CN106244665A - Method and device for quickly detecting 10-50um organisms in ship ballast water - Google Patents
Method and device for quickly detecting 10-50um organisms in ship ballast water Download PDFInfo
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- CN106244665A CN106244665A CN201610776270.0A CN201610776270A CN106244665A CN 106244665 A CN106244665 A CN 106244665A CN 201610776270 A CN201610776270 A CN 201610776270A CN 106244665 A CN106244665 A CN 106244665A
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
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- 238000010079 rubber tapping Methods 0.000 claims description 5
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- 239000000126 substance Substances 0.000 description 4
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- 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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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
A method and a device for quickly detecting 10-50um organisms in ship ballast water relate to a method and a device for detecting ballast water, and the method comprises the following steps: filtering out living organisms with the diameter not less than 50um in the ballast water sample by using a filter to obtain filtrate; filtering and retaining no less than 10um of viable organisms in the filtrate using a small pore size filter; the cleaning solution back-washes the small-aperture filter and washes the living organisms of 10-50um into the sample tank; adding a living cell fluorescent stain into the sample groove for dyeing; adding cell lysate to the sample tank; detecting the relative fluorescence intensity of the liquid in the sample groove by using a fluorometer; comparing the relative fluorescence intensity with a reference value to judge whether the relative fluorescence intensity reaches the standard or not; the detection device is an automatic detection system built by using a container, a liquid pump and a fluorometer. The invention has simple structure, convenient use, high detection speed, high precision and real and reliable data.
Description
Technical field
The present invention relates to detection method and the device of biological living in the ballast water that boats and ships carry, say it is a kind of detection in detail
10-50um biology method for quick and device in the ballast water for ship that precision is high, data are true and reliable.
Background technology
It is known that transport by sea boats and ships are in order to ensure stability of ship, typically install ballast water additional originating harbour, wait to stop
Discharging ballast water when leaning against purpose harbour again, its entrained biology can bring biotic intrusion risk to purpose Port State.In order to
Tackling this problem, International Maritime Organization (IMO), United States Coasts Guard (USCG) and Environmental Protection Agency (EPA) are all formulated
Corresponding biotic intrusion counter-measure and discharge of ballast water control pact, it is desirable to most commercial transportation boats and ships must install ballast additional
Water treatment facilities (BWTS) are to process ballast water.The ETV draft clear stipulaties of IMO G8 directive/guide and USCG reference discharge ballast
The size of organism in water and viable biological concentration, as follows:
The minimum dimension viable biological more than or equal to 50 m is less than 10/m3；
Minimum dimension is less than 50 m but is more than or equal to the viable biological of 10 m less than 10/ml；
And as health standard, target microorganism is less than following concentration:
Poisonous vibrio cholera (serotype O1 and O139) is less than 1 cfu/100ml；
Escherichia coli: less than 250 cfu/100ml；
Enterococcus: less than 100 cfu/100ml.
At present, IMO world compressive effect also Pending The Entry Into Force, but the effective date the most close on.USCG the most unilaterally announces
Its specification comes into force, and the commercial transportation boats and ships of mandatory provision all entrance U.S. waters must be installed and meet USCG specification and discharge standard
BWTS, otherwise cannot reach port and carry out relevant operation.Because of water quality and the bio distribution situation difference in each waters, boats and ships institute
The BWTS installed is it cannot be guaranteed that the ballast water of discharge every time can meet discharge standard, it is necessary to before discharging near Port State voluntarily
Detection target organism content, therefore, on ship, testing staff must have abundant biological basis knowledge and detection experience, joins simultaneously
Standby advanced biological detection instrument.For most ship's repairs & maintenance facilities and crewman's know-how, it is difficult to meet this requirement.
In general, 10-50um biology is mainly unicellular alga, because of its species particularity, and can not be by filtration side
Method is removed completely, can not be killed completely by ultra-vioket radiation or chemical method, and of a great variety, it has also become ballast organism in water
The difficult point of detection.Existing device for fast detecting is to estimate frustule quantity according to the chlorophyllous content of algae, our experiments show that,
Chlorophyll in part dead frustule still can keep complete, the method judging cell survival conditions according to chlorophyll content
Error is big, reliability is low, erroneous judgement even occurs.On the other hand, existing detection equipment can be by thin for the algae of some below 10um
Born of the same parents also count, and detection data are the most accurate.
Summary of the invention
Present invention aim to address above-mentioned the deficiencies in the prior art, it is provided that a kind of simple in construction, easy to use, detection speed
Degree is fast, precision is high, 10-50um biology method for quick and device in the ballast water for ship that data are true and reliable.
The present invention solves above-mentioned the deficiencies in the prior art and be the technical scheme is that
10-50um biology method for quick in a kind of ballast water for ship, it is characterised in that comprise the steps:
(1) large aperture filter is used will (to be discharged) the ballast water sample after ballast water treatment equipment processes to be not less than
The viable biological of 50um leaches, and obtains filter liquor；
(2) take Aml filter liquor, use small-bore filter to filter and retain and filter liquor is not less than the viable biological of 10um, little
Viable biological in 10um leaches with liquid；
(3) use the cleanout fluid backwash small-bore filter of 3-10%Aml, by the size that retains at the viable biological of 10-50um
It is flushed in sample cell；
(4) adding live cell fluorescent stain in sample cell, dye 5-30(10) minute；
(5), after dyeing, in sample cell, the cell pyrolysis liquid of liquid volume 0.5-2 times in it is added；
(6) after cell (film) fully splits (molten) solution, the relative intensity of fluorescence of liquid in use exometer detection sample cell；
The acquisition methods of the strong reference value of relative fluorescence is as follows:
A, selection known symbols are closed in ballast water treatment D-2 standard raw to regulation, the survival containing 10-50um of 10-50um viable biological
Thing number reaches the water sample of the upper limit and will be not less than in detection sample as reference detection sample, use large aperture filter
The viable biological of 50um leaches, and obtains filter liquor；
B, take Aml filter liquor, use small-bore filter to filter and retain and filter liquor is not less than the viable biological of 10um, is less than
The viable biological of 10um leaches with liquid；
C, use the cleanout fluid backwash small-bore filter of 3-10%Aml, by the size that retains at the viable biological of 10-50um
It is flushed in sample cell；
D, in sample cell add live cell fluorescent stain, dye 5-30(10) minute；
After e, dyeing, in sample cell, add the cell pyrolysis liquid of liquid volume 0.5-2 times in it；
F, after cell fully cracks, use the relative intensity of fluorescence of liquid in exometer detection sample cell；
G, by step a with reference to detection sample selection standard choose 1-100 kind different reference detection sample, every kind with reference to examine
Test sample product are detected at least one times by the operational approach of step a, b, c, d, e, f, obtain average relative fluorescence according to the data obtained strong
Angle value, this average relative fluorescence intensity level is relative intensity of fluorescence reference value；
The relative intensity of fluorescence reference value contrast that the relative intensity of fluorescence value (6th) step obtained and g step obtain, it is judged that this pressure
Carry whether water sample meets discharge standard.
In the present invention, reference the detection sample described in a step obtains through following method: use large aperture filter will be through
In (to be discharged) ballast water sample after the process of ballast water treatment equipment, the viable biological not less than 50um leaches, and must leach
Liquid；Take Aml filter liquor, use small-bore filter to filter and retain and filter liquor is not less than the viable biological of 10um, is less than
The viable biological of 10um leaches with liquid；The cleanout fluid backwash small-bore filter using 3-10%Aml, the size that will retain
Viable biological at 10-50um is flushed in sample cell；By the liquid in sample cell through FDA-CMFDA method at fluorescence microscopy
Detect under mirror, when wherein the content of 10-50um viable biological meets the requirements, can become with reference to detection sample.
In the present invention, the reference detection sample described in a step also can obtain through following method: artificial preparation algae solution
Or nature seawater processes through ultraviolet (UV) or processes through sodium hypochlorite (NaClO), obtain test fluid；Use large aperture filter will
In test fluid, the viable biological not less than 50um leaches, and obtains filter liquor；Take Aml filter liquor, use small-bore filter to filter also
Retain the viable biological not less than 10um in filter liquor, the viable biological less than 10um leaches with liquid；Use 3-10%Aml's
Cleanout fluid backwash small-bore filter, the size retained is flushed in sample cell at the viable biological of 10-50um；Will
Liquid in sample cell detects under FDA-CMFDA method is at fluorescence microscope, and wherein the content of 10-50um viable biological conforms to
When asking, can become with reference to detection sample.
Heretofore described large aperture filter is that the nylon bolting silk using mesh catercorner length to be 35-50um is made
Filter；Described small-bore filter be use absolute hole diameter be that the microporous filter membrane of 10um is made, can be with backwash
Filter；
Heretofore described live cell fluorescent stain is FDA or/and CMFDA, its addition according to liquid volume in sample cell,
With 1-3mg/ml(2mg/ml) ratio add.The staining reaction time is 5-30(10) minute.FDA is fluorescein(e) diacetate.
Heretofore described cleanout fluid is BES, MOPS, PBS(pH=7.0), Tris-HCl (0.2-1.0M, pH=6.0-
7.2) at least one in.Can effectively prevent the abiotic hydrolysis of FDA or CMFDA.
Heretofore described cell pyrolysis liquid is methanol and in the mixed liquor of chloroform (volume ratio 1:1-1:3) or acetone
Kind.
10-50um biology device for fast detecting in a kind of ballast water for ship, it is characterised in that include sales kit (SK), flushing liquor
Case, lysate case, waste water tank, detection case, grade one filter and two grades of filters, the outlet of grade one filter and sales kit (SK) import phase
Even, detection case is provided with stain and adds entrance, the outlet of sales kit (SK), flushing liquor case and stain case is respectively equipped with sample pump,
Flushing pump and cracking pump, sample delivery side of pump is connected with the Single port of secondary filter through strainer valve, another of secondary filter
Port is connected with flushing pump outlet through flushing valve, and the connecting tube between secondary filter with flushing valve is through tapping valve and waste water tank phase
Even, the inlet of detection case connecting line through between liquid feed valve with strainer valve and secondary filter is connected, cracking pump discharge and inspection
Measuring tank is connected, and detection case is provided with exometer.
Heretofore described grade one filter be mesh catercorner length be the nylon bolting silk of 35-50um；Two grades of filters
It is the microporous filter membrane of 10um for absolute hole diameter；The wavelength of transmitted light of exometer is 460-490nm, and the excitation light wave of reception is a length of
510-550nm.Described detection case bottom is connected with waste water tank through drain valve.
Heretofore described 10-50um biology refers to: is smaller in size than 50um but is more than or equal to the viable biological of 10um.
Use assembly of the invention and method to detect the 10-50um biochron in ballast water for ship to be discharged, first press
Use certain cleanout fluid specific, live cell fluorescent stain, thin is tested out according to the acquisition methods of the strong reference value of relative fluorescence
Relative intensity of fluorescence reference value during the combination of cellular lysate liquid；When boats and ships need detection, use this specific cleanout fluid, living cells
Fluorescent dye, the combination of cell pyrolysis liquid detect relative intensity of fluorescence according to step 1-6, when relative intensity of fluorescence is not more than
During relative intensity of fluorescence reference value, illustrate that ballast water for ship to be discharged meets discharge standard, otherwise boats and ships to be discharged are described
Ballast water does not meets discharge standard, and in the case of using identical various reagent, relative intensity of fluorescence reference value only needs to check one
Secondary, can be repeatedly, the most many times inspection become reference value, in the case of having this reference value to obtain, present configuration is simple, make
With convenient, speed is fast, precision is high in detection, and data are true and reliable.
Accompanying drawing explanation
Fig. 1 is the structural representation of speed detector in the present invention.
Fig. 2 is the corresponding relation figure in the present invention between relative intensity of fluorescence and four slit bamboo or chopped wood algae living cells content.
Detailed description of the invention
10-50um biology method for quick in a kind of ballast water for ship, comprises the steps:
(1) large aperture filter is used will the ballast water sample to be discharged after ballast water treatment equipment processes to be not less than
The viable biological of 50um leaches, and obtains filter liquor；
(2) take Aml filter liquor, use small-bore filter to filter and retain and filter liquor is not less than the viable biological of 10um, little
Viable biological in 10um leaches with liquid；
(3) use the cleanout fluid backwash small-bore filter of 3-10%Aml, by the size that retains at the viable biological of 10-50um
It is flushed in sample cell；
(4) in sample cell, add live cell fluorescent stain, dye 5-30 minute；
(5), after dyeing, in sample cell, the cell pyrolysis liquid of liquid volume 0.5-2 times in it is added；
(6) after cell fully cracks, the relative intensity of fluorescence of liquid in use exometer detection sample cell；
The acquisition methods of the strong reference value of relative fluorescence is as follows:
A, selection known symbols are closed in ballast water treatment D-2 standard raw to regulation, the survival containing 10-50um of 10-50um viable biological
Thing number reaches the water sample of the upper limit as with reference to detection sample；Use large aperture filter will be not less than in detection sample
The viable biological of 50um leaches, and obtains filter liquor；
B, take Aml filter liquor, use small-bore filter to filter and retain and filter liquor is not less than the viable biological of 10um, is less than
The viable biological of 10um flows out with liquid；
C, use the cleanout fluid backwash small-bore filter of 3-10%Aml, by the size that retains at the viable biological of 10-50um
It is flushed in sample cell；
D, in sample cell add live cell fluorescent stain, dye 5-30(10) minute；
After e, dyeing, in sample cell, add the cell pyrolysis liquid of liquid volume 0.5-2 times in it；
F, after cell (film) fully splits (molten) solution, use the relative intensity of fluorescence of liquid in exometer detection sample cell；
G, by step a with reference to detection sample selection standard choose 1-100 kind different reference detection sample, every kind with reference to examine
Test sample product are detected at least one times by the operational approach of step a, b, c, d, e, f, obtain average relative fluorescence according to the data obtained strong
Angle value, this average relative fluorescence intensity level is relative intensity of fluorescence reference value；
The relative intensity of fluorescence reference value contrast that the relative intensity of fluorescence value (6th) step obtained and g step obtain, it is judged that this pressure
Carry whether water sample meets discharge standard.
Wherein A is the natural number of any numerical value, preferably 100-500.
In the present invention, reference the detection sample described in a step obtains through following method: use large aperture filter will be through
In ballast water sample to be discharged after the process of ballast water treatment equipment, the viable biological not less than 50um leaches, and obtains filter liquor；
Take Aml filter liquor, use small-bore filter to filter and retain and filter liquor is not less than the viable biological of 10um, less than 10um's
Viable biological leaches with liquid；Use the cleanout fluid backwash small-bore filter of 3-10%Aml, by the size that retains at 10-
The viable biological of 50um is flushed in sample cell；By the liquid in sample cell under FDA-CMFDA method is at fluorescence microscope
Detection, when wherein the content of 10-50um viable biological meets the requirements, this pressure to be discharged after ballast water treatment equipment processes
Carry water sample can become with reference to detection sample.
In the present invention, the reference detection sample described in a step also can obtain through following method: artificial preparation algae solution
Or nature seawater processes through ultraviolet (UV) or processes through sodium hypochlorite (NaClO), obtain test fluid；Use large aperture filter will
In test fluid, the viable biological not less than 50um leaches, and obtains filter liquor；Take Aml filter liquor, use small-bore filter to filter also
Retain the viable biological not less than 10um in filter liquor, the viable biological less than 10um leaches with liquid；Use 3-10%Aml's
Cleanout fluid backwash small-bore filter, the size retained is flushed in sample cell at the viable biological of 10-50um；Will
Liquid in sample cell detects under FDA-CMFDA method is at fluorescence microscope, and wherein the content of 10-50um viable biological conforms to
When asking, this ballast water sample to be discharged after ballast water treatment equipment processes can become with reference to detection sample.
Further, described large aperture filter is the nylon bolting silk using mesh catercorner length to be 35-50um
The filter made；Described small-bore filter be use absolute hole diameter be that the microporous filter membrane of 10um is made, can backwash
Filter.Described live cell fluorescent stain is FDA or/and CMFDA, its addition according to liquid volume in sample cell, with
Ratio 1-3mg/ml(2mg/ml) adds.The staining reaction time is 5-30(10) minute.FDA is fluorescein(e) diacetate；Institute
The cleanout fluid stated is BES, MOPS, PBS(pH=7.0), at least one in Tris-HCl (0.2-1.0M, pH=6.0-7.2), can
Effectively prevent the abiotic hydrolysis of FDA or CMFDA.Wherein PBS is: vulcanized lead, Tris-HCl be: three (methylol) amino first
Alkane.Described cell pyrolysis liquid is methanol and the one in the mixed liquor of chloroform (volume ratio 1:1-1:3) or acetone.
10-50um biology device for fast detecting in the ballast water for ship realizing above-mentioned detection method, structure such as Fig. 1 institute
Show: include sales kit (SK) 2, flushing liquor case 13, lysate case 3, waste water tank 14, detection case 17, grade one filter 1 and two grades of filters 8,
Grade one filter 1 is the filter that the nylon bolting silk using mesh catercorner length to be 50um is made；Two grades of filters are to use absolutely
The filter that the microporous filter membrane that aperture is 10um is made.The outlet of grade one filter is connected with sales kit (SK) 2 import, through one-level mistake
Liquid after filter filters enters and can be provided with primary pump between grade one filter and sales kit (SK) 2 on connecting line in sales kit (SK) 2,
Can also be that grade one filter is located at above sales kit (SK) 2, utilize the gravity of liquid naturally to flow into.Detection case 17 is provided with stain
Add entrance 12, the outlet of sales kit (SK) 2, flushing liquor case 14 and lysate case 3 is respectively equipped with sample pump 4, flushing pump 13 and cracking
Pump 5, the outlet of sample pump 4 is connected through the Single port of strainer valve 6 with secondary filter 8, and the another port of secondary filter 8 is through punching
Washing valve 11 to be connected with flushing pump 13 outlet, the connecting tube between secondary filter 8 and flushing valve 11 is through tapping valve 10 and waste water tank 15
Being connected, the inlet of detection case 17 connecting line through between liquid feed valve 9 with strainer valve 6 and secondary filter 8 is connected, and cracks pump 5
Exporting and be connected with detection case 17 through cracking valve 7, detection case 17 is provided with exometer 18, and the detection probe of exometer 18 is located at detection
In case 17；The wavelength of transmitted light of exometer is 460-490nm, a length of 510-550nm of excitation light wave of reception；Described detection case
Bottom is connected with waste water tank 15 through drain valve 16.
Heretofore described 10-50um biology refers to: is smaller in size than 50um but is more than or equal to the viable biological of 10um.
In ballast water for ship during the work of 10-50um biology device for fast detecting, ballast water sample to be detected is put into one
In level filter, the viable biological being not less than 50um in ballast water sample is leached by grade one filter, leaching after being filtered
Liquid enters in sales kit (SK), is separately added into cleanout fluid and cell pyrolysis liquid in flushing liquor case and lysate case；Opened trap valve and
Tapping valve, other valve closing, sample pump works, and quantitative filters the filter liquor in sales kit (SK) through secondary filter, leaches
Liquid enter in waste water tank, and retained by secondary filter not less than the viable biological of 10um；Close strainer valve and tapping valve,
Now dyeing valve and drain valve are also at closed mode, open flushing valve and liquid feed valve, and flushing pump works, and uses quantitative flushing
Secondary filter backwash, the 10-50um biology retained are flushed in detection case by liquid；Closing flushing valve and liquid feed valve,
Warp-wise stain adds entrance and adds appropriate live cell fluorescent stain in sales kit (SK), living cells is carried out fluorescence staining；Right
Living cells has dyeed after (about 10 minutes), opens cracking valve, cracks pump work, is inputted by cell pyrolysis liquid in detection case, treat
After cell fully cracks, exometer is used to measure the relative intensity of fluorescence value in detection case.Choose 80 kinds and pass through laboratory method
It has been determined that, ballast water water sample to be discharged containing 9 10-50um viable biological as ballast water sample to be detected, pass through
Above-mentioned using method uses this device to detect, and averaged is as relative intensity of fluorescence reference value；By relative fluorescence
Intensity level contrasts with relative intensity of fluorescence reference value, when relative intensity of fluorescence is not more than relative intensity of fluorescence reference value, and explanation
Ballast water for ship to be discharged meets discharge standard, otherwise illustrates that ballast water for ship to be discharged does not meets discharge standard, uses
In the case of identical various reagent, relative intensity of fluorescence reference value only demand takes once, can be repeatedly, examine the most many times
Becoming reference value in testing, in the case of having this reference value, present configuration is simple, easy to use, and speed is fast, precision is high in detection,
Data are true and reliable.
Example 1
10-50um biology method for quick in a kind of ballast water for ship, comprises the steps: that use mesh catercorner length is
The filter that the nylon bolting silk of 50um is made is by the least in the ballast water sample to be discharged after ballast water treatment equipment processes
Viable biological in 50um leaches, and obtains filter liquor；Taking 200ml filter liquor, the viable biological less than 10um leaches with liquid；Use
The substance withdrawl syndrome of 10ml is the Tris-HCl backwash small-bore filter of 0.8mol/L, pH=7, the size retained is existed
The viable biological of 10-50um is flushed in sample cell；Live cell fluorescent stain fluorescein diethyl is added in sample cell
Acid esters 20mg, dyes 10 minutes；After having dyeed, (cell pyrolysis liquid is first to add the cell pyrolysis liquid of 10ml in sample cell
The liquid that alcohol and chloroform 1:2 by volume mixes), stand 5 minutes；After cell fully cracks, use exometer detection sample
The relative intensity of fluorescence of liquid in product groove.
The acquisition methods of the strong reference value of relative fluorescence is as follows: known to selecting 60 kinds, every milliliter containing 9 10-50um survival
Biological ballast water water sample to be discharged is as with reference to detection sample；Every kind is proceeded as follows with reference to detection sample: use net
Hole catercorner length is the filter made of the nylon bolting silk of 50um by with reference to viable biological not less than 50um in detection sample
Leach, obtain filter liquor；Taking 200ml filter liquor, the viable biological less than 10um leaches with liquid；The amount using the material of 10ml is dense
Tris-HCl backwash small-bore filter that degree is 0.8mol/L, pH=7, by the size that retains at the viable biological of 10-50um
It is flushed in sample cell；In sample cell, add live cell fluorescent stain fluorescein(e) diacetate 20mg, dye 10 points
Clock；After having dyeed, add in sample cell 10ml cell pyrolysis liquid (cell pyrolysis liquid is methanol and chloroform by volume 1:
2 liquid mixed), stand 5 minutes；After cell fully cracks, in use exometer detection sample cell, liquid is the most glimmering
Light intensity value.Calculating these the 60 kinds meansigma methodss with reference to the relative intensity of fluorescence obtained after detection sample treatment, this meansigma methods is
Relative intensity of fluorescence reference value.By the relative intensity of fluorescence value obtained after ballast water treatment to be detected and relative intensity of fluorescence reference
Value contrast, i.e. can determine whether whether this ballast water sample to be detected meets discharge standard.
Obtain through following method with reference to detection sample: use large aperture filter by after ballast water treatment equipment processes
In ballast water sample to be discharged, the viable biological not less than 50um leaches, and obtains filter liquor；Take Aml filter liquor, use small-bore
Filter filters and retains the viable biological not less than 10um in filter liquor, the viable biological less than 10um leaches with liquid；Make
With the cleanout fluid backwash small-bore filter of 3-10%Aml, the size retained is flushed at the viable biological of 10-50um
In sample cell；Being detected under FDA-CMFDA method is at fluorescence microscope by liquid in sample cell, wherein 10-50um survival is raw
When the content of thing meets the requirements, can become with reference to detection sample.The present invention is respectively adopted cleanout fluid select BES, MOPS,
PBS(pH=7.0), at least one in Tris-HCl (0.2-1.0M, pH=6.0-7.2), it is possible to reduce FDA's to greatest extent
Abiotic hydrolysis.Cell pyrolysis liquid can make the fluorescein within living cells leach, and improves the accuracy of testing result.
FDA is connected with the acetic acid free radical of two conjugation, is a kind of apolar substance, can pass freely through alga cells film, quilt
The non-specific enzymatic hydrolysis such as esterase in cell, protease, lipase become fluorescein.FDA is leuco-compounds, and itself does not has
Have fluorescence, and product fluorescein be a kind of polarity and the material with fluorescence radiation performance, stable chemical nature, be difficult to by
Decompose, it is difficult to through cell membrane, in intracellular accumulation.When the fluorescein of intracellular storage exceed a certain amount of after be discharged into environment
In, owing to reactant is different from the fluorescent characteristic of product, even if therefore reactant is superfluous, the most not mensuration of interference product.
Relation between relative intensity of fluorescence and 10-50um biology content
Four slit bamboo or chopped wood algae solution of preparation variable concentrations, its survivaling cell content uses the detection of FDA-CMFDA method to determine.Each solution is added
Enter in assembly of the invention, use the method for the present invention to carry out the test of relative intensity of fluorescence.With four slit bamboo or chopped wood algae living cells content
For x-axis, with the relative intensity of fluorescence that records as y-axis, making Fig. 2, as shown in Figure 2, the testing result of the present invention and reality are lived thin
Born of the same parents' content has preferable one-to-one relationship, it is possible to reflection
Go out the living cells content in sample.
Claims (9)
1. 10-50um biology method for quick in a ballast water for ship, it is characterised in that comprise the steps:
(1) large aperture filter is used the ballast water sample after ballast water treatment equipment processes will to be not less than the survival of 50um
Biology leaches, and obtains filter liquor；
(2) take Aml filter liquor, use small-bore filter to filter and retain and filter liquor is not less than the viable biological of 10um, little
Viable biological in 10um leaches with liquid；
(3) use the cleanout fluid backwash small-bore filter of 3-10%Aml, by the size that retains at the viable biological of 10-50um
It is flushed in sample cell；
(4) in sample cell, add live cell fluorescent stain, dye 5-30 minute；
(5), after dyeing, in sample cell, the cell pyrolysis liquid of liquid volume 0.5-2 times in it is added；
(6) after cell fully cracks, the relative intensity of fluorescence of liquid in use exometer detection sample cell；
The acquisition methods of the strong reference value of relative fluorescence is as follows:
Known symbols is selected to close in ballast water treatment D-2 standard to the regulation of 10-50um viable biological, containing 10-50um viable biological
Number reaches the water sample of the upper limit will be not less than 50um as reference detection sample, use large aperture filter in detection sample
Viable biological leach, obtain filter liquor；
B, take Aml filter liquor, use small-bore filter to filter and retain and filter liquor is not less than the viable biological of 10um, is less than
The viable biological of 10um leaches with liquid；
C, use the cleanout fluid backwash small-bore filter of 3-10%Aml, by the size that retains at the viable biological of 10-50um
It is flushed in sample cell；
D, in sample cell add live cell fluorescent stain, dye 5-30 minute；
After e, dyeing, in sample cell, add the cell pyrolysis liquid of liquid volume 0.5-2 times in it；
F, after cell fully cracks, use the relative intensity of fluorescence of liquid in exometer detection sample cell；
G, by step a with reference to detection sample selection standard choose 1-100 kind different reference detection sample, every kind with reference to examine
Test sample product are detected at least one times by the operational approach of step a, b, c, d, e, f, obtain average relative fluorescence according to the data obtained strong
Angle value, this average relative fluorescence intensity level is relative intensity of fluorescence reference value；
The relative intensity of fluorescence reference value contrast that the relative intensity of fluorescence value (6th) step obtained and g step obtain, it is judged that this pressure
Carry whether water sample meets discharge standard.
10-50um biology method for quick in a kind of ballast water for ship the most according to claim 1, it is characterised in that:
Reference detection sample described in a step obtains through following method: use large aperture filter will be at ballast water treatment equipment
In ballast water sample to be discharged after reason, the viable biological not less than 50um leaches, and obtains filter liquor；Take Aml filter liquor, use
Small-bore filter filters and retains the viable biological not less than 10um in filter liquor, the viable biological less than 10um is filtered with liquid
Go out；Use the cleanout fluid backwash small-bore filter of 3-10%Aml, the size retained is rinsed at the viable biological of 10-50um
Enter in sample cell；Being detected under FDA-CMFDA method is at fluorescence microscope by liquid in sample cell, wherein 10-50um deposits
When the content of living organism meets the requirements, can become with reference to detection sample.
10-50um biology method for quick in a kind of ballast water for ship the most according to claim 1, it is characterised in that:
Reference detection sample described in a step obtains through following method: artificial preparation algae solution or nature seawater are at ultraviolet
Manage or process through sodium hypochlorite, obtain test fluid；Use large aperture filter that test fluid will be not less than the viable biological filter of 50um
Go out, obtain filter liquor；Take Aml filter liquor, use small-bore filter to filter and retain the survival life being not less than 10um in filter liquor
Thing, viable biological less than 10um leach with liquid；Use the cleanout fluid backwash small-bore filter of 3-10%Aml, will retain
Size be flushed in sample cell at the viable biological of 10-50um；By the liquid in sample cell through FDA-CMFDA method glimmering
Detect under light microscope, when wherein the content of 10-50um viable biological meets the requirements, can become with reference to detection sample.
4. according to 10-50um biology method for quick, its feature in a kind of ballast water for ship described in claim 1 or 2 or 3
It is: described large aperture filter is the filter that the nylon bolting silk using mesh catercorner length to be 35-50um is made；Institute
The small-bore filter stated be use absolute hole diameter be that the microporous filter membrane of 10um is made, can be with the filter of backwash.
10-50um biology method for quick in a kind of ballast water for ship the most according to claim 4, it is characterised in that:
Described live cell fluorescent stain is FDA or/and CMFDA, and its addition is according to liquid volume in sample cell, with 1-3mg/ml's
Ratio adds.
10-50um biology method for quick in a kind of ballast water for ship the most according to claim 5, it is characterised in that:
Described cleanout fluid be BES, MOPS, PBS, concentration be at least one in the Tris-HCl of the pH=6.0-7.2 of 0.2-1.0M.
7., according to 10-50um biology method for quick in a kind of ballast water for ship described in claim 5 or 6, its feature exists
It is methanol and the one in the mixed liquor of chloroform 1:1-1:3 by volume or acetone in: described cell pyrolysis liquid.
8. 10-50um biology device for fast detecting in a ballast water for ship, it is characterised in that: include sales kit (SK), flushing liquor case,
Lysate case, waste water tank, detection case, grade one filter and two grades of filters, the outlet of grade one filter is connected with sales kit (SK) import,
Detection case is provided with stain and adds entrance, and the outlet of sales kit (SK), flushing liquor case and stain case is respectively equipped with sample pump, flushing
Pump and cracking pump, sample delivery side of pump is connected with the Single port of secondary filter through strainer valve, the another port of secondary filter
Being connected with flushing pump outlet through flushing valve, the connecting tube between secondary filter with flushing valve is connected with waste water tank through tapping valve, inspection
The inlet of measuring tank connecting line through between liquid feed valve with strainer valve and secondary filter is connected, cracking pump discharge and detection case phase
Even, detection case is provided with exometer.
10-50um biology device for fast detecting in ballast water for ship the most according to claim 8, it is characterised in that: described
Grade one filter be mesh catercorner length be the nylon bolting silk of 35-50um；Two grades of filters be absolute hole diameter be the micro-of 10um
Hole filter membrane；The wavelength of transmitted light of exometer is 460-490nm, a length of 510-550nm of excitation light wave of reception.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610776270.0A CN106244665A (en) | 2016-08-31 | 2016-08-31 | Method and device for quickly detecting 10-50um organisms in ship ballast water |
| PCT/CN2017/099893 WO2018041215A1 (en) | 2016-08-31 | 2017-08-31 | Fast detection method and device for organisms of 10-50 μm in ship ballast water |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610776270.0A CN106244665A (en) | 2016-08-31 | 2016-08-31 | Method and device for quickly detecting 10-50um organisms in ship ballast water |
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| Publication Number | Publication Date |
|---|---|
| CN106244665A true CN106244665A (en) | 2016-12-21 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610776270.0A Pending CN106244665A (en) | 2016-08-31 | 2016-08-31 | Method and device for quickly detecting 10-50um organisms in ship ballast water |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN106244665A (en) |
| WO (1) | WO2018041215A1 (en) |
Cited By (5)
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| CN107192655A (en) * | 2017-07-26 | 2017-09-22 | 连云港市质量技术综合检验检测中心 | A kind of household purified water machine Cryptosporidium rejection rate method of inspection |
| WO2018041215A1 (en) * | 2016-08-31 | 2018-03-08 | 威海中远造船科技有限公司 | Fast detection method and device for organisms of 10-50 μm in ship ballast water |
| 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 |
| CN109374584A (en) * | 2018-08-27 | 2019-02-22 | 九江精密测试技术研究所 | A kind of ballast water for ship biological effectiveness real time on-line monitoring device |
| CN111413479A (en) * | 2020-04-14 | 2020-07-14 | 诸暨市金桥实业有限公司 | Water quality detection method and system |
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| JP2007135582A (en) * | 2005-10-19 | 2007-06-07 | Jfe Engineering Kk | Method and apparatus for detecting microorganisms in ballast water |
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| CN109374584A (en) * | 2018-08-27 | 2019-02-22 | 九江精密测试技术研究所 | A kind of ballast water for ship biological effectiveness real time on-line monitoring device |
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Also Published As
| Publication number | Publication date |
|---|---|
| WO2018041215A1 (en) | 2018-03-08 |
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Application publication date: 20161221 |