CN105158035B - The preparation method that a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis is cut into slices - Google Patents
The preparation method that a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis is cut into slices Download PDFInfo
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- CN105158035B CN105158035B CN201510514184.8A CN201510514184A CN105158035B CN 105158035 B CN105158035 B CN 105158035B CN 201510514184 A CN201510514184 A CN 201510514184A CN 105158035 B CN105158035 B CN 105158035B
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Abstract
The present invention relates to the preparation method that a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis is cut into slices, including：(1) win；(2) mark；(3) dispensing；(4) embed；(5) cut；(6) polish；(7) cut；(8) clean；(9) dry.Present invention ensures that the maximum sagittal plane of otolith micro-chemical analysis, the degree of accuracy of raising otolith micro-chemical analysis, have a good application prospect.
Description
Technical field
The invention belongs to otolith process field, more particularly to a kind of little yellow croaker larva and juvenile otolith for micro-chemical analysis is sweared
The preparation method of shape face section.
Background technology
The larva and juvenile phase is the important stage of fish early life history, and habitat close relation, larva and juvenile it is dead
Die the supplement that dynamic directly affects fishery resources.
Traditional fishery resources survey method and biotelemetry can not be implemented to inhabit ring in the migration fishes full history of life
The continuous monitoring in border.Otolith information provides the environmental change undergone in the fish history of life.From otolith core to rim area
Element composition generally record in temporal sequence fish from birth to it is captured when the different niches feature that is undergone.Nearly more than 40 years
Come, otolith is able to develop rapidly simultaneously extensively as the particular feature of habitat " fingerprint " label in the research field of fish space-time dynamic
Using.
Reflect while the frame-type to fish early life history is confined into fish otolith macro result, and the otolith of larva and juvenile
Macro is combined with different growing ages and carries out analysis and can accurately illustrate between fish early growth critical stage and habitat
Corresponding relation.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of little yellow croaker larva and juvenile otolith for micro-chemical analysis and sweared
The preparation method of shape face section, this method guarantees that the maximum sagittal plane of otolith micro-chemical analysis, raising otolith micro-chemical analysis
The degree of accuracy.
The preparation method 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, bag
Include：
(1) win：The sagitta in larva and juvenile statocyst is taken out with tip tweezers under anatomical lens, is then placed in and fills
Cleaned in the culture dish of deionized water, dried at room temperature, is put into centrifuge tube, and put on otolith numbering；
(2) mark：After differentiating left otolith and auris dextra stone under anatomical lens, it is respectively placed in centrifuge tube, and on centrifuge tube
Left or right is marked uniformly to take left otolith or auris dextra stone to carry out subsequent operation to show left and right otolith；
(3) dispensing：By A glue and B glue by volume 1:0.5~2 is coated on masking foil, heats while stirring 30~40 seconds；
Numbered otolith embedded box is taken, picks AB glue point in embedded box cassette bottom；Larva and juvenile otolith is placed in AB glue, and presses clamp lug
Stone surface, record the numbering of otolith and the numbering of corresponding embedded box；
(4) embed：In mass ratio 6~8:1 stirs and evenly mixs resin and curing agent, after being mixed toward the injection of each embedded box
3~4ml of resin；Dried after embedding, resin mass is taken out after to be hardened；
(5) cut：Otolith resin mass is sticked on the sheet glass for indicating otolith numbering using AB glue；After to be solidified, adopt
Excess resin is cut off with otolith cutting machine (Discoplan-TS, Si Teer company), and otolith is carried out using diamond disk
Corase grind, exposed to close to otolith core；Use 1200 mesh waterproof abrasive papers instead again and carry out manual fine grinding, be completely exposed to otolith core；
(6) polish：Polished manually using diamond polishing liquid and MD polishing cloths, untill confirming otolith surface without scratch；
(7) cut：Using the excess portion of otolith cutting machine (Discoplan-TS, Si Teer company) cutting otolith sheet glass
Point, it is ensured that otolith slice size can be put into the sample stage of micro-chemical analysis instrument；
(8) clean：Larva and juvenile otolith grinding and polishing is face-up, using deionized water rinsing, after to be placed in ultrasound in ultra-pure water clear
Wash；After ultrasonic cleaning, otolith grinding and polishing face order is rinsed respectively using deionized water, ultra-pure water；
(9) dry：Finally the resin mass after cleaning is dried, for otolith micro-chemical analysis.
A glue and B glue in the step (3) are chemical reaction adhesive.
Resin in the step (4) is epoxy resin, curing agent IPTG.
Drying temperature in the step (4) and (9) is 38~40 DEG C, and drying time is 12~15 hours.
Ultra-pure water in the step (8) was cleaned by ultrasonic the time for 2~3 minutes.
Beneficial effect
(1) present invention solve because larva and juvenile otolith gently and it is small cause use resin embedding when movement even inclination, use a little
Glue method carries out embedding again after otolith is fixed can effectively avoid this problem；
(2) use cuts method and avoids the problem of pure hand lapping process of otolith causes otolith sagittal plane to wear into inclined-plane,
It ensure that the maximum sagittal plane of otolith micro-chemical analysis；
(3) hand-polished method avoid when machine polishes rotating speed it is fast cause larva and juvenile thin and small otolith is ground
The problem of broken；
(4) method of cleaning can effectively remove the impurity of otolith sagittal plane, avoid micro- while ensureing that otolith is not lost
Interference during chemical analysis；
(5) above-mentioned series of steps, it is ensured that the reliability of larva and juvenile otolith micro-chemical analysis result.
Brief description of the drawings
Fig. 1 is the otolith Sr/Ca ratio variation diagrams of the little yellow croaker larva and juvenile on May 7；
Fig. 2 is the otolith Sr/Ca ratio variation diagrams of the little yellow croaker larva and juvenile on May 28；
Fig. 3 is the otolith Sr/Ca ratio variation diagrams of the little yellow croaker larva and juvenile on June 23.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
1) win：The sagitta in little yellow croaker larva and juvenile statocyst is taken out with tweezers under anatomical lens, pays attention to wanting light tweezer
Put down gently, in order to avoid damage otolith；After the otolith of larva and juvenile is taken out, it is put into the culture dish for containing ultra-pure water and is cleaned, go ear falling
Stone surface organic matter, dries at room temperature, is put into centrifuge tube and is used as analysis of material stand-by, and puts on otolith numbering；
2) mark：After differentiating left otolith and auris dextra stone under anatomical lens, it is respectively placed in centrifuge tube, and in centrifuge tube subscript
Remember the numbering of otolith, cover mark left (L) or right (R) in centrifuge tube to show left and right otolith.Left otolith is uniformly taken subsequently to be grasped
Make；
3) dispensing：A glue and B glue extrusion is a small amount of, by volume 1：1 ratio is carried out on masking foil on alcolhol burner
Heating, heats while stirring 30 seconds；Numbered otolith embedding circle box is taken, the AB glue point of mixing is picked in embedded box box with toothpick
Bottom；Larva and juvenile otolith is placed in AB glue rapidly, and otolith surface is pressed with tweezers, ensures that otolith sagittal plane is horizontal；Treat cold
But solidify 45 minutes；Record the numbering of otolith and the numbering of corresponding embedded box；Wherein, the A glue and B glue, chemical name used is
Chemically react adhesive (Amada Co., Ltd.)；
4) embed：In mass ratio 7:1 mixes epoxy resin and curing agent, and deployed resin is injected toward each embedded box
3ml, resin need to cover larva and juvenile otolith；Dried 12 hours in 38 DEG C of baking ovens after embedding；Taking-up resin mass after to be hardened, and
With the numbering of the dissection good each otolith of drypoint on resin mass；Wherein, the resin and curing agent used, chemical name are asphalt mixtures modified by epoxy resin respectively
Fat and IPTG (Si Teer companies, Copenhagen, Denmark)；
5) cut：Otolith resin mass is sticked on the sheet glass for indicating otolith numbering using AB glue, and ensures resin mass
Otolith numbering can be covered；After to be solidified, Excess resin is cut off using otolith cutting machine (Discoplan-TS, Si Teer company),
And otolith is roughly ground using diamond disk, exposed to close to otolith core；1200 mesh waterproof abrasive papers are used instead again to refine by hand,
Period at any time under upright metallurgical microscope with saturating, viewed in reflected light surface appearance, is completely exposed to otolith core；
6) polish：Use diamond polishing liquid and the manual polishing grinding face of MD polishing cloths；Used under upright metallurgical microscope
Untill reflected light confirms otolith surface without scratch；
7) clean：Larva and juvenile otolith grinding and polishing is face-up, using deionized water rinsing 6 times, after be placed in ultra-pure water it is ultrasonic
Cleaning 2~3 minutes；After ultrasonic cleaning, 8 times flushings are carried out respectively using deionized water, ultra-pure water to otolith grinding and polishing face order；For
Prevent from washing out larva and juvenile otolith, the thin mouth shower nozzle of water bottle avoids being directed at otolith face；
8) dry：Finally the resin mass after cleaning is placed in 38 DEG C of baking ovens, otolith macro is can be used for after 12 hours
Analysis.
9) carbon film is plated：Larva and juvenile otolith sample is placed in vacuum coating equipment (JEE-420, Jeol Ltd.) and steamed
Carbon film is plated, electric current is arranged to 35A, and plated film time is 25 seconds.
10) otolith micro-chemical analysis：Utilize electron probe microanalyser (english abbreviation EPMA, JXA-8100 type, Japan
Electronics Co., Ltd) from core along most long axial edge carry out it is continuous get measuring point ready, at intervals of 5 μm between point, EPMA accelerating potentials
It is respectively 15kV with electron beam current, 2.0 × 10-8A, beam spot diameter, are 5 μm, and every residence time is 15s.Take calcium carbonate
(CaCO3) and strontium titanates (SrTiO3) it is standard sample.Obtained each point is represented with the ratio of Sr contents and Ca contents, unified to use
Sr contents：Ca content × 103Ratio represent (abbreviation Sr/Ca ratios).
(May 7, May 28, June 23), the larva and juvenile ear of Different Individual size are sampled with the interval in about January
The micro- chemical contrast of stone：
(1) same growth phase compares.3 periods juvenile fish from otolith core at 15~35 μm, show
High Sr/Ca values (table 1), examine through one-way analysis of variance, the high Sr/Ca values near the little yellow croaker otolith core of 3 periods
Between without significant difference (P ﹥ 0.05).
(2) Sr/Ca ratios variation tendency compares.The little yellow croaker otolith Sr/Ca ratios in May 7, May 28 and June 23
Variation tendency is consistent, and in the otolith Sr/Ca (see Fig. 1-3) more substantially overlapping than value changes of identical growth phase：On May 7
Body is 9.11 ± 0.80 from core to average Sr/Ca at 20 μm；The individual on May 28 is from core to average Sr/Ca between 30 μm
9.48±1.11；The individual on June 23 is 8.90 ± 0.76 from core to average Sr/Ca between 20 μm；Hereafter between about 400 μm,
Show as Sr/Ca ratios and drop to 6：The juvenile fish Sr/Ca of three periods is followed successively by 5.87 ± 0.84,6.15 ± 0.50,5.62
±0.57。
The method as disclosed in embodiment as can be seen here, larva and juvenile otolith micro-chemical analysis result is accurately and reliably.
The high Sr values of little yellow croaker larva and juvenile otolith core space of the Different Individual size of table 1
Claims (5)
1. the preparation method that a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis is cut into slices, including：
(1) win：Under anatomical lens with tip tweezers by larva and juvenile statocyst sagitta take out, be then placed in fill from
Cleaned in the culture dish of sub- water, dried at room temperature, is put into centrifuge tube, and put on otolith numbering；
(2) mark：After differentiating left otolith and auris dextra stone under anatomical lens, it is respectively placed in centrifuge tube, and marked on centrifuge tube
Left or right uniformly takes left otolith or auris dextra stone to carry out subsequent operation to show left and right otolith；
(3) dispensing：By A glue and B glue by volume 1:0.5~2 is coated on masking foil, heats while stirring 30~40 seconds；Take
The otolith embedded box of numbering, AB glue point is picked in embedded box cassette bottom；Larva and juvenile otolith is placed in AB glue, and presses otolith table
Face, record the numbering of otolith and the numbering of corresponding embedded box；
(4) embed：In mass ratio 6~8:1 stirs and evenly mixs resin and curing agent, injects mixed resin toward each embedded box
3~4ml；Dried after embedding, resin mass is taken out after to be hardened；
(5) cut：Otolith resin mass is sticked on the sheet glass for indicating otolith numbering using AB glue；After to be solidified, using ear
Stone cutting machine cuts off Excess resin, and otolith is roughly ground using diamond disk, is exposed to close to otolith core；Use instead again
1200 mesh waterproof abrasive papers carry out manual fine grinding, are completely exposed to otolith core；
(6) polish：Polished manually using diamond polishing liquid and MD polishing cloths, untill confirming otolith surface without scratch；
(7) cut：Chopped off the ears using otolith cutting machine the redundance of stone sheet glass；
(8) clean：Larva and juvenile otolith grinding and polishing is face-up, using deionized water rinsing, after be placed in ultra-pure water and be cleaned by ultrasonic；It is super
After sound cleaning, otolith grinding and polishing face order is rinsed respectively using deionized water, ultra-pure water；
(9) dry：Finally the resin mass after cleaning is dried, for otolith micro-chemical analysis.
2. the making that a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis according to claim 1 is cut into slices
Method, it is characterised in that：A glue and B glue in the step (3) are chemical reaction adhesive.
3. the making that a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis according to claim 1 is cut into slices
Method, it is characterised in that：Resin in the step (4) is epoxy resin.
4. the making that a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis according to claim 1 is cut into slices
Method, it is characterised in that：Drying temperature in the step (4) and (9) is 38~40 DEG C, and drying time is 12~15 hours.
5. the making that a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis according to claim 1 is cut into slices
Method, it is characterised in that：Ultra-pure water in the step (8) was cleaned by ultrasonic the time for 2~3 minutes.
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| CN201510514184.8A CN105158035B (en) | 2015-08-20 | 2015-08-20 | The preparation method that a kind of little yellow croaker larva and juvenile otolith sagittal plane for micro-chemical analysis is cut into slices |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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 |
| 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 |
| 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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|---|---|---|---|---|
| CN101539495A (en) * | 2009-03-04 | 2009-09-23 | 上海海洋大学 | Preparation of mackerel otolith cross-sectional slices and method for determining age thereof |
| CN104374615A (en) * | 2014-09-16 | 2015-02-25 | 中国水产科学研究院黑龙江水产研究所 | Schizothoracinae otolith slice production method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101539495A (en) * | 2009-03-04 | 2009-09-23 | 上海海洋大学 | Preparation of mackerel otolith cross-sectional slices and method for determining age thereof |
| CN104374615A (en) * | 2014-09-16 | 2015-02-25 | 中国水产科学研究院黑龙江水产研究所 | Schizothoracinae otolith slice production method |
Non-Patent Citations (2)
| Title |
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| Juvenile Salmonid Otolith Extraction and Preparation Techniques for Microscopic Examination;W. J. Glick et al.;《FRED Reports》;19930731;第5页、7-8、10-13、18页 * |
| 利用耳石鉴定头足类年龄与生长研究进展;马金 等;《海洋渔业》;20090831;第31卷(第3期);第316-324页 * |
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