A method for extracting nematodes directly from agar culture medium for morphological identification

Abstract

The application discloses a method for extracting nematodes directly from agar culture medium for morphological identification, and relates to the technical field of nematode separation and identification; the method comprises five core steps of agar block treatment, nematode release, filtration and purification, fixation and transparency, and slide preparation; through low-temperature softening, gentle centrifugation, and gradient filtration combination, the problems of low extraction rate of nematodes, easy damage of worm bodies, and interference of agar residues in observation in the prior art are solved, efficient and complete recovery of nematodes from agar culture is realized, and the requirements of morphological identification on worm body integrity and definition are met; the method is simple in operation, low in cost, and high in universality, and is suitable for morphological identification of various agar culture nematodes such as Caenorhabditis elegans and plant parasitic nematodes.

Description

Technical Field

[0001] This invention relates to the field of nematode isolation and identification technology, and more particularly to a method for directly extracting nematodes from agar culture medium for morphological identification. Background Technology

[0002] Nematodes are a group of tiny invertebrates widely used in research fields such as developmental biology, genetics, and ecology. Among these, agar plate culture is the most commonly used culture method for nematodes (e.g., NGM plate culture of Caenorhabditis elegans). Morphological identification is the gold standard for nematode classification and species identification, requiring the worm to be intact, with a clear body surface and no damage to internal structures.

[0003] Currently, traditional methods for extracting nematodes from agar mainly include: 1. Manual picking with a needle, which is extremely inefficient, only suitable for small quantities of nematodes, and easily damages the nematodes; 2. Direct washing of agar blocks with water, resulting in incomplete nematode release and significant agar residue, interfering with microscopic observation; 3. Melting agar at high temperatures, which causes nematode shrinkage and tissue damage, rendering them unusable for morphological identification. Existing technologies suffer from low extraction rates, poor nematode integrity, and severe interference from impurities, making it difficult to meet the needs of large-scale, high-quality morphological identification of nematodes cultured in agar.

[0004] Therefore, developing an efficient, mild extraction method that can completely recover nematodes without agar residue is of great significance for the morphological identification of nematodes cultured in agar. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for directly extracting nematodes from agar culture medium for morphological identification, relating to the field of nematode isolation and identification technology. This method includes five core steps: agar block processing, nematode release, filtration and purification, fixation and clearing, and slide preparation. Through a combination of low-temperature softening, gentle centrifugation, and gradient filtration, it solves the problems of low nematode extraction rate, easy damage to nematode bodies, and interference from agar residue in existing technologies. This method achieves efficient and complete recovery of nematodes from agar cultures, meeting the requirements for morphological identification regarding the integrity and clarity of the nematode bodies. The method is simple to operate, low in cost, and highly versatile, applicable to the morphological identification of various agar-cultured nematodes such as *C. elegans* and *Nematoda*.

[0006] To achieve the above technical effects, the following technical solution is adopted: A method for directly extracting nematodes from agar culture medium for morphological identification includes the following steps: Step S1: Agar block treatment Take an agar plate containing nematodes, cut the agar into small pieces under aseptic conditions, transfer them into centrifuge tubes, add sterile buffer, and let stand to soften the agar. Step S2: Nematode Release Place the centrifuge tube from step S1 in a vortex mixer and vortex at low speed to free the nematodes from the agar. Then centrifuge at low speed, discard the upper agar debris, and retain the lower nematode suspension containing the nematodes. Step S3: Filtration and purification The nematode suspension in step S2 is diluted with sterile water and passed through the first and second double-layer sterile screens in sequence. The nematodes on the second screen are collected. The inner wall of the second sterile screen is rinsed with sterile water and the rinsing liquid is collected to obtain the purified nematode suspension. Step S4: Fix transparency Add an equal amount of fixative to the purified nematode suspension obtained in step S3, fix at room temperature, and then transfer to a drying oven for transparent treatment until the nematode is completely transparent to obtain transparent nematodes. Step S5: Slide Preparation and Morphological Identification Take the transparent nematode obtained in step S4 and place it in a drop of glycerol on a glass slide. Place 3 glass beads on the edge, cover with a coverslip, seal with resin, and after drying, observe under a differential interference microscope for morphological identification.

[0007] Furthermore, in step S1, agar plates containing well-grown nematodes cultured at 20-25℃ for 3-7 days are used, and the agar plates are NGM agar medium.

[0008] Furthermore, in step S1, the agar is cut into 0.5-1cm pieces. 3 After transferring the uniform small pieces into centrifuge tubes, add 2-5 times the volume of pre-cooled sterile M9 buffer, mix well, and let stand at 4°C for 10-15 minutes to soften the agar.

[0009] Furthermore, the low-speed vortex conditions in step S2 are: rotation speed 500-800 rpm, low-speed vortexing for 3-5 minutes, after vortexing, coarse filtration can be performed using 60-mesh sterile gauze to remove unbroken agar blocks, and then centrifugation can be performed.

[0010] Furthermore, the low-speed centrifugation conditions in step S2 are: 300-500g low-speed centrifugation for 1-2 minutes.

[0011] Furthermore, in step S3, the first layer of sterile screen has a mesh size of 500 μm; the second layer of sterile screen has a mesh size of 31 μm; and in step S3, the inner wall of the second layer of sterile screen is rinsed with sterile water three times.

[0012] Furthermore, in step S4, the fixative is either a glycerol-alcohol-water fixative or a paraformaldehyde fixative, wherein the volume ratio of glycerol, alcohol and water in the glycerol-alcohol-water fixative is 5:5:90; and the mass concentration of paraformaldehyde in the paraformaldehyde fixative is 4%.

[0013] Furthermore, in step S4, when the fixative is glycerol-alcohol-water fixative, fix at room temperature in the dark for 24 hours, and then transfer to a 37°C constant temperature drying oven for transparent treatment for 7-14 days.

[0014] Furthermore, in step S4, when the fixative is paraformaldehyde fixative, the fixation time is shortened to 2 hours, while the transparency time remains unchanged.

[0015] Furthermore, in step S5, the glycerol droplet concentration is 50%, and each droplet contains 4-5 adult worms.

[0016] The beneficial effects of this invention are as follows: 1. High extraction efficiency: The combination of low-temperature softening, low-speed vortexing, and gradient filtration ensures complete release of nematodes, with an extraction rate of over 90%, far exceeding that of the traditional water washing method (50%-60%). 2. Good worm integrity: Low temperature and low speed operation throughout the process to avoid high temperature damage and mechanical breakage, worm integrity rate ≥95%, no damage to the body surface and clear internal structure; 3. No interference from impurities: The double-layer sieve gradient filtration thoroughly removes impurities such as agar residue and bacterial clumps, and the background of the insects is clean after fixation and transparency, so there is no interference during microscopic observation; 4. High versatility: It is suitable for various agar culture nematodes such as Caenorhabditis elegans and plant parasitic nematodes (such as root-knot nematodes and cyst nematodes), and can meet the morphological identification needs of different types of nematodes; 5. Simple operation and low cost: No special instruments are required; conventional laboratory equipment can be used. Reagents are inexpensive and readily available, making it suitable for large-scale sample processing. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. The drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 These are photographs of *Caenorhabditis elegans* identified by the method provided in Embodiment 1 of this invention. Figure 2 These are photographs of *C. elegans* identified by the method provided in Comparative Example 1 of this invention. Figure 3 These are photographs of *C. elegans* identified by the method provided in Comparative Example 2 of this invention. Figure 4 The image shows a photograph of *Caenorhabditis elegans* identified by the method provided in Comparative Example 3 of this invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0022] The present invention provides a method for directly extracting nematodes from agar culture medium for morphological identification: 1. Agar block treatment Take NGM medium (Near-Gross Mushroom) agar plates containing well-grown nematodes that have been cultured at 20-25℃ for 3-7 days, and cut the agar into 0.5-1cm pieces using a scalpel in a sterile laminar flow hood. 3 Cut the agar blocks into small, uniform pieces to avoid compressing the nematodes. Transfer the agar blocks to a 15mL sterile centrifuge tube, add 3 times the volume of pre-cooled sterile M9 buffer, gently invert to mix, and let stand at 4°C for 10-15 minutes to soften the agar structure at low temperature, reduce nematode encapsulation, and avoid heat damage to the nematodes.

[0023] 2. Nematode release Place the softened centrifuge tubes in a vortex mixer and set the speed to 500-800 rpm. Vortex at low speed for 3-5 minutes to gently release the nematodes from the agar gaps into the buffer solution, avoiding high-speed vortexing that could cause the nematodes to break apart. If large pieces of agar remain after vortexing, filter through 60-mesh sterile gauze to remove impurities. Transfer the filtrate to a new centrifuge tube. Then centrifuge at 300-500g at low speed for 1-2 minutes. At this point, agar debris will float to the top, and the nematodes will settle to the bottom. Carefully discard the turbid upper layer, retaining 1-2 mL of the lower nematode suspension.

[0024] 3. Filtration and purification The nematode suspension was diluted to 5 mL with sterile water and then subjected to a double-layer sterile sieve gradient filtration: first, it was passed through a 500 μm sieve to remove large particles such as unbroken agar blocks and bacterial clumps; then, it was passed through a 31 μm sieve to retain the nematodes (nematode body length is usually > 50 μm), while smaller impurities passed through the sieve with the filtrate. The inner wall of the 31 μm sieve was gently rinsed three times with sterile water, and the nematodes on the sieve were washed into a sterile petri dish to obtain a purified nematode suspension free of agar residue.

[0025] 4. Fixed transparency Add an equal volume of glycerol-alcohol-water fixative (glycerol: anhydrous ethanol: sterile water = 5:5:90, by volume) to the nematode suspension in the culture dish, mix gently, and fix at room temperature in the dark for 24 hours to coagulate the nematode proteins and maintain morphological stability. After fixation, transfer to a 37℃ constant temperature drying oven for clearing treatment for 7-14 days, until the nematode body is completely transparent and the internal organs are clearly visible, facilitating observation of morphology and structure under a microscope. Alternatively, add an equal volume of 4% paraformaldehyde fixative to the nematode suspension in the culture dish, mix gently, and fix at room temperature in the dark for 2 hours to coagulate the nematode proteins and maintain morphological stability. After fixation, transfer to a 37℃ constant temperature drying oven for 7-14 days for transparent treatment until the nematode body is completely transparent and the internal organs are clearly visible, facilitating observation of morphology and structure under a microscope. 5. Slide preparation and morphological identification Take a clean glass slide and add one drop of 50% sterile glycerol solution to the center. Under a stereomicroscope, use a nematode-picking needle to select 4-5 intact, uniformly sized, transparent nematodes (a combination of male and female adults), and transfer them to the glycerol drop, arranging them neatly. Place three sterile glass beads (1-2 mm in diameter) symmetrically around the edge of the glycerol drop (to prevent the coverslip from compressing the nematodes). Slowly cover the slide with the coverslip, avoiding air bubbles. Seal the slide with neutral resin along the edge of the coverslip. After drying at room temperature for 24 hours, place it under a differential interference phase-contrast microscope and observe the morphological characteristics of the nematodes' body surface, stylet, esophagus, and reproductive organs at 400-1000x magnification. Identify the species by referring to a nematode classification atlas.

[0026] Example 1: Extraction and Identification of Caenorhabditis elegans 1. Agar block treatment: Take an NGM agar plate (containing OP50 E. coli bacterial growth) that has been incubated at 20℃ for 5 days, and aseptically cut it into 0.8cm pieces. 3 Transfer the small pieces to a 15mL centrifuge tube, add 3mL of pre-cooled M9 buffer, and incubate at 4℃ for 12min. 2. Nematode release: Vortex at 600 rpm for 4 min, centrifuge at 300 g for 1.5 min, discard the supernatant, and keep 1.5 mL of nematode suspension; 3. Filtration and purification: Pass the nematode suspension through a 500μm sieve and a 31μm sieve in sequence, rinse the 31μm sieve three times with sterile water, and collect the nematode suspension; 4. Fixation and clearing: Add equal volumes of glycerol-alcohol-water fixative (volume ratio of glycerol: anhydrous ethanol: sterile water = 5:5:90), fix at room temperature for 24 hours, and clear at 37℃ for 10 days; 5. Slide identification: Prepare a slide using 50% glycerol solution, support it with glass beads, mount it with resin, and observe it under a differential interference microscope. Figure 1 As shown, the nematode has a smooth body surface, a rod-shaped esophagus, and clearly defined male and female reproductive organs; this is *Caenorhabditis elegans*.

[0027] Comparative Example 1: Same as Example 1, the only difference being the absence of the low-temperature softening step, i.e., the lack of standing at 4°C for 12 minutes, with the same effect. Figure 2 As shown.

[0028] Comparative Example 2: Same as Example 1, the only difference being the absence of gentle centrifugation (500-800 rpm, low-speed vortexing for 3-5 minutes followed by low-speed centrifugation at 300-500g for 1-2 minutes), replaced by high-speed centrifugation for 1-2 minutes, with the same effect. Figure 3 As shown.

[0029] Comparative Example 3: Same as Example 1, the only difference being the absence of gradient filtration, replaced by direct 31μm sieve filtration, with the same effect. Figure 4 As shown.

[0030] Comparative Example 4: Traditional manual picking methods have low extraction rates (approximately 30%-50%), are prone to insect damage (damage rate >20%), leave significant agar residue, and are time-consuming, failing to meet the needs of large-scale morphological identification. Compared to the method of this invention, the extraction efficiency and insect integrity are significantly lower.

[0031] The identification results of Example 1 and Comparative Examples 1-4 are shown in Table 1: Table 1 Comparison of identification effects between Example 1 and Comparative Examples 1-4

[0032] In summary, this invention discloses a method for directly extracting nematodes from agar culture medium for morphological identification, relating to the field of nematode isolation and identification technology. The method comprises five core steps: agar block processing, nematode release, filtration and purification, fixation and clearing, and slide preparation. Through a combination of low-temperature softening, gentle centrifugation, and gradient filtration, it solves the problems of low nematode extraction rate, easy damage to nematode bodies, and interference from agar residue in existing technologies. This method achieves efficient and complete recovery of nematodes from agar cultures, meeting the requirements for morphological identification regarding nematode integrity and clarity. The method is simple to operate, low in cost, and highly versatile, applicable to the morphological identification of various agar-cultured nematodes, such as *C. elegans* and *Nematoda*.

[0033] Therefore, those skilled in the art will recognize that although embodiments of the present invention have been shown and described in detail herein, many other variations or modifications conforming to the principles of the present invention can be directly determined or derived from the disclosure of the present invention without departing from the spirit and scope of the invention. Therefore, the scope of the present invention should be understood and recognized as covering all such other variations or modifications.

Claims

1. A method for directly extracting nematodes from agar culture medium for morphological identification, characterized in that, The method includes the following steps: Step S1: Agar block treatment Take an agar plate containing nematodes, cut the agar into small pieces under aseptic conditions, transfer them into centrifuge tubes, add sterile buffer, and let stand to soften the agar. Step S2: Nematode Release Place the centrifuge tube from step S1 in a vortex mixer and vortex at low speed to free the nematodes from the agar. Then centrifuge at low speed, discard the upper agar debris, and retain the lower nematode suspension containing the nematodes. Step S3: Filtration and purification The nematode suspension in step S2 is diluted with sterile water and passed through the first and second double-layer sterile screens in sequence. The nematodes on the second screen are collected. The inner wall of the second sterile screen is rinsed with sterile water and the rinsing liquid is collected to obtain the purified nematode suspension. Step S4: Fix transparency Add an equal amount of fixative to the purified nematode suspension obtained in step S3, fix at room temperature, and then transfer to a drying oven for transparent treatment until the nematode is completely transparent to obtain transparent nematodes. Step S5: Slide Preparation and Morphological Identification Take the transparent nematode obtained in step S4 and place it in a drop of glycerol on a glass slide. Place 3 glass beads on the edge, cover with a coverslip, seal with resin, and after drying, observe under a differential interference microscope for morphological identification.

2. The method for directly extracting nematodes from agar culture medium for morphological identification as described in claim 1, characterized in that, In step S1, take an agar plate containing well-grown nematodes cultured at 20-25℃ for 3-7 days. The agar plate is NGM agar medium.

3. The method for directly extracting nematodes from agar culture medium for morphological identification as described in claim 1, characterized in that, In step S1, the agar is cut into 0.5-1cm pieces. 3 After transferring the uniform small pieces into centrifuge tubes, add 2-5 times the volume of pre-cooled sterile M9 buffer, mix well, and let stand at 4°C for 10-15 minutes to soften the agar.

4. The method for directly extracting nematodes from agar culture medium for morphological identification as described in claim 1, characterized in that, In step S2, the low-speed vortex conditions are: rotation speed 500-800 rpm, low-speed vortex for 3-5 minutes. After vortexing, coarse filtration can be performed using 60-mesh sterile gauze to remove unbroken agar blocks before centrifugation.

5. The method for directly extracting nematodes from agar culture medium for morphological identification as described in claim 1, characterized in that, The low-speed centrifugation conditions in step S2 are: 300-500g, low-speed centrifugation for 1-2 minutes.

6. The method for directly extracting nematodes from agar culture medium for morphological identification as described in claim 1, characterized in that, In step S3, the first layer of sterile screen has a mesh size of 500 μm; the second layer of sterile screen has a mesh size of 31 μm; and the inner wall of the second layer of sterile screen is rinsed with sterile water 3 times in step S3.

7. The method for directly extracting nematodes from agar culture medium for morphological identification as described in claim 1, characterized in that, In step S4, the fixative is either a glycerol-alcohol-water fixative or a paraformaldehyde fixative, wherein the volume ratio of glycerol, alcohol and water in the glycerol-alcohol-water fixative is 5:5:90; and the mass concentration of paraformaldehyde in the paraformaldehyde fixative is 4%.

8. The method for directly extracting nematodes from agar culture medium for morphological identification as described in claim 1, characterized in that, In step S4, when the fixative is glycerol-alcohol-water fixative, fix at room temperature in the dark for 24 hours, and then transfer to a 37°C constant temperature drying oven for transparent treatment for 7-14 days.

9. A method for directly extracting nematodes from agar culture medium for morphological identification as described in claim 1, characterized in that, In step S4, when the fixative is paraformaldehyde fixative, the fixation time is shortened to 2 hours, while the transparency time remains unchanged.

10. A method for directly extracting nematodes from agar culture medium for morphological identification as described in claim 1, characterized in that, In step S5, the glycerol drop concentration is 50%, and 4-5 adult insects are placed in each drop.

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