CN105821111B - Application of glucometer in rapid detection of biotoxicity of environmental pollutants - Google Patents
Application of glucometer in rapid detection of biotoxicity of environmental pollutants Download PDFInfo
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- CN105821111B CN105821111B CN201510012584.9A CN201510012584A CN105821111B CN 105821111 B CN105821111 B CN 105821111B CN 201510012584 A CN201510012584 A CN 201510012584A CN 105821111 B CN105821111 B CN 105821111B
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- 238000001514 detection method Methods 0.000 title claims abstract description 52
- 239000003344 environmental pollutant Substances 0.000 title abstract description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 70
- 239000008103 glucose Substances 0.000 claims abstract description 70
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- 239000007788 liquid Substances 0.000 claims abstract description 11
- 230000000813 microbial effect Effects 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 47
- 241000588724 Escherichia coli Species 0.000 claims description 45
- 230000002401 inhibitory effect Effects 0.000 claims description 43
- 238000000034 method Methods 0.000 claims description 31
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- 244000063299 Bacillus subtilis Species 0.000 claims description 24
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 22
- 241000894006 Bacteria Species 0.000 claims description 16
- 239000000356 contaminant Substances 0.000 claims description 14
- 230000007613 environmental effect Effects 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 13
- 241000235342 Saccharomycetes Species 0.000 claims description 12
- 239000002068 microbial inoculum Substances 0.000 claims description 8
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- 239000013049 sediment Substances 0.000 claims description 6
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- 238000011156 evaluation Methods 0.000 claims description 3
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- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 2
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims 2
- 241000588769 Proteus <enterobacteria> Species 0.000 claims 1
- 230000001376 precipitating effect Effects 0.000 claims 1
- 239000002689 soil Substances 0.000 abstract description 19
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- 230000001988 toxicity Effects 0.000 abstract description 8
- 231100000419 toxicity Toxicity 0.000 abstract description 8
- LLEMOWNGBBNAJR-UHFFFAOYSA-N biphenyl-2-ol Chemical compound OC1=CC=CC=C1C1=CC=CC=C1 LLEMOWNGBBNAJR-UHFFFAOYSA-N 0.000 description 30
- 235000010292 orthophenyl phenol Nutrition 0.000 description 15
- 239000000126 substance Substances 0.000 description 15
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 11
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- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 4
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an application of a glucometer in rapidly detecting biotoxicity of environmental pollutants, and a detection method comprises the following steps: 1) preparing a microbial liquid; 2) mixing and incubating a microbial solution, a glucose solution, a culture medium and a sample to be tested; 3) detecting the concentration of glucose in a sample to be detected by a glucometer; 4) and evaluating the biological toxicity of the sample to be tested. The glucometer has the characteristics of short detection time, low cost, convenient operation and the like when being used for detecting the concentration of the glucose in the sample. Therefore, the glucometer is combined with the microorganism to be used for detecting the biotoxicity of the sample, has the characteristics of time saving, economy and easy operation, provides a new way for detecting the biotoxicity, and simultaneously provides a basis for determining a treatment scheme of sewage and polluted soil.
Description
Technical field
The present invention relates to acute biological toxicity detection fields, more particularly, to the journey based on microorganism to glucose consumption
Degree, the bio-toxicity of environmental contaminants is quickly detected using blood glucose meter.
Background technique
With outstanding day by day, the bio-toxicity detection of problem of environmental pollution, the especially quick detection of bio-toxicity is heavy
The property wanted more highlights.Bio-toxicity is traditionally detected with fish and water flea, but often low-response, time-consuming, costly.Microorganism
Reproduction speed is fast, cheap, is the ideal material for detecting bio-toxicity.
Method currently with microorganism detection environmental contaminants bio-toxicity can be divided into two major classes: colorimetric method and electrochemistry
Method.
As its name suggests, colorimetric method judges the relative size of bio-toxicity according to the depth of coloration, and most successful example isAnd Toxi-ChromoThat utilize is vibrio fischeri (Vibrio fischeri).
Vibrio fischeri is one kind of photobacteria, and main existence can naturally issue fluorescence in briny environment.When noxious material exists
When, the remitted its fury of the fluorescence issued, thus the relatively large of a certain substance toxicity can be judged according to the power of fluorescence
It is small.Toxi-ChromoUtilize the Escherichia coli induced.The Escherichia coli induced can express beta galactosidase,
The enzyme has higher susceptibility to heavy metal ion, and can hydrolyze specific substrate and then generate color.When noxious material is deposited
When, beta galactosidase is suppressed, so that hydrolyzed amount of substrate is reduced, the color of formation is shallower, according to the depth of color
It can judge the relative size of a certain substance bio-toxicity.AlthoughAnd Toxi-ChromoQuotient
Industry, but still only exist very big limitation: firstly, it is necessary to which special spectrophotometer, at high cost, it is unfavorable for being widely applied；
Secondly, vibrio fischeri strain is rare, it is expensive.The Induction Process of Escherichia coli is complicated, and to operator, there is also potential
Security risk.
Electrochemical process mainly utilizes the electron mediators such as benzoquinones, the potassium ferricyanide.The breathing that electron mediator can participate in microorganism is made
With being reduced, the electron mediator of reduction-state is aoxidized in electrode surface generates oxidation current.The presence of noxious material can inhibit micro- life
The respiration of object causes final oxidation current to reduce so that the electron mediator amount being reduced is reduced.Therefore, according to oxidation
The size of electric current may determine that the relative size of a certain substance bio-toxicity.But electrochemical method needs electrochemical workstation etc. special
Industry instrument, it is expensive.Meanwhile electrochemical process requires operator to have certain understanding to Electrochemistry Knowledge, and to electrification
Learning work station has higher proficiency.These all limit the application and popularization of electrochemical method.
Accordingly, it is desirable to provide a kind of low in cost, the method for easily operated detection environmental contaminants acute biological toxicity.
Summary of the invention
The purpose of the present invention is to provide a kind of application of blood glucose meter in quickly detection environmental contaminants bio-toxicity.
Preferably, blood glucose meter is portable glucose meter, more preferably commercialized portable glucose meter.
Application of the blood glucose meter in detection environmental contaminants bio-toxicity, detection method include the following steps:
1) microbial inoculum is prepared；
2) microbial inoculum, glucose solution, culture medium and sample to be tested are mixed and is incubated for；
3) with the concentration of glucose in blood glucose meter detection sample to be tested；
4) bio-toxicity of sample to be tested is evaluated.
The microorganism is Gram-negative bacteria, gram-positive bacteria or fungi；Wherein the Gram-negative bacteria is big
Enterobacteria, Psychrobacter, nitrosomonas, preferably Escherichia coli；The gram-positive bacteria is bacillus subtilis, becomes
Shape bacillus, actinomyces, preferably bacillus subtilis；The fungi is saccharomycete, Candida albicans, the stealthy bacterium of white, preferably
For saccharomycete.
The method for wherein preparing microbial inoculum includes:
1) microbial inoculant is cultivated in culture medium to logarithmic growth phase；
2) thallus is collected, bacterial sediment is obtained；
3) washing thalline precipitates；
4) disperse bacterial sediment with dispersion liquid.
In the above method, the culture medium of the microorganism culture medium for being suitable for microorganism growth well known in the art is cultivated,
Such as the culture medium of Escherichia coli inoculation is LB culture medium, SOB culture medium or SOC culture medium etc., preferably LB culture medium；It is described withered
The culture medium of careless bacillus is beef-protein medium, LBG culture medium, preferably beef-protein medium；Institute
The culture medium for stating saccharomycete is YPD culture medium, YPED culture medium, YPEG culture medium etc., preferably YPD culture medium.
Further, the condition for cultivating microorganism is also it is known in the art that including but is not limited to, the culture of Escherichia coli
Condition is 37 DEG C and cultivates 16h in LB culture medium；The condition of culture of bacillus subtilis is 37 DEG C in beef extract-peptone culture
Base culture 24 hours；The condition of culture of saccharomycete is 30 DEG C and cultivates 16 hours in YPD culture medium.
In the above-mentioned methods, it is preferable that collect the thallus of culture, with centrifugal process to obtain bacterial sediment.
Conventional buffer can be used in the washing solution of bacterial sediment, and follow-up test may be influenced in thallus culture to remove
As a result substance.As phosphate buffer, Tris-Hcl buffer, HEPEs buffer, boric acid-borate buffer solution or sodium chloride are molten
Liquid.Preferably, sodium chloride solution, PBS solution, TBS solution or TBE solution that the washing solution is 0.85%.
Preferably, the dispersion liquid is aqueous solution, sodium chloride solution or culture medium, preferably aqueous solution.
Glucose is nutriment necessary to most of microorganism grows.The presence of noxious material can inhibit microorganism
Growth and breeding, so that microorganism reduces the consumption of glucose.Therefore, according to the number of glucose utilization, can sentence
The relative size of disconnected a certain substance toxicity out.Blood glucose meter be at present be mass produced popularization dedicated for Portugal in blood of human body
The portable device of grape sugared content can quickly detect the concentration of glucose in blood, thus it is proposed that utilizing blood glucose meter
The concentration of glucose in sample to be tested is detected, preferably with commercialized blood glucose meter detection pollutant to the bio-toxicity of thallus.
Preferably, as follows with bio-toxicity, the calculation formula of inhibiting rate in inhibiting rate evaluation sample to be tested: inhibiting rate
(%)=(Nc-Ne)/Nc × 100%
Wherein NCFor the concentration of glucose of the first sample to be tested, Ne is the concentration of glucose of the second sample to be tested.
The incubation time of first sample to be tested and the second sample to be tested is identical as incubation temperature.Preferably, detection environment is dirty
When microorganism used in the method for dye object bio-toxicity is Escherichia coli or bacillus subtilis, incubation temperature is 37 DEG C；It is preferred that
Ground, detect environmental contaminants bio-toxicity method used in microorganism be fungi when, incubation temperature be 30 DEG C.
Preferably incubation time is 30-90 minutes.Preferably, when microorganism is Escherichia coli, incubation time is 60 minutes；
Preferably, when microorganism is bacillus subtilis, incubation time is 30-90 minutes；Preferably, it when microorganism is fungi, is incubated for
Time is 30-90 minutes.
When the first sample to be tested and the second sample to be tested are different environmental contaminants samples, inhibiting rate indicates that environment is dirty
The relative size of bio-toxicity between dye object.
First sample to be tested can be check sample, and the check sample refers to microbial inoculum, glucose solution, culture
Base and contrast solution are mixed and are incubated for, and wherein contrast solution is aqueous solution, culture medium, sodium chloride solution, preferably aqueous solution.This
When inhibiting rate indicate be size of second sample to be tested relative to the bio-toxicity of check sample.
In addition this method can also be used for the bio-toxicity of detection different material, test substance can be configured to various concentration
Sample to be tested, detects the concentration of glucose of different samples to be tested, and calculates the inhibiting rate of each sample, draws concentration of glucose-
The curve of inhibiting rate can calculate maximum half-inhibitory concentration IC according to inhibiting rate curve50(maximum half
Inhibition concentration), i.e., when inhibiting rate be 50% when substance concentration.IC50It is a certain substance biology of evaluation
The important indicator of toxicity size.
The environmental contaminants are the mixture of organic pollutant, inorganic pollution or both；Preferably, the inorganic dirt
Dye object is soluble in the inorganic compound of water, such as heavy metallic salt, inorganic acid, inorganic base noxious material；Preferably, described organic
Pollutant is the organic compound for being insoluble in water, such as benzene, phenol, o-phenyl phenol, chlorophenesic acid.
In addition, the present invention can also be used in the detection for the bio-toxicity for being administered sewage, containing there are many bio-toxicities in sewage
Substance, such as heavy metal, organic pollutant.The improvement of present sewage includes improving water quality with aquatic animal, but work as sewage
Bio-toxicity it is high when, launching aquatic animal can not survive, not only cannot purify sewage also and will cause waste economically, thus
When the bio-toxicity of sewage reaches reasonable range, launching aquatic animal can be only achieved the purpose for further improving water quality.This
The method of invention is capable of the bio-toxicity of very convenient efficiently detection sewage, and the determination for waste water control scheme provides support.
Moreover, the present invention can also be used in the detection of the bio-toxicity of contaminated soil.Containing more in polluted soil
Kind bio-toxicity substance, such as heavy metal ion, organic pollutant.At present soil remediation include plant absorption, microorganism remediation,
The methods of Co-bioremediation.But these methods can only inform which kind of journey the content reparation of pollutant in soil has arrived in amount
Degree, without be able to tell that people's soil remediation to it is different degrees of when corresponding toxicity.Therefore, pass through the bio-toxicity of soil, energy
The repair for further instructing soil combines the reparation degree of soil and its influence to biology, is soil remediation
More reference informations are provided.
Beneficial effects of the present invention are as follows:
Present invention utilizes have been commercialized the blood glucose meter used at present to detect the content of glucose in sample.Blood glucose
Instrument for have the characteristics that in sample when examination of glucose concentration detection time it is short, it is low in cost, convenient for operating.Thus blood glucose meter
It is used for the detection of sample bio-toxicity in conjunction with microorganism, there is time saving, economic, easily operated feature, and examine for bio-toxicity
That surveys provides new approach, while also providing foundation for the resolution determination of sewage.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is shown in embodiment 1 based on microorganism and commercialization portable glucose meter detection environmental contaminants bio-toxicity
Feasibility verifying.
Fig. 2 shows various concentration Cd is detected in embodiment 22+To the inhibiting rate curve of Escherichia coli bio-toxicity.
Fig. 3 shows detection various concentration As in embodiment 32+To the inhibiting rate curve of Escherichia coli bio-toxicity.
Fig. 4 shows detection various concentration Pb in embodiment 42+To the inhibiting rate curve of Escherichia coli bio-toxicity.
Fig. 5, which is shown, detects various concentration 3,5- chlorophenesic acid to the inhibiting rate of Escherichia coli bio-toxicity in embodiment 5 five
Curve.
Fig. 6, which is shown, detects various concentration o-phenyl phenol to the inhibiting rate curve of Escherichia coli bio-toxicity in embodiment 6.
Fig. 7, which is shown, detects various concentration phenol to the inhibiting rate curve of Escherichia coli bio-toxicity in embodiment 7.
Fig. 8 shows detection various concentration Cd in embodiment 82+To the inhibiting rate curve of bacillus subtilis bio-toxicity.
Fig. 9 shows detection various concentration Pb in embodiment 92+To the inhibiting rate curve of bacillus subtilis bio-toxicity.
Figure 10, which is shown, detects various concentration phenol to the inhibiting rate curve of bacillus subtilis bio-toxicity in embodiment 10.
Figure 11, which is shown, detects inhibition of the various concentration o-phenyl phenol to bacillus subtilis bio-toxicity in embodiment 11
Rate curve.
Figure 12 shows detection various concentration 3 in embodiment 12, and 5- chlorophenesic acid is bent to the inhibiting rate of saccharomycete bio-toxicity
Line
Figure 13, which is shown, detects various concentration o-phenyl phenol to the inhibiting rate curve of saccharomycete bio-toxicity in embodiment 13
Figure 14, which is shown, detects different sewage sample to the inhibiting rate of Escherichia coli bio-toxicity in embodiment 14
Figure 15, which is shown, detects different soils sample to the inhibiting rate of Escherichia coli bio-toxicity in embodiment 15
Specific embodiment
In order to illustrate more clearly of the present invention, the present invention is done further below with reference to preferred embodiments and drawings
It is bright.Similar component is indicated in attached drawing with identical appended drawing reference.It will be appreciated by those skilled in the art that institute is specific below
The content of description is illustrative and be not restrictive, and should not be limited the scope of the invention with this.
Embodiment 1: Escherichia coli and blood glucose meter detect Cu2+Bio-toxicity
Escherichia coli (ATCC 25922) cultivate 16 hours at 37 DEG C in LB culture medium, wherein the configuration side of LB culture medium
Method is as follows: it takes 0.5g beef extract, 1g peptone and 0.5g sodium chloride to be dissolved in 100mL deionized water, adjusts pH to 7.0-7.4,
120 DEG C of sterilizing 20min, natural cooling in high pressure steam sterilization.
Escherichia coli are collected by centrifugation, 6000 revs/min of centrifugation 6min collect thallus；The Escherichia coli collected are washed, are used
0.85% sodium chloride solution cleans thallus resuspension, then collects thallus with 5000 revs/min of centrifugation 5min, is repeated once above-mentioned
Cleaning operation；Thallus will finally be obtained after cleaning to be scattered in 0.85% sodium chloride solution, obtaining concentration is OD600=2.5
Bacterium solution.
Taking 5 volumes is the centrifuge tube of 1.5ml, and number is 1,2,3,4,5 respectively.Wherein 1 (it is not added as check sample
Any poisonous substance, but the sodium chloride solution with noxious material same volume is added), remaining is testing tube.Successively to each Guan Zhongjia
Enter the LB culture medium of 100 μ L, 10 μ L glucose solution (2%w/V), 80 μ L concentration are OD600=2.5 Escherichia coli bacteria liquids and 10 μ
The Cu of L various concentration2+Solution.Make Cu in each centrifuge tube2+Final concentration be followed successively by 0,2,4,6,8mg/L.Each centrifuge tube is placed in
60 minutes in 37 DEG C of constant incubators, the concentration of glucose in each centrifuge tube is then detected with commercialization portable glucose meter.Root
According to the difference of concentration, the Cu of various concentration can be calculated2+To the inhibiting rate of Escherichia coli Growth breeding, and then learn Cu2+Biology
The relative size of toxicity.
Embodiment 1 the results show that with copper ion concentration increase, concentration of glucose is also stepped up in sample, table
Bright Cu2+To the growth and breedings of Escherichia coli, there are inhibiting effect, so that it reduces the consumption of glucose, i.e., inhibiting rate is rising
It is high.This also indicates that the bio-toxicity based on microorganism and commercialization portable glucose meter detection environmental contaminants is feasible.
Embodiment 2: Escherichia coli and blood glucose meter detect Cd2+Bio-toxicity
Cd is detected according to the method for embodiment 12+To the bio-toxicity of Escherichia coli.Cd2+Final concentration is respectively 0,5,10,
15, when 20,25mg/L caused by inhibiting rate be respectively 0,18%, 35%, 53%, 59%, 65%, and calculate to obtain Cd2+IC50For
14.2mg/L。
Embodiment 3: Escherichia coli and blood glucose meter detect As2+Bio-toxicity
As is detected according to the method for embodiment 12+To the bio-toxicity of Escherichia coli.As2+Final concentration is respectively 0,1,2,3,
4, when 5,6,7mg/L caused by inhibiting rate be respectively 0,10%, 15%, 30%, 40%, 50%, 60%, 83%, and calculate to obtain As2+
IC50For 5mg/L.
Embodiment 4: Escherichia coli and blood glucose meter detect Pb2+Bio-toxicity
Pb is detected according to the method for embodiment 12+To the bio-toxicity of Escherichia coli.Pb2+Final concentration is respectively 0,25,50,
75, when 100,125,150,175mg/L caused by inhibiting rate be respectively 0,16%, 21%, 27%, 37%, 42%, 53%,
68%, and calculate to obtain Pb2+IC50For 143mg/L.
Embodiment 5: the bio-toxicity of Escherichia coli and blood glucose meter detection 3,5- parachlorophenol
Bio-toxicity of the detection 3,5- parachlorophenol to Escherichia coli according to the method for embodiment 1.3,5- parachlorophenol is whole
Inhibiting rate caused by when concentration is 0,5,10,15,20mg/L is respectively 0,13.7%, 36.4%, 59%, 72.7%, finally
The IC of 3,5- parachlorophenol50For 13mg/L.
Embodiment 6: the bio-toxicity of Escherichia coli and blood glucose meter detection o-phenyl phenol
Bio-toxicity of the detection o-phenyl phenol to Escherichia coli according to the method for embodiment 1.O-phenyl phenol final concentration
Respectively 0, when 5,10,15,20,25,30,35,40,45mg/L caused by inhibiting rate be respectively 0,5.9%, 12%, 17.6%,
23.5%, 30%, 41.2%, 52.9%, 71%, 82%, and calculate o-phenyl phenol IC50For 33.7mg/L.
Embodiment 7: the bio-toxicity of Escherichia coli and blood glucose meter detection phenol
Bio-toxicity of the detection phenol to Escherichia coli according to the method for embodiment 1.Phenol final concentration is respectively 0,10,
20, when 40,60,80,120mg/L caused by inhibiting rate be respectively 0,4.2%, 16.7%, 33.3%, 41.7%, 50%,
62.5%, 75%, and calculate phenol IC50For 80mg/L.
Embodiment 8: bacillus subtilis and blood glucose meter detect Cd2+Bio-toxicity
Bacillus subtilis (CGMCC 1.1086) is cultivated 24 hours in beef-protein medium at 37 DEG C, wherein
The configuration method of bacillus subtilis bacterium culture medium is as follows: 1g peptone, 0.3g beef extract and 0.5g sodium chloride being taken to be dissolved in 100ml
In ionized water, pH to 7.0-7.4,120 DEG C of sterilizing 20min, natural cooling in high pressure steam sterilization are adjusted.
Bacillus subtilis is collected by centrifugation, 6000 revs/min of centrifugation 6min collect thallus；Wash the bacillus subtilis collected
Bacterium is cleaned thallus resuspension with PBS solution, then with 5000 revs/min of centrifugation 5min collection thallus, is repeated once above-mentioned clear
Wash operation；Thallus will finally be obtained after cleaning to be scattered in 0.85% sodium chloride solution, obtaining concentration is OD600=2.5 bacterium
Liquid.
Detect Cd2+To the bio-toxicity of bacillus subtilis.Taking 7 volumes is the centrifuge tube of 1.5mL, respectively number be 1,
2,3,4,5,6,7, wherein 1 conduct check sample (any poisonous substance is not added, but the water with noxious material same volume is added),
Remaining is test group.The culture solution of 100 μ L, 10 μ L glucose solution (2%W/V), 80 μ are successively added into each centrifuge tube
L concentration is OD600The Cd of=2.5 bacillus subtilis bacterium solution and 10 μ L various concentrations2+Solution.Make Cd in final each centrifuge tube2 +Final concentration be followed successively by 0,5,10,15,20,25,30mg/L.Each centrifuge tube is placed in 37 DEG C of constant incubators 60 minutes, so
The concentration for detecting glucose in each centrifuge tube with commercialization portable glucose meter afterwards can calculate difference according to the difference of concentration
The Cd of concentration2+To the inhibiting rate of Escherichia coli Growth breeding, above-mentioned different Cd2+Counted inhibiting rate is respectively 0,9.3%,
10%, 14%, 16.3%, 18.6%, 21%.
Embodiment 9: bacillus subtilis and blood glucose meter detect Pb2+Bio-toxicity
Pb is detected according to the method for embodiment 82+To the bio-toxicity of bacillus subtilis.Pb2+Final concentration be respectively 0,
30, when 60,90,120,150,180mg/L caused by inhibiting rate be respectively 0,11.6%, 14%, 25.6%, 34.9%,
48.9%, 53.5%, and calculate to obtain Pb2+IC50For 157mg/L.
Embodiment 10: the bio-toxicity of bacillus subtilis and blood glucose meter detection phenol
Phenol is detected to the bio-toxicity of bacillus subtilis according to the method for embodiment 8.The final concentration of phenol is respectively
0, when 10,20,40,60,80,100,120mg/L caused by inhibiting rate be respectively 0,6.4%, 8.5%, 12.8%, 17%,
19.1%, 20%, 21.3%.
Embodiment 11: the bio-toxicity of bacillus subtilis and blood glucose meter detection o-phenyl phenol
O-phenyl phenol is detected to the bio-toxicity of bacillus subtilis according to the method for embodiment 8.O-phenyl phenol
Inhibiting rate caused by when final concentration is respectively 0,10,20,30,40,50,60mg/L is respectively 0,10.6%, 15%, 17%,
19.1%, 21.3%, 23.4%.
Embodiment 12: the bio-toxicity of yeast and blood glucose meter detection 3,5- chlorophenesic acid
Yeast (S288C) is cultivated 16 hours in YPD culture medium at 30 DEG C, and wherein the configuration method of YPD culture medium is as follows:
2% glucose solution and 120 DEG C of sterilizing 20min in high-pressure steam sterilizing pan containing 1% yeast extract solution will be contained respectively, from
It is so cooling, then the two is aseptically mixed.
Yeast is collected by centrifugation, 6000 revs/min of centrifugation 6min collect thallus；The saccharomycete collected is washed, with TBS solution by bacterium
Weight is outstanding to be cleaned, then collects thallus with 5000 revs/min of centrifugation 5min, is repeated once above-mentioned cleaning operation；It finally will be clear
It obtains thallus after washing to be scattered in 0.85% sodium chloride solution, obtaining concentration is OD600=2.5 bacterium solution.
3,5- chlorophenesic acid is detected to the bio-toxicity of saccharomycete.Taking 7 volumes is the centrifuge tube of 1.5mL, is numbered respectively
It is 1,2,3,4,5.Wherein 1 as check sample (any poisonous substance is not added, but the water with noxious material same volume is added),
Remaining is testing tube.The culture solution of 100 μ L, 10 μ L glucose solution (2%W/V), 80 μ L are successively added into each centrifuge tube
Concentration is OD600The 3,5- chlorophenesic acid solution of=2.5 saccharomycete bacterium solution and 10 μ L various concentrations.Make 3,5- in each centrifuge tube
The ultimate density of chlorophenesic acid is followed successively by 0,5,10,15,20mg/L.It is small that each centrifuge tube is placed in 30 DEG C of constant incubators 16
When, inhibiting rate caused by the concentration of above-mentioned different 3,5- chlorophenesic acids is respectively 0,26%, 39%, 52%, 78%, and calculate 3,
The IC of 5- chlorophenesic acid50For 14.2mg/L.
Embodiment 13: the bio-toxicity of yeast and blood glucose meter detection o-phenyl phenol
O-phenyl phenol is detected to the bio-toxicity of saccharomycete according to the method for embodiment 12.The concentration of o-phenyl phenol point
Not Wei 0,10,20,30,40,50,60mg/L when caused by inhibiting rate be respectively 0,17.4%, 26%, 30%, 39.1%,
43.5%, 52.1%, and the IC of o-phenyl phenol is obtained finally50For 57.6mg/L.
As seen from the above-described embodiment, Escherichia coli, hay bacillus and yeast can be detected with blood glucose meter combination heavy metal and
The bio-toxicity of organic compound.
Embodiment 14: the detection (sewage) of environmental contaminants
The bacterium solution preparation such as embodiment 1 of Escherichia coli.
Sewage 10mL is obtained respectively from refuse landfill, chemical laboratory inorganic waste liquids bucket, electroplate factory.6 sample cells are taken,
1,2,3,4 are respectively labeled as, No. 1 is control tube, and No. 2-4 pipe is sewage sample to be tested pipe, is successively added 100 μ L's into each pipe
LB culture medium, 10 μ L glucose solution (2%w/V), 80 μ L concentration are the Escherichia coli bacteria liquid of OD600=2.5, in No. 1 pipe
10 μ L distilled water are added, 10 μ L are sequentially added in 2-4 pipe from refuse landfill, chemical laboratory inorganic waste liquids bucket, plating
The sewage that factory obtains.Each centrifuge tube is placed in 37 DEG C of constant incubators 60 minutes, then with commercialization portable glucose meter inspection
Survey the concentration of glucose in each centrifuge tube.According to the difference of concentration, it is numerous to Escherichia coli Growth that different sewage sample can be calculated
The inhibiting rate grown, respectively 20%, 32% and 60%, and then learn the bio-toxicity of different sewage sample, while passing through comparison
Concentration of glucose in different sewage sample can compare the height of different sewage sample bio-toxicity.
Embodiment 15: the detection of environmental contaminants
The bacterium solution preparation such as embodiment 1 of Escherichia coli.
Contaminated soil 5g is obtained respectively near Beijing North 4th Ring Road gas station, Beijing experimental plot, Hainan mining area (to divide
Bian Hao not be sample 1, sample 2, sample 3), it is separately added into the volumetric flask of 1,2, No. 3 10ml, distilled water is added and is settled to
10ml is mixed by inversion sufficiently to dissolve the polluter in soil；Then solution is transferred to centrifuge tube, 12000rpm centrifugation 5
Minute, supernatant is transferred in new centrifuge tube, as contaminated soil sample to be tested.1,2,3,4 sample cell is taken, successively to each
The LB culture medium of 100 μ L, 10 μ L glucose solution (2%w/V) are added in pipe, 80 μ L concentration are the Escherichia coli of OD600=2.5
10 μ L distilled water are added in No. 1 pipe, sequentially adds in 2-4 pipe and is derived from Beijing North 4th Ring Road gas station, Beijing for bacterium solution
10 μ L of contaminated soil sample to be tested near experimental plot, Hainan mining area.Each centrifuge tube is placed in 60 in 37 DEG C of constant incubators
Minute, the concentration of glucose in each centrifuge tube is then detected with commercialization portable glucose meter.According to the difference of concentration, can calculate
The inhibiting rate that this three kinds different contaminated soil samples breed Escherichia coli Growth out, respectively 23.8%, 4.8%, 16.7%,
And then learn the bio-toxicities of different contaminated soil samples, while by concentration of glucose in comparison different soils sample, it can be with
Compare the height of different contaminated soil sample bio-toxicities.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention may be used also on the basis of the above description for those of ordinary skill in the art
To make other variations or changes in different ways, all embodiments can not be exhaustive here, it is all to belong to this hair
The obvious changes or variations that bright technical solution is extended out are still in the scope of protection of the present invention.
Claims (11)
1. quickly detecting the detection method of environmental contaminants bio-toxicity using blood glucose meter, which is characterized in that detection method is as follows:
1) microbial inoculum is prepared；
2) two samples to be tested are mixed respectively with microbial inoculum, glucose solution and culture medium and is incubated for 30-60 min；
3) concentration of glucose in two samples to be tested is detected respectively with commercially available portable glucose meter；
4) bio-toxicity of sample to be tested is evaluated；
With the bio-toxicity of inhibiting rate evaluation sample to be tested in step 4), the calculation formula of the inhibiting rate be inhibiting rate (%)=
(Nc-Ne)/Nc × 100%；Wherein NCFor the concentration of glucose of the first sample to be tested, Ne is the grape to the second sample to be tested
Sugared concentration.
2. detection method according to claim 1, which is characterized in that the method for preparing microbial inoculum includes:
1) microbial inoculant is cultivated in culture medium to logarithmic growth phase；
2) thallus is collected, bacterial sediment is obtained；
3) washing thalline precipitates；
4) disperse bacterial sediment with dispersion liquid, microbial inoculum is made.
3. detection method according to claim 1, which is characterized in that the microorganism is Gram-negative bacteria, gram
Positive bacteria or fungi.
4. detection method according to claim 1, which is characterized in that the Gram-negative bacteria is Escherichia coli, thermophilic cold
Bacillus or nitrosomonas.
5. detection method according to claim 4, which is characterized in that the Gram-negative bacteria is Escherichia coli.
6. detection method according to claim 1, which is characterized in that the gram-positive bacteria be bacillus subtilis,
Proteus or actinomyces.
7. detection method according to claim 6, which is characterized in that the gram-positive bacteria is bacillus subtilis.
8. detection method according to claim 1, which is characterized in that the fungi is saccharomycete, Candida albicans or white
Color stealth bacterium.
9. detection method according to claim 8, which is characterized in that the fungi is saccharomycete.
10. detection method according to claim 1, which is characterized in that the solution of washing thalline precipitating is in step 2
0.85% sodium chloride solution, PBS solution, TBS solution or TBE solution.
11. detection method according to claim 1, which is characterized in that dispersion liquid is aqueous solution, culture medium in step 4)
Or sodium chloride solution.
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