CN102453743A - Method for preparing source water quality acute biotoxicity detection reagent and detection method of detection reagent - Google Patents
Method for preparing source water quality acute biotoxicity detection reagent and detection method of detection reagent Download PDFInfo
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- CN102453743A CN102453743A CN2010105232115A CN201010523211A CN102453743A CN 102453743 A CN102453743 A CN 102453743A CN 2010105232115 A CN2010105232115 A CN 2010105232115A CN 201010523211 A CN201010523211 A CN 201010523211A CN 102453743 A CN102453743 A CN 102453743A
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Abstract
The invention discloses a method for preparing a source water quality acute biotoxicity detection reagent. The method comprises the following steps of: 1) inoculating an inclined surface strain of a vibrioqinghaiensis Q67 bacterial strain to a fresh inclined surface, and culturing at the temperature of between 15 and 25 DEG C for 12 to 18 hours, wherein bacteria emit bright light at the moment; 2) tenderly scouring bacterial lawn down from the inclined surface by using 10ml of 10.85 percent normal saline, uniformly shaking, and adjusting the concentration of a bacterial solution by using a small amount of normal saline; and 3) adding 0.1ml of adjusted bacterial solution into a sample test tube filled with 2ml of 0.85 percent normal saline, and detecting luminescence by using a photometer, wherein the bacterial solution with the luminous intensity of 2 to 5 million photons per second is used as the detection reagent of a fresh bacterial solution. Compared with the prior art, the invention has the advantages that: the vibrioqinghaiensis can grow and emit the light without sodium chloride, so that high-concentration sodium chloride is not required to be added into a test sample, the physicochemical characteristics of the sample cannot be changed, and the accuracy of a test is ensured; and the method is used for security detection of source water.
Description
Technical field
The present invention relates to a kind of toxicity and detect detection reagent, be specially a kind of preparation source water water quality acute biological toxicity detection reagent and detection method thereof.Belong to source water water quality inspection technique field.
Background technology
Water is Source of life.Along with the development of progress of science and technology, industrial and agricultural production, as the water body environment of industrial and agricultural waste Rendezvous Point, the pollution that receives is more and more serious.By poor management, reason such as scarcity of resources, environmental change and Infrastructure insufficient investment, the whole world has 1/5th people can't obtain safe tap water approximately.How being human tap water and the domestic water that safety, cleaning are provided, is the urgent problem that countries in the world face, and in order to protect the aquatic ecosystem of drinking water source and health, adopts chemical process and biological method that source water water quality is monitored and estimated usually.
The chemical process evaluating water quality is to adopt various instrument direct analysis to measure kind and the concentration and the relevant with it parameter (like colourity, COD, BOD etc.) of objectionable impurities in the water body, serves as according to the control criterion of making Pollutant levels with surveyed data.Biological method is based on biology, ecology and toxicology principle and carries out water quality assessment.Like biological assessment benchmark (Biocriteria) is exactly to utilize responsive biology to test the bio-toxicity of chemical pollutant.
Index system is perfect when chemical process monitoring and evaluating water quality, foundation is abundant, it is with clearly defined objective to subdue, and can accurately measure the kind of objectionable impurities, can accurately measure their concentration or content to most of pollutents, so be the method that generally adopts.But the chemical monitoring method exists not enough, and it is difficult to predict from the concentration analysis data of different pollutents the real toxicity of waste water, and chemico-analytic data can not reflect the quality of water quality on the whole, or the reflection pollutent is to biology and Ecosystem System Influence.
The bio-toxicity experiment of monitoring and comprehensive evaluation water quality has long term toxicity test and acute toxicity test.Biological long term toxicity test is observed the influence of poisonous substance to its physiological activity hydrobiont in whole life cycle from be born to breeding.Duration of experiment is from several thoughtful several years.Biological acute toxicity test is to observe the biological experiment that the stimulation of poisonous substance is produced significant reaction at short notice.The indicator organism of biological acute toxicity has fish, algae, benthic mollusc, planktonic organism and mikrobe etc.Photogenic bacterium because of its unique physiological property, be prone to and modern optical electro-detection means are complementary and test fast.Owing to photogenic bacterium than the other biological kind more fast, economical, save advantages such as space and good reliability and receive much concern, therefore developed and utilized photogenic bacterium to carry out the bio-toxicity testing method of water quality toxicity monitoring and comprehensive evaluation.
At present, photogenic bacterium toxicity detection method has been widely used in pollution and the bio-toxicity of water quality to be judged, simple and easy to do with it, detect advantage such as with low cost and receive publicity, can be referring to the mensuration of acute toxicity of water quality---photogenic bacterium method, GB/T15441-1995; But if detect, then need in sample, to add sodium-chlor and reach 3% final concentration, might change the physicochemical property of sample after so the sodium-chlor of high density adds, the exactness of influence test with the ocean luminescent bacteria.But Shang Weijian is useful on the detection fast and safely of source water.
Summary of the invention
The objective of the invention is for a kind of method for preparing source water water quality acute biological toxicity detection reagent is provided.Employing Qinghai Vibrion Q67 substratum is processed fresh bacterium liquid or is adopted the Qinghai Vibrion lyophilized powder to process test and use bacterium liquid, to overcome the deficiency of prior art.
Another object of the present invention aims to provide the detection method based on this detection reagent.
The object of the invention can be realized through following technical scheme.
A kind of method for preparing source water water quality acute biological toxicity detection reagent, concrete steps are following:
1) adopt fresh bacterium liquid: with the slant strains of Qinghai Vibrion Q67 bacterial strain, move and receive on the fresh inclined-plane, cultivate 12h-18h for 15-25 ℃, this moment, bacterial luminescence was bright;
2) with 10ml 0.85% saline water lawn is softly washed away from the inclined-plane, shake up, with a small amount of saline water adjustment bacterial concentration;
3) get the adjusted bacterium liquid of 0.1ml, add and to be equipped with in the sample test cell of 2ml 0.85% saline water, detect luminously with photometer, select for use luminous intensity in the bacterium liquid of 200-500 ten thousand photons/second detection reagent as fresh bacterium liquid.
Step 2) in, saline water adjustment bacterial concentration is OD
660Be 0.2～0.5.
In the step 1), can also in Qinghai Vibrion Q67 lyophilisate, add the 5mL resuscitation fluid through lyophilisate preparation, thorough mixing under the room temperature held 8-15 minute, makes bacterium recover luminous.
Described Qinghai Vibrion Q67 lyophilisate is bought by market, is stored among-20 ℃ of refrigerators, uses front opening, makes it dissolving with an amount of resuscitation fluid.
Described resuscitation fluid is that volumetric molar concentration is 0.05-0.2M, and the pH value is the phosphoric acid buffer of 6.0-9.0, or quality-concentration of volume percent is the Glucose Liquid of 0.5-8.0%.
The detection method of above-mentioned detection reagent, its concrete steps are following:
1) get source water to be checked, add NaCl to final concentration be 0.8% as the tap water sample liquid; The inferior boiling point zero(ppm) water that adopts secondary heavily to steam, add NaCl to final concentration be 0.8% as control sample liquid;
2) to the luminous detection of sample: get six test cuvettes, wherein three test cuvettes add 2ml source water sample liquid respectively as one group of parallel appearance of sample, and three test cuvettes are put into 2ml control sample liquid respectively also as one group of parallel appearance of contrast in addition; Every separated 15s adds fresh bacterium liquid of 0.1ml or 0.05ml lyophilisate bacterium liquid successively in three test cuvettes in every group of parallel appearance; After placing 15min respectively; Just adopt the luminous detection appearance to test the luminous intensity of sample in the cuvette at a distance from 15s between every group of parallel appearance like this, calculate relative luminous intensity then.
The relative luminous intensity of source water is 80%～120% safety, and＞120% is suspicious, and＜80% is dangerous.
The present invention compares with prior art, utilize photogenic bacterium to external world poisonous substance the characteristics of sensitive reaction are arranged, adopt the exclusive a kind of fresh water photogenic bacterium of China---Qinghai Vibrion is used bacterial classification as detecting; Adopt the exclusive a kind of fresh water photogenic bacterium Qinghai Vibrion Q67 bacterial strain of China that all kinds of poisonous substances are all had the sensitive reaction, and used Qinghai Vibrion is fresh water type luminescent bacteria, need not that sodium-chlor just can be grown and luminous; Therefore need not to add high density chlorination sodium in the specimen; Can not change the physicochemical property of sample, guarantee the exactness of test, be used for the safety detection of source water; Detection speed is fast: can in 0.5 hour, can obtain a result, judge whether safety of water quality.
Embodiment
Adopt the distinctive fresh water photogenic bacterium of China-Qinghai Vibrion Q67 bacterial strain Vibrio qinghaiensis Q67 among the present invention; Be to being present in the reflection of the comprehensive toxicity of institute's Toxic in the sample, even the new synthetics that never occurred needs only poisonous to organism; Can make a difference to photogenic bacterium; And reflect from the luminous power of photogenic bacterium delicately, modern photon detection technique is very sensitive, and atomic weak light intensity has taken place to change and also can detect; So extremely sensitive with the luminous detection of photogenic bacterium to the reaction of poisonous substance, than the general biomass cells several magnitude that is quick on the draw.
Luminous detection is used instrument: the BHP95114 type water quality toxicity analyser that adopts Binsong Photon Technology Co., Ltd. Beijing to produce is used instrument for test.
The Qinghai Vibrion Q67 bacterial strain Vibrio qinghaiensis Q67 that adopts among the present invention is outstanding by red legend, Wang Jie; The novel species of a kind of photogenic bacterium that Chen Xiaoyun etc. find; Be published in document: Oceanologia et Limnologia Sinica, 25 (3): 273-278, this Qinghai Vibrion Q67 medium component is following:
MgSO
42.47g, MgCO
30.79g, MgBr
20.09g, MgCl
20.109g, CaCO
30.103g, KCl 0.122g, NaCl 8.29g, Mg (HCO
3)
20.50g, yeast extract paste 5g, tryptone 5g, glycerine 3g, agar 20g, be dissolved in the 1000mL zero(ppm) water, with its bevel, in 121 ℃ of sterilization 20min, it is subsequent use to store in 4 ℃ of refrigerators.
The luminescent bacteria lyophilisate adopts the lyophilisate that is made into by above-mentioned bacterial classification, and the patent No. of this lyophilisate: 97106203.X can be bought by market; Be stored among-20 ℃ of refrigerators, use front opening, make it dissolving with an amount of resuscitation fluid, about 10 minutes; Recover luminous vigor, just can be used for detecting, this resuscitation fluid is with reference to East China Normal University's journal (natural science edition; 2008,4, " isolation identification of Qinghai Lake photogenic bacterium and electron microscopic observation " article that 58-65 delivers.
The luminous detection data computing adopts following formula:
Source water water evaluation of result:
| The effect toxicity grading | Relative luminous intensity |
| Safety | 80％～120％ |
| Suspicious | ＞120％ |
| Dangerous | ＜80％ |
One, test is prepared with bacterium liquid
1, the preparation of fresh bacterium liquid:
With the slant strains of above-mentioned Qinghai Vibrion Q67 bacterial strain, move and receive on the fresh inclined-plane, cultivate 12h-18h for 15-25 ℃; This moment, bacterial luminescence was bright, with 10ml 0.85%NaCl solution, lawn was softly washed away from the inclined-plane; Shake up,, make bacterial concentration OD with a small amount of saline water adjustment bacterial concentration
660About ≈ 0.3, get 0.1ml bacterium liquid, adding is equipped with in the sample test cell of 2ml saline water, and is luminous with the photometer detection, and luminous intensity is when 200-500 ten thousand photons/second, and this fresh bacterium liquid just can be used as the bacterium liquid of test usefulness.
2, the preparation of lyophilisate bacterium liquid:
In Qinghai Vibrion Q67 bacterial strain lyophilisate, add the 5ml resuscitation fluid, thorough mixing, held made bacterium recover luminous about 10 minutes under the room temperature, obtained lyophilisate bacterium liquid, also can be used as test and used bacterium liquid.
Embodiment 1, and certain source water water quality toxicity detects
One, sampling
(1) it is an amount of to get the source water in Shanghai waterhead area June and July, is labeled as A and B respectively, add NaCl to final concentration be 0.8%.
(2) contrast water sample liquid: with the inferior boiling point zero(ppm) water that secondary heavily steams, add NaCl to final concentration be 0.8%.
Two, luminous detection
Be ready to three test cuvettes, each cuvette respectively adds the 2ml water sample, is made as three parallel appearance; Control sample liquid is also got three test cuvettes; Each adds 2ml, and three parallel appearance are set equally, adds fresh bacterium liquid 0.1ml or lyophilized powder bacterium liquid 0.05ml successively and uses bacterium liquid as test; The timed interval that each test cuvette adds bacterium liquid is 15s; After placing 15min, measure the luminous intensity of each measuring cup, calculating relative luminous intensity with the luminous detection appearance 15s of being separated by successively.
Three, luminous detection data computing:
Four, test result evaluation:
Embodiment 2, and certain source water water quality toxicity detects
One, sampling
(1) it is an amount of to get the sample in certain source water August and September, is labeled as C and D respectively, add NaCl to final concentration be 0.8%.
(2) blank water sample contrast, with the inferior boiling point zero(ppm) water that secondary heavily steams, add NaCl to final concentration be 0.8%.
Two, luminous detection
The BHP95114 type water quality toxicity analyser that adopts Binsong Photon Technology Co., Ltd. Beijing to produce is used instrument for test.
Can use lyophilized powder bacterium liquid, also can use fresh bacterium liquid.
Be ready to three test cuvettes, each cuvette respectively adds 2ml zero(ppm) water sample, is made as three parallel appearance; Three test cuvettes are also got in blank water sample contrast, and each adds 2ml, and three parallel appearance are set equally; Each test cuvette 15s at interval adds fresh bacterium liquid 0.1ml or lyophilisate bacterium liquid 0.05ml successively, uses bacterium liquid as test, after each test cuvette is placed 15min; Measure the luminous intensity respectively test cuvette with the luminous detection appearance 15s of being separated by successively, calculate relative luminous intensity.
Three, luminous detection data computing
Four, test result evaluation.
Claims (6)
1. method for preparing source water water quality acute biological toxicity detection reagent, it is characterized in that: concrete steps are following:
1) adopt fresh bacterium liquid: with the slant strains of Qinghai Vibrion Q67 bacterial strain, move and receive on the fresh inclined-plane, cultivate 12h-18h for 15-25 ℃, this moment, bacterial luminescence was bright;
2) with 10ml 0.85% saline water lawn is softly washed away from the inclined-plane, shake up, with a small amount of saline water adjustment bacterial concentration;
3) get the adjusted bacterium liquid of 0.1ml, add and to be equipped with in the sample test cell of 2ml 0.85% saline water, detect with photometer luminous, select for use luminous intensity at the bacterium liquid of 200-500 ten thousand photons/second as detection reagent.
2. the method for preparing source water water quality acute biological toxicity detection reagent according to claim 1 is characterized in that: step 2) in, use saline water adjustment bacterial concentration to be OD
660Be 0.2～0.5.
3. the method for preparing source water water quality acute biological toxicity detection reagent according to claim 1; It is characterized in that: in the step 1); When preparing, in Qinghai Vibrion Q67 lyophilisate, add 5mL resuscitation fluid, thorough mixing through lyophilisate; Under the room temperature held 8-15 minute, make bacterium recover luminous.
4. the method for preparing source water water quality acute biological toxicity detection reagent according to claim 3; It is characterized in that: described Qinghai Vibrion Q67 lyophilisate is bought by market; Be stored among-20 ℃ of refrigerators, use front opening, make it dissolving with an amount of resuscitation fluid.
5. the method for preparing source water water quality acute biological toxicity detection reagent according to claim 4; It is characterized in that: described resuscitation fluid is that volumetric molar concentration is 0.05-0.2M; The pH value is the phosphoric acid buffer of 6.0-9.0, or quality-concentration of volume percent is the Glucose Liquid of 0.5-8.0%.
6. the detection method of the detection reagent of claim 1 preparation, it is characterized in that: concrete steps are following:
1) get source water to be checked, add NaCl to final concentration be 0.8% as the tap water sample liquid; The inferior boiling point zero(ppm) water that adopts secondary heavily to steam, add NaCl to final concentration be 0.8% as control sample liquid;
2) to the luminous detection of sample: get six test cuvettes, wherein three test cuvettes add 2ml source water sample liquid respectively as one group of parallel appearance of sample, and three test cuvettes are put into 2ml control sample liquid respectively also as one group of parallel appearance of contrast in addition; Every separated 15s adds fresh bacterium liquid of 0.1ml or 0.05ml lyophilisate bacterium liquid successively in three test cuvettes in every group of parallel appearance; After placing 15min respectively; Just adopt the luminous detection appearance to test the luminous intensity of sample in the cuvette at a distance from 15s between every group of parallel appearance like this, calculate relative luminous intensity then.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105588831A (en) * | 2016-01-04 | 2016-05-18 | 中国科学院城市环境研究所 | Method for detecting acute toxicity of rare earth tailing pond surrounding groundwater pollution by using freshwater luminescent bacteria |
| CN105987898A (en) * | 2015-03-05 | 2016-10-05 | 上海市南洋模范中学 | Detection method for acute biological toxicities of pollutants on dust-haze day |
| CN106568902A (en) * | 2016-11-09 | 2017-04-19 | 暨南大学 | Surgical smoke biological toxicity evaluation method |
| CN107764965A (en) * | 2017-10-17 | 2018-03-06 | 中国科学院生态环境研究中心 | Combined pollutant biological genetic toxicity is quick in a kind of drinking water, the method for high flux detection |
| CN108507999A (en) * | 2018-03-26 | 2018-09-07 | 成都飞航智库科技有限公司 | One kind being applied to bio-toxicity detection method in biotechnology |
| CN109946433A (en) * | 2019-03-18 | 2019-06-28 | 天津市宇驰检测技术有限公司 | Sewage detection method |
| CN113340884A (en) * | 2021-06-23 | 2021-09-03 | 同济大学 | Buoy type online water quality detection device based on vibrio qinghai |
| CN117487793A (en) * | 2023-10-31 | 2024-02-02 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | Combined acute toxicity test method for field water quality on-site detection |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101698862A (en) * | 2009-10-09 | 2010-04-28 | 华东师范大学 | Detection method for acute biological toxicity of drinking water contained in plastic bottle |
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2010
- 2010-10-28 CN CN2010105232115A patent/CN102453743A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101698862A (en) * | 2009-10-09 | 2010-04-28 | 华东师范大学 | Detection method for acute biological toxicity of drinking water contained in plastic bottle |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105987898A (en) * | 2015-03-05 | 2016-10-05 | 上海市南洋模范中学 | Detection method for acute biological toxicities of pollutants on dust-haze day |
| CN105588831A (en) * | 2016-01-04 | 2016-05-18 | 中国科学院城市环境研究所 | Method for detecting acute toxicity of rare earth tailing pond surrounding groundwater pollution by using freshwater luminescent bacteria |
| CN106568902A (en) * | 2016-11-09 | 2017-04-19 | 暨南大学 | Surgical smoke biological toxicity evaluation method |
| CN107764965A (en) * | 2017-10-17 | 2018-03-06 | 中国科学院生态环境研究中心 | Combined pollutant biological genetic toxicity is quick in a kind of drinking water, the method for high flux detection |
| CN108507999A (en) * | 2018-03-26 | 2018-09-07 | 成都飞航智库科技有限公司 | One kind being applied to bio-toxicity detection method in biotechnology |
| CN109946433A (en) * | 2019-03-18 | 2019-06-28 | 天津市宇驰检测技术有限公司 | Sewage detection method |
| CN113340884A (en) * | 2021-06-23 | 2021-09-03 | 同济大学 | Buoy type online water quality detection device based on vibrio qinghai |
| CN117487793A (en) * | 2023-10-31 | 2024-02-02 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | Combined acute toxicity test method for field water quality on-site detection |
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Application publication date: 20120516 |