CN105158035A - Production method of little yellow croaker larval otolith sagittal plane sections for micro-chemical analysis - Google Patents
Production method of little yellow croaker larval otolith sagittal plane sections for micro-chemical analysis Download PDFInfo
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- CN105158035A CN105158035A CN201510514184.8A CN201510514184A CN105158035A CN 105158035 A CN105158035 A CN 105158035A CN 201510514184 A CN201510514184 A CN 201510514184A CN 105158035 A CN105158035 A CN 105158035A
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Abstract
The invention relates to a production method of little yellow croaker larval otolith sagittal plane sections for micro-chemical analysis. The production method comprises steps as follows: (1) picking; (2) marking; (3) glue dispensing; (4) embedment; (5) cutting and grinding; (6) polishing; (7) cutting; (8) washing; (9) drying. With the adoption of the method, maximum sagittal planes for micro-chemical analysis of otoliths are guaranteed, and the accuracy of the micro-chemical analysis of otoliths is improved, so that the method has the bright application prospect.
Description
Technical field
The invention belongs to otolith process field, particularly a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis method for making of cutting into slices.
Background technology
The larva and juvenile phase is the important stage of fish early life history, and with habitat close relation, the death of larva and juvenile dynamically directly affects supplementing of fishery resources.
Traditional fishery resources survey method and biotelemetry cannot implement the continuous monitoring of habitat in migration fishes full history of life.Otolith information provides the environmental change experienced in fish history of life.Fish from birth to the different niches feature experienced time captured from otolith core to the usual temporally sequential recording of the element of rim area composition.Over nearly more than 40 years, otolith is developed rapidly and widespread use in the research field of fish space-time dynamic as the particular feature of habitat " fingerprint " label.
Become fish otolith macro result to be confined to reflect the frame-type of fish early life history, and the otolith macro of larva and juvenile combine with different growing ages and carries out analyzing the corresponding relation can accurately illustrated between fish early growth critical stage and habitat.
Summary of the invention
Technical matters to be solved by this invention is to provide the method for making that a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis is cut into slices, and this method guarantees that the maximum sagittal plane of otolith micro-chemical analysis, improves the accuracy of otolith micro-chemical analysis.
The method for making that a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis of the present invention is cut into slices, comprising:
(1) win: with tip tweezers, the sagitta in larva and juvenile statocyst is taken out under anatomical lens, then put into the double dish filling deionized water and clean, dry under room temperature, put into centrifuge tube, and put on otolith numbering;
(2) mark: differentiate left otolith and auris dextra stone under anatomical lens after, be placed in centrifuge tube respectively, and on centrifuge tube, mark left or right to show left and right otolith, left otolith is got in unification or auris dextra stone carries out subsequent operation;
(3) glue is put: be coated on masking foil in 1:0.5 ~ 2 by volume by A glue and B glue, heat while stirring 30 ~ 40 seconds; Get numbered otolith embedded box, pick AB glue point at the bottom of embedded box box; Larva and juvenile otolith is placed on AB glue, and presses otolith surface, the record numbering of otolith and the numbering of corresponding embedded box;
(4) embed: resin and hardening agent stir and evenly mix by 6 ~ 8:1 in mass ratio, inject the resin 3 ~ 4ml after mixing toward each embedded box; Embedding post-drying, rear taking-up resin mass to be hardened;
(5) cut: on the glass sheet using AB glue to be sticked in by otolith resin mass to indicate otolith numbering; After to be solidified, adopt otolith cutting machine (Discoplan-TS, Si Teer company) to excise Excess resin, and use diamond disk to roughly grind otolith, to exposing close to otolith core; Use 1200 order waterproof abrasive papers again instead and carry out manual fine grinding, expose completely to otolith core;
(6) polishing: use diamond polishing liquid and the manual polishing of MD polishing cloth, confirms that otolith surface is without till scratch;
(7) cut: adopt otolith cutting machine (Discoplan-TS, Si Teer company) to cut the redundance of otolith glass sheet, guarantee that otolith slice size can put into the sample stage of micro-chemical analysis instrument;
(8) clean: the grinding and polishing of larva and juvenile otolith is faced up, use deionized water rinsing, be placed on ultrasonic cleaning in ultrapure water; After ultrasonic cleaning, deionized water is adopted to otolith grinding and polishing face order, ultrapure water rinses respectively;
(9) dry: finally the resin mass after cleaning is dried, for otolith micro-chemical analysis.
A glue in described step (3) and B glue are chemical reaction bonding agent.
Resin in described step (4) is epoxy resin, and hardening agent is IPTG.
Bake out temperature in described step (4) and (9) is 38 ~ 40 DEG C, and drying time is 12 ~ 15 hours.
The ultrapure water ultrasonic cleaning time in described step (8) is 2 ~ 3 minutes.
beneficial effect
(1) the invention solves because larva and juvenile otolith is light and little and cause even tilting with moving during resin embedding, carrying out embedding again after otolith is fixing with dotting glue method and effectively can avoid this problem;
(2) adopting the method that cuts to avoid otolith pure hand lapping process causes otolith sagittal plane to grind problem in the slope, ensure that the maximum sagittal plane of otolith micro-chemical analysis;
(3) hand-polished method avoids the problem of the otolith abrasive disc fragmentation causing larva and juvenile thin and little because rotating speed during machine polishing is fast;
(4) method of cleaning, while guarantee otolith is not lost, effectively can be removed the sagittal impurity of otolith, avoid interference during micro-chemical analysis;
(5) above-mentioned series of steps, ensure that the reliability of larva and juvenile otolith micro-chemical analysis result.
Accompanying drawing explanation
Fig. 1 is the otolith Sr/Ca ratio variation diagram of the little yellow croaker larva and juvenile on May 7;
Fig. 2 is the otolith Sr/Ca ratio variation diagram of the little yellow croaker larva and juvenile on May 28;
Fig. 3 is the otolith Sr/Ca ratio variation diagram of the little yellow croaker larva and juvenile on June 23.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
1) win: with tweezers, the sagitta in little yellow croaker larva and juvenile statocyst is taken out under anatomical lens, note wanting light tweezer to put down gently, in order to avoid damage otolith; After the otolith of larva and juvenile is taken out, put into the double dish containing ultrapure water and clean, remove otolith surface organic, dry under room temperature, put into centrifuge tube stand-by as analysis of material, and put on otolith numbering;
2) mark: differentiate left otolith and auris dextra stone under anatomical lens after, be placed in centrifuge tube respectively, and on centrifuge tube, mark the numbering of otolith, cover at centrifuge tube and mark left (L) or right (R) to show left and right otolith.Unification is got left otolith and is carried out subsequent operation;
3) glue is put: extrude on a small quantity by A glue and B glue, the ratio of 1:1 is on masking foil by volume, and spirit lamp heats, and heats while stirring 30 seconds; Get numbered otolith embedding circle box, pick the AB glue point of mixing at the bottom of embedded box box with toothpick; Rapidly larva and juvenile otolith is placed on AB glue, and with tweezers pressing otolith surface, ensures otolith sagittal plane level; To be cooledly solidify 45 minutes; The record numbering of otolith and the numbering of corresponding embedded box; Wherein, the A glue of use and B glue, chemical name is chemical reaction bonding agent (Amada Co., Ltd.);
4) embed: 7:1 is by epoxy resin and hardening agent mixing in mass ratio, injects deployed resin 3ml toward each embedded box, and resin needs to cover larva and juvenile otolith; Dry 12 hours in 38 DEG C of baking ovens after embedding; Rear taking-up resin mass to be hardened, and by the numbering of dissecting the good each otolith of drypoint on resin mass; Wherein, the resin of use and hardening agent, chemical name is epoxy resin and IPTG (Si Teer company, Copenhagen, Denmark) respectively;
5) cut: on the glass sheet using AB glue to be sticked in by otolith resin mass to indicate otolith numbering, and guarantee that resin mass can cover otolith numbering; After to be solidified, adopt otolith cutting machine (Discoplan-TS, Si Teer company) to excise Excess resin, and use diamond disk to roughly grind otolith, to exposing close to otolith core; Use the manual fine grinding of 1200 order waterproof abrasive papers again instead, period uses saturating, viewed in reflected light surface appearance at any time under upright metallurgical microscope, exposes completely to otolith core;
6) polishing: use diamond polishing liquid and the manual polishing abrasive surface of MD polishing cloth; Confirm that otolith surface is without scratch with reflected light under upright metallurgical microscope till;
7) clean: the grinding and polishing of larva and juvenile otolith is faced up, use deionized water rinsing 6 times, to be placed in ultrapure water ultrasonic cleaning 2 ~ 3 minutes; After ultrasonic cleaning, adopt deionized water, ultrapure water to carry out 8 times respectively to otolith grinding and polishing face order and rinse; For preventing being washed out by larva and juvenile otolith, the thin mouth shower nozzle of water bottle is avoided aiming at otolith face;
8) dry: finally the resin mass after cleaning is placed in 38 DEG C of baking ovens, after 12 hours, can be used for otolith micro-chemical analysis.
9) carbon film is plated: larva and juvenile otolith sample is placed in evaporation carbon film in vacuum coating equipment (JEE-420, Jeol Ltd.), electric current is set to 35A, and the plated film time is 25 seconds.
10) otolith micro-chemical analysis: utilize electron probe microanalyser (english abbreviation EPMA, JXA-8100 type, Jeol Ltd.) get measuring point from core continuously ready along the longest axial edge, 5 μm are spaced apart between point, EPMA accelerating potential and electron beam current are respectively 15kV, and 2.0 × 10
-8a, beam spot diameter, is 5 μm, and often some residence time is 15s.Take calcium carbonate (CaCO
3) and strontium titanates (SrTiO
3) be standard model.Obtain each point ratio of Sr content and Ca content and represent, unifiedly use Sr content: Ca content × 10
3ratio represent (be called for short Sr/Ca ratio).
Carry out sampling (May 7, May 28, June 23) with the about interval in January, the micro-chemical contrast of larva and juvenile otolith of Different Individual size:
(1) same growth phase comparison.The juvenile fish of 3 time periods from otolith core to 15 ~ 35 μm, all show high Sr/Ca value (table 1), through one-way analysis of variance inspection, without significant difference (P ﹥ 0.05) between the high Sr/Ca value near the little yellow croaker otolith core of 3 time periods.
(2) Sr/Ca ratio variation tendency comparison.May 7, May 28 are consistent with the little yellow croaker otolith Sr/Ca ratio variation tendency on June 23, and in the otolith Sr/Ca ratio change substantially overlapping (see Fig. 1-3) of identical growth phase: the individuality on May 7 is 9.11 ± 0.80 from core to a 20 μm average Sr/Ca; The individuality on May 28 is 9.48 ± 1.11 from average Sr/Ca between core to 30 μm; The individuality on June 23 is 8.90 ± 0.76 from average Sr/Ca between core to 20 μm; After this between about 400 μm, show as Sr/Ca ratio and drop to the juvenile fish Sr/Ca of 6: three time periods and be followed successively by 5.87 ± 0.84,6.15 ± 0.50,5.62 ± 0.57.
As can be seen here by method disclosed in embodiment, larva and juvenile otolith micro-chemical analysis result accurately and reliably.
The little yellow croaker larva and juvenile otolith core space height Sr value of table 1 Different Individual size
Claims (5)
1., for the method for making that the little yellow croaker larva and juvenile otolith sagittal plane of micro-chemical analysis is cut into slices, comprising:
(1) win: with tip tweezers, the sagitta in larva and juvenile statocyst is taken out under anatomical lens, then put into the double dish filling deionized water and clean, dry under room temperature, put into centrifuge tube, and put on otolith numbering;
(2) mark: differentiate left otolith and auris dextra stone under anatomical lens after, be placed in centrifuge tube respectively, and on centrifuge tube, mark left or right to show left and right otolith, left otolith is got in unification or auris dextra stone carries out subsequent operation;
(3) glue is put: be coated on masking foil in 1:0.5 ~ 2 by volume by A glue and B glue, heat while stirring 30 ~ 40 seconds; Get numbered otolith embedded box, pick AB glue point at the bottom of embedded box box; Larva and juvenile otolith is placed on AB glue, and presses otolith surface, the record numbering of otolith and the numbering of corresponding embedded box;
(4) embed: resin and hardening agent stir and evenly mix by 6 ~ 8:1 in mass ratio, inject mixed resin 3 ~ 4ml toward each embedded box; Embedding post-drying, rear taking-up resin mass to be hardened;
(5) cut: on the glass sheet using AB glue to be sticked in by otolith resin mass to indicate otolith numbering; After to be solidified, adopt otolith cutting machine excision Excess resin, and use diamond disk to roughly grind otolith, to exposing close to otolith core; Use 1200 order waterproof abrasive papers again instead and carry out manual fine grinding, expose completely to otolith core;
(6) polishing: use diamond polishing liquid and the manual polishing of MD polishing cloth, confirms that otolith surface is without till scratch;
(7) cut: adopt otolith cutting machine to chop off the ears the redundance of stone glass sheet;
(8) clean: the grinding and polishing of larva and juvenile otolith is faced up, use deionized water rinsing, be placed on ultrasonic cleaning in ultrapure water; After ultrasonic cleaning, deionized water is adopted to otolith grinding and polishing face order, ultrapure water rinses respectively;
(9) dry: finally the resin mass after cleaning is dried, for otolith micro-chemical analysis.
2. a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis according to claim 1 method for making of cutting into slices, is characterized in that: the A glue in described step (3) and B glue are chemical reaction bonding agent.
3. a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis according to claim 1 method for making of cutting into slices, it is characterized in that: the resin in described step (4) is epoxy resin, hardening agent is IPTG.
4. a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis according to claim 1 method for making of cutting into slices, it is characterized in that: the bake out temperature in described step (4) and (9) is 38 ~ 40 DEG C, and drying time is 12 ~ 15 hours.
5. a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis according to claim 1 method for making of cutting into slices, is characterized in that: the ultrapure water ultrasonic cleaning time in described step (8) is 2 ~ 3 minutes.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105738175A (en) * | 2016-03-04 | 2016-07-06 | 中国水产科学研究院北戴河中心实验站 | Method for preparing otolith sample for early life history of fish |
| CN106970079A (en) * | 2017-02-27 | 2017-07-21 | 上海海洋大学 | A kind of method that utilization inner casing identifies squids age in days |
| CN112335716A (en) * | 2020-11-03 | 2021-02-09 | 中国水产科学研究院北戴河中心实验站 | Method for rapidly picking otoliths of flounder larvae |
| CN113588328A (en) * | 2021-08-10 | 2021-11-02 | 中国水产科学研究院黑龙江水产研究所 | Micro-sampling method for micro-chemical analysis of otolith |
| CN114486442A (en) * | 2022-01-28 | 2022-05-13 | 公安部物证鉴定中心 | Sample preparation method for trace material evidence element analysis |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105738175A (en) * | 2016-03-04 | 2016-07-06 | 中国水产科学研究院北戴河中心实验站 | Method for preparing otolith sample for early life history of fish |
| CN105738175B (en) * | 2016-03-04 | 2018-07-31 | 中国水产科学研究院北戴河中心实验站 | A kind of fish early life history otolith sample preparation methods |
| CN106970079A (en) * | 2017-02-27 | 2017-07-21 | 上海海洋大学 | A kind of method that utilization inner casing identifies squids age in days |
| CN106970079B (en) * | 2017-02-27 | 2019-12-27 | 上海海洋大学 | Method for identifying day age of teleost by using inner shell |
| CN112335716A (en) * | 2020-11-03 | 2021-02-09 | 中国水产科学研究院北戴河中心实验站 | Method for rapidly picking otoliths of flounder larvae |
| CN112335716B (en) * | 2020-11-03 | 2022-04-15 | 中国水产科学研究院北戴河中心实验站 | Method for rapidly picking otoliths of flounder larvae |
| CN113588328A (en) * | 2021-08-10 | 2021-11-02 | 中国水产科学研究院黑龙江水产研究所 | Micro-sampling method for micro-chemical analysis of otolith |
| CN113588328B (en) * | 2021-08-10 | 2022-04-19 | 中国水产科学研究院黑龙江水产研究所 | Micro-sampling method for micro-chemical analysis of otolith |
| CN114486442A (en) * | 2022-01-28 | 2022-05-13 | 公安部物证鉴定中心 | Sample preparation method for trace material evidence element analysis |
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