WO2018041215A1 - Fast detection method and device for organisms of 10-50 μm in ship ballast water - Google Patents
Fast detection method and device for organisms of 10-50 μm in ship ballast water Download PDFInfo
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- WO2018041215A1 WO2018041215A1 PCT/CN2017/099893 CN2017099893W WO2018041215A1 WO 2018041215 A1 WO2018041215 A1 WO 2018041215A1 CN 2017099893 W CN2017099893 W CN 2017099893W WO 2018041215 A1 WO2018041215 A1 WO 2018041215A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
- C12N1/125—Unicellular algae isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/89—Algae ; Processes using algae
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- the invention relates to a method and a device for detecting a biological living body in a ballast water carried by a ship, and in particular relates to a method and a device for detecting a 10-50 um biological rapid detection in a ship ballast water with high detection precision and reliable data.
- Surviving organisms having a minimum size greater than or equal to 50 ⁇ m are less than 10/m3; less than 10 ⁇ g/ml of living organisms having a minimum size of less than 50 ⁇ m but greater than or equal to 10 ⁇ m;
- the indicator microorganism is less than the following concentrations:
- Toxic Vibrio cholerae (serotype 01 and 0139) is less than 1 cfu / 100 ml;
- E. coli less than 250 cfu / 100 ml
- Enterococci less than 100 cfu / 100ml.
- the object of the present invention is to solve the above-mentioned deficiencies of the prior art, and provide a 10-50um biological rapid detection method and device for ship ballast water with simple structure, convenient use, high detection speed, high precision and reliable data.
- a 10-50um biological rapid detection method for ship ballast water characterized in that the method comprises the following steps:
- the 10 um surviving organism is filtered out with the liquid;
- the method for obtaining the relative fluorescence strong reference value is as follows:
- the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer
- each reference test sample is tested at least once according to the operation methods of steps a, b, c, d, e, f, according to the obtained
- the data is used to determine an average relative fluorescence intensity value, the average relative fluorescence intensity value being a relative fluorescence intensity reference value;
- the relative fluorescence intensity value obtained in the step (6) is compared with the relative fluorescence intensity reference value obtained in the step g, and it is judged whether the ballast water sample meets the emission standard.
- the reference test sample described in the step a of the present invention is obtained by using a large-aperture filter to viable a living biofilter of not less than 50 ⁇ m in the ballast water sample (to be discharged) treated by the ballast water treatment apparatus. Out, the filtrate is obtained; the Aml filtrate is taken, and the small-aperture filter is used to filter and retain the living organism of not less than 10um in the filtrate, and the living organisms less than 10um are filtered with the liquid; using 3-10% Aml for cleaning Liquid backwashing small pore size filter, rinsing the living organism with a size of 10-50 um into the sample tank; detecting the liquid in the sample tank by a FDA-CMFDA method under a fluorescence microscope, wherein 10-50 um of living organisms When the content meets the requirements, it can be used as a reference test sample.
- the reference test sample described in the step a of the present invention can also be obtained by manually preparing an algae solution or natural seawater by ultraviolet (UV) treatment or sodium hypochlorite (NaClO) to obtain a test liquid; using a large pore size filter The viable organism of not less than 50 um in the test solution is filtered to obtain a filtrate; the Aml filtrate is taken, and a small-aperture filter is used to filter and retain a living organism of not less than 10 ⁇ m in the filtrate, and a living organism of less than 10 ⁇ m with the liquid Filtration; backwash the small pore size filter with 3-10% Aml cleaning solution, flush the trapped living organism with the size of 10-50um into the sample tank; the liquid in the sample tank is fluoresced by FDA-CMFDA method Microscopically, when the content of 10-50um surviving organisms meets the requirements, It can be used as a reference test sample.
- UV ultraviolet
- NaClO sodium hypochlorite
- the large-aperture filter described in the present invention is a filter made of a nylon mesh having a mesh diagonal length of 35-50 um; the small-diameter filter is a microporous filter having an absolute pore diameter of 10 ⁇ m. a filter that can be backwashed;
- the living cell fluorescent staining agent in the present invention is FDA or/and CMFDA, and the amount thereof is added in a ratio of 1-3 mg/ml (2 mg/ml) according to the liquid volume in the sample tank.
- the staining reaction time is 5-30 (10) minutes.
- the FDA is fluorescein diacetate.
- the cell lysate described in the present invention is one of a mixture of methanol and chloroform (volume ratio of 1:1 to 1:3) or acetone.
- the utility model relates to a 10-50um biological rapid detecting device for ship ballast water, which comprises a sample box, a flushing liquid tank, a lysing liquid tank, a waste water tank, a detecting box, a primary filter and a secondary filter, and an outlet of the primary filter.
- the dyeing agent inlet port is arranged on the detection box, and the sample pump, the flushing pump and the cracking pump are respectively arranged on the outlets of the sample box, the flushing liquid tank and the dyeing agent tank, and the outlet of the sample pump passes through the filtering valve and the second One port of the stage filter is connected, and the other port of the second stage filter is connected to the outlet of the flushing pump via a flushing valve, and the connecting pipe between the secondary filter and the flushing valve is connected to the waste water tank through the drain valve, and the liquid of the detecting box is connected.
- the mouth inlet valve is connected with the connection line between the filter valve and the secondary filter, and the outlet of the cracking pump is connected to the detection box, and the detection box is provided with a fluorometer.
- the primary filter described in the present invention is a nylon mesh having a mesh diagonal length of 35-50 um; the secondary filter is a microporous filter having an absolute pore diameter of 10 um; the fluorometer emits light at a wavelength of 460-490 nm.
- the received excitation light has a wavelength of 510-550 nm.
- the lower part of the detection box is connected to the waste water tank via a discharge valve.
- the 10-50 um organism described in the present invention refers to a living organism having a size of less than 50 um but greater than or equal to 10 um.
- the relative fluorescence intensity reference value only needs to be tested once, and it can become in multiple or even countless inspections.
- the reference value has the advantages of simple structure, convenient use, high detection speed, high precision and reliable data.
- Figure 1 is a schematic view showing the structure of a medium speed detecting device of the present invention.
- Figure 2 is a graph showing the relationship between the relative fluorescence intensity and the viable cell content of Tetrahymena in the present invention. .
- a 10-50um biological rapid detection method for ship ballast water comprises the following steps: a filter made of a nylon mesh having a mesh diagonal length of 50 um and a pressure to be discharged after being treated by a ballast water treatment device
- the Tris-HCl backwashing small pore size filter, the trapped living organism with a size of 10-50 um is flushed into the sample tank; the live cell fluorescent stain fluorescein diacetate 20 mg is added to the sample tank, and staining is performed for 10 minutes.
- cell lysate is a mixture of methanol and chloroform in a volume ratio of 1:2
- the fluorometer is used for detection. The relative fluorescence intensity of the liquid in the sample cell.
- the relative fluorescence strong reference value is obtained as follows: 60 known ballast water samples to be discharged containing 9 10-50 um surviving organisms per ml are selected as reference test samples; for each reference test sample, the following operations are performed: A filter made of a nylon mesh with a mesh length of 50 ⁇ m is used to filter out the living organism of not less than 50 ⁇ m in the reference test sample to obtain a filtrate; 200 ml of the filtrate is taken, and the living organism is less than 10 ⁇ m.
- the average of the relative fluorescence intensities obtained after the treatment of the 60 reference test samples was calculated, which is the relative fluorescence intensity reference value. Comparing the relative fluorescence intensity value obtained after the ballast water to be treated with the relative fluorescence intensity reference value, it can be determined whether the ballast water sample to be tested meets the discharge standard.
- the reference test sample is obtained by filtering the viable organism of not less than 50 um in the ballast water sample to be discharged after being treated by the ballast water treatment apparatus using a large-aperture filter to obtain a filtrate; taking Aml filtrate Use a small pore size filter to filter and intercept the living organisms of not less than 10um in the filtrate, and the living organisms less than 10um are filtered out with the liquid; backwash the small pore size filter with 3-10% Aml cleaning solution, the size will be intercepted
- the 10-50um surviving organism is flushed into the sample tank; the liquid in the sample tank is detected by a FDA-CMFDA method under a fluorescence microscope, and when the content of the 10-50um surviving organism meets the requirements, it can be used as a reference test sample.
- Cell lysate can leaching fluorescein inside living cells, improving the accuracy of the test results.
- the FDA has two conjugated acetic acid free radicals. It is a non-polar substance that can pass freely through the algal cell membrane and is hydrolyzed to fluorescein by non-specific enzymes such as esterase, protease and lipase in the cell.
- the FDA is a colorless compound that does not have fluorescence itself, and the reaction product fluorescein is a polar and fluorescent luminescent material that is chemically stable, is not easily decomposed, and is difficult to pass through the cell membrane and accumulate in cells.
- the relationship between the relative fluorescence intensity and the biological content of 10-50 um was used to prepare different concentrations of tetraterpene solution, and the viable cell content was determined by FDA-CMFDA method. Each solution was added to the apparatus of the present invention and tested for relative fluorescence intensity using the method of the present invention. Taking the living cell content of tetradium algae as the x-axis and the measured relative fluorescence intensity as the y-axis, FIG. 2 is made. As can be seen from FIG. 2, the detection result of the present invention has a good one-to-one correspondence with the actual living cell content. It reflects the amount of viable cells in the sample.
- a 10-50um biological rapid detection method for ship ballast water comprises the following steps:
- the method for obtaining the relative fluorescence strong reference value is as follows:
- the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer
- each reference test sample is tested at least once according to the operation methods of steps a, b, c, d, e, f, according to the obtained
- the data is used to determine an average relative fluorescence intensity value, the average relative fluorescence intensity value being a relative fluorescence intensity reference value;
- A is any number, preferably a natural number from 100 to 500.
- the reference test sample described in the step a in the present invention is obtained by filtering a surviving organism of not less than 50 ⁇ m in the ballast water sample to be discharged after being treated by the ballast water treatment apparatus using a large-aperture filter, The filtrate is obtained; the Aml filtrate is taken, and the small-aperture filter is used to filter and retain the living organisms of not less than 10 ⁇ m in the filtrate, and the living organisms less than 10 ⁇ m are filtered with the liquid; the cleaning solution of 3-10% Aml is used.
- the ballast water sample to be discharged after being treated by the ballast water treatment equipment can be used as a reference test sample.
- the reference test sample described in the step a of the present invention can also be obtained by manually preparing an algae solution or natural seawater by ultraviolet (UV) treatment or sodium hypochlorite (NaClO) to obtain a test liquid; using a large pore size filter The viable organism of not less than 50 um in the test solution is filtered to obtain a filtrate; the Aml filtrate is taken, and a small-aperture filter is used to filter and retain a living organism of not less than 10 ⁇ m in the filtrate, and a living organism of less than 10 ⁇ m with the liquid Filtration; backwash the small pore size filter with 3-10% Aml cleaning solution, flush the trapped living organism with the size of 10-50um into the sample tank; the liquid in the sample tank is fluoresced by FDA-CMFDA method Under the microscope, when the content of 10-50 um surviving organisms meets the requirements, the ballast water sample to be discharged after being treated by the ballast water treatment equipment can be used as a reference test sample.
- UV ultraviolet
- the large-aperture filter is a filter made of a nylon mesh having a mesh diagonal length of 35-50 um; and the small-aperture filter is a microporous filter having an absolute pore diameter of 10 ⁇ m.
- Membrane-made, backwashable filter The living cell fluorescent staining agent is FDA or/and CMFDA, and the amount thereof is added in a ratio of 1-3 mg/ml (2 mg/ml) according to the liquid volume in the sample tank.
- the staining reaction time is 5-30 (10) minutes.
- the PBS is: lead sulfide, and Tris-HCl is: tris(hydroxymethyl)aminomethane.
- the cell lysate is one of a mixture of methanol and chloroform (1:1:3 by volume) or acetone.
- FIG. 1 shows: including sample box 2, flushing liquid tank 13, cracking liquid tank 3, waste water tank 14, detecting tank 17, primary filter 1 and secondary filter 8, and primary filter 1 is using mesh diagonal
- a secondary filter is a filter made of a microporous membrane with an absolute pore size of 10 um.
- the outlet of the primary filter is connected to the inlet of the sample box 2, and the liquid filtered by the primary filter enters the connection line between the primary filter and the sample box 2 in the sample box 2, and may be provided with a primary pump or a first pump.
- the stage filter is placed above the sample box 2 and naturally flows in by the gravity of the liquid.
- the detection tank 17 is provided with a dye inlet port 12, and the sample tank 4, the flushing pump 13 and the cracking pump 5 are respectively disposed at the outlets of the sample tank 2, the flushing liquid tank 14 and the lysing tank 3, and the outlet of the sample pump 4 is filtered.
- the valve 6 is connected to one port of the secondary filter 8, the other port of the secondary filter 8 is connected to the outlet of the flushing pump 13 via the flushing valve 11, and the connecting pipe between the secondary filter 8 and the flushing valve 11 is discharged through the draining valve 10 is connected to the waste water tank 15, and the liquid inlet of the detection tank 17 is connected to the connection line between the filter valve 6 and the secondary filter 8 via the inlet valve 9, and the outlet of the cracking pump 5 is connected to the detection tank 17 via the cracking valve 7.
- the detection box 17 is provided with a fluorometer 18, and the detection probe of the fluorometer 18 is disposed in the detection box 17; the emission wavelength of the fluorometer is 460-490 nm, and the wavelength of the received excitation light is 510-550 nm; the lower part of the detection box
- the discharge valve 16 is connected to the waste water tank 15.
- the 10-50 um organism described in the present invention refers to a living organism having a size of less than 50 um but greater than or equal to 10 um.
- the ballast water sample to be tested is placed in the primary filter, and the primary filter filters out the living organism of not less than 50um in the ballast water sample.
- the filtered filtrate enters the sample box, and the cleaning liquid and the cell lysate are respectively added to the rinsing liquid tank and the lysing liquid tank; the filter valve and the liquid discharging valve are opened, the other valves are closed, the sample pump is operated, and the sample is quantitatively sampled.
- the filtrate in the tank is filtered through a secondary filter, and the filtered liquid is discharged into the waste water tank.
- the living organisms not less than 10um are trapped by the secondary filter; the filter valve and the drain valve are closed, and the dye valve and the dye valve are The discharge valve is also closed, the flush valve and the inlet valve are opened, the flushing pump is operated, the secondary filter is backwashed with a quantitative flushing liquid, and the 10-50 um of the trapped organism is flushed into the test chamber; the flushing valve is closed and Into the liquid inlet valve, add appropriate amount of live cell fluorescent stain to the sample box through the inlet of the staining agent, and fluorescently stain the living cells; after the living cells are dyed (about 10 minutes), the cracking valve is opened, and the pyrolysis pump works.
- the relative fluorescence intensity reference value is only required to be taken once, and it can be used as a reference value in multiple or even countless tests.
- the invention has the advantages of simple structure, convenient use, high detection speed, high precision and reliable and reliable data.
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Abstract
Disclosed are a fast detection method and device for organisms of 10-50 μm in ship ballast water, relating to detection methods and devices for ballast water, wherein the method comprises the following steps: filtering a ballast water sample with a filter to filter out live organisms larger than 50 μm, and obtaining the filtrate; filtering with a filter having a small bore diameter, and trapping live organisms of no less than 10 μm in the filtrate; backwashing the filter having a small bore diameter with a cleaning fluid, and flushing the live organisms with a size of 10-50 μm into a sample groove; adding a living cell fluorescence dye into the sample groove to stain; adding a cell lysis solution into the sample groove; detecting the relative fluorescence intensity of the liquid in the sample groove with a fluorometer; and determining whether the standard is reached by means of comparison of the relative fluorescence intensity and the reference value, the detection device being an automatic detection system with a container, a liquid pump and a fluorometer built therein.
Description
本发明涉及船舶携带的压载水中生物活体的检测方法及装置,详细讲是一种检测精度高、数据真实可靠的船舶压载水中10-50um生物快速检测方法及装置。The invention relates to a method and a device for detecting a biological living body in a ballast water carried by a ship, and in particular relates to a method and a device for detecting a 10-50 um biological rapid detection in a ship ballast water with high detection precision and reliable data.
我们知道,远洋运输船舶为了保证船舶稳定性,一般在始发港口加装压载水,待停靠在目的港口时再排放压载水,其所携带的生物会给目的港口国带来生物入侵风险。为了应对此问题,国际海事组织(IMO)、美国海岸警卫队(USCG)和美国环境保护署(EPA)均制定了相应的生物入侵应对措施和压载水排放控制公约,要求多数商业运输船舶必须加装压载水处理设备(BWTS)以处理压载水。IMO G8导则和USCG参考的ETV草案明确规定了排放压载水中生物的尺寸及存活生物浓度,如下所示:We know that in order to ensure the stability of ships, ocean-going vessels generally install ballast water at the port of origin. When they are docked at the destination port, they discharge ballast water. The organisms carried by them will bring the risk of biological invasion to the port of destination. . In response to this problem, the International Maritime Organization (IMO), the US Coast Guard (USCG) and the US Environmental Protection Agency (EPA) have developed corresponding bio-invasion response measures and ballast water emission control conventions, requiring most commercial transport vessels to A ballast water treatment unit (BWTS) is installed to treat the ballast water. The IMO G8 Guidelines and the USCG reference ETV draft specify the size and viable bioconcentration of organisms in the ballast water discharged as follows:
最小尺寸大于或等于50μm的存活生物少于10个/m3;最小尺寸小于50μm但大于或等于10μm的存活生物少于10个/ml;Surviving organisms having a minimum size greater than or equal to 50 μm are less than 10/m3; less than 10 μg/ml of living organisms having a minimum size of less than 50 μm but greater than or equal to 10 μm;
且作为人体健康标准,指标微生物小于下述浓度:And as a standard of human health, the indicator microorganism is less than the following concentrations:
有毒霍乱弧菌(血清型01和0139)少于1cfu/100ml;Toxic Vibrio cholerae (serotype 01 and 0139) is less than 1 cfu / 100 ml;
大肠杆菌:少于250cfu/100ml;E. coli: less than 250 cfu / 100 ml;
肠道球菌:少于100cfu/100ml。Enterococci: less than 100 cfu / 100ml.
目前,IMO国际压载水公约还未生效,但生效日期也已临近。SCG已经单方面宣布其规范生效,强制规定凡进入美国水域的商业运输船舶必须安装符合USCG规范和排放标准的BWTS,否则无法靠港进行相关作业。因各个水域的水质和生物分布情况有所差异,船舶所安装的BWTS不能保证每次排放的压载水都能满足排放标准,必须在靠近港口国排放前自行检测目标生物含量。At present, the IMO International Ballast Water Convention has not yet entered into force, but the effective date is approaching. SCG has unilaterally announced that its specifications are in force, and it is mandatory that commercial transport vessels entering US waters must install BWTS that meet USCG specifications and emission standards, otherwise they will not be able to rely on ports for related operations. Due to the difference in water quality and bio-distribution of each water area, the BWTS installed on the ship cannot guarantee that the ballast water discharged each time can meet the discharge standard, and the target biological content must be self-detected before discharge from the port country.
船上检测人员必须具有丰富的生物基础知识和检测经验,同时配备先进的生物检测仪器;对于绝大多数船舶设施和船员知识水平来说,很难满足此要求。一般
来说,10-50um生物主要为单细胞藻类,因其物种特殊性,既不能通过过滤方法完全去除,也不能通过紫外照射或化学方法完全灭杀,且种类繁多,已成为压载水中生物检测的难点。现有的快速检测装置是根据藻类叶绿素的含量预估藻细胞数量,经实验表明,部分已死亡藻细胞内的叶绿素仍然能保持完整,依据叶绿素含量判定细胞存活状态的方法误差大、可靠性低,甚至出现误判。另一方面,现有的的检测设备会将一些10um以下的藻细胞也计入,检测数据不精准。On-board inspectors must have extensive biological basic knowledge and testing experience, as well as advanced biometric instruments; it is difficult to meet the requirements of most ship facilities and crew knowledge. General
For example, 10-50um organisms are mainly unicellular algae. Due to their species specificity, they can neither be completely removed by filtration, nor completely eliminated by ultraviolet irradiation or chemical methods, and they have a wide variety of species. Difficulties. The existing rapid detection device estimates the number of algae cells based on the content of algae chlorophyll. Experiments show that the chlorophyll in some dead algae cells can still remain intact, and the method for determining cell survival state according to chlorophyll content has large error and low reliability. And even misjudgment. On the other hand, the existing detection equipment will also count some algae cells below 10um, and the detection data is not accurate.
问题的解决方案Problem solution
本发明的目的是解决上述现有技术的不足,提供一种结构简单、使用方便,检测速度快、精度高,数据真实可靠的船舶压载水中10-50um生物快速检测方法及装置。The object of the present invention is to solve the above-mentioned deficiencies of the prior art, and provide a 10-50um biological rapid detection method and device for ship ballast water with simple structure, convenient use, high detection speed, high precision and reliable data.
本发明解决上述现有技术的不足所采用的技术方案是:The technical solution adopted by the present invention to solve the above deficiencies of the prior art is:
一种船舶压载水中10-50um生物快速检测方法,其特征在于包括如下步骤:A 10-50um biological rapid detection method for ship ballast water, characterized in that the method comprises the following steps:
(1)使用大孔径过滤器将经压载水处理设备处理后的(待排放的)压载水样品中不小于50um的存活生物滤出,得滤出液;(1) using a large-aperture filter to filter out viable organisms of not less than 50 μm in the ballast water sample (to be discharged) treated by the ballast water treatment equipment to obtain a filtrate;
(2)取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10um的存活生物、小(2) Take Aml filtrate, filter with a small pore size filter and intercept the living organisms of not less than 10um in the filtrate, small
于10um的存活生物随液体滤出;The 10 um surviving organism is filtered out with the liquid;
(3)使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物(3) Backwash the small pore size filter with 3-10% Aml cleaning solution, and the living organisms with a size of 10-50um will be trapped.
冲洗进入到样品槽内;Flush into the sample tank;
(4)向样品槽内加入活细胞荧光染色剂,染色5-30(10)分钟;(4) adding a living cell fluorescent stain to the sample tank and staining for 5-30 (10) minutes;
(5)染色后,向样品槽内加入其内液体体积0.5-2倍的细胞裂解液;(5) after dyeing, adding 0.5-2 times the volume of the liquid lysate to the sample tank;
(6)待细胞(膜)充分裂(溶)解后,使用荧光计检测样品槽内液体的相对荧光强度;(6) After the cell (membrane) is sufficiently cleaved (dissolved), the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer;
相对荧光强参考值的获取方法如下:The method for obtaining the relative fluorescence strong reference value is as follows:
a、选用已知符合压载水处理D-2标准中对10-50um存活生物的规定、含10-50um
存活生物个数达到上限的水样作为参考检测样品,使用大孔径过滤器将参考检测样品中不小于50um的存活生物滤出,得滤出液;a. Use the known 10-50um surviving organisms in the D-2 standard for ballast water treatment, including 10-50um
A water sample having an upper limit of the number of surviving organisms is used as a reference test sample, and a viable liquid of not less than 50 μm in the reference test sample is filtered out using a large-aperture filter to obtain a filtrate;
b、取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10um的存活生物、小于10um的存活生物随液体滤出;b, taking Aml filtrate, using a small pore size filter to filter and retaining not less than 10um of surviving organisms in the filtrate, less than 10um of living organisms are filtered with liquid;
c、使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;c. Backwash the small pore size filter with 3-10% Aml cleaning solution, and rinse the trapped living organism with the size of 10-50um into the sample tank;
d、向样品槽内加入活细胞荧光染色剂,染色5-30(10)分钟;d, adding a living cell fluorescent stain to the sample tank, staining for 5-30 (10) minutes;
e、染色后,向样品槽内加入其内液体体积0.5-2倍的细胞裂解液;e, after dyeing, adding 0.5-2 times the volume of the liquid lysate to the sample tank;
f、待细胞充分裂解后,使用荧光计检测样品槽内液体的相对荧光强度;f. After the cells are fully lysed, the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer;
g、按步骤a中参考检测样品的选取标准选取1-100种不同的参考检测样品,每种参考检测样品按步骤a、b、c、d、e、f的操作方法检测至少一次,根据所得数据求出平均相对荧光强度值,该平均相对荧光强度值为相对荧光强度参考值;g, according to the selection criteria of the reference test sample in step a, 1-100 different reference test samples are selected, and each reference test sample is tested at least once according to the operation methods of steps a, b, c, d, e, f, according to the obtained The data is used to determine an average relative fluorescence intensity value, the average relative fluorescence intensity value being a relative fluorescence intensity reference value;
将第(6)步获得的相对荧光强度值与第g步获得的相对荧光强度参考值对比,判断该压载水样品是否符合排放标准。The relative fluorescence intensity value obtained in the step (6) is compared with the relative fluorescence intensity reference value obtained in the step g, and it is judged whether the ballast water sample meets the emission standard.
本发明中第a步中所述的参考检测样品经如下方法获得:使用大孔径过滤器将经压载水处理设备处理后的(待排放的)压载水样品中不小于50um的存活生物滤出,得滤出液;取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10um的存活生物、小于10um的存活生物随液体滤出;使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;将样品槽内的液体经FDA-CMFDA法在荧光显微镜下检测,其中10-50um存活生物的含量符合要求时,即可成为参考检测样品。The reference test sample described in the step a of the present invention is obtained by using a large-aperture filter to viable a living biofilter of not less than 50 μm in the ballast water sample (to be discharged) treated by the ballast water treatment apparatus. Out, the filtrate is obtained; the Aml filtrate is taken, and the small-aperture filter is used to filter and retain the living organism of not less than 10um in the filtrate, and the living organisms less than 10um are filtered with the liquid; using 3-10% Aml for cleaning Liquid backwashing small pore size filter, rinsing the living organism with a size of 10-50 um into the sample tank; detecting the liquid in the sample tank by a FDA-CMFDA method under a fluorescence microscope, wherein 10-50 um of living organisms When the content meets the requirements, it can be used as a reference test sample.
本发明中第a步中所述的参考检测样品也可经如下方法获得:人工配制藻类溶液或自然海水经紫外线(UV)处理或经次氯酸钠(NaClO)处理,得测试液;使用大孔径过滤器将测试液中不小于50um的存活生物滤出,得滤出液;取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10um的存活生物、小于10um的存活生物随液体滤出;使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;将样品槽内的液体经FDA-CMFDA法在荧光显微镜下检测,其中10-50um存活生物的含量符合要求时,
即可成为参考检测样品。本发明中所述的大孔径过滤器为使用网孔对角线长度为35-50um的尼龙筛绢制成的过滤器;所述的小孔径过滤器为使用绝对孔径为10um的微孔滤膜制成的、可以反冲洗的过滤器;The reference test sample described in the step a of the present invention can also be obtained by manually preparing an algae solution or natural seawater by ultraviolet (UV) treatment or sodium hypochlorite (NaClO) to obtain a test liquid; using a large pore size filter The viable organism of not less than 50 um in the test solution is filtered to obtain a filtrate; the Aml filtrate is taken, and a small-aperture filter is used to filter and retain a living organism of not less than 10 μm in the filtrate, and a living organism of less than 10 μm with the liquid Filtration; backwash the small pore size filter with 3-10% Aml cleaning solution, flush the trapped living organism with the size of 10-50um into the sample tank; the liquid in the sample tank is fluoresced by FDA-CMFDA method Microscopically, when the content of 10-50um surviving organisms meets the requirements,
It can be used as a reference test sample. The large-aperture filter described in the present invention is a filter made of a nylon mesh having a mesh diagonal length of 35-50 um; the small-diameter filter is a microporous filter having an absolute pore diameter of 10 μm. a filter that can be backwashed;
本发明中所述活细胞荧光染色剂为FDA或/和CMFDA,其加入量根据样品槽内液体体积、以1-3mg/ml(2mg/ml)的比例加入。染色反应时间为5-30(10)分钟。FDA为荧光素二乙酸酯。The living cell fluorescent staining agent in the present invention is FDA or/and CMFDA, and the amount thereof is added in a ratio of 1-3 mg/ml (2 mg/ml) according to the liquid volume in the sample tank. The staining reaction time is 5-30 (10) minutes. The FDA is fluorescein diacetate.
本发明中所述的清洗液是BES、MOPS、PBS(pH=7.0)、ris-HCl(0.2-1.0M,p H=6.0-7.2)中的至少一种。可以有效防止FDA或CMFDA的非生物水解。The cleaning solution described in the present invention is at least one of BES, MOPS, PBS (pH = 7.0), ris-HCl (0.2 - 1.0 M, p H = 6.0 - 7.2). It can effectively prevent abiotic hydrolysis of FDA or CMFDA.
本发明中所述细胞裂解液为甲醇与氯仿(体积比1∶1-1∶3)的混合液或丙酮中的一种。The cell lysate described in the present invention is one of a mixture of methanol and chloroform (volume ratio of 1:1 to 1:3) or acetone.
一种船舶压载水中10-50um生物快速检测装置,其特征在于包括样品箱、冲洗液箱、裂解液箱、废水箱、检测箱、一级过滤器和二级滤器,一级过滤器的出口与样品箱进口相连,检测箱上设有染色剂加入口,样品箱、冲洗液箱和染色剂箱的出口上分别设有样品泵、冲洗泵和裂解泵,样品泵的出口经过滤阀与二级过滤器的一端口相连,二级过滤器的另一端口经冲洗阀与冲洗泵出口相连,二级过滤器与冲洗阀间的连接管经排液阀与废水箱相连,检测箱的进液口经进液阀与过滤阀和二级过滤器间的连接管路相连,裂解泵出口与检测箱相连,检测箱上设有荧光计。The utility model relates to a 10-50um biological rapid detecting device for ship ballast water, which comprises a sample box, a flushing liquid tank, a lysing liquid tank, a waste water tank, a detecting box, a primary filter and a secondary filter, and an outlet of the primary filter. Connected to the inlet of the sample box, the dyeing agent inlet port is arranged on the detection box, and the sample pump, the flushing pump and the cracking pump are respectively arranged on the outlets of the sample box, the flushing liquid tank and the dyeing agent tank, and the outlet of the sample pump passes through the filtering valve and the second One port of the stage filter is connected, and the other port of the second stage filter is connected to the outlet of the flushing pump via a flushing valve, and the connecting pipe between the secondary filter and the flushing valve is connected to the waste water tank through the drain valve, and the liquid of the detecting box is connected. The mouth inlet valve is connected with the connection line between the filter valve and the secondary filter, and the outlet of the cracking pump is connected to the detection box, and the detection box is provided with a fluorometer.
本发明中所述的一级过滤器为网孔对角线长度为35-50um的尼龙筛绢;二级滤器为绝对孔径为10um的微孔滤膜;荧光计的发射光波长为460-490nm,接收的激发光波长为510-550nm。所述的检测箱下部经排放阀与废水箱相连。The primary filter described in the present invention is a nylon mesh having a mesh diagonal length of 35-50 um; the secondary filter is a microporous filter having an absolute pore diameter of 10 um; the fluorometer emits light at a wavelength of 460-490 nm. The received excitation light has a wavelength of 510-550 nm. The lower part of the detection box is connected to the waste water tank via a discharge valve.
本发明中所述的10-50um生物是指:尺寸小于50um但大于或等于10um的存活生物。The 10-50 um organism described in the present invention refers to a living organism having a size of less than 50 um but greater than or equal to 10 um.
发明的有益效果Advantageous effects of the invention
使用本发明的装置及方法来检测待排放的船舶压载水中10-50um生物时,首先按照相对荧光强参考值的获取方法来测试出使用特定的某种清洗液、活细胞荧光染色剂、细胞裂解液组合时的相对荧光强度参考值;在船舶需要检测时,使
用该特定的清洗液、活细胞荧光染色剂、细胞裂解液的组合按照步骤1-6检测出相对荧光强度,当相对荧光强度不大于相对荧光强度参考值时,说明待排放的船舶压载水符合排放标准,反之说明待排放的船舶压载水不符合排放标准,使用相同的各种试剂的情况下,相对荧光强度参考值仅需检验一次,即可在多次、甚至无数次检验中成为参考值,有此参考值得情况下,本发明结构简单、使用方便,检测速度快、精度高,数据真实可靠。When using the apparatus and method of the present invention to detect 10-50 um of organisms in the ballast water of a ship to be discharged, firstly, according to the method of obtaining a relative fluorescence strong reference value, a specific cleaning liquid, a living cell fluorescent stain, and a cell are tested. Relative fluorescence intensity reference value for lysate combination; when the ship needs to be tested,
The relative fluorescence intensity is detected according to steps 1-6 using the combination of the specific cleaning solution, the living cell fluorescent stain, and the cell lysate. When the relative fluorescence intensity is not greater than the relative fluorescence intensity reference value, the ship ballast water to be discharged is indicated. Compliance with emission standards, and vice versa indicates that the ship's ballast water to be discharged does not meet the emission standards. When using the same various reagents, the relative fluorescence intensity reference value only needs to be tested once, and it can become in multiple or even countless inspections. The reference value has the advantages of simple structure, convenient use, high detection speed, high precision and reliable data.
对附图的简要说明Brief description of the drawing
图1是本发明中速检测装置的结构示意图。BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic view showing the structure of a medium speed detecting device of the present invention.
图2是本发明中相对荧光强度与四爿藻活细胞含量之间的对应关系图。。Figure 2 is a graph showing the relationship between the relative fluorescence intensity and the viable cell content of Tetrahymena in the present invention. .
实施该发明的最佳实施例BEST MODE FOR CARRYING OUT THE INVENTION
实例1Example 1
一种船舶压载水中10-50um生物快速检测方法,包括如下步骤:使用网孔对角线长度为50um的尼龙筛绢制成的过滤器将经压载水处理设备处理后的待排放的压载水样品中不小于50um的存活生物滤出,得滤出液;取200ml滤出液,小于10um的存活生物随液体滤出;使用10ml的物质的量浓度为0.8mol/L、pH=7的Tris-HCl反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;向样品槽内加入活细胞荧光染色剂荧光素二乙酸酯20mg,染色10分钟;染色完成后,向样品槽内加入10ml的细胞裂解液(细胞裂解液为甲醇与氯仿按体积比1∶2混合而成的液体),静置5分钟;细胞充分裂解后,使用荧光计检测样品槽内液体的相对荧光强度。A 10-50um biological rapid detection method for ship ballast water comprises the following steps: a filter made of a nylon mesh having a mesh diagonal length of 50 um and a pressure to be discharged after being treated by a ballast water treatment device The surviving organism of not less than 50um in the water-carrying sample is filtered out to obtain the filtrate; 200ml of the filtrate is taken, and the living organisms less than 10um are filtered out with the liquid; the concentration of the substance using 10ml is 0.8mol/L, pH=7 The Tris-HCl backwashing small pore size filter, the trapped living organism with a size of 10-50 um is flushed into the sample tank; the live cell fluorescent stain fluorescein diacetate 20 mg is added to the sample tank, and staining is performed for 10 minutes. After the staining is completed, 10 ml of cell lysate (the cell lysate is a mixture of methanol and chloroform in a volume ratio of 1:2) is added to the sample tank, and allowed to stand for 5 minutes; after the cells are fully lysed, the fluorometer is used for detection. The relative fluorescence intensity of the liquid in the sample cell.
相对荧光强参考值的获取方法如下:选用60种已知的、每毫升含9个10-50um存活生物的待排放压载水水样作为参考检测样品;对每种参考检测样品进行如下操作:使用网孔对角线长度为50um的尼龙筛绢制成的过滤器将参考检测样品中不小于50um的存活生物滤出,得滤出液;取200ml滤出液,小于10um的存活生物随液体滤出;使用10ml的物质的量浓度为0.8mol/L、pH=7的Tris-HCl反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;向
样品槽内加入活细胞荧光染色剂荧光素二乙酸酯20mg,染色10分钟;染色完成后,向样品槽内加入10ml的细胞裂解液(细胞裂解液为甲醇与氯仿按体积比1∶2混合而成的液体),静置5分钟;细胞充分裂解后,使用荧光计检测样品槽内液体的相对荧光强度值。计算出该60种参考检测样品处理后得到的相对荧光强度的平均值,该平均值为相对荧光强度参考值。将待检测压载水处理后得到的相对荧光强度值与相对荧光强度参考值对比,即可判断该待检测压载水样品是否符合排放标准。The relative fluorescence strong reference value is obtained as follows: 60 known ballast water samples to be discharged containing 9 10-50 um surviving organisms per ml are selected as reference test samples; for each reference test sample, the following operations are performed: A filter made of a nylon mesh with a mesh length of 50 μm is used to filter out the living organism of not less than 50 μm in the reference test sample to obtain a filtrate; 200 ml of the filtrate is taken, and the living organism is less than 10 μm. Filtration; backwashing the small pore size filter with a concentration of 0.8 ml/L of Tris-HCl at a concentration of 0.8 mol/L, pH=7, and rinsing the living organism with a cut-off size of 10-50 um into the sample tank;
Add 20mg of live fluorescent dye fluorescein diacetate to the sample tank and stain for 10 minutes. After the staining is completed, add 10ml of cell lysate to the sample tank (the cell lysate is mixed with methanol and chloroform in a volume ratio of 1:2). The resulting liquid is allowed to stand for 5 minutes; after the cells are fully lysed, the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer. The average of the relative fluorescence intensities obtained after the treatment of the 60 reference test samples was calculated, which is the relative fluorescence intensity reference value. Comparing the relative fluorescence intensity value obtained after the ballast water to be treated with the relative fluorescence intensity reference value, it can be determined whether the ballast water sample to be tested meets the discharge standard.
参考检测样品经如下方法获得:使用大孔径过滤器将经压载水处理设备处理后的待排放的压载水样品中不小于50um的存活生物滤出,得滤出液;取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10um的存活生物、小于10um的存活生物随液体滤出;使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;将样品槽内的液体经FDA-CMFDA法在荧光显微镜下检测,其中10-50um存活生物的含量符合要求时,即可成为参考检测样品。本发明中分别采用清洗液选用BES、MOPS、PBS(pH=7.0)、Tris-HCl(0.2-1.0M,pH=6.0-7.2)中的至少一种,能够最大限度的减少FDA的非生物水解。细胞裂解液能够使活细胞内部的荧光素浸出,提高检测结果的准确性。The reference test sample is obtained by filtering the viable organism of not less than 50 um in the ballast water sample to be discharged after being treated by the ballast water treatment apparatus using a large-aperture filter to obtain a filtrate; taking Aml filtrate Use a small pore size filter to filter and intercept the living organisms of not less than 10um in the filtrate, and the living organisms less than 10um are filtered out with the liquid; backwash the small pore size filter with 3-10% Aml cleaning solution, the size will be intercepted The 10-50um surviving organism is flushed into the sample tank; the liquid in the sample tank is detected by a FDA-CMFDA method under a fluorescence microscope, and when the content of the 10-50um surviving organism meets the requirements, it can be used as a reference test sample. In the present invention, at least one of BES, MOPS, PBS (pH=7.0), Tris-HCl (0.2-1.0M, pH=6.0-7.2) is used in the cleaning solution, respectively, which can minimize the abiotic hydrolysis of FDA. . Cell lysate can leaching fluorescein inside living cells, improving the accuracy of the test results.
FDA连有两个共轭的醋酸自由基,是一种非极性物质,能自由通过藻类细胞膜,被细胞中的酯酶、蛋白酶、脂肪酶等非专一性酶催化水解成荧光素。FDA是无色化合物,本身没有荧光,而反应产物荧光素是一种极性且具有荧光发光性能的物质,化学性质稳定、不易被分解,难以穿过细胞膜,在细胞内积累。当细胞内储存的荧光素超过一定量后释放到环境中,由于反应物与产物的荧光特性不同,因此即使反应物过剩,也不干扰产物的测定。相对荧光强度与10-50um生物含量之间的关系配制不同浓度的四爿藻溶液,其存活细胞含量使用FDA-CMFDA法检测确定。将各溶液加入到本发明的装置中,使用本发明的方法进行相对荧光强度的测试。以四爿藻活细胞含量为x轴,以测得的相对荧光强度为y轴,作出图2,由图2可知,本发明的检测结果与实际活细胞含量具有较好的一一对应关系,能够反映出样品中的活细胞含量。
The FDA has two conjugated acetic acid free radicals. It is a non-polar substance that can pass freely through the algal cell membrane and is hydrolyzed to fluorescein by non-specific enzymes such as esterase, protease and lipase in the cell. The FDA is a colorless compound that does not have fluorescence itself, and the reaction product fluorescein is a polar and fluorescent luminescent material that is chemically stable, is not easily decomposed, and is difficult to pass through the cell membrane and accumulate in cells. When the fluorescein stored in the cells exceeds a certain amount and is released into the environment, since the fluorescence characteristics of the reactants and the product are different, even if the reactants are excessive, the measurement of the product is not disturbed. The relationship between the relative fluorescence intensity and the biological content of 10-50 um was used to prepare different concentrations of tetraterpene solution, and the viable cell content was determined by FDA-CMFDA method. Each solution was added to the apparatus of the present invention and tested for relative fluorescence intensity using the method of the present invention. Taking the living cell content of tetradium algae as the x-axis and the measured relative fluorescence intensity as the y-axis, FIG. 2 is made. As can be seen from FIG. 2, the detection result of the present invention has a good one-to-one correspondence with the actual living cell content. It reflects the amount of viable cells in the sample.
发明实施例Invention embodiment
一种船舶压载水中10-50um生物快速检测方法,包括如下步骤:A 10-50um biological rapid detection method for ship ballast water comprises the following steps:
(1)使用大孔径过滤器将经压载水处理设备处理后的待排放的压载水样品中不小于50um的存活生物滤出,得滤出液;(1) using a large-aperture filter to filter out living organisms of not less than 50 μm in the ballast water sample to be discharged after being treated by the ballast water treatment equipment, to obtain a filtrate;
(2)取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10um的存活生物、小于10um的存活生物随液体滤出;(2) taking Aml filtrate, filtering and intercepting the living organism of not less than 10um in the filtrate, and the living organisms less than 10um in the filtrate are filtered out with the liquid;
(3)使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;(3) backwashing the small-aperture filter with a cleaning solution of 3-10% Aml, and rinsing the trapped living organism with a size of 10-50 um into the sample tank;
(4)向样品槽内加入活细胞荧光染色剂,染色5-30分钟;(4) adding a living cell fluorescent stain to the sample tank and staining for 5-30 minutes;
(5)染色后,向样品槽内加入其内液体体积0.5-2倍的细胞裂解液;(5) after dyeing, adding 0.5-2 times the volume of the liquid lysate to the sample tank;
(6)待细胞充分裂解后,使用荧光计检测样品槽内液体的相对荧光强度;(6) After the cells are sufficiently lysed, the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer;
相对荧光强参考值的获取方法如下:The method for obtaining the relative fluorescence strong reference value is as follows:
a、选用已知符合压载水处理D-2标准中对10-50um存活生物的规定、含10-50um存活生物个数达到上限的水样作为参考检测样品;使用大孔径过滤器将参考检测样品中不小于50um的存活生物滤出,得滤出液;a. Select a water sample that meets the requirements of 10-50um surviving organisms in the D-2 standard for ballast water treatment, and the upper limit of the number of living organisms with 10-50um as the reference test sample; use the large-aperture filter to refer to the test. The viable organism of not less than 50 um in the sample is filtered out to obtain a filtrate;
b、取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10um的存活生物、小于10um的存活生物随液体流出;b, taking Aml filtrate, using a small pore filter to filter and intercepting the surviving organisms of not less than 10um in the filtrate, the living organisms less than 10um flow out with the liquid;
c、使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;c. Backwash the small pore size filter with 3-10% Aml cleaning solution, and rinse the trapped living organism with the size of 10-50um into the sample tank;
d、向样品槽内加入活细胞荧光染色剂,染色5-30(10)分钟;d, adding a living cell fluorescent stain to the sample tank, staining for 5-30 (10) minutes;
e、染色后,向样品槽内加入其内液体体积0.5-2倍的细胞裂解液;e, after dyeing, adding 0.5-2 times the volume of the liquid lysate to the sample tank;
f、待细胞(膜)充分裂(溶)解后,使用荧光计检测样品槽内液体的相对荧光强度;f. After the cell (membrane) is sufficiently cleaved (dissolved), the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer;
g、按步骤a中参考检测样品的选取标准选取1-100种不同的参考检测样品,每种参考检测样品按步骤a、b、c、d、e、f的操作方法检测至少一次,根据所得数据求出平均相对荧光强度值,该平均相对荧光强度值为相对荧光强度参考值;g, according to the selection criteria of the reference test sample in step a, 1-100 different reference test samples are selected, and each reference test sample is tested at least once according to the operation methods of steps a, b, c, d, e, f, according to the obtained The data is used to determine an average relative fluorescence intensity value, the average relative fluorescence intensity value being a relative fluorescence intensity reference value;
将第(6)步获得的相对荧光强度值与第g步获得的相对荧光强度参考值对比,
判断该压载水样品是否符合排放标准。Comparing the relative fluorescence intensity values obtained in step (6) with the relative fluorescence intensity reference values obtained in step g,
Determine if the ballast water sample meets the emission standards.
其中A为任何数值,优选100-500的自然数。Wherein A is any number, preferably a natural number from 100 to 500.
本发明中第a步中所述的参考检测样品经如下方法获得:使用大孔径过滤器将经压载水处理设备处理后的待排放的压载水样品中不小于50um的存活生物滤出,得滤出液;取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10u m的存活生物、小于10um的存活生物随液体滤出;使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;将样品槽内的液体经FDA-CMFDA法在荧光显微镜下检测,其中10-50um存活生物的含量符合要求时,该经压载水处理设备处理后的待排放的压载水样品即可成为参考检测样品。The reference test sample described in the step a in the present invention is obtained by filtering a surviving organism of not less than 50 μm in the ballast water sample to be discharged after being treated by the ballast water treatment apparatus using a large-aperture filter, The filtrate is obtained; the Aml filtrate is taken, and the small-aperture filter is used to filter and retain the living organisms of not less than 10 μm in the filtrate, and the living organisms less than 10 μm are filtered with the liquid; the cleaning solution of 3-10% Aml is used. Backwashing the small-aperture filter, flushing the trapped living organisms with a size of 10-50um into the sample tank; and checking the liquid in the sample tank by the FDA-CMFDA method under the fluorescence microscope, wherein the content of 10-50um surviving organisms When the requirements are met, the ballast water sample to be discharged after being treated by the ballast water treatment equipment can be used as a reference test sample.
本发明中第a步中所述的参考检测样品也可经如下方法获得:人工配制藻类溶液或自然海水经紫外线(UV)处理或经次氯酸钠(NaClO)处理,得测试液;使用大孔径过滤器将测试液中不小于50um的存活生物滤出,得滤出液;取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10um的存活生物、小于10um的存活生物随液体滤出;使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;将样品槽内的液体经FDA-CMFDA法在荧光显微镜下检测,其中10-50um存活生物的含量符合要求时,该经压载水处理设备处理后的待排放的压载水样品即可成为参考检测样品。The reference test sample described in the step a of the present invention can also be obtained by manually preparing an algae solution or natural seawater by ultraviolet (UV) treatment or sodium hypochlorite (NaClO) to obtain a test liquid; using a large pore size filter The viable organism of not less than 50 um in the test solution is filtered to obtain a filtrate; the Aml filtrate is taken, and a small-aperture filter is used to filter and retain a living organism of not less than 10 μm in the filtrate, and a living organism of less than 10 μm with the liquid Filtration; backwash the small pore size filter with 3-10% Aml cleaning solution, flush the trapped living organism with the size of 10-50um into the sample tank; the liquid in the sample tank is fluoresced by FDA-CMFDA method Under the microscope, when the content of 10-50 um surviving organisms meets the requirements, the ballast water sample to be discharged after being treated by the ballast water treatment equipment can be used as a reference test sample.
更进一步的,所述的大孔径过滤器为使用网孔对角线长度为35-50um的尼龙筛绢制成的过滤器;所述的小孔径过滤器为使用绝对孔径为10um的微孔滤膜制成的、可反冲洗的过滤器。所述活细胞荧光染色剂为FDA或/和CMFDA,其加入量根据样品槽内液体体积、以1-3mg/ml(2mg/ml)的比例加入。染色反应时间为5-30(10)分钟。FDA为荧光素二乙酸酯;所述的清洗液是BES、MOPS、PBS(pH=7.0)、Tris-HCl(0.2-1.0M,pH=6.0-7.2)中的至少一种,可有效防止FDA或CMFDA的非生物水解。其中PBS为:硫化铅、Tris-HCl为:三(羟甲基)氨基甲烷。所述的细胞裂解液为甲醇与氯仿(体积比1∶1-1∶3)的混合液或丙酮中的一种。Further, the large-aperture filter is a filter made of a nylon mesh having a mesh diagonal length of 35-50 um; and the small-aperture filter is a microporous filter having an absolute pore diameter of 10 μm. Membrane-made, backwashable filter. The living cell fluorescent staining agent is FDA or/and CMFDA, and the amount thereof is added in a ratio of 1-3 mg/ml (2 mg/ml) according to the liquid volume in the sample tank. The staining reaction time is 5-30 (10) minutes. The FDA is fluorescein diacetate; the cleaning solution is at least one of BES, MOPS, PBS (pH=7.0), Tris-HCl (0.2-1.0 M, pH=6.0-7.2), which can effectively prevent Abiotic hydrolysis by the FDA or CMFDA. The PBS is: lead sulfide, and Tris-HCl is: tris(hydroxymethyl)aminomethane. The cell lysate is one of a mixture of methanol and chloroform (1:1:3 by volume) or acetone.
用于实现上述检测方法的船舶压载水中10-50um生物快速检测装置,结构如图
1所示:包括样品箱2、冲洗液箱13、裂解液箱3、废水箱14、检测箱17、一级过滤器1和二级滤器8,一级过滤器1为使用网孔对角线长度为50um的尼龙筛绢制成的过滤器;二级滤器是使用绝对孔径为10um的微孔滤膜制成的过滤器。一级过滤器的出口与样品箱2进口相连,经一级过滤器过滤后的液体进入与样品箱2内一级过滤器和样品箱2间连接管路上可以设有一级泵,也可以是一级过滤器设在样品箱2上方,利用液体的重力自然流入。检测箱17上设有染色剂加入口12,样品箱2、冲洗液箱14和裂解液箱3的出口上分别设有样品泵4、冲洗泵13和裂解泵5,样品泵4的出口经过滤阀6与二级过滤器8的一端口相连,二级过滤器8的另一端口经冲洗阀11与冲洗泵13出口相连,二级过滤器8与冲洗阀11间的连接管经排液阀10与废水箱15相连,检测箱17的进液口经进液阀9与过滤阀6和二级过滤器8间的连接管路相连,裂解泵5出口经裂解阀7与检测箱17相连,检测箱17上设有荧光计18,荧光计18的检测探头设在检测箱17内;荧光计的发射光波长为460-490nm,接收的激发光波长为510-550nm;所述的检测箱下部经排放阀16与废水箱15相连。10-50um biological rapid detection device for ship ballast water used to realize the above detection method, the structure is as shown in the figure
1 shows: including sample box 2, flushing liquid tank 13, cracking liquid tank 3, waste water tank 14, detecting tank 17, primary filter 1 and secondary filter 8, and primary filter 1 is using mesh diagonal A filter made of nylon sifter with a length of 50 um; a secondary filter is a filter made of a microporous membrane with an absolute pore size of 10 um. The outlet of the primary filter is connected to the inlet of the sample box 2, and the liquid filtered by the primary filter enters the connection line between the primary filter and the sample box 2 in the sample box 2, and may be provided with a primary pump or a first pump. The stage filter is placed above the sample box 2 and naturally flows in by the gravity of the liquid. The detection tank 17 is provided with a dye inlet port 12, and the sample tank 4, the flushing pump 13 and the cracking pump 5 are respectively disposed at the outlets of the sample tank 2, the flushing liquid tank 14 and the lysing tank 3, and the outlet of the sample pump 4 is filtered. The valve 6 is connected to one port of the secondary filter 8, the other port of the secondary filter 8 is connected to the outlet of the flushing pump 13 via the flushing valve 11, and the connecting pipe between the secondary filter 8 and the flushing valve 11 is discharged through the draining valve 10 is connected to the waste water tank 15, and the liquid inlet of the detection tank 17 is connected to the connection line between the filter valve 6 and the secondary filter 8 via the inlet valve 9, and the outlet of the cracking pump 5 is connected to the detection tank 17 via the cracking valve 7. The detection box 17 is provided with a fluorometer 18, and the detection probe of the fluorometer 18 is disposed in the detection box 17; the emission wavelength of the fluorometer is 460-490 nm, and the wavelength of the received excitation light is 510-550 nm; the lower part of the detection box The discharge valve 16 is connected to the waste water tank 15.
本发明中所述的10-50um生物是指:尺寸小于50um但大于或等于10um的存活生物。The 10-50 um organism described in the present invention refers to a living organism having a size of less than 50 um but greater than or equal to 10 um.
船舶压载水中10-50um生物快速检测装置工作时,将待检测的压载水样品放入一级过滤器内,一级过滤器将压载水样品中不小于50um的存活生物滤出,经其过滤后的滤出液进入样品箱内,向冲洗液箱和裂解液箱内分别加入清洗液和细胞裂解液;打开过滤阀和排液阀,其它阀门关闭,样品泵工作,定量的将样品箱内的滤出液经二级过滤器过滤,滤出的液体排入废水箱内,而不小于10um的存活生物被二级过滤器截留;关闭过滤阀和排液阀,此时染色阀和排放阀也处于关闭状态,打开冲洗阀和进液阀,冲洗泵工作,使用定量的冲洗液对二级过滤器反冲洗,将其截留的10-50um生物冲洗进检测箱内;关闭冲洗阀和进液阀,经向染色剂加入口向样品箱内加入适量的活细胞荧光染色剂、对活细胞进行荧光染色;对活细胞染色完成(约10分钟)后,打开裂解阀,裂解泵工作,将细胞裂解液输入检测箱内,待细胞充分裂解后,使用荧光计测出检测箱内的
相对荧光强度值。选取80种通过实验室方法已经确定的、含9个10-50um存活生物的待排放压载水水样作为待检测的压载水样品,通过上述的使用方法使用该装置进行检测,求取平均值作为相对荧光强度参考值;将相对荧光强度值与相对荧光强度参考值对比,当相对荧光强度不大于相对荧光强度参考值时,说明待排放的船舶压载水符合排放标准,反之说明待排放的船舶压载水不符合排放标准,使用相同的各种试剂的情况下,相对荧光强度参考值仅需求取一次,即可在多次、甚至无数次检验中成为参考值,有此参考值的情况下,本发明结构简单、使用方便,检测速度快、精度高,数据真实可靠。
When the 10-50um biological rapid detection device in the ship ballast water works, the ballast water sample to be tested is placed in the primary filter, and the primary filter filters out the living organism of not less than 50um in the ballast water sample. The filtered filtrate enters the sample box, and the cleaning liquid and the cell lysate are respectively added to the rinsing liquid tank and the lysing liquid tank; the filter valve and the liquid discharging valve are opened, the other valves are closed, the sample pump is operated, and the sample is quantitatively sampled. The filtrate in the tank is filtered through a secondary filter, and the filtered liquid is discharged into the waste water tank. The living organisms not less than 10um are trapped by the secondary filter; the filter valve and the drain valve are closed, and the dye valve and the dye valve are The discharge valve is also closed, the flush valve and the inlet valve are opened, the flushing pump is operated, the secondary filter is backwashed with a quantitative flushing liquid, and the 10-50 um of the trapped organism is flushed into the test chamber; the flushing valve is closed and Into the liquid inlet valve, add appropriate amount of live cell fluorescent stain to the sample box through the inlet of the staining agent, and fluorescently stain the living cells; after the living cells are dyed (about 10 minutes), the cracking valve is opened, and the pyrolysis pump works. Cell lysates input detection tank, until sufficient cell lysis, fluorescence is measured using the detection tank
Relative fluorescence intensity values. 80 samples of ballast water to be discharged containing 9 10-50 um surviving organisms determined by laboratory methods were selected as the ballast water samples to be tested, and the device was used for detection by the above-mentioned method of use to obtain an average The value is used as a relative fluorescence intensity reference value; the relative fluorescence intensity value is compared with the relative fluorescence intensity reference value, and when the relative fluorescence intensity is not greater than the relative fluorescence intensity reference value, it indicates that the ship ballast water to be discharged meets the emission standard, and vice versa The ship's ballast water does not meet the discharge standard. When the same reagent is used, the relative fluorescence intensity reference value is only required to be taken once, and it can be used as a reference value in multiple or even countless tests. In case, the invention has the advantages of simple structure, convenient use, high detection speed, high precision and reliable and reliable data.
Claims (9)
- 一种船舶压载水中10-50um生物快速检测方法,其特征在于包括如下步骤:A 10-50um biological rapid detection method for ship ballast water, characterized in that the method comprises the following steps:(1)使用大孔径过滤器将经压载水处理设备处理后的压载水样品中不小于50um的存活生物滤出,得滤出液;(1) using a large-aperture filter to filter out viable organisms of not less than 50 μm in the ballast water sample treated by the ballast water treatment equipment to obtain a filtrate;(2)取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10um的存活生物、小于10um的存活生物随液体滤出;(2) taking Aml filtrate, filtering and intercepting the living organism of not less than 10um in the filtrate, and the living organisms less than 10um in the filtrate are filtered out with the liquid;(3)使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;(3) backwashing the small-aperture filter with a cleaning solution of 3-10% Aml, and rinsing the trapped living organism with a size of 10-50 um into the sample tank;(4)向样品槽内加入活细胞荧光染色剂,染色5-30分钟;(4) adding a living cell fluorescent stain to the sample tank and staining for 5-30 minutes;(5)染色后,向样品槽内加入其内液体体积0.5-2倍的细胞裂解液;(5) after dyeing, adding 0.5-2 times the volume of the liquid lysate to the sample tank;(6)待细胞充分裂解后,使用荧光计检测样品槽内液体的相对荧光强度;(6) After the cells are sufficiently lysed, the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer;相对荧光强参考值的获取方法如下:The method for obtaining the relative fluorescence strong reference value is as follows:a、选用已知符合压载水处理D-2标准中对10-50um存活生物的规定、含10-50um存活生物个数达到上限的水样作为参考检测样品,使用大孔径过滤器将参考检测样品中不小于50um的存活生物滤出,得滤出液;a. Select a water sample that meets the requirements of 10-50um surviving organisms in the D-2 standard for ballast water treatment, and the upper limit of the number of living organisms with 10-50um as the reference test sample, and use the large-aperture filter to refer to the test. The viable organism of not less than 50 um in the sample is filtered out to obtain a filtrate;b、取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10um的存活生物、小于10um的存活生物随液体滤出;b, taking Aml filtrate, using a small pore size filter to filter and retaining not less than 10um of surviving organisms in the filtrate, less than 10um of living organisms are filtered with liquid;c、使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;c. Backwash the small pore size filter with 3-10% Aml cleaning solution, and rinse the trapped living organism with the size of 10-50um into the sample tank;d、向样品槽内加入活细胞荧光染色剂,染色5-30分钟;d, adding a living cell fluorescent stain to the sample tank, staining for 5-30 minutes;e、染色后,向样品槽内加入其内液体体积0.5-2倍的细胞裂解液;e, after dyeing, adding 0.5-2 times the volume of the liquid lysate to the sample tank;f、待细胞充分裂解后,使用荧光计检测样品槽内液体的相对荧光强度;f. After the cells are fully lysed, the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer;g、按步骤a中参考检测样品的选取标准选取1-100种不同的参考检 测样品,每种参考检测样品按步骤a、b、c、d、e、f的操作方法检测至少一次,根据所得数据求出平均相对荧光强度值,该平均相对荧光强度值为相对荧光强度参考值;g. Select 1-100 different reference tests according to the selection criteria of the reference test samples in step a. The sample is tested, and each reference test sample is detected at least once according to the operation methods of steps a, b, c, d, e, and f, and the average relative fluorescence intensity value is obtained according to the obtained data, and the average relative fluorescence intensity value is a relative fluorescence intensity reference. value;将第(6)步获得的相对荧光强度值与第g步获得的相对荧光强度参考值对比,判断该压载水样品是否符合排放标准。The relative fluorescence intensity value obtained in the step (6) is compared with the relative fluorescence intensity reference value obtained in the step g, and it is judged whether the ballast water sample meets the emission standard.
- 根据权利要求1所述的一种船舶压载水中10-50um生物快速检测方法,其特征在于:第a步中所述的参考检测样品经如下方法获得:使用大孔径过滤器将经压载水处理设备处理后的待排放的压载水样品中不小于50um的存活生物滤出,得滤出液;取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10um的存活生物、小于10um的存活生物随液体滤出;使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;将样品槽内的液体经FDA-CMFDA法在荧光显微镜下检测,其中10-50um存活生物的含量符合要求时,即可成为参考检测样品。A 10-50 um biological rapid detection method for a ship's ballast water according to claim 1, wherein the reference test sample described in the step a is obtained by using a large-aperture filter to pass the ballast water. The surviving organism of not less than 50um in the ballast water sample to be discharged after the treatment equipment is filtered out, and the filtrate is obtained; the Aml filtrate is taken, and the filter is filtered using a small pore size filter and the survival of not less than 10um in the filtrate is intercepted. Biological, less than 10um surviving organisms are filtered out with liquid; backwash the small pore size filter with 3-10% Aml cleaning solution, and the trapped living organisms with the size of 10-50um are flushed into the sample tank; The liquid is tested under the fluorescence microscope by the FDA-CMFDA method, and when the content of 10-50 um surviving organism meets the requirements, it can be used as a reference test sample.
- 根据权利要求1所述的一种船舶压载水中10-50um生物快速检测方法,其特征在于:第a步中所述的参考检测样品经如下方法获得:人工配制藻类溶液或自然海水经紫外线处理或经次氯酸钠处理,得测试液;使用大孔径过滤器将测试液中不小于50um的存活生物滤出,得滤出液;取Aml滤出液,使用小孔径过滤器过滤并截留滤出液中不小于10um的存活生物、小于10um的存活生物随液体滤出;使用3-10%Aml的清洗液反冲洗小孔径过滤器、将截留的尺寸在10-50um的存活生物冲洗进入到样品槽内;将样品槽内的液体经FDA-CMFDA法在荧光显微镜下检测,其中10-50um存活生物的含量符合要求时,即可成为参考检测样品。The method for detecting rapid detection of 10-50 um in ship ballast water according to claim 1, wherein the reference test sample described in the step a is obtained by manually preparing an algae solution or natural seawater by ultraviolet treatment. Or the test solution is obtained by sodium hypochlorite treatment; the viable liquid of not less than 50 um in the test liquid is filtered out by using a large-aperture filter to obtain a filtrate; the Aml filtrate is taken, filtered using a small-diameter filter and the filtrate is intercepted. Surviving organisms of not less than 10um, viable organisms of less than 10um are filtered out with liquid; backwash the small-aperture filter with 3-10% Aml of washing solution, and flush the retained organisms with a size of 10-50um into the sample tank. The liquid in the sample tank is detected by a FDA-CMFDA method under a fluorescence microscope, and when the content of the 10-50 um surviving organism meets the requirements, it can be used as a reference test sample.
- 根据权利要求1或2或3所述的一种船舶压载水中10-50um生物快速检测方法,其特征在于:所述的大孔径过滤器为使用网孔对角线长度为35-50um的尼龙筛绢制成的过滤器;所述的小孔径过滤器为 使用绝对孔径为10um的微孔滤膜制成的、可以反冲洗的过滤器。The method for rapid detection of 10-50 um in a ballast water of a ship according to claim 1 or 2 or 3, wherein the large-aperture filter is a nylon having a mesh diagonal length of 35-50 um. a filter made of sieve; the small pore filter is A backwashable filter made of a microporous membrane with an absolute pore size of 10 um.
- 根据权利要求4所述的一种船舶压载水中10-50um生物快速检测方法,其特征在于:所述活细胞荧光染色剂为FDA或/和CMFDA,其加入量根据样品槽内液体体积、以1-3mg/ml的比例加入。The method according to claim 4, wherein the living cell fluorescent staining agent is FDA or/and CMFDA, and the adding amount is based on the liquid volume in the sample tank. Add in a ratio of 1-3 mg/ml.
- 根据权利要求5所述的一种船舶压载水中10-50um生物快速检测方法,其特征在于:所述的清洗液是BES、MOPS、PBS(pH=7.0)、Tris-HCl中的至少一种。The method according to claim 5, wherein the cleaning solution is at least one of BES, MOPS, PBS (pH=7.0) and Tris-HCl. .
- 根据权利要求5或6所述的一种船舶压载水中10-50um生物快速检测方法,其特征在于:所述细胞裂解液为甲醇与氯仿按体积比1∶1-1∶3的混合液或丙酮中的一种。The method for rapid detection of 10-50 um in a ship's ballast water according to claim 5 or 6, wherein the cell lysate is a mixture of methanol and chloroform in a volume ratio of 1:1 to 1:3 or One of the acetone.
- 一种船舶压载水中10-50um生物快速检测装置,其特征在于:包括样品箱、冲洗液箱、裂解液箱、废水箱、检测箱、一级过滤器和二级滤器,一级过滤器的出口与样品箱进口相连,检测箱上设有染色剂加入口,样品箱、冲洗液箱和染色剂箱的出口上分别设有样品泵、冲洗泵和裂解泵,样品泵的出口经过滤阀与二级过滤器的一端口相连,二级过滤器的另一端口经冲洗阀与冲洗泵出口相连,二级过滤器与冲洗阀间的连接管经排液阀与废水箱相连,检测箱的进液口经进液阀与过滤阀和二级过滤器间的连接管路相连,裂解泵出口与检测箱相连,检测箱上设有荧光计。The utility model relates to a 10-50um biological rapid detecting device for ship ballast water, which comprises: a sample box, a flushing liquid tank, a lysing liquid tank, a waste water tank, a detecting box, a primary filter and a secondary filter, and a primary filter. The outlet is connected to the inlet of the sample box, and the dye inlet is provided on the detection box. The sample pump, the flushing pump and the cracking pump are respectively arranged at the outlets of the sample box, the flushing liquid tank and the dyeing tank, and the outlet of the sample pump passes through the filtering valve and One port of the secondary filter is connected, and the other port of the secondary filter is connected to the outlet of the flushing pump via a flushing valve, and the connecting pipe between the secondary filter and the flushing valve is connected to the waste water tank through the drain valve, and the inlet of the detecting box The liquid port is connected to the connecting line between the filter valve and the secondary filter through the inlet valve, and the outlet of the cracking pump is connected to the detecting box, and a fluorescent meter is arranged on the detecting box.
- 根据权利要求8所述的船舶压载水中10-50um生物快速检测装置,其特征在于:所述的一级过滤器为网孔对角线长度为35-50um的尼龙筛绢;二级滤器为绝对孔径为10um的微孔滤膜;荧光计的发射光波长为460-490nm,接收的激发光波长为510-550nm。 The 10-50 um biological rapid detecting device for ship ballast water according to claim 8, wherein the primary filter is a nylon mesh having a mesh diagonal length of 35-50 um; the secondary filter is A microporous membrane with an absolute pore size of 10 um; the fluorometer emits at a wavelength of 460-490 nm and the received excitation light has a wavelength of 510-550 nm.
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CN108827891A (en) * | 2018-06-21 | 2018-11-16 | 上海海事大学 | Ballast water for ship microalgae cell biology amount detection systems and method |
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