fluid nutrient medium of step 1) mix, obtain detecting the cultivation bacterium liquid of Fluoride Concentration In Waters;
The cultivation bacterium liquid of the detection Fluoride Concentration In Waters that four, the water sample of different fluorine content and step 3 is obtained is (1 ~ 2) by volume: 1000 mix respectively, are under 34 ~ 40 ℃ the condition in temperature, and concussion cultivation 4 ~ 6h obtains the fluorine-containing bacterium liquid of variable concentrations; Then get the fluorine-containing bacterium liquid of each concentration, carrying out fluorescence intensity under fluorescent microscope detects, utilize image software microscopic analysis system quantifies fluorescence intensity to characterize the bacterial growth situation, set up relation curve and the regression equation y=-8.6575x+994.36 of bacterium liquid fluorescence intensity and fluorine content; Wherein x is the fluorine concentration of fluorine-containing water sample, and y is the fluorescence intensity of fluorine-containing bacterium liquid; Fluorine concentration is respectively 0.01mg/L, 0.05mg/L, 0.1mg/L, 0.5mg/L, 1mg/L, 5mg/L, 10mg/L, 20mg/L and 50mg/L in the water sample of different fluorine content;
Five, get water sample to be measured, joining in the cultivation bacterium liquid of the detection Fluoride Concentration In Waters that step 3 obtains, is that 4 ~ 6h is cultivated in concussion under 34 ~ 40 ℃ the condition in temperature, measure bacterium liquid fluorescence intensity, utilize the fluorine content in the regression equation calculation water sample to be measured that step 4 obtains; The volume ratio (1 ~ 2) of the cultivation bacterium liquid of the detection Fluoride Concentration In Waters that obtains with step 3 of water sample to be measured wherein: 1000.
A kind of advantage of utilizing photobacteria to detect the method for Fluoride Concentration In Waters of present embodiment is, within a short period of time, detect the concentration of fluorine-containing water body and estimate its toxicity by bacterial growth, compare with conventional arts such as instrumental analysis with the chemical detection method that cost is low, step is few, simple to operate.A kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters of present embodiment, utilize photobacteria sensitive fluorescent degree to be better than the advantage of traditional OD value, by fluorescent microscope and image software microscopic analysis system quantifies fluorescence intensity to characterize the bacterial growth situation, then set up relation curve and the regression equation of bacterium liquid fluorescence intensity and fluorine content, utilize regression equation can calculate fast the content of fluorine in the water sample, compare with traditional agar diffusion method, the method of utilizing photobacteria detection Fluoride Concentration In Waters of present embodiment is faster and accurate, and convenient experimental operation, degree of accuracy is high, do not need special high-precision analytical instrument and checkout equipment, can be applicable to the environmental water quality monitoring field.
Embodiment two: what present embodiment and embodiment one were different is: the volume ratio of the LB fluid nutrient medium of OP50-GFP coli strain bacterium liquid and step 1 is 1.5:1000 in the step 2, and other step is identical with embodiment one with parameter.
Embodiment three: what present embodiment was different from embodiment one or two is: cultivation temperature is 37 ℃ in the step 2, and incubation time is 10h, and other step is identical with embodiment one or two with parameter.
Embodiment four: one of present embodiment and embodiment one to three be: the plateau OP50-GFP coli strain bacterium liquid that step 2 obtains in the step 3 and the volume ratio of LB fluid nutrient medium are 1:100, and other step is identical with one of parameter and embodiment one to three.
Embodiment five: present embodiment from one of embodiment one to four is: the water in the step 4 in the water sample of different fluorine content is distilled water, and other step is identical with one of parameter and embodiment one to four.
Embodiment six: what present embodiment was different from one of embodiment one to five is: the volume ratio of the cultivation bacterium liquid of the detection Fluoride Concentration In Waters that the water sample of different fluorine content and step 3 obtain in the step 4 is 1.5:1000, and other step is identical with one of embodiment one or five with parameter.
Embodiment seven: one of present embodiment and embodiment one to six be: the temperature conditions of shaken cultivation is 37 ℃ in step 4 and the step 5, and the time is 5h, and other step is identical with one of parameter and embodiment one to six.
With following verification experimental verification beneficial effect of the present invention:
Embodiment 1, a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters are carried out according to the following steps:
One, configuration LB fluid nutrient medium: take by weighing the 1g yeast extract, 2g peptone and 2g NaCl join in the 1000mL distilled water, are under 120 ℃ of conditions in temperature, and heating 20min sterilizes, and obtains the LB fluid nutrient medium;
Two, getting the 2mL bacterial concentration is 10
9The OP50-GFP coli strain bacterium liquid of cuf/mL joins in the LB fluid nutrient medium of step 1, is under 37 ℃ the condition in temperature, cultivates 10h, to plateau, obtains plateau OP50-GFP coli strain bacterium liquid; Wherein the OP50-GFP coli strain is bought the international nematode germplasm center (CGC) of subsidizing from America NI H;
Three, the plateau OP50-GFP coli strain bacterium liquid that step 2 is obtained and the LB fluid nutrient medium of step 1 obtain detecting the cultivation bacterium liquid of Fluoride Concentration In Waters by volume for 1:100 mixes;
The cultivation bacterium liquid of the detection Fluoride Concentration In Waters that four, the water sample of different fluorine content and step 3 is obtained mixes respectively for 1.5:1000 by volume, is that 5h is cultivated in concussion, obtains the fluorine-containing bacterium liquid of variable concentrations under 37 ℃ the condition in temperature; Then get the fluorine-containing bacterium liquid of each concentration of 0.1mL, drip respectively on microslide, with the cover glass capping of tiling, be to carry out fluorescence intensity under the zeiss fluorescent microscope of Axio ObserverA1 to detect in model, utilize AxioVision LE image software microscopic analysis system quantifies fluorescence intensity to characterize the bacterial growth situation, the relation curve of setting up bacterium liquid fluorescence intensity and fluorine content as shown in Figure 1, regression equation: y=-8.6575x+994.36(x is the fluorine concentration of fluorine-containing water sample, and y is the fluorescence intensity of fluorine-containing bacterium liquid); Wherein water is distilled water in the water sample of different fluorine content, fluorine concentration is respectively 0.01mg/L, 0.05mg/L, 0.1mg/L, 0.5mg/L, 1mg/L, 5mg/L in the water sample,, 10mg/L, 20mg/L and 50mg/L, canonical reference Drinking Water water quality standard and sewage discharge concentration are set;
Five, get the water sample to be measured that known fluorine concentration is 0.05mg/L, it is joined in the cultivation bacterium liquid of the detection Fluoride Concentration In Waters that step 3 obtains, be under 37 ℃ the condition in temperature, 5h is cultivated in concussion, measure bacterium liquid fluorescence intensity, the fluorine content in the regression equation calculation of utilizing step 4 the to obtain water sample to be measured; Wherein the cultivation bacterium liquid of the detection Fluoride Concentration In Waters that obtains of water sample to be measured and step 3 is 1.5:1000 by volume.
By the method for present embodiment, the fluorine content that calculates water sample to be measured is 0.048mg/L, and is very nearly the same with actual value, and accuracy is high.
A kind of advantage of utilizing photobacteria to detect the method for Fluoride Concentration In Waters of present embodiment is, within a short period of time, detect the concentration of fluorine-containing water body and estimate its toxicity by bacterial growth, compare with conventional arts such as instrumental analysis with the chemical detection method that cost is low, step is few, simple to operate.A kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters of present embodiment, utilize photobacteria sensitive fluorescent degree to be better than the advantage of traditional OD value, by fluorescent microscope and image software microscopic analysis system quantifies fluorescence intensity to characterize the bacterial growth situation, then set up relation curve and the regression equation of bacterium liquid fluorescence intensity and fluorine content, utilize regression equation can calculate fast the content of fluorine in the water sample, compare with traditional agar diffusion method, the method of utilizing photobacteria detection Fluoride Concentration In Waters of present embodiment is faster and accurate, and convenient experimental operation, degree of accuracy is high, do not need special high-precision analytical instrument and checkout equipment, can be applicable to the environmental water quality monitoring field.
Embodiment 2, a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters are carried out according to the following steps: present embodiment as different from Example 1: water sample concentration to be measured is 0.1mg/L in the step 7, and other processes and parameter are identical with embodiment 1.
Utilize the regression equation of embodiment 1, the fluorine content that calculates water sample to be measured is 0.116mg/L, and is very nearly the same with actual value, and accuracy is high.
Embodiment 3, a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters are carried out according to the following steps: present embodiment as different from Example 1: water sample concentration to be measured is 0.5mg/L in the step 7, and other processes and parameter are identical with embodiment 1.
Utilize the regression equation of embodiment 1, the fluorine content that calculates water sample to be measured is 0.485mg/L, and is very nearly the same with actual value, and accuracy is high.
Embodiment 4, a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters are carried out according to the following steps: present embodiment as different from Example 1: water sample concentration to be measured is 1mg/L in the step 7, and other processes and parameter are identical with embodiment 1.
Utilize the regression equation of embodiment 1, the fluorine content that calculates water sample to be measured is 1.212mg/L, and is very nearly the same with actual value, and accuracy is high.
Utilize the regression equation of embodiment 1, the fluorine content that calculates water sample to be measured is 5.626mg/L, and is very nearly the same with actual value, and accuracy is high.
Embodiment 6, a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters are carried out according to the following steps: present embodiment as different from Example 1: water sample concentration to be measured is 10mg/L in the step 7, and other processes and parameter are identical with embodiment 1.
Utilize the regression equation of embodiment 1, the fluorine content that calculates water sample to be measured is 11.302mg/L, and is very nearly the same with actual value, and accuracy is high.
Embodiment 7, a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters are carried out according to the following steps: present embodiment as different from Example 1: water sample concentration to be measured is 20mg/L in the step 7, and other processes and parameter are identical with embodiment 1.
Utilize the regression equation of embodiment 1, the fluorine content that calculates water sample to be measured is 21.446mg/L, and is very nearly the same with actual value, and accuracy is high.
Embodiment 8, a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters are carried out according to the following steps: present embodiment as different from Example 1: water sample concentration to be measured is 50mg/L in the step 7, and other processes and parameter are identical with embodiment 1.
Utilize the regression equation of embodiment 1, the fluorine content that calculates water sample to be measured is 52.181mg/L, and is very nearly the same with actual value, and accuracy is high.
Claims (7)
1. method of utilizing photobacteria to detect Fluoride Concentration In Waters is characterized in that the method carries out according to the following steps:
One, configuration LB fluid nutrient medium;
Two, be 10 with bacterial concentration
6~ 10
9The OP50-GFP coli strain bacterium liquid of cuf/mL is (1 ~ 2) by volume with the LB fluid nutrient medium of step 1: 1000 mix, be under 34 ~ 40 ℃ the condition in temperature, cultivate 9 ~ 11h, to plateau, obtain plateau OP50-GFP coli strain bacterium liquid;
Three, the plateau OP50-GFP coli strain bacterium liquid that step 2 is obtained is 1:(90 ~ 110 by volume with the LB fluid nutrient medium of step 1) mix, obtain detecting the cultivation bacterium liquid of Fluoride Concentration In Waters;
The cultivation bacterium liquid of the detection Fluoride Concentration In Waters that four, the water sample of different fluorine content and step 3 is obtained is (1 ~ 2) by volume: 1000 mix respectively, are under 34 ~ 40 ℃ the condition in temperature, and concussion cultivation 4 ~ 6h obtains the fluorine-containing bacterium liquid of variable concentrations; Then get the fluorine-containing bacterium liquid of each concentration, carrying out fluorescence intensity under fluorescent microscope detects, utilize image software microscopic analysis system quantifies fluorescence intensity to characterize the bacterial growth situation, set up relation curve and the regression equation y=-8.6575x+994.36 of bacterium liquid fluorescence intensity and fluorine content; Wherein x is the fluorine concentration of fluorine-containing water sample, and y is the fluorescence intensity of fluorine-containing bacterium liquid; Fluorine concentration is respectively 0.01mg/L, 0.05mg/L, 0.1mg/L, 0.5mg/L, 1mg/L, 5mg/L, 10mg/L, 20mg/L and 50mg/L in the water sample of different fluorine content;
Five, get water sample to be measured, joining in the cultivation bacterium liquid of the detection Fluoride Concentration In Waters that step 3 obtains, is that 4 ~ 6h is cultivated in concussion under 34 ~ 40 ℃ the condition in temperature, measure bacterium liquid fluorescence intensity, utilize the fluorine content in the regression equation calculation water sample to be measured that step 4 obtains; The volume ratio (1 ~ 2) of the cultivation bacterium liquid of the detection Fluoride Concentration In Waters that obtains with step 3 of water sample to be measured wherein: 1000.
2. a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters claimed in claim 1 is characterized in that the volume ratio of the LB fluid nutrient medium of OP50-GFP coli strain bacterium liquid and step 1 in the step 2 is 1.5:1000.
3. claim 1 or 2 described a kind of methods of utilizing photobacteria to detect Fluoride Concentration In Waters is characterized in that cultivation temperature is 37 ℃ in the step 2, and incubation time is 10h.
4. a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters claimed in claim 3 is characterized in that step 2 obtains in the step 3 plateau OP50-GFP coli strain bacterium liquid and the volume ratio of LB fluid nutrient medium are 1:100.
5. a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters claimed in claim 3 is characterized in that the water in the water sample of different fluorine content in the step 4 is distilled water.
6. a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters claimed in claim 3 is characterized in that the volume ratio of the cultivation bacterium liquid of the water sample of different fluorine content in the step 4 and the detection Fluoride Concentration In Waters that step 3 obtains is 1.5:1000.
7. a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters claimed in claim 3 is characterized in that the temperature conditions of shaken cultivation in step 4 and the step 5 is 37 ℃, and the time is 5h.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105505964A (en) * | 2015-12-24 | 2016-04-20 | 清华大学 | Method for monitoring biogas fermentation system in real time and application |
| CN108344847A (en) * | 2018-02-05 | 2018-07-31 | 环境保护部华南环境科学研究所 | A method of monitoring water quality toxicity using tetrahymena |
| CN112034021A (en) * | 2020-09-08 | 2020-12-04 | 南京农业大学 | Method for detecting fluorine content in escherichia coli |
| CN116626001A (en) * | 2023-05-24 | 2023-08-22 | 中国矿业大学 | Method for measuring adsorption rate of sulfate reducing bacteria on coal surface based on fluorescence spectrophotometry |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505964A (en) * | 2015-12-24 | 2016-04-20 | 清华大学 | Method for monitoring biogas fermentation system in real time and application |
| CN105505964B (en) * | 2015-12-24 | 2020-02-14 | 清华大学 | Method for monitoring biogas fermentation system in real time and application |
| CN108344847A (en) * | 2018-02-05 | 2018-07-31 | 环境保护部华南环境科学研究所 | A method of monitoring water quality toxicity using tetrahymena |
| CN112034021A (en) * | 2020-09-08 | 2020-12-04 | 南京农业大学 | Method for detecting fluorine content in escherichia coli |
| CN116626001A (en) * | 2023-05-24 | 2023-08-22 | 中国矿业大学 | Method for measuring adsorption rate of sulfate reducing bacteria on coal surface based on fluorescence spectrophotometry |
| CN116626001B (en) * | 2023-05-24 | 2024-04-05 | 中国矿业大学 | Method for measuring adsorption rate of sulfate reducing bacteria on coal surface based on fluorescence spectrophotometry |
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