CN102914528B - 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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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 discharge 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 parathyroid gland compensatory hypertrophy, disturbs the alcium and phosphor metabolization of bone.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 paralysis, paralysis.Fluorine also can suppress endocrine effect, and gonad, adrenal gland and pancreas are produced to harmful effect.High concentration fluoride pollution can chafe and mucous membrane, causes 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 owing to lacking special standard specimen, cannot obtain accurate numerical value.Chemical analysis comprises the method for the mensuration fluorine such as ammonium persulfate spectrophotometric method, periodic acid silver potassium spectrophotometric method and formaldoxime spectrophotometric method, and 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, grown cultures operation is simple and easy, there is various mutations body bacterial strain, can be by the OP50-GFP coli strain bacterium liquid indication water pollution degree the quantization signifying that have green fluorescent protein peptone GFP to express, and the variation of spreading by nutrient culture media, evaluate the quality of its life condition, be therefore widely used in environmental water quality monitoring field.
Summary of the invention
The present invention requires the technical matters of height, complicated operation, cost costliness for what solve that the method for existing detection Fluoride Concentration In Waters exists 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, by bacterial concentration, be 10
6~ 10
9the OP50-GFP coli strain bacterium liquid of cuf/mL is (1 ~ 2) with the LB fluid nutrient medium of step 1 by volume: 1000 mix, in temperature, be under the condition of 34 ~ 40 ℃, cultivate 9 ~ 11h, to plateau, obtain plateau OP50-GFP coli strain bacterium liquid;
Three, plateau OP50-GFP coli strain bacterium liquid step 2 being obtained is 1:(90 ~ 110 with the LB fluid nutrient medium of step 1 by volume) mix, obtain detecting the cultivation bacterium liquid of Fluoride Concentration In Waters;
The cultivation bacterium liquid of the detection Fluoride Concentration In Waters four, the water sample of different fluorine content being obtained with step 3 is by volume for (1 ~ 2): 1000 mix respectively, is under the condition of 34 ~ 40 ℃ 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, under fluorescent microscope, carry out fluorescence intensity detection, utilize image software microscopic analysis system quantifies fluorescence intensity to characterize bacterial growth situation, set up relation curve and the regression equation y=-8.6575x+994.36 of bacterium liquid fluorescence intensity and fluorine content; The fluorine concentration that wherein x is fluorine-containing water sample, y is the fluorescence intensity of fluorine-containing bacterium liquid; 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, 10mg/L, 20mg/L and 50mg/L;
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 under the condition of 34 ~ 40 ℃, 4 ~ 6h is cultivated in concussion 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 obtaining 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, by bacterial growth, detect the concentration of fluorine-containing water body and evaluate its toxicity, compare with conventional arts such as instrumental analysis with 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 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 water sample to be measured, with traditional agar diffusion method comparison, 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 environmental water quality monitoring field.
Accompanying drawing explanation
Fig. 1 is the graph of relation of bacterium liquid fluorescence intensity and fluorine content in 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, by bacterial concentration, be 10
6~ 10
9the OP50-GFP coli strain bacterium liquid of cuf/mL is (1 ~ 2) with the LB fluid nutrient medium of step 1 by volume: 1000 mix, in temperature, be under the condition of 34 ~ 40 ℃, cultivate 9 ~ 11h, to plateau, obtain plateau OP50-GFP coli strain bacterium liquid;
Three, plateau OP50-GFP coli strain bacterium liquid step 2 being obtained is 1:(90 ~ 110 with the LB fluid nutrient medium of step 1 by volume) mix, obtain detecting the cultivation bacterium liquid of Fluoride Concentration In Waters;
The cultivation bacterium liquid of the detection Fluoride Concentration In Waters four, the water sample of different fluorine content being obtained with step 3 is by volume for (1 ~ 2): 1000 mix respectively, is under the condition of 34 ~ 40 ℃ 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, under fluorescent microscope, carry out fluorescence intensity detection, utilize image software microscopic analysis system quantifies fluorescence intensity to characterize bacterial growth situation, set up relation curve and the regression equation y=-8.6575x+994.36 of bacterium liquid fluorescence intensity and fluorine content; The fluorine concentration that wherein x is fluorine-containing water sample, y is the fluorescence intensity of fluorine-containing bacterium liquid; 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, 10mg/L, 20mg/L and 50mg/L;
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 under the condition of 34 ~ 40 ℃, 4 ~ 6h is cultivated in concussion 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 obtaining 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, by bacterial growth, detect the concentration of fluorine-containing water body and evaluate its toxicity, compare with conventional arts such as instrumental analysis with 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 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 water sample, with traditional agar diffusion method comparison, 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 environmental water quality monitoring field.
Embodiment two: present embodiment is different from embodiment one: in step 2, the volume ratio of the LB fluid nutrient medium of OP50-GFP coli strain bacterium liquid and step 1 is 1.5:1000, other step is identical with embodiment one with parameter.
Embodiment three: present embodiment is different from embodiment one or two: in step 2, cultivation temperature is 37 ℃, incubation time is 10h, other step is identical with embodiment one or two with parameter.
Embodiment four: one of present embodiment and embodiment one to three: the plateau OP50-GFP coli strain bacterium liquid that in step 3, step 2 obtains and the volume ratio of LB fluid nutrient medium are 1:100, other step is identical with one of parameter and embodiment one to three.
Embodiment five: present embodiment and one of embodiment one to four: the water in step 4 in the water sample of different fluorine content is distilled water, other step is identical with one of parameter and embodiment one to four.
Embodiment six: present embodiment is different from one of embodiment one to five: the volume ratio of the cultivation bacterium liquid of the detection Fluoride Concentration In Waters that in step 4, the water sample of different fluorine content and step 3 obtain 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: in step 4 and step 5, the temperature conditions of shaken cultivation is 37 ℃, the time is 5h, 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 1g yeast extract, 2g peptone and 2g NaCl, join in 1000mL distilled water, is under 120 ℃ of conditions in temperature, heating 20min, carries out sterilizing, obtains LB fluid nutrient medium;
Two, getting 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 the condition of 37 ℃ in temperature, cultivates 10h, to plateau, obtains plateau OP50-GFP coli strain bacterium liquid; Wherein OP50-GFP coli strain is bought the international nematode germplasm center (CGC) of subsidizing from America NI H;
Three, plateau OP50-GFP coli strain bacterium liquid step 2 being obtained by volume for 1:100 mixes, obtains detecting the cultivation bacterium liquid of Fluoride Concentration In Waters with the LB fluid nutrient medium of step 1;
The cultivation bacterium liquid of the detection Fluoride Concentration In Waters four, the water sample of different fluorine content being obtained with step 3 mixes respectively for 1.5:1000 by volume, is under the condition of 37 ℃ in temperature, and 5h is cultivated in concussion, obtains the fluorine-containing bacterium liquid of variable concentrations; Then get the fluorine-containing bacterium liquid of each concentration of 0.1mL, drip respectively on microslide, with the cover glass capping of tiling, in model, be to carry out fluorescence intensity detection under the zeiss fluorescent microscope of Axio ObserverA1, utilize AxioVision LE image software microscopic analysis system quantifies fluorescence intensity to characterize bacterial growth situation, the relation curve of setting up bacterium liquid fluorescence intensity and fluorine content as shown in Figure 1, regression equation: the y=-8.6575x+994.36(x fluorine concentration that is fluorine-containing water sample, y is the fluorescence intensity of fluorine-containing bacterium liquid); Wherein in the water sample of different fluorine content, water is distilled water, in water sample, 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, 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, joined in the cultivation bacterium liquid of the detection Fluoride Concentration In Waters that step 3 obtains, in temperature, be under the condition of 37 ℃, 5h is cultivated in concussion, measure bacterium liquid fluorescence intensity, the fluorine content in the regression equation calculation water sample to be measured that utilizes step 4 to obtain; The cultivation bacterium liquid of the detection Fluoride Concentration In Waters that wherein water sample to be measured and step 3 obtain is 1.5:1000 by volume.
By the method for the present embodiment, the fluorine content that calculates water sample to be measured is 0.048mg/L, and very nearly the same with actual value, accuracy is high.
A kind of advantage of utilizing photobacteria to detect the method for Fluoride Concentration In Waters of the present embodiment is, within a short period of time, by bacterial growth, detect the concentration of fluorine-containing water body and evaluate its toxicity, compare with conventional arts such as instrumental analysis with 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 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 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 water sample, with traditional agar diffusion method comparison, the method of utilizing photobacteria detection Fluoride Concentration In Waters of the 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 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: the present embodiment as different from Example 1: in step 7, water sample concentration to be measured is 0.1mg/L, 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 very nearly the same with actual value, 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: the present embodiment as different from Example 1: in step 7, water sample concentration to be measured is 0.5mg/L, 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 very nearly the same with actual value, 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: the present embodiment as different from Example 1: in step 7, water sample concentration to be measured is 1mg/L, 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 very nearly the same with actual value, accuracy is high.
Embodiment 5, a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters are carried out according to the following steps: the present embodiment as different from Example 1: in step 7, water sample concentration to be measured is 5mg/L, and other processes and parameter are identical with test one.
Utilize the regression equation of embodiment 1, the fluorine content that calculates water sample to be measured is 5.626mg/L, and very nearly the same with actual value, 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: the present embodiment as different from Example 1: in step 7, water sample concentration to be measured is 10mg/L, 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 very nearly the same with actual value, 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: the present embodiment as different from Example 1: in step 7, water sample concentration to be measured is 20mg/L, 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 very nearly the same with actual value, 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: the present embodiment as different from Example 1: in step 7, water sample concentration to be measured is 50mg/L, 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 very nearly the same with actual value, accuracy is high.
Claims (7)
1. utilize photobacteria to detect the method for Fluoride Concentration In Waters, it is characterized in that the method carries out according to the following steps:
One, configuration LB fluid nutrient medium;
Two, by bacterial concentration, be 10
6~ 10
9the OP50-GFP coli strain bacterium liquid of cuf/mL is (1 ~ 2) with the LB fluid nutrient medium of step 1 by volume: 1000 mix, in temperature, be under the condition of 34 ~ 40 ℃, cultivate 9 ~ 11h, to plateau, obtain plateau OP50-GFP coli strain bacterium liquid;
Three, plateau OP50-GFP coli strain bacterium liquid step 2 being obtained is 1:(90 ~ 110 with the LB fluid nutrient medium of step 1 by volume) mix, obtain detecting the cultivation bacterium liquid of Fluoride Concentration In Waters;
The cultivation bacterium liquid of the detection Fluoride Concentration In Waters four, the water sample of different fluorine content being obtained with step 3 is by volume for (1 ~ 2): 1000 mix respectively, is under the condition of 34 ~ 40 ℃ 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, under fluorescent microscope, carry out fluorescence intensity detection, utilize image software microscopic analysis system quantifies fluorescence intensity to characterize bacterial growth situation, set up relation curve and the regression equation y=-8.6575x+994.36 of bacterium liquid fluorescence intensity and fluorine content; The fluorine concentration that wherein x is fluorine-containing water sample, y is the fluorescence intensity of fluorine-containing bacterium liquid; 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, 10mg/L, 20mg/L and 50mg/L;
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 under the condition of 34 ~ 40 ℃, 4 ~ 6h is cultivated in concussion 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 obtaining 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 step 2 is 1.5:1000.
3. a kind of method of utilizing photobacteria to detect Fluoride Concentration In Waters described in claim 1 or 2, is characterized in that in step 2, cultivation temperature is 37 ℃, 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 plateau OP50-GFP coli strain bacterium liquid that in step 3, step 2 obtains 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 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 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 step 5 is 37 ℃, and the time is 5h.
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| CN112034021B (en) * | 2020-09-08 | 2022-02-01 | 南京农业大学 | Method for detecting fluorine content in escherichia coli |
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