CN108226493A - A kind of industrial wastewater bio-toxicity detection method based on genetic recombination photogen - Google Patents
A kind of industrial wastewater bio-toxicity detection method based on genetic recombination photogen Download PDFInfo
- Publication number
- CN108226493A CN108226493A CN201810023633.2A CN201810023633A CN108226493A CN 108226493 A CN108226493 A CN 108226493A CN 201810023633 A CN201810023633 A CN 201810023633A CN 108226493 A CN108226493 A CN 108226493A
- Authority
- CN
- China
- Prior art keywords
- photogen
- sample
- industrial wastewater
- toxicity
- dilution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of industrial wastewater bio-toxicity detection method based on genetic recombination photogen, belongs to environmental monitoring technology field.This method selects photogen Escherichia coli HB101 pUCD607 to use bacterium as detection, includes the following steps：The first step prepares the bacteria suspension of photogen.Second step, sample gradient dilution.Third walks, and sample comprehensive toxicity measures.The method of the present invention is not stringent to pH area requirements, does not need to adjust salinity, is suitable for the photogen comprehensive toxicity test of industrial wastewater under condition of different pH, very sensitive to the comprehensive toxicity of industrial wastewater.The present invention establishes the micropore board measuring method of photogen, white 96 orifice plates of sampling carry out the detection of bioluminescence intensity for experiment container in microplate reader, 96 holes of every block of plate can measure simultaneously, and required sample volume can be reduced into 250 μ L from 10mL, and experimental implementation is simple and convenient, quick.
Description
Technical field
The invention belongs to environmental monitoring technology fields, and in particular to a kind of Industry Waste based on genetic recombination photogen is aquatic
Object detection method of toxicity.
Background technology
Industrial wastewater generally characterizes the extent of injury of aquatic ecosystem with comprehensive toxicity index, and the index is in waste water
Processing, discharge and monitoring party mask are significant.Conventional Physico-chemical tests method can only provide a small number of specific objective pollutants
Content data, it is difficult to evaluate the comprehensive toxicity of waste water.Bio-toxicity detection method can reflect pollutional load and biologically
Between quantitative relationship, embodiment be all components in complex system comprehensive function, including that may be present between each component
Adduction, antagonism or synergistic effect.
When photogen is exposed in poisonous and hazardous industrial wastewater, luminous intensity is suppressed, and luminous intensity is suppressed
Degree and comprehensive toxicity intensity there is correlation.Photogen is can to emit visible fluorescence under a kind of normal physiological condition
Bacterium.Luminescence process is a kind of metabolic processes in bacterial body, i.e., the light respiration process on oxidation-respiration chain.It is glimmering in bacterium
Under the catalytic action of light enzyme, oxidation occurs for reduced form flavine monokaryon glycosides (FMNH2) and molecular oxygen and a long-chain aldehyde in thalline also
Original reaction, generation flavine monokaryon glycosides (FMN), carboxylic acid (RCOOH) and water, and with the release of light.When photogen touches industry
In waste water during toxic contaminants substance, the metabolism of bacterium can be influenced or interfere, so as to decline the luminous intensity of bacterium.It utilizes
This characteristic of photobacteria, using the variation of luminous intensity as index, to detect the biology of poisonous and harmful substance in industrial wastewater
Toxicity.
It is concerned when Photobacterium Phosphoreum Toxicity experiment is because its reaction time is short, high sensitivity the advantages that, is detected with photogen
Water body toxicity has become a kind of simple, quick bio-toxicity detection means.It is bright hair to study both at home and abroad at present more
Polished rod bacterium, Qinghai Vibrion and Fermi operator etc..The Microtox methods to grow up the 1980s are with ocean photogen
Vibrio fishieri are to indicate biology progress toxotest, this method fast, economical, and application is relatively broad.But its pH range
It is required that stringent (6~8), and ocean photogen be because it need to add a certain amount of NaCl (2%~3%) in test, and be not suitable for light
The toxotest of water water body.Photobacterium phosphoreum is the common ocean photogen in China, and pH accommodations are narrow, it is necessary to by sample pH value
It could be measured after being adjusted to 7.3~7.5, sample toxicity may be caused to be distorted.Industrial wastewater complicated component, pH ranges are big, because
It is necessary to develop a kind of photogen comprehensive toxicity test method more suitable for industrial wastewater for this.
Invention content
The shortcomings that in order to overcome existing seawater luminescent bacteria toxicity test technology and deficiency, the purpose of the present invention is to provide
A kind of industrial wastewater bio-toxicity detection method based on genetic recombination photogen.This method is not stringent to pH area requirements, no
It needs to adjust salinity, is suitable for the photogen comprehensive toxicity test of industrial wastewater under condition of different pH.The method overcome seawater
Photogen requires the limitation stringent, acute toxicity testing result is caused to be distorted to pH and salt.Specific method selects photogen
Escherichia coli HB101 pUCD607 use bacterium as detection, include the following steps：The first step prepares photogen
Bacteria suspension.Second step, sample gradient dilution.Third walks, and sample comprehensive toxicity measures.
The purpose of the present invention is achieved through the following technical solutions：
A kind of industrial wastewater bio-toxicity detection method based on genetic recombination photogen, the method are selected
Escherichia coli HB101 pUCD607 use bacterium as detection, include the following steps：
The first step prepares the bacteria suspension of photogen：It takes and freezes strain, centrifuged after defrosting, cultivated after removing supernatant,
First time resuscitation fluid renewed vaccination cultivates again to certain absorbance (OD) value to new culture solution, liquid is discarded supernatant after centrifugation, make
It is spare into bacteria suspension；
Second step, sample gradient dilution：First industrial wastewater is diluted successively from stoste by 10 times of dilution ratios, Preliminary detection
After toxicity, best dilution range is determined, then carry out sample dilution by 2 times of gradients successively from the upper limit of best dilution range；
Third walks, and sample comprehensive toxicity measures：For sample after transfer dilution to ELISA Plate, blank control and standard control are same
Shi Jinhang is added in after bacteria suspension and is measured bioluminescence intensity by microplate reader.
Photogen used in the present invention is a strain gene engineering bacterium, entitled Escherichia coli HB101
pUCD607.A plasmid pUCD607 for carrying luxCDABE genes is inserted in this strain gene group.
Preferably, the absorbance described in the first step (OD) value is OD550=0.6~1.
Preferably, the ELISA Plate described in third step is 96 hole elisa Plates.
The present invention is had the following advantages relative to the prior art and effect：
(1) photogen Escherichia coli HB101pUCD607 used in the present invention are current domestic suitable different pH
Under the conditions of industrial wastewater photogen, it is very sensitive to the comprehensive toxicity of industrial wastewater.
(2) present invention establishes the micropore board measuring method of photogen, and white 96 orifice plates of sampling are experiment container in enzyme mark
The detection of bioluminescence intensity is carried out on instrument, 96 holes of every block of plate can measure simultaneously, and required sample volume can be from
10mL is reduced into 250 μ L, and experimental implementation is simple and convenient, quick.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, but the implementation of the present invention is not limited to this.
The general embodiment of the present invention：
Key instrument and consumptive material：Microplate reader, constant temperature air bath oscillation shaking table, superclean bench, liquid-transfering gun, multichannel liquid relief
Device, vortex mixer, oscillator plate, 96 orifice plate of white flat bottom, loading slot, timer, EP pipes, conical flask.
Main agents：Kanamycin sulfate, tryptone, dusty yeast, sodium chloride, glucose, potassium chloride, zinc sulfate.
Main solution is prepared：
This method uses reagent unless otherwise stated, should be the analytical reagents for meeting national standard, distilled water or
The water of comparable purity.
(1) kanamycin mother liquid of 0.3g/L：3mg kanamycins is dissolved in 10mL distilled water, independent filtration sterilization, packing
In 1.5mL EP pipes, -20 DEG C of preservations.A pipe is taken to be placed on after 4 DEG C of defrostings for use.
(2) the LBG culture solutions containing 30 μ g/L kanamycins：10g tryptones, 5g dusty yeasts, 10g sodium chloride, 1g Portugals
Grape sugar is dissolved in 1000mL distilled water, 121 DEG C of high pressure sterilization 20min；It is to be cooled to after room temperature, add 100 μ L kanamycins
(0.3g/L), 4 DEG C of refrigerators preserve.
(3) 0.1M KCl solution：7.45g KCl are dissolved in 1000mL water, and high pressure sterilization is placed on 4 DEG C for use.
(4) 2M KCl solution：14.9g KCl are dissolved in 100mL water, and high pressure sterilization is placed on 4 DEG C for use.
(5)ZnSO4Mother liquor：10000mg Zn/L (every liter plus 1M of HCl 1mL acidifications).With mother liquor various concentration
Zn standard solution is as photogen quality control of the experiment.
The LB solid mediums of (6) 30 μ g/L kanamycins：100mL LB fluid nutrient mediums are prepared, add in 1.2-1.5g fine jades
Cosmetics, 121 DEG C of high pressure sterilization 20min；To be cooled to 50 DEG C, 10 μ L kanamycins (0.3g/L) are added, mixing is down flat plate.
The recovery of strain：This operation is carried out, and maintain the temperature at 25 DEG C under aseptic condition in superclean bench.
E.coli HB101pUCD607 cryopreservation tubes are taken out from -20 DEG C of refrigerators, 4 DEG C of defrostings is placed in, removes supernatant after centrifugation, are added in
1mL LB culture solutions, after mixing in LB fluid nutrient mediums of the access 10mL containing 30 μ g/L kanamycins.Or a small spoon is taken to shine
Bacterium freeze-dried powder is added in the sterile 0.1M KCl of 10mL gently shakes recovery 1h in 25 DEG C.In the LB for being added to 30 μ g/L kanamycins
It crosses on solid medium, 25 DEG C of 24~48h of culture.Picking single bacterium colony is inoculated in the LB liquid that 10mL contains 30 μ g/L kanamycins
In culture medium.On shaking table 16h or so is cultivated in 25 DEG C, 180r/min.
Then it takes in 200 μ L bacterium solutions to 200mL LB culture solutions, 25 DEG C on shaking table, 180r/min culture 16h or so, directly
To OD550It is 0.6~1, luminous value>100000.Then gained bacterium solution at 4000g, 4 DEG C is centrifuged 10 minutes, discarded supernatant
Liquid.Photogen is resuspended in spare in 20mL 0.1M KCl.It can determine to expand the amount cultivated according to actual needs.
Sample gradient dilutes：Industrial wastewater is diluted to successively from stoste by 10 times of dilution ratios using 100mL graduated cylinders
0.01% (being citing herein, the specific maximum ratio that dilutes is regarding wastewater toxicity intensity depending on) after Preliminary detection toxicity, determines most preferably
Dilution range.Then sample dilution is carried out by 2 times of gradients successively from the upper limit of best dilution range, it is to be measured.
Toxotest：Experiment uses flat 96 hole elisa Plates of White-opalescent as reaction vessel.In order to avoid in experiment
Time error, first is horizontally-arranged for blank control, that is, usesWater replaces sample, and each column sample well is done with first hole of row
Compare.It is parallel per Kong Weiyi, each sample do 4 it is parallel.The KCl solution and 210 μ L samples of 10 μ L 2M is added in per hole, so
Then mixing 1 minute on oscillator plate afterwards adds in 30 μ L bacterium solutions since first row with Multi-channel liquid transfer device toward every hole.Sample
The specific location mode of product is as shown in table 1.Since photogen is by time effects are larger and the time of microplate reader scanning entire plate is
50s, therefore speed is fast during addition bacterium solution, add entire plate in 1min as possible.Start to use timing during to first row plus bacterium solution
Device timing, after 15min by microplate reader (such as：Omega BMG companies, Germany) measure bioluminescence.Entirely
Experimentation controls temperature at 25 DEG C.
Quality control：The conditions such as temperature and pH in photogen experiment may all have an impact experimental result.The present invention
Choose Zn to be used as with reference to poisonous substance, be shown in Table 1, preceding 4 row of every piece of ELISA Plate be all measure a series of concentration Zn solution (0,0.05,
0.1st, 0.5,1,2,6 and 10mg/L) luminous inhibiting rate, and calculate its EC50 value, it is higher that dose-effect curve should be degree of fitting
S type curves, and EC50 values should (0.6~1.2mg/L, the range be repeatedly measured by this laboratory in certain range
EC50 values determine) to ensure validity, accuracy and the repeatability of experiment.
Data analysis：The relative luminous inhibiting rate calculation formula of photogen is as follows：
Shine relative inhibition (%)=(control luminous intensity-sample luminous intensity)/control luminous intensity × 100%.
Escherichia coli HB101 pUCD607 used in embodiment in document, " comment by industrial wastewater toxicity appraisal
The suggestion of valency method system and its application example .2011,20 (3):Disclosed in 549-559 ".
Location mode of the sample that table 1 is recommended in 96 hole elisa Plates
Note：First horizontally-arranged C is blank control, i.e., replaces sample with pure water.Preceding 4 row of every piece of ELISA Plate are all a series of
The Zn solution of concentration.It is parallel per Kong Weiyi, each sample do 4 it is parallel.KCl solution, the 210 μ L of 10 μ L 2M are added in per hole
Sample and 30 μ L bacterium solutions.
Embodiment 1
The comprehensive toxicity of coking wastewater is measured with photogen.
Water sample is acquired from certain segment process in certain Treatment of Coking Effluent workshop, each sampling point acquires 4 samples and repeats.Original is useless
Water sample is usedWater presses 1：10 dilution be configured to a series of concentration waste water solution (100%, 10%, 1%, 0.1%,
0.01%) it is spare.
E.coli HB101 pUCD607 cryopreservation tubes is taken to remove after centrifuging, 1mL LB culture solutions are added in after supernatant, after mixing
It accesses in LB fluid nutrient mediums of the 10mL containing 30 μ g/L kanamycins.25 DEG C on shaking table, 180r/min cultures 16h or so.So
After take in 200 μ L bacterium solutions to 200mL LB culture solutions, 25 DEG C on shaking table, 180r/min culture 16h or so, until OD550For
0.6~1, luminous value>100000.Then gained bacterium solution at 4000g, 4 DEG C is centrifuged 10 minutes, discards supernatant liquid.By bacterium
It is resuspended in spare in 20mL 0.1M KCl.
Experiment uses flat 96 hole elisa Plates of White-opalescent as reaction vessel.The specific location mode of sample is as shown in table 1.
All for use sample wells add in the KCl solution of 10 μ L 2M per hole.First is horizontally-arranged for blank control, the 210 μ L of addition per holeWater.ELISA Plate it is preceding 4 row B-H holes sequentially add 210 μ L various concentrations Zn solution (0.05,0.1,0.5,1,
2nd, 6 and 10mg/L).The B-F holes of 5-12 row sequentially add the sample of 210 μ L difference diluted concentrations.Then on oscillator plate
Mixing 1 minute adds in 30 μ L bacterium solutions since first row with Multi-channel liquid transfer device toward each sample well.During to first row plus bacterium solution
Start to use timer timing, passing through microplate reader after 15min measures bioluminescence value.Calculate the rear each sample compared with blank control
Shine inhibiting rate.
Best dilution range is determined according to measurement result, 100% concentration and 10% the concentration inhibiting rate that shines are respectively greater than>
80% He<20%, it is thus determined that most preferably the dilution upper limit is 100% concentration, i.e. stoste.Then 1 is pressed to the waste water of 100% concentration:2
A series of waste water solution (100%, 50%, 25%, 12.5%, 6.3%, 3.1%) that dilution is configured to concentration is spare.By above-mentioned
The bioluminescence value of method determination sample.Calculate the luminous inhibiting rate of the rear each sample compared with blank control.It the results are shown in Table 2.
2 coking wastewater sample of table is to the luminous inhibiting rate (%) of photogen
| Waste strength | Repeat 1 | Repeat 2 | Repeat 3 | Average value |
| 1.6% | 14.11 | 15.95 | 18.40 | 16.15 |
| 3.1% | 27.67 | 21.28 | 18.61 | 22.52 |
| 6.3% | 25.20 | 23.37 | 39.44 | 29.34 |
| 12.5% | 25.64 | 30.24 | 29.66 | 28.51 |
| 25% | 45.08 | 40.30 | 35.67 | 40.35 |
| 50% | 54.54 | 56.08 | 54.86 | 55.16 |
| 100% | 82.12 | 82.92 | 82.69 | 82.58 |
Embodiment 2
The comprehensive toxicity of final draining is bleachinged and dyeing with the different factories' printing and dyeing of photogen screening.
The final draining of the effluent treatment plant of 5 printing and dyeing mills is acquired, each sampling point acquires 4 samples and repeats.Original is useless
Water sample is usedWater presses 1：2 dilution be configured to a series of concentration waste water solution (100%, 50%, 25%, 12.5%,
6.3%th, 3.1%) it is spare.
E.coli HB101pUCD607 cryopreservation tubes is taken to remove after centrifuging, 1mL LB culture solutions are added in after supernatant, after mixing
It accesses in LB fluid nutrient mediums of the 10mL containing 30 μ g/L kanamycins.25 DEG C on shaking table, 180r/min cultures 16h or so.So
After take in 200 μ L bacterium solutions to 200mL LB culture solutions, 25 DEG C on shaking table, 180r/min culture 16h or so, until OD550For
0.6~1, luminous value>100000.Then gained bacterium solution at 4000g, 4 DEG C is centrifuged 10 minutes, discards supernatant liquid.By bacterium
It is resuspended in spare in 20mL 0.1M KCl.
Experiment uses flat 96 hole elisa Plates of White-opalescent as reaction vessel.The specific location mode of sample is as shown in table 1.
All for use sample wells add in the KCl solution of 10 μ L 2M per hole.First is horizontally-arranged for blank control, the 210 μ L of addition per holeWater.ELISA Plate it is preceding 4 row B-H holes sequentially add 210 μ L various concentrations Zn solution (0.05,0.1,0.5,1,
2nd, 6 and 10mg/L).The B-F holes of 5-12 row sequentially add the sample of 210 μ L difference diluted concentrations.Then on oscillator plate
Mixing 1 minute adds in 30 μ L bacterium solutions since first row with Multi-channel liquid transfer device toward each sample well.During to first row plus bacterium solution
Start to use timer timing, passing through microplate reader after 15min measures bioluminescence value.Calculate the rear each sample compared with blank control
Shine inhibiting rate.It the results are shown in Table 3.
The printing and dyeing bleaching and dyeing wastewater sample of table 3 is to the luminous inhibiting rate (%) of photogen
| Final draining sample | Repeat 1 | Repeat 2 | Average value |
| Printing and dyeing mill 1 | 10.84 | 21.06 | 15.95 |
| Printing and dyeing mill 2 | 49.52 | 51.34 | 50.43 |
| Printing and dyeing mill 3 | 68.18 | 69.62 | 68.90 |
| Printing and dyeing mill 4 | 64.57 | 56.37 | 60.47 |
| Printing and dyeing mill 5 | 12.74 | 24.42 | 18.58 |
Embodiment 3
The comprehensive toxicity of the different final drainings in paper mill is screened with photogen.
The final draining of the effluent treatment plant in 6 paper mills is acquired, each sampling point acquires 4 samples and repeats.Original is useless
Water sample is usedWater presses 1：2 dilution be configured to a series of concentration waste water solution (100%, 50%, 25%, 12.5%,
6.3%th, 3.1%) it is spare.
E.coli HB101pUCD607 cryopreservation tubes is taken to remove after centrifuging, 1mL LB culture solutions are added in after supernatant, after mixing
It accesses in LB fluid nutrient mediums of the 10mL containing 30 μ g/L kanamycins.25 DEG C on shaking table, 180r/min cultures 16h or so.So
After take in 200 μ L bacterium solutions to 200mL LB culture solutions, 25 DEG C on shaking table, 180r/min culture 16h or so, until OD550For
0.6~1, luminous value>100000.Then gained bacterium solution at 4000g, 4 DEG C is centrifuged 10 minutes, discards supernatant liquid.By bacterium
It is resuspended in spare in 20mL 0.1M KCl.
Experiment uses flat 96 hole elisa Plates of White-opalescent as reaction vessel.The specific location mode of sample is as shown in table 1.
All for use sample wells add in the KCl solution of 10 μ L 2M per hole.First is horizontally-arranged for blank control, the 210 μ L of addition per holeWater.ELISA Plate it is preceding 4 row B-H holes sequentially add 210 μ L various concentrations Zn solution (0.05,0.1,0.5,1,
2nd, 6 and 10mg/L).The B-F holes of 5-12 row sequentially add the sample of 210 μ L difference diluted concentrations.Then on oscillator plate
Mixing 1 minute adds in 30 μ L bacterium solutions since first row with Multi-channel liquid transfer device toward each sample well.During to first row plus bacterium solution
Start to use timer timing, passing through microplate reader after 15min measures bioluminescence value.Calculate the rear each sample compared with blank control
Shine inhibiting rate.It the results are shown in Table 4.
4 paper waste sample of table is to the luminous inhibiting rate (%) of photogen
| Final draining sample | Repeat 1 | Repeat 2 | Average value |
| Paper mill 1 | 60.71 | 64.66 | 62.69 |
| Paper mill 2 | 72.29 | 67.54 | 69.92 |
| Paper mill 3 | 86.89 | 88.58 | 87.74 |
| Paper mill 4 | 58.08 | 55.75 | 56.91 |
| Paper mill 5 | 51.33 | 50.85 | 51.09 |
| Paper mill 6 | 39.13 | 51.94 | 45.54 |
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (3)
1. a kind of industrial wastewater bio-toxicity detection method based on genetic recombination photogen, it is characterised in that including following step
Suddenly：
The first step prepares the bacteria suspension of photogen：It takes and freezes strain, centrifuged after defrosting, cultivated after removing supernatant, first
Secondary resuscitation fluid renewed vaccination cultivates again to certain OD values to new culture solution, discards supernatant liquid after centrifugation, it is standby that bacteria suspension is made
With；
Second step, sample gradient dilution：First industrial wastewater is diluted successively from stoste by 10 times of dilution ratios, Preliminary detection toxicity
Afterwards, it determines best dilution range, then carries out sample dilution by 2 times of gradients successively from the upper limit of best dilution range；
Third walks, and sample comprehensive toxicity measures：Sample after transfer dilution to ELISA Plate, blank control and standard control simultaneously into
Row adds in after bacteria suspension and measures bioluminescence intensity by microplate reader；
The photogen is Escherichia coli HB101 pUCD607.
2. the industrial wastewater bio-toxicity detection method according to claim 1 based on genetic recombination photogen, feature
It is：
OD values described in the first step are OD550=0.6~1.
3. the industrial wastewater bio-toxicity detection method according to claim 1 based on genetic recombination photogen, feature
It is：
ELISA Plate described in third step is 96 hole elisa Plates.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810023633.2A CN108226493A (en) | 2018-01-10 | 2018-01-10 | A kind of industrial wastewater bio-toxicity detection method based on genetic recombination photogen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810023633.2A CN108226493A (en) | 2018-01-10 | 2018-01-10 | A kind of industrial wastewater bio-toxicity detection method based on genetic recombination photogen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108226493A true CN108226493A (en) | 2018-06-29 |
Family
ID=62640732
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810023633.2A Pending CN108226493A (en) | 2018-01-10 | 2018-01-10 | A kind of industrial wastewater bio-toxicity detection method based on genetic recombination photogen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108226493A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109680033A (en) * | 2018-12-29 | 2019-04-26 | 华南师范大学 | A kind of comprehensive water-body toxicity quick detection kit |
| CN111103412A (en) * | 2018-10-25 | 2020-05-05 | 中国石油化工股份有限公司 | Rapid determination method for anaerobic feasibility of petrochemical wastewater |
| CN111830226A (en) * | 2020-07-31 | 2020-10-27 | 福建安格思安全环保技术有限公司 | Industrial wastewater toxicity detection method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127588A (en) * | 2010-12-09 | 2011-07-20 | 济南市供排水监测中心 | Method for quantitatively detecting toxicity of water quality |
| CN103105388A (en) * | 2013-01-29 | 2013-05-15 | 复旦大学 | Luminescent bacteria test paper for rapidly detecting water quality biotoxicity and preparation method thereof |
| CN106755286A (en) * | 2016-12-17 | 2017-05-31 | 桂林理工大学 | A kind of method that utilization Vibrio-qinghaiensis sp. Q67 tests oil extraction waste water bio-toxicity |
-
2018
- 2018-01-10 CN CN201810023633.2A patent/CN108226493A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127588A (en) * | 2010-12-09 | 2011-07-20 | 济南市供排水监测中心 | Method for quantitatively detecting toxicity of water quality |
| CN103105388A (en) * | 2013-01-29 | 2013-05-15 | 复旦大学 | Luminescent bacteria test paper for rapidly detecting water quality biotoxicity and preparation method thereof |
| CN106755286A (en) * | 2016-12-17 | 2017-05-31 | 桂林理工大学 | A kind of method that utilization Vibrio-qinghaiensis sp. Q67 tests oil extraction waste water bio-toxicity |
Non-Patent Citations (1)
| Title |
|---|
| YI-XIANG FANG ET AL: "Use of TIE techniques to characterize industrial effluents in the Pearl River Delta region", 《ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111103412A (en) * | 2018-10-25 | 2020-05-05 | 中国石油化工股份有限公司 | Rapid determination method for anaerobic feasibility of petrochemical wastewater |
| CN111103412B (en) * | 2018-10-25 | 2024-01-23 | 中国石油化工股份有限公司 | Rapid determination method for anaerobic feasibility of petrochemical wastewater |
| CN109680033A (en) * | 2018-12-29 | 2019-04-26 | 华南师范大学 | A kind of comprehensive water-body toxicity quick detection kit |
| CN111830226A (en) * | 2020-07-31 | 2020-10-27 | 福建安格思安全环保技术有限公司 | Industrial wastewater toxicity detection method |
| CN111830226B (en) * | 2020-07-31 | 2022-08-23 | 福建安格思安全环保技术有限公司 | Industrial wastewater toxicity detection method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ica et al. | Characterization of mono-and mixed-culture Campylobacter jejuni biofilms | |
| CA1125152A (en) | Selective determination of viable somatic and microbial cells | |
| JP3270722B2 (en) | Bacteria detection method and detection device | |
| AU660048B2 (en) | Apparatus for monitoring liquids | |
| EP0574977A1 (en) | Direct method for detecting very low levels of coliform contamination | |
| AU2005217026B2 (en) | Measuring contamination | |
| Lehtola et al. | Survival of Campylobacter jejuni in potable water biofilms: a comparative study with different detection methods | |
| CN108226493A (en) | A kind of industrial wastewater bio-toxicity detection method based on genetic recombination photogen | |
| CN101570783A (en) | Detection kit and detection method for 9 species of pathogenic organisms in marine products | |
| CN108120812A (en) | In a kind of dyeing waste water in groups biological toxicity tests and differentiate appraisal procedure | |
| Ramalho et al. | Improved methods for the enumeration of heterotrophic bacteria in bottled mineral waters | |
| Bonadonna et al. | Innovative analytical methods for monitoring microbiological and virological water quality | |
| CA1201963A (en) | Method of concentrating and measuring unicellular organisms | |
| WO2002008454A2 (en) | Method and device for viable and nonviable prokaryotic and eukaryotic cell quantitation | |
| CN105277535B (en) | Ammonia nitrogen field fast detection method in a kind of water for eliminating reagent blank influence | |
| CN102288653A (en) | Online biochemical oxygen demand (BOD) detector and detection method of same | |
| JP2003052392A (en) | Method for detecting microorganism and kit for detecting microorganism | |
| Apte et al. | Rapid detection of sewage contamination in marine waters using a fluorimetric assay of β-D-galactosidase activity | |
| McFeters et al. | Rapid direct methods for enumeration of specific, active bacteria in water and biofilms | |
| CN107447023A (en) | A kind of method of polyP bacteria in FISH joint Flow cytometry sewage disposal system | |
| Lee et al. | A rapid method for detecting bacteria in drinking water | |
| CN107167443A (en) | A kind of method that utilization ultraviolet spectrometer detects PCB77 | |
| RU2576030C1 (en) | Method for detecting danger of microbiological water pollution | |
| CN108018365A (en) | A kind of absolute quantitation detects the kit and detection method of total comma bacillus and pathogenic comma bacillus | |
| Garland et al. | Physiological profiling of microbial communities |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180629 |
|
| RJ01 | Rejection of invention patent application after publication |