CN102323266A - Method for identifying phytoplankton living organisms discharged from ballast water - Google Patents
Method for identifying phytoplankton living organisms discharged from ballast water Download PDFInfo
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- CN102323266A CN102323266A CN201110142336A CN201110142336A CN102323266A CN 102323266 A CN102323266 A CN 102323266A CN 201110142336 A CN201110142336 A CN 201110142336A CN 201110142336 A CN201110142336 A CN 201110142336A CN 102323266 A CN102323266 A CN 102323266A
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Abstract
The invention provides a method for identifying phytoplankton living organisms discharged from ballast water, which comprises the following steps of: 1, acquiring a phytoplankton water sample; 2, dropping 1.0-2.0 mol of 1-1.5 percent neutral red solution in each litre of the phytoplankton water sample, and dyeing; 3, dropping a formaldehyde solution in the water sample for fixing the sample; and 4, taking the sample for carrying out microscopic examination, and counting. Compared with the prior art, the method has the advantages of more rapidness and simplification of the whole dyeing process, lower cost, remarkable effect and easiness in identifying whether phytoplanktons are living organisms.
Description
Technical field
The present invention relates to the discrimination method of plant living body, particularly, the present invention relates to the discrimination method of the phytoplankton live body of discharge of ballast water.
Background technology
Ballast Water is meant water and the suspension thereof that installs additional for control boats and ships trim, heel, drinking water, stability or stress aboard ship.Boats and ships for vacating tonnage, must be drained into Ballast Water in the marine site of bank state during to bank.Show that according to related data the Ballast Water that global boats and ships carry every year has 12,000,000,000 tons approximately, contains a large amount of biologies and microorganism in the sediment of Ballast Water and ballast tank, has comprised bacterium, algae, protozoan, planktonic organism and other biological.The animals and plants that swim that average every cubic metre of Ballast Water contains are above 100,000,000; The whole world is present in is diffused into all over the world with boats and ships in the ballast water for ship that the sea life in marine site have 7000 kinds approximately every day; Many bacteriums, plant and animal still survive in Ballast Water and the sediment thereof through the voyage of several months; When boats and ships when freight in the harbour, these biologies are along with Ballast Water is discharged into harbour state waters.If these biological invasions are dealt with improperly, can make a big impact:
Aspect ecological, the biology of invasion can be preyed on local species, changes living environment or changes environmental baseline (as reducing the transparency of water), replaces local species, reduces local bio-diversity, even causes the extinction of local species, the destruction of whole ecological system etc.;
At economic aspect, invasive species possibly cause direct economic losses such as reducing marine product output, also can cause the indirect economic loss relevant with the human health ecology influence;
Aspect human beings'health; The microorganism of ballast water for ship discharging shifts and may form fearful threat; Ballast Water is except carrying bacterium and virus; Also can transmit some microalgaes, comprise the poisonous kind that possibly form harmful water bloom or red tide, these pathogen and microorganism possibly cause human serious disease even death.
The tiny organism that the marine ship Ballast Water carries causes the species invasion problem through discharging, more and more receives the concern of International Maritime Organization (IMO).According to " ballast water for ship and sediment control and management international convention (draft) ", D-2 bar Ballast Water operative norm.Be set to limit value according to this discharge capacity of carrying out living body biological in the ballast water for ship of Ballast Management.Article one regulation wherein: individual bulk concentration＜10 of living body biological minimum dimension>=50 μ m/m
3, the living body biological minimum dimension is individual bulk concentration＜10/ml of 10～50 μ m.
What require in the standard is living body biological concentration, and this just requires at short notice, miscellaneous biological species in the Ballast Water is distinguished anyway, and with the number of biology add with, under existing detection technique condition, this is a very big challenge.
Yet present stage for the Ballast Water phytoplankton whether the evaluation of live body then be key one ring to the management ballast water for ship.
In the common employed authentication method, be divided into reagent usually and identify and fluorescence method evaluation dual mode at present.The former can only identify phytoplankton in the fresh water by a common cover reagent, and the latter then cost is higher, is difficult to popularize in the practical operation of China's research work.As adopt fluorescence method to identify the live body phytoplankton, must detect algae cell activity through the index of measuring biomasss such as cell density or chlorophyll a.Institute's elapsed time is longer, needs usually to cultivate after 5-7 days, could understand the algae cell activity situation, and whether frustule all kills, and the mensuration granting of chlorophyll a is also very loaded down with trivial details.
Therefore need a kind of can the fast detecting Ballast Water in the discrimination method of discharging phytoplankton.
Summary of the invention
The objective of the invention is to prior art when detecting the halonereid activity; The common length consuming time of institute's method of application and loaded down with trivial details, also have higher cost; It is also relatively poor that it detects effect; Detect the also unsettled defective of back result, the discrimination method of the phytoplankton live body of ballast water for ship discharging is provided.
The technical matters that will solve required for the present invention, can realize through following technical scheme:
The discrimination method of the phytoplankton live body of discharge of ballast water may further comprise the steps:
(1) gathers the phytoplankton water sample;
(2) in every liter of phytoplankton water sample, drip 1～1.5% neutral red solution 1.0-2.0ml, dye;
(3) in water sample, dripping formalin solution again carries out sample and fixes;
(4) sample thief carries out microscopy, and counting.
The preparation method of said 1～1.5% neutral red solution is:
Take by weighing 0.5g～0.75g dimethyl diaminophenazine chloride and be dissolved in 50ml Ringer solution, hot a little in 30 ℃-40 ℃ the temperature range, make it dissolving, filter, preserve in the dark place in the brown bottle of packing into.
Said Ringer solution is prepared from following component:
The time of the dyeing in the said step (2) is 10-15min.
The concentration of volume percent of the formalin solution in the said step (3) is 4-10%.
According to the discrimination method of the phytoplankton live body of discharge of ballast water of the present invention, more preferably, said Ringer solution is prepared from following component:
Above-mentioned water can be distilled water or deionized water.
According to the discrimination method of the phytoplankton live body of discharge of ballast water of the present invention, preferably, the time of the dyeing in the said step (2) is 15min.The concentration of volume percent of the formalin solution in the said step (3) is 5%.
Dimethyl diaminophenazine chloride of the present invention is meant " 3-amino-7-methylamino-2-toluphenazine hydrochloride ".
" % " of neutral red solution of the present invention is meant mass percent.
The invention has the beneficial effects as follows, compared with prior art, the whole dyeing course of method of the present invention more fast, simplify, and cost is lower, effect is obvious, very easily distinguishes whether live body of phytoplankton.
Embodiment
In order to make technological means of the present invention, creation characteristic, to reach purpose and effect and be easy to understand and understand,, further set forth the present invention below in conjunction with specific embodiment.
The detection that the phytoplankton live body of 1 pair of SHOU-BWDL-03 sample of embodiment (the simulation ballast for cruising water sample that sample is provided by middle ship heavy industry the 7th O four research institutes) carries out.
1 reagent
1.1 preparation Ringer solution:
Earlier sodium chloride, potassium chloride, lime chloride are dissolved in respectively in a small amount of distilled water, mix the back and arrive 100ml with distilled water diluting.
1.21% neutral red solution
Take by weighing the 0.5g dimethyl diaminophenazine chloride and be dissolved in 50ml Ringer liquid,, make it to dissolve very soon, filter with filter paper (qualitative filter paper) 30 ℃-40 ℃ heat a little, in the brown bottle of packing in dark place preservation (solution is prone to oxidation precipitation, loses colouring power).
2 staining procedures:
2.1 from Ballast Water, gather phytoplankton water sample (3 groups of parallel water samples), drip 1% neutral red solution 1.0ml dyeing 15min in every premium on currency appearance;
2.2 Dropwise 5 % formalin solution carries out sample and fixes in water sample again;
2.3 after sample shaken up, sample thief 0.1ml observed the live body phytoplankter under 400 * optical microscope, and counting.Can be observed live body phytoplankton cell red-dyed in the visual field, dead volume dye-free phenomenon.Testing result for SHOU-BWDL-03 sample (the simulation ballast for cruising water sample that sample is provided by middle ship heavy industry the 7th O four research institutes) finds that the live body phytoplankton is 168, and dead phytoplankter number is 129, and the microscopy result is consistent after 2 hours.
Embodiment 2
The reagent of present embodiment is identical with the cardinal principle of embodiment 1 with method, and difference is,
Said Ringer solution is prepared from following component:
The time of the dyeing in the said step (2) is 14-15min.
In staining procedure 2.1, in every group of every premium on currency appearance, dripping 1.5% neutral red solution is 1.0ml (the 0.75g dimethyl diaminophenazine chloride is dissolved in 50ml Ringer liquid).Testing result for SHOU-BWDL-03 sample (the simulation ballast for cruising water sample that sample is provided by middle ship heavy industry the 7th O four research institutes) is found; The live body phytoplankton is 168; Dead phytoplankter number is 127; After 2 hours once more microscopy live body phytoplankton be 168, dead phytoplankter number is 128.
Embodiment 3
The reagent of present embodiment is identical with the cardinal principle of embodiment 1 with method, and difference is,
The concentration of volume percent of the formalin solution in the said step (3) is 5-8%.
1% neutral red solution that in staining procedure 2.1, in every group of every premium on currency appearance, drips is 2.0ml (the 0.5g dimethyl diaminophenazine chloride is dissolved in 50ml Ringer liquid).Testing result for SHOU-BWDL-03 sample (the simulation ballast for cruising water sample that sample is provided by middle ship heavy industry the 7th O four research institutes) is found; The live body phytoplankton is 167; Dead phytoplankter number is 127, and the continued microscopy came to the same thing in 2 hours.
Control Example
Direct microscopy result for SHOU-BWDL-03 sample (the simulation ballast for cruising water sample that sample is provided by middle ship heavy industry the 7th O four research institutes) finds; Because dead organelles such as phytoplankton cell body inner chlorophyll soon are still complete relatively, be prone to it is judged to be live body; But along with the prolongation of set time, organelles such as chloroplast can be with the variation of osmotic pressure inside and outside the phytoplankton cell disappearance of breaking.Thereby same group of sample different time microscopy occur and have certain error; For the first time find that the live body phytoplankton is 196 during microscopy; Dead phytoplankter number is 99, and microscopy finds that the live body phytoplankton is 168 after 2 hours, and dead phytoplankter number is 129.And microscopy length consuming time.
By embodiment 1-3 and control Example relatively, can draw method of the present invention and have advantages such as " simplification fast,, effect obviously, are very easily distinguished phytoplankton, and whether live body and good stability, error amount are little " to the individual bulk concentration testing result of same group of sample phytoplankton live body.And the reagent that the present invention adopts is commercially available cheap reagent, thereby makes the cost that method of the present invention consumed reduce greatly.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the instructions just explains principle of the present invention; The present invention also has various changes and modifications under the prerequisite that does not break away from spirit and scope of the invention, and these variations and improvement all fall in the scope of the invention that requires protection.The present invention requires protection domain to be defined by appending claims and equivalent thereof.
Claims (6)
1. the discrimination method of the phytoplankton live body of discharge of ballast water may further comprise the steps:
(1) gathers the phytoplankton water sample;
(2) in every liter of phytoplankton water sample, drip 1～1.5% neutral red solution 1.0-2.0ml, dye;
(3) in water sample, dripping formalin solution again carries out sample and fixes;
(4) sample thief carries out microscopy, and counting.
2. method according to claim 1; It is characterized in that; The preparation method of said 1～1.5% neutral red solution is: takes by weighing 0.5g～0.75g dimethyl diaminophenazine chloride and is dissolved in 50ml Ringer solution, and hot a little in 30 ℃-40 ℃ the temperature range, make it dissolving; Filter, preserve in the dark place in the brown bottle of packing into.
3. method according to claim 1 is characterized in that, said Ringer solution is prepared from following component:
4. method according to claim 3 is characterized in that, said Ringer solution is prepared from following component:
5. method according to claim 1 is characterized in that, the time of the dyeing in the said step (2) is 10-15min.
6. method according to claim 1 is characterized in that, the concentration of volume percent of the formalin solution in the said step (3) is 4-10%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102636487A (en) * | 2012-03-23 | 2012-08-15 | 上海海洋大学 | Method for identifying discharged live zooplanktons in blast water |
| CN106244669A (en) * | 2016-10-18 | 2016-12-21 | 上海海洋大学 | A kind of method of 10 50 microns of living body biologicals in quick discriminating water |
| CN106442488A (en) * | 2016-08-31 | 2017-02-22 | 威海中远造船科技有限公司 | Method for detecting living organisms with size of more than 50 micrometers in ballast water |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100072144A1 (en) * | 2006-09-27 | 2010-03-25 | Tsugiyoshi Osakabe | Method of treating ballast water of ship |
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- 2011-05-30 CN CN201110142336A patent/CN102323266A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100072144A1 (en) * | 2006-09-27 | 2010-03-25 | Tsugiyoshi Osakabe | Method of treating ballast water of ship |
Non-Patent Citations (2)
| Title |
|---|
| 李伟才: "日照港和邻近锚地及其入境船舶压舱水中浮游植物群集结构的特征", 《海洋科学》, vol. 30, no. 12, 31 December 2006 (2006-12-31), pages 52 - 57 * |
| 李炳乾 等: "福建外来船舶压舱水中浮游植物种类组成与丰度及其影响因素的初步研究", 《台湾海峡》, vol. 28, no. 2, 31 May 2009 (2009-05-31) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102636487A (en) * | 2012-03-23 | 2012-08-15 | 上海海洋大学 | Method for identifying discharged live zooplanktons in blast water |
| CN106442488A (en) * | 2016-08-31 | 2017-02-22 | 威海中远造船科技有限公司 | Method for detecting living organisms with size of more than 50 micrometers in ballast water |
| CN106442488B (en) * | 2016-08-31 | 2019-12-10 | 威海中远造船科技有限公司 | method for detecting living organisms with size of more than 50 mu m in ballast water |
| CN106244669A (en) * | 2016-10-18 | 2016-12-21 | 上海海洋大学 | A kind of method of 10 50 microns of living body biologicals in quick discriminating water |
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Application publication date: 20120118 |