CN112514857A - Preparation and use method of tool for picking model animal caenorhabditis elegans - Google Patents

Abstract

The invention provides a preparation method and a using method of a caenorhabditis elegans tool for picking a model animal, wherein the preparation method of the caenorhabditis elegans tool comprises the following steps: selecting a handle with the length of about 12-15 cm, connecting the hair strands or eyelashes with the handle, and setting an included angle between a straight line where the hair strands or eyelashes are located and the axis of the handle to be 120-130 degrees; the step of picking the adult nematodes comprises the following steps: and (3) selecting a tool, soaking and sterilizing, pinching a handle of the tool by hand, and scratching the hair with the worms on a new culture medium for several times to enable the nematodes to fall off from the hair to realize transfer. The invention has the beneficial effects that: the invention can solve the problems that the existing platinum metal wire is high in processing and using cost, the platinum wire is easy to break and inconvenient to use, eyelashes in the eyelashes are soft and inconvenient for adult picking and limited in material taking, and realizes quick, accurate, direct and efficient nematode picking operation.

Description

Preparation and use method of tool for picking model animal caenorhabditis elegans

Technical Field

The invention relates to the technical field of picking model animal caenorhabditis elegans, in particular to a preparation method and a use method of a tool for picking model animal caenorhabditis elegans.

Background

Caenorhabditis elegans was first studied in 1963 in Sydney Brenner in the uk and has been widely used as a model organism in life science research and made a major breakthrough. In the laboratory, nematodes were grown on NGM plates containing OP50 E.coli and incubated at 20 ℃ for a 3.5 day growth cycle of about 3 weeks. The nematode is simple in structure, and is small in nematode individual formed by 959 cells, the length of an adult is only about 1 mm, as shown in figure 1, the larva individual is small and is difficult to be detected by naked eyes and needs to be observed by a microscope. The experimental operation of the nematodes comprises culture, passage, experiment and the like, individual nematodes need to be picked, the nematodes with the length of 1 mm are difficult to be picked by using conventional tools such as tweezers, medicine spoons, glass rods and the like, and the picking of the nematodes needs special tools.

The method is characterized in that the device is required to be utilized firstly, the method is more remarkable in the nematode picking work, a convenient tool is required to be provided for picking the nematodes smoothly, accurately, quickly and conveniently, the convenient nematode picking tool can enable the experiment to be more smooth and has the effect of being doubled with half the effort, for example, after nematode transgenic injection, a very small number of nematode individuals with successful transgenes need to be picked from the offspring with many transgenic failures, if the individuals with fluorescent marks need to be picked and operated under a fluorescent microscope, the moving direction of an object in an ocular lens is inconsistent with the actual moving direction, contrary to the habit recognition, the nematode picking operation is more difficult, the operation is higher in difficulty in experimental operation in nematode research, time and labor are consumed, a convenient nematode picking tool is particularly important, the operation can be completed within 10 minutes, and the use of the unusable tool usually needs 1 hour, greatly reducing the experimental efficiency. For another example, the nematodes are biological individuals, have nerve centers, have physiological behaviors of benefiting and avoiding pests, are mostly in a feeding state or in a dormant state on the culture medium, are convenient to pick, and when trying to pick, if the picking is unsuccessful for the first time, the nematodes can be stimulated to generate the behavior of escaping and crawling, the difficulty of picking the crawling nematodes is increased, a good tool can be competent for the operation, and more the nematodes can be successful at one time, so that the nematode escape opportunity is not given. Nematode picking requires working experience, but a good tool and method of nematode picking is more important.

There are reports of picking nematodes with platinum wires, as shown in FIG. 2, or eyelashes. Platinum belongs to inert metal, is not easy to oxidize and is resistant to high temperature of flame, and is often used for picking monoclonal bacterial plaque. The platinum wire used for picking the nematodes needs to be subjected to wire drawing processing, so that the platinum wire is thinner, the tip needs to be passivated to be used for picking the nematodes, but the tip can be processed into a hook shape, and the patterns are various. However, platinum is soft, easy to break and durable, and has a high price, the manufacturing process is relatively complex, the cost is high, and a convenient device for picking nematodes is yet to be developed. Referring to the method of platinum wire, picking up the worm with the filament is more convenient, there is a report that picking up the nematode with the eyelash, the root of the eyelash is sticked on the rod, the tip of the eyelash can be used for picking up the nematode, the cost is low, the use is convenient, but the tip of the eyelash is very thin and soft, picking up the larva is appropriate, because the length of eyelash is about 1 cm, the defect that the eyelash is soft and is not easy to control is more obvious, the individual is longer and the deformation amount is larger, the movement of the tip is difficult to control by using the handle, picking up the larger adult force with the tip of the eyelash which is soft is not good, and the defect needs to be solved by using the filament with a slightly larger bearing force. Eyelashes become softer for prolonged use due to aging and are no longer suitable for use. In addition, the eyelashes used are human eyelashes, and the material is limited. According to our long-term nematode research work, we developed a tool suitable for picking nematodes on the basis of eyelashes.

How to solve the above technical problems is the subject of the present invention.

Disclosure of Invention

The invention mainly aims to provide a simple and convenient tool for picking individual monomers of model animal nematodes, and aims to provide a preparation method and a use method of the tool for picking model animal caenorhabditis elegans, so as to solve the problems that the existing platinum wire is high in processing and use cost, the platinum wire is easy to break and inconvenient to use, eyelashes are soft and inconvenient to pick adults, and the material is limited, and realize quick, accurate, direct and efficient nematode picking operation.

The invention is realized by the following measures: preparation of a tool for picking a model animal caenorhabditis elegans, which comprises the following steps:

s1, selecting a handle with the length of about 12-15 cm;

s2, the handle in the step S1 consists of two parts, and the handle consists of a rod with uniform thickness and a micropipette plastic gun head sleeved at the end of the rod, wherein the micropipette plastic gun head is a yellow gun head of 200 microliter;

s3, debugging a microscope, under the microscope, connecting hairlines or eyelashes after the tip end of a plastic gun head is subjected to thermal capacity, cooling and connecting, arranging the hairlines or eyelashes, baking the plastic gun head with open fire to melt the plastic gun head into a mass, connecting the mass with the end points of the hairlines or eyelashes under the microscope before the mass is melted and not solidified, embedding the hairlines or eyelashes of 1-2 mm into the viscous hot-melt plastic mass for firm connection, adjusting the angle between the hairlines or eyelashes and a rod, completing the connection after cooling, and cutting off the hairlines or eyelashes and leaving the hairlines or eyelashes of 2-3 mm;

s4, making the included angle between the straight line of the hair or eyelash and the axis of the handle be 120-130 degrees, thus completing the preparation of the tool for picking the model animal caenorhabditis elegans.

In order to better achieve the above object, the present invention further provides a method for using the tool for picking the model animal caenorhabditis elegans, the method comprises the following steps:

picking up nematode adults:

(1) operating an ultra-clean room, preparing a culture dish with an NGM nematode culture medium, preparing a nematode food OP50 Escherichia coli fresh and sufficient, keeping the environmental temperature at 21 ℃, and preparing a fresh 200 mg/L chlorine dioxide solution;

(2) before using a nematode picking tool, soaking the hair ends of the nematodes in a fresh 200 mg/L chlorine dioxide solution for 2 minutes, sterilizing, and wiping the hair ends with sterile paper after the picking tool is soaked and sterilized;

(3) when picking the nematodes, before replacing each nematode, the picking tool needs to be sterilized, when preparing to pick the nematodes, the handle of the picking tool is pinched by hand, a culture dish cultured with the nematodes is placed under the body type microscope, and the brightness, the focal length and the magnification of the body type microscope are adjusted; picking up nematodes in an inclined way under a microscope with the magnification of 400 times;

(4) the tool handle is picked up by pinching with hands, the inclination is kept, and the included angle between the straight line of the hair and the plane of the culture medium is small;

(5) inserting short hair tips into the middle section of the adult nematodes, picking up the short hair tips, and transferring the short hair tips to a new culture medium;

(6) the hair with the selected insects is scratched on a new culture medium for several times, so that the nematodes fall off from the hair to realize transfer;

(7) the transfer to fresh medium should also be done under microscope to ensure successful nematode transfer;

(8) repeating the operation until the insects with the quantity required by the task are picked;

(9) after finishing, picking the hair ends of the tool, soaking and sterilizing the hair ends in a fresh 200 mg/L chlorine dioxide solution for 2 minutes, and then putting the hair ends back to the original position;

picking of nematode larvae:

(1) picking eyelashes, wherein the included angle between the eyelashes and the straight line of the main body of the nematode is 30-45 degrees;

(2) confirming transfer to a new plate under a microscope;

(3) when a plurality of nematode larvae are transferred at one time, the nematode tool for picking up the adults is adopted to sweep for several times on the NGM flat plate with more larvae, the larvae are scraped off, a plurality of larvae are adhered by the viscous acting force of the escherichia coli on the culture medium, and then the larvae are transferred to a new culture dish.

In order to better achieve the above object, the present invention further provides a method for preparing a tool for picking a model animal caenorhabditis elegans, which specifically comprises the following steps:

the nematode tool is used for picking model animal caenorhabditis elegans and single nematodes, adult nematodes are picked by using hair strands which are not human eyelashes and have certain hardness relative to the hair strands of the human eyelashes, the hair strands are not easy to control unlike the individual slender and soft human eyelashes, the hair strands are convenient to obtain, the cost is low, the chemical property is stable, the service life is long, the biological toxicity is avoided, the nematode individual is tiny, the length of the adult is only 1 mm, the adult nematodes can only be picked by using the tip of the hair, the nematode has the functions of adult and larva, the efficiency is improved by picking two different types of hair for two different types of individuals, the adult is picked to use the hair strands, the human eyes which are conventionally used for picking the nematodes are used for picking the larvae of the nematodes, the tool used for the targeted design of the nematodes with different sizes can improve the picking experimental efficiency, and the labor is better than that different types of hair are separated, the purpose of making the best use of things is achieved, and the purpose that an adult picking tool can pick adults and is suitable for larvae and is difficult to pick larvae is achieved, the problem can be effectively solved by designing and using different tools, the adult nematodes are mature, perfect in individual development, complete in function and high in activity, the adult nematodes are used in a nematode experiment, the larvae are used less because of not being developed and perfect, the whole eyelash is used for picking nematodes conventionally, the adult nematodes are picked by using hair filaments instead of the adult nematodes with larger experimental demand, the adult picking efficiency of short hair filaments is far higher than that of human eyelashes, the human eyelashes are restricted by sources greatly, the number of the human eyelashes is limited, and the hair filament raw materials are abundant. The adult picking tool for picking adult and larva of nematodes aiming at different purposes can be distinguished and distinguished by handles with different colors, the adult picking tool is shorter in hair wire, about 2-4 mm in length, has certain hardness and rigidity compared with the existing more eyelash, has smaller deformation than the tip of the eyelash when the tip of the adult is contacted with the nematode and a culture medium, can better realize the operation intention, is softer and larger in deformation amount, and has uncontrollable displacement of the tip, particularly on a nematode culture medium with certain viscosity, the adult picking tool for picking adult nematodes by soft and longer eyelash appears weak, picks the adult nematodes by using 2-4 mm short hair wires, and can fly the nematode by the deformation recovery action at the moment when the nematode is picked by using slightly longer hair wires and is separated from the culture medium, the short hair (2-4 mm) has small deformation, the hair has certain hardness, the deformation of the hair is small when the nematodes are picked, the contained potential energy is small, the recovery of the picked instant hair deformation, namely the release of the potential energy is not enough to enable the insects to fly, the loss of the insects is reduced, the tip of the hair is closer to the handle when the hair is short (2-4 mm), the hair has certain rigidity, the movement of the tip of the hair is more flexible when the handle is used for operating the hair, the movement of the tip of the hair is easy to control, the displacement condition of the tip of the hair is similar to that of the tip of the handle, the movement intention of the handle is directly reflected, the tip of the hair can be accurately and quickly sent to a specified position according to the intention of people, such as the center of a nematode worm body, the insects can be directly, accurately and quickly picked up, and the success rate of picking up the long and thin eyelashes by using the short hair to pick up the insects can be improved compared with the soft and thin human eyes which are difficult to control The operation time is reduced, the operation stability is improved, the short hair strands have certain rigidity, the operation controllability of the short hair strands is stronger, and the nematodes cannot be flicked off, so the nematodes are high in first picking success rate, picking of the nematodes can be completed at one time, the nematodes can be neatly fallen, the first picking means that the nematodes can be picked by first contacting, the nematodes are not picked easily by second picking, the nematodes are still on the culture medium and are picked again, the difficulty in secondary picking is large, the nematodes can climb to escape, the nematodes in motion are not easy to pick, the first picking success rate of picking the nematodes by using the short hair strands is high and exceeds 80%, the first picking success rate of picking the nematodes by using eyelashes is less than 30%, the highest speed of picking the nematodes by using the picking tool can be realized, about 10-12 nematodes can be picked per minute, and the operation stability is good, the tool has good performance and can be developed into an experimental consumable product. The speed of picking the nematodes by using the eyelashes is greatly influenced by the tool, the consumed time is long, the operation stability is poor, the nematodes can be picked up from the angle vertical to the body of the nematodes or from a certain included angle or other angles, the picking operation is not influenced by the body position of the nematodes, the nematodes can be picked up from any angle, the diameter of the used hair is about 0.1 mm in order to reduce the elasticity and the diameter size of the hair, the texture is relatively soft, the hardness of the hair with the diameter of 0.1 mm can support the nematodes, and the hair has certain toughness but is not enough to bounce the picked up nematodes due to the short length;

using human eyelashes to pick up nematode larvae: the eyelash structure is tapered and evenly thinned, the hardness of the eyelash structure is gradually reduced along with the diameter thinning, the hair quality is soft and is not easy to control, a hardness area suitable for picking up the larva is close to the tip, a hardness area suitable for picking up the adult insect is close to the root, the maximum potential energy contained in the eyelash structure is sequentially reduced from thick to thin, the insect cannot easily fly away even if the deformation amount is large, the larva can be picked up by the eyelash structure, the eyelash has the tip, the integral eyelash tip is soft, small objects are difficult to pick up on the surface of a semisolid and certain-viscosity nematode culture medium, the eyelash tip can be directly inserted into the culture medium or curled when contacting with the culture medium, the moving direction of the eyelash can be changed, the eyelash is difficult to control, the curling of the tip can be amplified and observed, the curled length is short and only limited to the tip position, but the movement of the eyelash can be difficult to control by, we processed the eyelash, cut the tip to make the operation movement intention reflect on the eyelash tip better, control the movement of the tip better, the operation belongs to the fine manipulation to be carried out under the body type microscope, cut the half length of the curly fragment can, should not cut too much, just made the eyelash instrument for picking up the nematode larva, after the hair selection is finished, began to pick up the preparation of the instrument: the length of the handle is about 12-15 cm, the handle is convenient for an adult to pinch, and the used handle consists of two parts: the method comprises the steps of arranging a rod with uniform thickness and a micropipette plastic gun head sleeved at the end of the rod, wherein the material of the rod is not limited, the plastic gun head is a 200 microliter yellow gun head (shown in figure 1), the design consistent with the function of the device also belongs to the protection range, the tip of the plastic gun head is heated to be melted and then connected with a hair, the connection is realized after cooling, the operation is carried out under a body microscope, the microscope is debugged, the hair is arranged, the plastic gun head is baked by naked flame to be melted into a mass, the mass is connected with a hair end point under the microscope before being melted into the mass and not solidified, the hair of 1-2 mm is embedded in a viscous hot melting plastic mass to ensure the firm connection, meanwhile, the angle between the hair and the rod is adjusted, the connection is completed after cooling, and the hair is cut off, and the hair of 2-. The plastic gun head is not suitable for connecting the hair immediately after melting, the hair is connected when the temperature is reduced to 60-70 ℃ and the melting body is not solidified so as to avoid damaging the hair by scalding, after solidification, if the plastic mass is larger, a blade can be used for trimming, the connection mode can be improved, for example, an adhesive method is used, an included angle is formed between the straight line of the hair and the axis of the handle, preferably between 120 and 130 ℃, when the included angle is formed, the included angle between the hair and the flat plate of the nematode culture medium is smaller when picking up the nematodes, the nematodes on the lying culture medium can be picked up by sticking to the culture surface so as to facilitate the power generation, if the included angle is not formed, the nematodes are difficult to pick up and inconvenient to generate the power, the shape of the hair can be straight line or slightly bent, the picking tool has simple manufacturing process, a plurality of hair can be manufactured at one time, and the picking tool can be used smoothly, the insect picking tool can be inversely inserted on a test tube rack or a special device, the protection of tip hair is well done, and fresh 200 mg/L chlorine dioxide solution is used for sterilizing for 2 minutes before and after use.

In order to better achieve the above object, the present invention further provides a method for using the tool for picking the model animal caenorhabditis elegans, the method comprises the following steps:

picking up nematode adults:

(1) preparing a culture dish with the NGM nematode culture medium, ensuring that the nematode food OP50 escherichia coli is fresh and sufficient, and keeping the environmental temperature at 21 ℃; preparing a fresh 200 mg/l chlorine dioxide solution;

(2) in order to avoid the pollution of the nematode culture medium by the mixed bacteria in the air, the operation is carried out in an ultra-clean room;

(3) before the tool is used and selected, the tool needs to be disinfected to prevent pollution, and the specific operation is to soak the hair ends in a fresh 200 mg/L chlorine dioxide solution for 2 minutes and sterilize;

(4) after the picking tool is soaked and sterilized, sterile paper is needed to be used for wiping, so that the nematode has developed taste function and is sensitive to odorous disinfectant;

(5) when picking nematodes, the nematode should be sterilized again every time a different nematode or picking tool is contaminated;

(6) when picking the nematodes, pinching the handle of the picking tool by hand, and keeping the handle inclined to pick the nematodes;

(7) the nematode is selected by using a body type microscope which has a continuous amplification function, and the maximum amplification factor is 400 times;

(8) placing the culture dish with the nematodes in the field of view of the body type microscope, and adjusting the brightness, the focal length and the magnification of the body type microscope;

(9) the target nematodes are found under a microscope, static insects are generally picked, and the picking difficulty of the crawling insects is high;

(10) the tool handle is picked up by pinching with hands, the inclination is kept, and the included angle between the straight line of the hair and the plane of the culture medium is small;

(11) the short hair tips are inserted into the lower part of the body of the nematodes at the middle section of the adult nematodes, the short hair tips are lifted and transferred to a new culture medium, the picking process is similar to that of picking up a rope on the ground by using a stick, the operation is simple, when the adult nematodes are picked by using soft and long filaments, the adult nematodes are easy to slip when being picked, the efficiency is reduced, because the picking tool has short hair and certain hardness and small elasticity, the slipping and bouncing of the nematodes cannot be caused, the success rate of picking for the first time is high, and the efficiency is high;

(12) the short hair is picked up vertically to the worm body and can form a certain inclination angle with the worm body;

(13) before picking, the nematodes can not be frightened to prevent the nematodes from escaping;

(14) the selection striving is successful once, and after the failure, the pest can escape due to the disturbance, and the selection difficulty is increased again;

(15) the nematodes should be transferred to a new culture dish immediately after picking, and are not suitable for staying in the air for a long time to prevent the nematodes from being dehydrated and dead in the air;

(16) the hair with the selected insects is scratched on a new culture medium for several times, so that the nematodes fall off from the hair to realize transfer;

(17) the transfer to fresh medium should also be done under microscope to ensure successful nematode transfer;

(18) repeating the operation until the insects with the quantity required by the task are picked;

(19) after finishing, picking the hair ends of the tool, soaking and sterilizing the hair ends in a fresh 200 mg/L chlorine dioxide solution for 2 minutes, and then putting the hair ends back to the original position;

picking of nematode larvae:

(20) the nematode larva individuals are relatively small, eyelashes need to be picked, and the picking difficulty is large;

(21) when the larva nematodes are picked up, the included angle of the eyelashes and the straight line of the main body of the nematode is 30-45 degrees to enlarge the contact area of the nematode and the eyelashes, increase the adhesion between the nematode and the eyelashes, reduce the contact area of the nematode and the culture medium and reduce the resistance, so the picking success rate can be improved, the picking mode vertical to the nematode does not have the advantage any more, because the eyelashes are long and thin and soft, the slippage phenomenon is easy to occur during picking up, and the efficiency is reduced;

(22) because the worm bodies are small, transfer to a new plate needs to be confirmed under a microscope to prevent transfer failure;

(23) when transferring a plurality of nematode larvae once, can use the above to choose the picker of the adult on the NGM flat board that has more larvae "sweep" several times, scrape the worm "down", borrow the comparatively viscous effort of escherichia coli on the culture medium in order to adhere a plurality of larvae simultaneously, transfer to in the new culture dish again.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention can solve the problem that picking of the nematode is difficult in operation, and the difficulty brought by the different sizes of the nematode bodies in different development stages is solved;

2. the nematode picking tool of the invention makes the advantages of the filamentous matters in the mechanical operation of the tiny organisms fully exerted, meanwhile, the hair replaces the platinum wire to effectively reduce the material and processing cost, the hair is convenient to obtain, the hair is tough and not easy to deform, the use is convenient, the relevant parameters of the manufacturing process of the tool of the invention can be quantized to ensure the quality of the product and the consistency of the result, through the microprocessing under a body type microscope and the quantitative control of the processing process, such as the hot melting time, the size of the melted plastic mass, the temperature during connection and the like, the hot melt adhesive can be used for convenient quantitative control in the gluing method connection, the development and the manufacture of relevant experimental consumables can be realized after the industrial fibers with appropriate parameters and performances replace the hair wires, the cost is lower, and a certain economic benefit is expected to be obtained.

3. The picking tool can realize the maximum speed of manually picking the nematodes, a skilled operator can pick one adult nematode in 5 to 6 seconds under a body microscope, picking can be realized by touching the nematode, the success rate is high, and the larva picking speed is about 15 to 20 seconds. Adult is picked out by using longer eyelashes, and the eyelashes are longer and not easy to control, and meanwhile, the front ends of the eyelashes are softer, so that the adult is picked out, and the adult has a component for bumping and transporting qi, and also needs the cooperation of insects, so that the first-time picking of the unsuccessful insects can enter the escaping crawling motion, and the difficulty is increased for the picking.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 is a schematic diagram of an adult caenorhabditis elegans of the present invention.

Fig. 2 is a schematic view of a conventional platinum wire insect picking tool.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. Of course, the specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Example 1

Referring to fig. 1 and fig. 2, the present invention provides a technical solution that the present invention is realized by the following measures: a preparation method and a use method of a tool for picking a model animal caenorhabditis elegans are disclosed, wherein the method comprises the following steps:

s1, selecting a handle with the length of about 12-15 cm;

s2, the handle in the step S1 consists of two parts, and the handle consists of a rod with uniform thickness and a micropipette plastic gun head sleeved at the end of the rod, wherein the micropipette plastic gun head is a yellow gun head of 200 microliter;

s3, debugging a microscope, under the microscope, connecting hairlines or eyelashes after the tip end of a plastic gun head is subjected to thermal capacity, cooling and connecting, arranging the hairlines or eyelashes, baking the plastic gun head with open fire to melt the plastic gun head into a mass, connecting the mass with the end points of the hairlines or eyelashes under the microscope before the mass is melted and not solidified, embedding the hairlines or eyelashes of 1-2 mm into the viscous hot-melt plastic mass for firm connection, adjusting the angle between the hairlines or eyelashes and a rod, completing the connection after cooling, and cutting off the hairlines or eyelashes and leaving the hairlines or eyelashes of 2-3 mm;

s4, making the included angle between the straight line of the hair or eyelash and the axis of the handle be 120-130 degrees, thus completing the preparation of the tool for picking the model animal caenorhabditis elegans.

In order to better achieve the above object, the present invention further provides a method for using the tool for picking the model animal caenorhabditis elegans, the method comprises the following steps:

picking up nematode adults:

(1) operating an ultra-clean room, preparing a culture dish with an NGM nematode culture medium, preparing a nematode food OP50 Escherichia coli fresh and sufficient, keeping the environmental temperature at 21 ℃, and preparing a fresh 200 mg/L chlorine dioxide solution;

(2) before using a nematode picking tool, soaking the hair ends of the nematodes in a fresh 200 mg/L chlorine dioxide solution for 2 minutes, sterilizing, and wiping the hair ends with sterile paper after the picking tool is soaked and sterilized;

(3) when picking the nematodes, before replacing each nematode, the picking tool needs to be sterilized, when preparing to pick the nematodes, the handle of the picking tool is pinched by hand, a culture dish cultured with the nematodes is placed under the body type microscope, and the brightness, the focal length and the magnification of the body type microscope are adjusted; picking up nematodes in an inclined way under a microscope with the magnification of 400 times;

(4) the tool handle is picked up by pinching with hands, the inclination is kept, and the included angle between the straight line of the hair and the plane of the culture medium is small;

(5) inserting short hair tips into the middle section of the adult nematodes, picking up the short hair tips, and transferring the short hair tips to a new culture medium;

(6) the hair with the selected insects is scratched on a new culture medium for several times, so that the nematodes fall off from the hair to realize transfer;

(7) the transfer to fresh medium should also be done under microscope to ensure successful nematode transfer;

(8) repeating the operation until the insects with the quantity required by the task are picked;

(9) after finishing, picking the hair ends of the tool, soaking and sterilizing the hair ends in a fresh 200 mg/L chlorine dioxide solution for 2 minutes, and then putting the hair ends back to the original position;

picking of nematode larvae:

(1) picking eyelashes, wherein the included angle between the eyelashes and the straight line of the main body of the nematode is 30-45 degrees;

(2) confirming transfer to a new plate under a microscope;

(3) when a plurality of nematode larvae are transferred at one time, the nematode tool for picking up the adults is adopted to sweep for several times on the NGM flat plate with more larvae, the larvae are scraped off, a plurality of larvae are adhered by the viscous acting force of the escherichia coli on the culture medium, and then the larvae are transferred to a new culture dish.

In order to better achieve the above object, the present invention further provides a method for preparing a tool for picking a model animal caenorhabditis elegans, which specifically comprises the following steps:

the nematode tool is used for picking model animal caenorhabditis elegans and single nematodes, adult nematodes are picked by using hair strands which are not human eyelashes and have certain hardness relative to the hair strands of the human eyelashes, the hair strands are not easy to control unlike the individual slender and soft human eyelashes, the hair strands are convenient to obtain, the cost is low, the chemical property is stable, the service life is long, the biological toxicity is avoided, the nematode individual is tiny, the length of the adult is only 1 mm, the adult nematodes can only be picked by using the tip of the hair, the nematode has the functions of adult and larva, the efficiency is improved by picking two different types of hair for two different types of individuals, the adult is picked to use the hair strands, the human eyes which are conventionally used for picking the nematodes are used for picking the larvae of the nematodes, the tool used for the targeted design of the nematodes with different sizes can improve the picking experimental efficiency, and the labor is better than that different types of hair are separated, the purpose of making the best use of things is achieved, and the purpose that an adult picking tool can pick adults and is suitable for larvae and is difficult to pick larvae is achieved, the problem can be effectively solved by designing and using different tools, the adult nematodes are mature, perfect in individual development, complete in function and high in activity, the adult nematodes are used in a nematode experiment, the larvae are used less because of not being developed and perfect, the whole eyelash is used for picking nematodes conventionally, the adult nematodes are picked by using hair filaments instead of the adult nematodes with larger experimental demand, the adult picking efficiency of short hair filaments is far higher than that of human eyelashes, the human eyelashes are restricted by sources greatly, the number of the human eyelashes is limited, and the hair filament raw materials are abundant. The adult picking tool for picking adult and larva of nematodes aiming at different purposes can be distinguished and distinguished by handles with different colors, the adult picking tool is shorter in hair wire, about 2-4 mm in length, has certain hardness and rigidity compared with the existing more eyelash, has smaller deformation than the tip of the eyelash when the tip of the adult is contacted with the nematode and a culture medium, can better realize the operation intention, is softer and larger in deformation amount, and has uncontrollable displacement of the tip, particularly on a nematode culture medium with certain viscosity, the adult picking tool for picking adult nematodes by soft and longer eyelash appears weak, picks the adult nematodes by using 2-4 mm short hair wires, and can fly the nematode by the deformation recovery action at the moment when the nematode is picked by using slightly longer hair wires and is separated from the culture medium, the short hair (2-4 mm) has small deformation, the hair has certain hardness, the deformation of the hair is small when the nematodes are picked, the contained potential energy is small, the recovery of the picked instant hair deformation, namely the release of the potential energy is not enough to enable the insects to fly, the loss of the insects is reduced, the tip of the hair is closer to the handle when the hair is short (2-4 mm), the hair has certain rigidity, the movement of the tip of the hair is more flexible when the handle is used for operating the hair, the movement of the tip of the hair is easy to control, the displacement condition of the tip of the hair is similar to that of the tip of the handle, the movement intention of the handle is directly reflected, the tip of the hair can be accurately and quickly sent to a specified position according to the intention of people, such as the center of a nematode worm body, the insects can be directly, accurately and quickly picked up, and the success rate of picking up the long and thin eyelashes by using the short hair to pick up the insects can be improved compared with the soft and thin human eyes which are difficult to control The operation time is reduced, the operation stability is improved, the short hair strands have certain rigidity, the operation controllability of the short hair strands is stronger, and the nematodes cannot be flicked off, so the nematodes are high in first picking success rate, picking of the nematodes can be completed at one time, the nematodes can be neatly fallen, the first picking means that the nematodes can be picked by first contacting, the nematodes are not picked easily by second picking, the nematodes are still on the culture medium and are picked again, the difficulty in secondary picking is large, the nematodes can climb to escape, the nematodes in motion are not easy to pick, the first picking success rate of picking the nematodes by using the short hair strands is high and exceeds 80%, the first picking success rate of picking the nematodes by using eyelashes is less than 30%, the highest speed of picking the nematodes by using the picking tool can be realized, about 10-12 nematodes can be picked per minute, and the operation stability is good, the tool has good performance and can be developed into an experimental consumable product. The speed of picking the nematodes by using the eyelashes is greatly influenced by the tool, the consumed time is long, the operation stability is poor, the nematodes can be picked up from the angle vertical to the body of the nematodes or from a certain included angle or other angles, the picking operation is not influenced by the body position of the nematodes, the nematodes can be picked up from any angle, the diameter of the used hair is about 0.1 mm in order to reduce the elasticity and the diameter size of the hair, the texture is relatively soft, the hardness of the hair with the diameter of 0.1 mm can support the nematodes, and the hair has certain toughness but is not enough to bounce the picked up nematodes due to the short length;

using human eyelashes to pick up nematode larvae: the eyelash structure is tapered and evenly thinned, the hardness of the eyelash structure is gradually reduced along with the diameter thinning, the hair quality is soft and is not easy to control, a hardness area suitable for picking up the larva is close to the tip, a hardness area suitable for picking up the adult insect is close to the root, the maximum potential energy contained in the eyelash structure is sequentially reduced from thick to thin, the insect cannot easily fly away even if the deformation amount is large, the larva can be picked up by the eyelash structure, the eyelash has the tip, the integral eyelash tip is soft, small objects are difficult to pick up on the surface of a semisolid and certain-viscosity nematode culture medium, the eyelash tip can be directly inserted into the culture medium or curled when contacting with the culture medium, the moving direction of the eyelash can be changed, the eyelash is difficult to control, the curling of the tip can be amplified and observed, the curled length is short and only limited to the tip position, but the movement of the eyelash can be difficult to control by, we processed the eyelash, cut the tip to make the operation movement intention reflect on the eyelash tip better, control the movement of the tip better, the operation belongs to the fine manipulation to be carried out under the body type microscope, cut the half length of the curly fragment can, should not cut too much, just made the eyelash instrument for picking up the nematode larva, after the hair selection is finished, began to pick up the preparation of the instrument: the length of the handle is about 12-15 cm, the handle is convenient for an adult to pinch, and the used handle consists of two parts: the method comprises the steps of arranging a rod with uniform thickness and a micropipette plastic gun head sleeved at the end of the rod, wherein the material of the rod is not limited, the plastic gun head is a 200 microliter yellow gun head (shown in figure 1), the design consistent with the function of the device also belongs to the protection range, the tip of the plastic gun head is heated to be melted and then connected with a hair, the connection is realized after cooling, the operation is carried out under a body microscope, the microscope is debugged, the hair is arranged, the plastic gun head is baked by naked flame to be melted into a mass, the mass is connected with a hair end point under the microscope before being melted into the mass and not solidified, the hair of 1-2 mm is embedded in a viscous hot melting plastic mass to ensure the firm connection, meanwhile, the angle between the hair and the rod is adjusted, the connection is completed after cooling, and the hair is cut off, and the hair of 2-. The plastic gun head is not suitable for connecting the hair immediately after melting, the hair is connected when the temperature is reduced to 60-70 ℃ and the melting body is not solidified so as to avoid damaging the hair by scalding, after solidification, if the plastic mass is larger, a blade can be used for trimming, the connection mode can be improved, for example, an adhesive method is used, an included angle is formed between the straight line of the hair and the axis of the handle, preferably between 120 and 130 ℃, when the included angle is formed, the included angle between the hair and the flat plate of the nematode culture medium is smaller when picking up the nematodes, the nematodes on the lying culture medium can be picked up by sticking to the culture surface so as to facilitate the power generation, if the included angle is not formed, the nematodes are difficult to pick up and inconvenient to generate the power, the shape of the hair can be straight line or slightly bent, the picking tool has simple manufacturing process, a plurality of hair can be manufactured at one time, and the picking tool can be used smoothly, the insect picking tool can be inversely inserted on a test tube rack or a special device, the protection of tip hair is well done, and fresh 200 mg/L chlorine dioxide solution is used for sterilizing for 2 minutes before and after use.

In order to better achieve the above object, the present invention further provides a method for using the tool for picking the model animal caenorhabditis elegans, the method comprises the following steps:

picking up nematode adults:

(1) preparing a culture dish with the NGM nematode culture medium, ensuring that the nematode food OP50 escherichia coli is fresh and sufficient, and keeping the environmental temperature at 21 ℃; preparing a fresh 200 mg/l chlorine dioxide solution;

(2) in order to avoid the pollution of the nematode culture medium by the mixed bacteria in the air, the operation is carried out in an ultra-clean room;

(3) before the tool is used and selected, the tool needs to be disinfected to prevent pollution, and the specific operation is to soak the hair ends in a fresh 200 mg/L chlorine dioxide solution for 2 minutes and sterilize;

(4) after the picking tool is soaked and sterilized, sterile paper is needed to be used for wiping, so that the nematode has developed taste function and is sensitive to odorous disinfectant;

(5) when picking nematodes, the nematode should be sterilized again every time a different nematode or picking tool is contaminated;

(6) when picking the nematodes, pinching the handle of the picking tool by hand, and keeping the handle inclined to pick the nematodes;

(7) the nematode is selected by using a body type microscope which has a continuous amplification function, and the maximum amplification factor is 400 times;

(8) placing the culture dish with the nematodes in the field of view of the body type microscope, and adjusting the brightness, the focal length and the magnification of the body type microscope;

(9) the target nematodes are found under a microscope, static insects are generally picked, and the picking difficulty of the crawling insects is high;

(10) the tool handle is picked up by pinching with hands, the inclination is kept, and the included angle between the straight line of the hair and the plane of the culture medium is small;

(11) the short hair tips are inserted into the lower part of the body of the nematodes at the middle section of the adult nematodes, the short hair tips are lifted and transferred to a new culture medium, the picking process is similar to that of picking up a rope on the ground by using a stick, the operation is simple, when the adult nematodes are picked by using soft and long filaments, the adult nematodes are easy to slip when being picked, the efficiency is reduced, because the picking tool has short hair and certain hardness and small elasticity, the slipping and bouncing of the nematodes cannot be caused, the success rate of picking for the first time is high, and the efficiency is high;

(12) the short hair is picked up vertically to the worm body and can form a certain inclination angle with the worm body;

(13) before picking, the nematodes can not be frightened to prevent the nematodes from escaping;

(14) the selection striving is successful once, and after the failure, the pest can escape due to the disturbance, and the selection difficulty is increased again;

(15) the nematodes should be transferred to a new culture dish immediately after picking, and are not suitable for staying in the air for a long time to prevent the nematodes from being dehydrated and dead in the air;

(16) the hair with the selected insects is scratched on a new culture medium for several times, so that the nematodes fall off from the hair to realize transfer;

(17) the transfer to fresh medium should also be done under microscope to ensure successful nematode transfer;

(18) repeating the operation until the insects with the quantity required by the task are picked;

(19) after finishing, picking the hair ends of the tool, soaking and sterilizing the hair ends in a fresh 200 mg/L chlorine dioxide solution for 2 minutes, and then putting the hair ends back to the original position;

picking of nematode larvae:

(20) the nematode larva individuals are relatively small, eyelashes need to be picked, and the picking difficulty is large;

(21) when the larva nematodes are picked up, the included angle of the eyelashes and the straight line of the main body of the nematode is 30-45 degrees to enlarge the contact area of the nematode and the eyelashes, increase the adhesion between the nematode and the eyelashes, reduce the contact area of the nematode and the culture medium and reduce the resistance, so the picking success rate can be improved, the picking mode vertical to the nematode does not have the advantage any more, because the eyelashes are long and thin and soft, the slippage phenomenon is easy to occur during picking up, and the efficiency is reduced;

(22) because the worm bodies are small, transfer to a new plate needs to be confirmed under a microscope to prevent transfer failure;

(23) when transferring a plurality of nematode larvae once, can use the above to choose the picker of the adult on the NGM flat board that has more larvae "sweep" several times, scrape the worm "down", borrow the comparatively viscous effort of escherichia coli on the culture medium in order to adhere a plurality of larvae simultaneously, transfer to in the new culture dish again.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (2)

1. The preparation method of the tool for picking the model animal caenorhabditis elegans is characterized by comprising the following steps:

s1, selecting a handle with the length of about 12-15 cm;

s2, the handle in the step S1 consists of two parts, and the handle consists of a rod with uniform thickness and a micropipette plastic gun head sleeved at the end of the rod, wherein the micropipette plastic gun head is a yellow gun head of 200 microliter;

s3, debugging a microscope, under the microscope, connecting hairlines or eyelashes after the tip end of a plastic gun head is subjected to thermal capacity, cooling and connecting, arranging the hairlines or eyelashes, baking the plastic gun head with open fire to melt the plastic gun head into a mass, connecting the mass with the end points of the hairlines or eyelashes under the microscope before the mass is melted and not solidified, embedding the hairlines or eyelashes of 1-2 mm into the viscous hot-melt plastic mass for firm connection, adjusting the angle between the hairlines or eyelashes and a rod, completing the connection after cooling, and cutting off the hairlines or eyelashes and leaving the hairlines or eyelashes of 2-3 mm;

s4, making the included angle between the straight line of the hair or eyelash and the axis of the handle be 120-130 degrees, thus completing the preparation of the tool for picking the model animal caenorhabditis elegans.

2. The use method of the tool for picking the model animal caenorhabditis elegans as claimed in claim 1 is characterized in that the use method comprises the following steps:

picking up nematode adults:

(1) operating an ultra-clean room, preparing a culture dish with an NGM nematode culture medium, preparing a nematode food OP50 Escherichia coli fresh and sufficient, keeping the environmental temperature at 21 ℃, and preparing a fresh 200 mg/L chlorine dioxide solution;

(2) before using a nematode picking tool, soaking the hair ends of the nematodes in a fresh 200 mg/L chlorine dioxide solution for 2 minutes, sterilizing, and wiping the hair ends with sterile paper after the picking tool is soaked and sterilized;

(3) when picking the nematodes, before replacing each nematode, the picking tool needs to be sterilized, when preparing to pick the nematodes, the handle of the picking tool is pinched by hand, a culture dish cultured with the nematodes is placed under the body type microscope, and the brightness, the focal length and the magnification of the body type microscope are adjusted; picking up nematodes in an inclined way under a microscope with the magnification of 400 times;

(4) the tool handle is picked up by pinching with hands, the inclination is kept, and the included angle between the straight line of the hair and the plane of the culture medium is small;

(5) inserting short hair tips into the middle section of the adult nematodes, picking up the short hair tips, and transferring the short hair tips to a new culture medium;

(6) the hair with the selected insects is scratched on a new culture medium for several times, so that the nematodes fall off from the hair to realize transfer;

(7) the transfer to fresh medium should also be done under microscope to ensure successful nematode transfer;

(8) repeating the operation until the insects with the quantity required by the task are picked;

(9) after finishing, picking the hair ends of the tool, soaking and sterilizing the hair ends in a fresh 200 mg/L chlorine dioxide solution for 2 minutes, and then putting the hair ends back to the original position;

picking of nematode larvae:

(1) picking eyelashes, wherein the included angle between the eyelashes and the straight line of the main body of the nematode is 30-45 degrees;

(2) confirming transfer to a new plate under a microscope;

(3) when a plurality of nematode larvae are transferred at one time, the nematode tool for picking up the adults is adopted to sweep for several times on the NGM flat plate with more larvae, the larvae are scraped off, a plurality of larvae are adhered by the viscous acting force of the escherichia coli on the culture medium, and then the larvae are transferred to a new culture dish.

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