WO2018041215A1

WO2018041215A1 - Fast detection method and device for organisms of 10-50 μm in ship ballast water

Info

Publication number: WO2018041215A1
Application number: PCT/CN2017/099893
Authority: WO
Inventors: 张灿; 王建; 詹世杰; 董晓林; 靳双亭; 韩德民
Original assignee: 威海中远造船科技有限公司; 张灿; 王建
Priority date: 2016-08-31
Filing date: 2017-08-31
Publication date: 2018-03-08
Also published as: CN106244665A

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

10-50 um biological rapid detection method and device for ship ballast water

Technical field

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.

Background technique

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:

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;

And as a standard of human health, 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.

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.

technical problem

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.

Problem solution

Technical solution

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.

The technical solution adopted by the present invention to solve the above deficiencies of the prior art is:

A 10-50um biological rapid detection method for ship ballast water, characterized in that the method comprises the following steps:

(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) Take Aml filtrate, filter with a small pore size filter and intercept the living organisms of not less than 10um in the filtrate, small

The 10 um surviving organism is filtered out with the liquid;

(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.

Flush into the sample tank;

(4) adding a living cell fluorescent stain to the sample tank and staining for 5-30 (10) minutes;

(5) after dyeing, adding 0.5-2 times the volume of the liquid lysate to the sample tank;

(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;

The method for obtaining the relative fluorescence strong reference value is as follows:

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, 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. 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, adding a living cell fluorescent stain to the sample tank, staining for 5-30 (10) minutes;

e, after dyeing, adding 0.5-2 times the volume of the liquid lysate to the sample tank;

f. After the cells are fully lysed, the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer;

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;

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 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.

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;

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 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.

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. 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.

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.

Advantageous effects of the invention

Beneficial effect

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.

Brief description of the drawing

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS 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. .

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1

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.

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.

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.

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.

Invention embodiment

Embodiments of the invention

A 10-50um biological rapid detection method for ship ballast water comprises the following steps:

(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) 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) 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) adding a living cell fluorescent stain to the sample tank and staining for 5-30 minutes;

(6) After the cells are sufficiently lysed, the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer;

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, 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;

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;

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.

Wherein 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 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.

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.

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.

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.

Industrial applicability

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

A 10-50um biological rapid detection method for ship ballast water, characterized in that the method comprises the following steps:

(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) 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) 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) adding a living cell fluorescent stain to the sample tank and staining for 5-30 minutes;

(5) after dyeing, adding 0.5-2 times the volume of the liquid lysate to the sample tank;

(6) After the cells are sufficiently lysed, the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer;

The method for obtaining the relative fluorescence strong reference value is as follows:

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, 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. 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, adding a living cell fluorescent stain to the sample tank, staining for 5-30 minutes;

e, after dyeing, adding 0.5-2 times the volume of the liquid lysate to the sample tank;

f. After the cells are fully lysed, the relative fluorescence intensity of the liquid in the sample tank is detected using a fluorometer;

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;

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.
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.
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.
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.
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.
The method according to claim 5, wherein the cleaning solution is at least one of BES, MOPS, PBS (pH=7.0) and Tris-HCl. .
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.
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.
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.