CN111849772B - Device and method for continuous culture and observation of model fish embryos - Google Patents
Device and method for continuous culture and observation of model fish embryos Download PDFInfo
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- CN111849772B CN111849772B CN202010764739.5A CN202010764739A CN111849772B CN 111849772 B CN111849772 B CN 111849772B CN 202010764739 A CN202010764739 A CN 202010764739A CN 111849772 B CN111849772 B CN 111849772B
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- 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
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/06—Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization
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- 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
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- C12M23/38—Caps; Covers; Plugs; Pouring means
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Abstract
A device for culturing and observing embryo of model fish comprises 3 parts including an incubation bottom plate, an embryo positioning plate device and a fixing device top cover; the device can be simultaneously used for continuous culture and real-time observation of model fish embryos, the culture solution is convenient and quick to replace, high-flux exposure and individualized continuous detection can be effectively realized, the operation is simple, and the artificial damage to the embryos is effectively reduced; the device disclosed by the invention can be compatible with continuous culture and observation of fish embryos in various modes, and can also adjust the size, the number and the variety collocation of the parts of the device according to experimental needs, so that the compatibility is strong.
Description
Technical Field
The invention belongs to the technical field of biological experimental supplies, and particularly relates to a device and a method for continuous culture and observation of model fish embryos.
Background
The model organism refers to a non-human species repeatedly used for a long time in the experimental process of researching life phenomena, wherein fishes such as zebrafish, medaka, gobiocypris rarus, rainbow trout and the like are widely applied to the research of ecotoxicology, molecular genetics, developmental biology and disease science as important model organisms. Compared with other organisms, the novel fish embryo model has the advantages of simple and convenient feeding, fast development, sensitivity to toxic components but insensitivity to changes of growth environment, suitability for large-scale culture in a laboratory environment, easy experimental operation, perennial oviposition, large oviposition amount and easy collection, and is very favorable for experimental observation and operation because the model fish embryo has optical transparency.
Usually, the culture of model fish embryos in experiments is mostly carried out in commercial cell culture dishes or porous cell culture plates, when the culture solution is replaced, a pipette or a plastic suction tube is usually used for sucking out the original culture solution, the suspended embryos move along with the suction force, the embryos are easily damaged by people through repeated liquid replacement operation, and the residual liquid in culture holes cannot be completely sucked out, so that experimental errors are caused. In microscopic observation, if the observation is directly carried out in a cell culture plate, because each culture hole usually has a plurality of embryos suspended in culture solution, the embryos are easy to move along with the shaking of the device and the liquid, which is not beneficial to the observation, and sometimes a plurality of cells are gathered together or close to the plate wall to influence the observation, and the simultaneous existence of a plurality of embryos cannot ensure that the continuous observation can be carried out on the same embryo in each experiment; if the embryos are transferred to an external device one by one for observation, the embryos need to be transferred back and forth, so that the time and the labor are consumed, and the embryos are easy to be infected with bacteria and cause artificial damage. Therefore, it is desirable to design a more convenient culture and observation device and method for model fish embryos.
Disclosure of Invention
Accordingly, the present invention is directed to a device and a method for continuous culture and observation of model fish embryos, which at least partially solve at least one of the above problems.
In order to achieve the above object, as one aspect of the present invention, there is provided an apparatus for continuous culture and observation of model fish embryos, comprising an incubation base plate, an apparatus top cover and an embryo positioning plate, wherein:
the incubation bottom plate, the device top cover and the embryo positioning plate are all made of semitransparent or transparent materials;
the front surface of the incubation bottom plate is provided with a plurality of grooves for correspondingly placing embryo positioning plates;
the wall and the bottom of the embryo positioning plate form a box-shaped space without a cover, and the wall of the embryo positioning plate is higher than the depth of the groove on the front surface of the incubation bottom plate; the bottom of the embryo positioning plate is provided with a micropore or an array of micropores for accommodating an embryo, and the micropore is communicated with the incubation bottom plate;
the device top cover is in a shape corresponding to that of the incubation bottom plate and can form a closed or semi-closed accommodating space with the incubation bottom plate.
As another aspect of the invention, the invention also provides a method for continuously culturing and observing the embryo of the model fish, which comprises the following steps:
selecting an embryo positioning plate with proper size, micropore quantity and embryo fixing support according to the process requirement and the size of the model fish embryo, placing the embryo positioning plate into a groove of an incubation bottom plate corresponding to the size of the embryo positioning plate, and adding a certain volume of culture solution containing specific pollutants, toxic components or target substances into the incubation bottom plate to completely submerge micropores in the embryo positioning plate; wherein the method for continuously culturing and observing the model fish embryos is carried out in the device for continuously culturing and observing the model fish embryos;
and respectively placing the fertilized model fish embryos into micropores of an embryo positioning plate of the device for continuously culturing and observing the model fish embryos by using a plastic dropper, covering a top cover of the device, and placing the device into an illumination incubator to continuously culture and expose the model fish embryos.
Based on the technical scheme, compared with the prior art, the culture and observation device and the culture and observation method have at least one of the following beneficial effects:
(1) the device can be used for continuously culturing and observing embryos and yolk fishes of small-sized fishes at the same time, and is efficient and reliable;
(2) the device can adjust the size, the type and the number of the device components according to the experimental requirements, and can realize high-flux operation;
(3) the device can conveniently and quickly replace the culture solution, reduces the artificial direct damage to the roes and the yellow croakers, and has high efficiency, rapidness, simplicity and convenience in the whole operation;
(4) the device provided by the invention is strong in compatibility, can be used for continuously culturing and observing embryos and vitellogenin of various small-sized fishes such as zebra fishes and medakas, can expand new functions according to experimental requirements, and has the possibility of continuous optimization.
Drawings
FIG. 1 is a schematic structural diagram of the apparatus for continuous culture and observation of model fish embryos of the present invention;
FIG. 2 is a schematic structural diagram of an incubation base plate in the device for continuous culture and observation of model fish embryos, wherein FIG. 2A is an oblique view and FIG. 2B is a front view;
FIG. 3 is a schematic structural diagram of an embryo positioning plate in the apparatus for continuous culture and observation of model fish embryos of the present invention, wherein FIG. 3A is a perspective view and FIG. 3B is a front view.
In the above drawings, the reference numerals have the following meanings:
1. incubating the bottom plate; 2. Positioning the embryo;
3. a device top cover;
11. marking characters; 12. A front groove;
21. an indication groove; 22. A strip channel;
23. fixing micropores for the round embryo; 24. An embryo fixing bracket;
25. a truncated cone shaped groove;
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
The term "and/or" is a conjunctive word describing the associative relationship of the associated objects, indicating that three relationships may exist: for example, taking "a and/or B" as an example, it can be expressed as: a exists alone, A and B exist simultaneously, and B exists alone. In addition, the character "a/B device" herein also means that the former and latter related objects are in an associated relationship of "and/or".
The invention discloses a device for continuously culturing and observing model fish embryos, which can be used for observing or culturing model fish embryos independently and can also be used for observing and culturing the whole hatching process of the fish embryos simultaneously. The device comprises an incubation bottom plate, a device top cover and an embryo positioning plate, wherein the incubation bottom plate, the device top cover and the embryo positioning plate are all three parts; wherein:
the three parts are all made of semitransparent or transparent materials so as to ensure illumination.
The incubation bottom plate can be in various shapes, and is in a common rectangular structure for convenient processing; the incubation bottom plate is provided with a mark so that a user can distinguish and identify the content type and record the observation position, for example, the left side and the upper side of the front surface of the incubation bottom plate are respectively provided with letters and numbers for the user to identify and record the observation position.
The front of the incubation bottom plate is provided with a plurality of grooves which are arranged in parallel and can be used for placing embryo positioning plates.
Embryo locating plate, its stave forms a square box-like space of uncovered with the bottom, this embryo locating plate stave is higher than the degree of depth of hatching the positive recess of bottom plate, so that take out or put into during the operation, side wall has the instruction recess on this embryo locating plate, a direction for instruct embryo locating plate during the experiment, this embryo locating plate bottom has the circular micropore and the array that are used for fixed embryo, this circular micropore and hatching bottom plate intercommunication, the inside support that is used for fixed embryo that has of circular micropore, communicate through the bar passageway between the adjacent circular micropore in embryo locating plate bottom, a circulation for the culture solution, there is the recess with hatching the bottom plate intercommunication between the circular micropore array in embryo locating plate bottom, be used for adding the culture solution.
The embryo positioning plate is characterized in that embryos are respectively placed in the round micropores through a plastic dropper during continuous culture, then the embryo positioning plate is placed on the absorbent paper, after residual solution is absorbed, the embryo positioning plate is placed in the groove in the front face of the incubation bottom plate, then culture solution is slowly added into the circular truncated cone-shaped groove between the round micropores through a solution transfer gun, the whole embryo positioning plate can be automatically covered by the culture solution, and the incubation bottom plate and the embryo positioning plate of the whole device can be placed on a microscope observation platform at any time for observation.
The device top cap, its shape corresponds with hatching the bottom plate shape, through hinge connection, avoids external pollution.
Preferably, the size of the wells on the front of the incubation base varies with the number, which can be adjusted according to the experimental arrangement, and in some embodiments of the invention, the number of wells is, for example, 8, divided into 2 rows of 4 parallel rows.
Preferably, the depth of the wells on the front of the incubation base may vary depending on the volume of culture used in the experiment, and in some embodiments of the invention the depth of the wells is 6 mm.
The grooves between the array of the circular micropores at the bottom of the embryo positioning plate and communicated with the incubation bottom plate can be in any shape, in some embodiments of the invention, the grooves are in a truncated cone shape, and the truncated cone-shaped grooves are tangent to the central axis of the strip-shaped channel between the micropores and gradually become smaller from top to bottom.
The size of the embryo positioning plate is changed with the number of micropores, and the number of the micropores is changed from 1 to 96. In some embodiments of the present invention, the number of the micropores of the embryo positioning plate is 12, so that 12 embryos can be placed at the same time. The depth range of the groove on the front surface of the incubation bottom plate is 3-25 mm. The depth range of the embryo positioning plate is 3-30 mm; the shape of the micropores at the bottom of the embryo positioning plate is circular, oval or polygonal; and/or the number of the micropores at the bottom of the embryo positioning plate is 1-96.
The inside of the circular micro-hole is provided with a bracket for fixing the embryo, and in some embodiments of the invention, the bracket is a triangular bracket which is gradually reduced from top to bottom and finally forms a small circle in the middle. Preferably, the support is a triangular support, a circular support or a pentagonal support.
The size of the micropore at the bottom of the embryo positioning plate and the size of the bracket for fixing the embryo in the micropore are set according to the size of the model fish embryo. In some embodiments of the invention, the embryo diameter using zebrafish and medaka is typically 1.5 mm.
The depth of the embryo positioning plate is changed along with the size of the model fish embryo. In some embodiments of the invention, the embryo location plate has a depth of 8 mm.
The model fish embryo is selected from zebra fish, medaka, Hemiculter nigrum, gobiocypris rarus, rainbow trout, carp, salmon, tilapia, catfish, sheep porgy, silver salmon and/or spiny fish embryo.
The invention also discloses a method for culturing/observing the model fish embryo, which comprises the following steps:
selecting an embryo positioning plate with proper size, micropore quantity and embryo fixing support according to the process requirement and the size of the model fish embryo, placing the embryo positioning plate into a groove of an incubation bottom plate corresponding to the size of the embryo positioning plate, and adding a certain volume of culture solution containing specific pollutants, toxic components or target substances into the incubation bottom plate to completely submerge micropores in the embryo positioning plate; wherein the method for culturing and observing the model fish embryo is carried out in the fish embryo culturing and observing device;
respectively placing fertilized model fish embryos into micropores of an embryo positioning plate of the fish embryo culturing and observing device by using a plastic dropper, covering a top cover of the device, and placing the device into an illumination incubator to continuously culture and expose the model fish embryos;
preferably, the solution in the incubation bottom plate is aspirated every 24 hours using a pipette or plastic pipette, and replaced with new solution.
The technical solution and effects of the present invention will be further explained by the following embodiments with reference to the accompanying drawings. It should be noted that the following specific examples are only for illustration and are not intended to limit the invention.
Examples
The structure of the device for continuous culture and observation of model fish embryos is shown in figures 1-3B, and mainly comprises an incubation bottom plate 1, an embryo positioning plate 2 and a device top cover 3, wherein:
the whole device and all parts are made of transparent resin to ensure illumination; the left side and the upper side of the front surface of the incubation bottom plate 1 are respectively provided with English letters and numbers 11 as marks, the front surface of the incubation bottom plate 1 is provided with 8 grooves 12 for placing the embryo positioning plate 2, the grooves 12 are divided into two rows, each row is provided with 4 parallel rows, and the depth of each groove 12 is 6 mm.
The degree of depth of 2 siding walls of embryo locating plate is 8 millimeters, there is instruction recess 21 side wall right side on embryo locating plate 2, indicate embryo locating plate 2's direction for when experimenting, there are 12 and the circular micropore 23 that are used for fixed embryo of hatching bottom plate 1 intercommunication in embryo locating plate 2 bottom, circular micropore 23 divide into 4 rows, 3 parallel arrangement of every row, communicate through bar passageway 22 between the adjacent micropore 23, there is the triangular support 24 that is used for fixed embryo in the circular micropore 23, triangular support 24 is dwindled down by last to gradually, form a little circular in the centre at last, there are 6 round platform shape recesses 25 with hatching bottom plate 1 intercommunication between the array of circular micropore 23, be used for adding the culture solution, the axis of the bar passageway 22 between round platform shape recess 25 and the micropore 23 is tangent, and by the top down diminishes gradually.
During continuous culture, firstly, a plastic dropper is used for respectively placing embryos with corresponding quantity into the round micropores 23, then the embryo positioning plates 2 are placed on absorbent paper, after residual solution is absorbed, the embryo positioning plates 2 are respectively placed in the front grooves 12 of the incubation bottom plate 1, then a liquid transfer gun is used for slowly adding culture liquid into the circular truncated cone-shaped grooves 25 among the round micropores 23, the culture liquid can automatically cover the whole embryo positioning plate 2, and at the moment, the incubation bottom plate 1 and the embryo positioning plate 2 of the whole device can be placed on a microscope observation platform at any time for observation.
The shape of the top cover 3 of the device corresponds to that of the incubation bottom plate 1, and the external pollution is avoided through hinge connection.
Through the design, the device for continuously culturing and observing the model fish embryos can be used for continuously culturing and observing the model fish embryos, has high flux and is efficient and reliable, the device can conveniently and quickly realize culture solution replacement, artificial damage to the embryos is reduced, and continuous observation of each embryo individual is realized.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The utility model provides a device that is used for model fish embryo continuous culture and observes, is including hatching bottom plate, device top cap and embryo locating plate, its characterized in that:
the incubation bottom plate, the device top cover and the embryo positioning plate are all made of semitransparent or transparent materials;
the front surface of the incubation bottom plate is provided with a plurality of grooves for correspondingly placing embryo positioning plates;
the plate wall and the bottom of the embryo positioning plate form a box-shaped space without a cover, the depth of the plate wall of the embryo positioning plate is higher than that of the groove on the front surface of the incubation bottom plate, the depth range of the embryo positioning plate is 3-30 mm, and the depth range of the groove on the front surface of the incubation bottom plate is 3-25 mm; the bottom of the embryo positioning plate is provided with an array of micropores for accommodating embryos, the micropores are communicated with the incubation bottom plate, a bracket for fixing the embryos is arranged in the micropores at the bottom of the embryo positioning plate, grooves which are communicated with the incubation bottom plate and used for adding culture solution are formed among the micropores at the bottom of the embryo positioning plate, adjacent micropores at the bottom of the embryo positioning plate are communicated through strip-shaped channels, and the central axes of the strip-shaped channels between the grooves and the micropores are tangent to be used for the circulation of the culture solution;
the shape of the top cover of the device corresponds to that of the incubation bottom plate, and a closed or semi-closed accommodating space can be formed between the top cover and the incubation bottom plate.
2. The apparatus for continuous culture and observation of model fish embryos of claim 1, wherein,
the number range of the grooves on the front surface of the incubation bottom plate is 1-96.
3. The apparatus for continuous culture and observation of model fish embryos of claim 1, wherein,
the shape of the micropores at the bottom of the embryo positioning plate is circular, oval or polygonal; and/or
The number of the micropores at the bottom of the embryo positioning plate is 1-96.
4. The apparatus for continuous culture and observation of model fish embryos of claim 3, wherein,
the support is a triangular support, a circular support or a pentagram-shaped support.
5. The apparatus for continuous culture and observation of model fish embryos of claim 1, wherein,
the size of the micropores at the bottom of the embryo positioning plate and the size of the bracket for fixing the embryo in the micropores are set according to the size of the model fish embryo.
6. The apparatus for continuous culture and observation of model fish embryos of claim 1, wherein,
the groove is in a round table shape and gradually becomes smaller from top to bottom.
7. The apparatus for continuous culture and observation of model fish embryos of claim 1, wherein,
the embryo positioning plate is provided with an indicating groove for indicating the direction of the corresponding positioning plate during the experiment; and/or
The embryo positioning plate is provided with a digital mark for distinguishing different embryo positioning plates and/or micropores on the bottom of the embryo positioning plate.
8. The apparatus for continuous culture and observation of model fish embryos of claim 1,
the top cover of the device is connected with the incubation bottom plate through a hinge.
9. The apparatus for continuous culture and observation of model fish embryos of claim 1, wherein,
the model fish embryo is selected from zebra fish, medaka, Hemiculter nigrum, gobiocypris rarus, rainbow trout, carp, salmon, tilapia, catfish, sheep porgy, silver salmon and/or spiny fish embryo.
10. A method for continuously culturing and observing model fish embryos, which is carried out in the device for continuously culturing and observing model fish embryos of any one of claims 1 to 9, and which comprises the following steps:
selecting an embryo positioning plate with proper size, micropore quantity and embryo fixing support according to the process requirement and the size of the model fish embryo, placing the embryo positioning plate into a groove of an incubation bottom plate corresponding to the size of the embryo positioning plate, and adding a certain volume of culture solution containing specific pollutants, toxic components or target substances into the incubation bottom plate to completely submerge micropores in the embryo positioning plate;
respectively placing fertilized model fish embryos into micropores of an embryo positioning plate of the fish embryo culturing and observing device by using a plastic dropper, covering a top cover of the device, and placing the device into an illumination incubator to continuously culture and expose the model fish embryos; during continuous culture, culture solution is slowly added into the grooves between the micropores to replace the solution.
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| CN106520541A (en) * | 2016-12-16 | 2017-03-22 | 常州市环境监测中心 | Kit for zebra fish embryo acute toxicity test |
| CN106719172A (en) * | 2017-01-23 | 2017-05-31 | 浙江师范大学 | Zebrafish embryo hatching apparatus are used in experiment |
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