A forest soil nematode separation method based on combination of bellman funnel method and centrifugal flotation method

Abstract

The application relates to a forest soil nematode separation method based on combination of Bellman funnel method and centrifugal flotation method, and relates to the field of nematode extraction.The application aims to solve the problem that traditional single separation method is difficult to consider efficiency and effect.The application provides a nematode separation method combining Bellman funnel method and centrifugal flotation method.Compared with the traditional single nematode separation method, the combined method not only improves the separation efficiency, reduces the loss and error in the operation process, but also better protects the integrity of the nematodes, ensures the purity of the separated samples, and thus provides a more accurate and reliable basis for subsequent nematode identification and counting.A forest soil nematode separation method based on combination of Bellman funnel method and centrifugal flotation method is obtained.

Description

Technical Field

[0001] This invention relates to the field of nematode extraction technology, specifically to a method for separating nematodes from forest soil based on a combination of the Bellman funnel method and centrifugal flotation. Background Technology

[0002] Against the backdrop of global change, the structure and function, biodiversity, and ecological balance of forest ecosystems are being disturbed by multiple factors, including climate warming, precipitation changes, biological invasions, and human activities. These disturbances, through direct and indirect feedback mechanisms, produce significant differences across different regions, threatening the stability of forest ecosystems and potentially weakening their crucial role in global carbon cycling and climate regulation. Soil nematodes are the most abundant metazoans in soil ecosystems, widely distributed across all major trophic levels of the soil food web, serving as a vital bridge connecting decomposers and higher trophic level organisms. Changes in their trophic group structure not only affect the stability of soil ecosystem processes but also sensitively reflect environmental disturbances, thus being considered ideal environmental indicator organisms (LIU TM, 2020; Hou Linxiu, 2022). Therefore, the accurate isolation and identification of soil nematodes is of great significance for revealing the dynamic changes in forest ecosystem processes under the background of climate change, assessing the impact of environmental disturbances on soil ecological functions, and conducting ecological research. Simultaneously, this process provides a scientific basis for biodiversity conservation, soil health monitoring, and the maintenance of ecosystem functions.

[0003] Isolation of soil nematodes is a crucial step in community analysis. Improving nematode isolation efficiency can more accurately reflect the quantity, structure, and diversity of soil nematode communities, which is of great significance for forest ecosystem research. Domestic and international scholars have conducted extensive research on nematode isolation techniques. Currently, commonly used isolation methods include wet sorting, shallow dish method, sucrose centrifugation, Carbomer sieve method, and semi-automatic washing method. When selecting a suitable isolation method, the specific characteristics of the soil and the research objectives should be comprehensively considered, and the isolation steps and parameters should be adjusted reasonably to ensure the accuracy and reliability of the results.

[0004] Forest soils are loose in structure and rich in organic matter, plant residues, and roots. These components easily mix with nematodes during the separation process, increasing the difficulty of separation. At the same time, some forest soils contain a high proportion of clay, and the strong adhesiveness of clay particles makes it easy for nematodes to be encapsulated, making complete separation difficult. Therefore, a single separation method is often insufficient to address these complex characteristics, resulting in incomplete separation. Summary of the Invention

[0005] The purpose of this invention is to solve the problem that traditional single separation methods are difficult to balance efficiency and effectiveness, and to provide a method for separating forest soil nematodes based on a combination of the Bellman funnel method and centrifugal flotation.

[0006] A method for isolating nematodes from forest soil based on a combination of the Bellman funnel method and centrifugal flotation is carried out according to the following steps:

[0007] Step S1:

[0008] Connect a rubber tube to the end of the funnel and clamp the end of the rubber tube tightly; place a layer of wire mesh inside the funnel, then place two layers of gauze on the wire mesh, and then place a layer of nematode filter paper on the gauze.

[0009] Step S2:

[0010] The fresh soil sample containing nematodes was evenly spread on the filter paper in step S1, and water was added to submerge the soil sample. After separation at a temperature of 20-22℃ for 24-48 hours, the clamp at the end of the rubber tube was opened to drain the water into a container for collection.

[0011] Step S3:

[0012] Add the remaining soil sample from the funnel and filter paper to the centrifuge tube, and also pour the washing liquid from the funnel, gauze and filter paper into the centrifuge tube. Stir well first, and then place it in the centrifuge for the first centrifugation.

[0013] Step S4:

[0014] After the first centrifugation, discard the supernatant and add sucrose solution; stir well and then place in a centrifuge for a second centrifugation; after centrifugation, collect the supernatant and mix it with the water collected in step S2, pass it through a sieve, and collect the separated nematodes.

[0015] The beneficial effects of this invention are:

[0016] (1) In the past, when using the Bellman funnel method alone to separate nematodes, it was necessary to frequently adjust the flow rate and medium concentration to adapt to different soil characteristics. The operation process was complex and time-consuming. At the same time, this method was inefficient in removing impurities and the separation effect was poor, resulting in low nematode purity. Centrifugal flotation has shown outstanding performance in improving nematode extraction efficiency, but due to the high requirements for equipment precision and operating conditions, its excessive centrifugal force may cause mechanical damage to nematodes, thereby affecting the accuracy of the separation results and the integrity of the nematodes. Therefore, traditional single separation methods are difficult to simultaneously meet the requirements of separation efficiency (number of nematodes extracted) and separation effect (nematode integrity). To this end, this invention provides a nematode separation method that combines the Bellman funnel method and centrifugal flotation. Compared with traditional single nematode separation methods, this combined method not only improves the separation efficiency and reduces losses and errors in the operation process, but also better protects the integrity of the nematodes and ensures the purity of the separated samples, thus providing a more accurate and reliable basis for subsequent nematode identification and counting.

[0017] (2) The method provided by the present invention has high separation efficiency, can meet the needs of soil sample analysis for various research purposes, and has good application prospects.

[0018] (3) The nematode separation method combining the Bellman funnel method and centrifugal flotation method provided by the present invention is simple to operate and solves the problem that a single separation method is difficult to balance efficiency and effectiveness.

[0019] This invention provides a method for separating nematodes from forest soil based on a combination of the Bellman funnel method and centrifugal flotation. Detailed Implementation

[0020] Specific Implementation Method 1: This implementation method is a forest soil nematode isolation method based on a combination of the Bellman funnel method and centrifugal flotation method, which is carried out according to the following steps:

[0021] Step S1:

[0022] Connect a rubber tube to the end of the funnel and clamp the end of the rubber tube tightly; place a layer of wire mesh inside the funnel, then place two layers of gauze on the wire mesh, and then place a layer of nematode filter paper on the gauze.

[0023] Step S2:

[0024] The fresh soil sample containing nematodes was evenly spread on the filter paper in step S1, and water was added to submerge the soil sample. After separation at a temperature of 20-22℃ for 24-48 hours, the clamp at the end of the rubber tube was opened to drain the water into a container for collection.

[0025] Step S3:

[0026] Add the remaining soil sample from the funnel and filter paper to the centrifuge tube, and also pour the washing liquid from the funnel, gauze and filter paper into the centrifuge tube. Stir well first, and then place it in the centrifuge for the first centrifugation.

[0027] Step S4:

[0028] After the first centrifugation, discard the supernatant and add sucrose solution; stir well and then place in a centrifuge for a second centrifugation; after centrifugation, collect the supernatant and mix it with the water collected in step S2, pass it through a sieve, and collect the separated nematodes.

[0029] The purpose of centrifugation in step S4 is to remove unwanted impurities, suspended solids, or liquid components dissolved in the supernatant, optimizing separation efficiency and purity. However, due to centrifugal force, some nematodes may remain in the supernatant. The proportion of these nematodes is relatively low, and from the perspective of experimental efficiency and target effect, the loss is acceptable. If there are special requirements, the supernatant can be added back to the centrifuge separately for re-centrifugation (at a lower speed), and only the nematodes can be recovered before being combined with subsequent steps to reduce nematode loss.

[0030] Specific Implementation Method Two: The difference between this implementation method and Specific Implementation Method One is that the funnel mentioned in step S1 is a plastic funnel with a diameter of 15-25cm.

[0031] The other steps are the same as in Specific Implementation Method 1.

[0032] Specific Implementation Method 3: The difference between this implementation method and Specific Implementation Method 1 or 2 is that the lower diameter of the plastic funnel is 3-4 cm and the height is 25-30 cm.

[0033] The other steps are the same as in Specific Implementation Method 1 or 2.

[0034] Specific Implementation Method Four: The difference between this implementation method and Specific Implementation Methods One to Three is that the fresh soil sample mentioned in step S2 needs to be manually broken into small pieces before use in order to maintain the natural structure of the soil sample and the integrity of the nematodes.

[0035] The other steps are the same as those in Specific Implementation Methods One to Three.

[0036] Specific Implementation Method 5: The difference between this implementation method and Specific Implementation Methods 1 to 4 is that: in step S3, a glass rod with an adsorption head is used for stirring to remove roots and organic matter from the soil.

[0037] The other steps are the same as those in Specific Implementation Methods One through Four.

[0038] Specific Implementation Method Six: The difference between this implementation method and Specific Implementation Methods One to Five is that the rotation speed of the first centrifugation in step S3 is 2000-3000 r / min, and the centrifugation time is 4-6 min.

[0039] The other steps are the same as those in Specific Implementation Methods 1 to 5.

[0040] Specific Implementation Method Seven: The difference between this implementation method and Specific Implementation Methods One to Six is ​​that the concentration of the sucrose solution mentioned in step S4 is 790-810 g / L.

[0041] The other steps are the same as those in Specific Implementation Methods 1 to 6.

[0042] Specific Implementation Method Eight: The difference between this implementation method and Specific Implementation Methods One to Seven is that the rotation speed of the second centrifugation in step S4 is 1500-2000 r / min, and the centrifugation time is 4-6 min.

[0043] The other steps are the same as those in Specific Implementation Methods 1 to 7.

[0044] Specific Implementation Method Nine: The difference between this implementation method and Specific Implementation Methods One to Eight is that after the second centrifugation in step S4, the collected supernatant is immediately mixed with the water collected in step S2 to prevent the nematodes from losing water in the high-density sucrose solution and to ensure the activity and integrity of the nematodes.

[0045] The other steps are the same as those in Specific Implementation Methods 1 to 8.

[0046] Specific Implementation Method 10: The difference between this implementation method and Specific Implementation Methods 1 to 9 is that the sieve set mentioned in step S4 is composed of 300 mesh, 400 mesh and 500 mesh sieves.

[0047] The other steps are the same as those in Specific Implementation Methods 1 to 9.

[0048] The beneficial effects of the present invention are verified using the following embodiments:

[0049] Example 1: A method for isolating nematodes from forest soil based on a combination of the Bellman funnel method and centrifugal flotation, comprising the following steps:

[0050] Step S1:

[0051] Connect a rubber tube to the end of a 20cm diameter plastic funnel and clamp the end of the rubber tube with a spring clip; place a layer of wire mesh inside the funnel, then place two layers of gauze on the wire mesh, and finally place a layer of nematode filter paper on the gauze.

[0052] The plastic funnel has a lower opening diameter of 3.5cm and a height of 27cm;

[0053] Step S2:

[0054] Weigh 100g of fresh soil sample, break it into small pieces by hand, spread it evenly on the filter paper in step S1, and add water until the soil sample is submerged; then, after separating at 20℃ for 48h, open the spring clamp at the end of the rubber tube and drain the water into a beaker for collection.

[0055] Step S3:

[0056] Add the remaining soil sample from the funnel and filter paper to a 250mL centrifuge tube, and pour the washing liquid from the funnel, gauze and filter paper into the centrifuge tube. Add water until the water-soil mixture weighs 200g. First, stir it evenly with a glass rod with an absorbent tip, and then place it in a centrifuge and centrifuge at 2500r / min for 5min.

[0057] Step S4:

[0058] After the first centrifugation, the supernatant was discarded, and sucrose solution (concentration of 800 g / L) was added until the soil suspension weighed 200 g. The soil suspension was first stirred evenly with a glass rod, and then placed in a centrifuge and centrifuged at 1500 r / min for 5 min. After centrifugation, the collected supernatant was immediately mixed with the water collected in step S2 using the Bellman funnel method to prevent the nematodes from losing water and deforming in the high-density sucrose solution. The separated nematodes were then collected by passing them through a nested sieve with 300 mesh, 400 mesh and 500 mesh screens.

[0059] As shown in Table 1, when the rotation speed was reduced to 1500 r / min for the second time, the number of nematodes increased to 339.57 / 100g. This indicates that the lower rotation speed protected the integrity of the nematodes, but the separation efficiency was slightly lower, and it may not be able to completely remove impurities around the nematodes. As a result, although the number of nematodes increased, the effect was not as good as in Example 3 with a higher rotation speed.

[0060] Example 2: A method for isolating nematodes from forest soil based on a combination of the Bellman funnel method and centrifugal flotation, comprising the following steps:

[0061] Step S1:

[0062] Connect a rubber tube to the end of a 20cm diameter plastic funnel and clamp the end of the rubber tube with a spring clip; place a layer of wire mesh inside the funnel, then place two layers of gauze on the wire mesh, and finally place a layer of nematode filter paper on the gauze.

[0063] The plastic funnel has a lower opening diameter of 3.5cm and a height of 27cm;

[0064] Step S2:

[0065] Weigh 100g of fresh soil sample, break it into small pieces by hand, spread it evenly on the filter paper in step S1, and add water until the soil sample is submerged; then, after separating at 20℃ for 48h, open the spring clamp at the end of the rubber tube and drain the water into a beaker for collection.

[0066] Step S3:

[0067] Add the remaining soil sample from the funnel and filter paper to a 250mL centrifuge tube, and pour the washing liquid from the funnel, gauze and filter paper into the centrifuge tube as well. Add water until the water-soil mixture weighs 200g. First, stir it evenly with a glass rod with an absorbent tip, and then place it in a centrifuge and centrifuge at 2000r / min for 5min.

[0068] Step S4:

[0069] After the first centrifugation, the supernatant was discarded, and sucrose solution (concentration of 800 g / L) was added until the soil suspension weighed 200 g. The soil suspension was first stirred evenly with a glass rod, and then placed in a centrifuge and centrifuged at 2000 r / min for 5 min. After centrifugation, the collected supernatant was immediately mixed with the water collected in step S2 using the Bellman funnel method to prevent the nematodes from losing water and deforming in the high-density sucrose solution. The separated nematodes were then collected by passing them through a nested sieve with 300 mesh, 400 mesh and 500 mesh screens.

[0070] As shown in Table 1, the second rotation speed was 1900 r / min, and the number of nematodes was 359.36 / 100g. The experimental results show that this rotation speed achieves near-efficient separation while controlling mechanical damage well, and the effect is better than the conditions of 1500 r / min and 2000 r / min.

[0071] Example 3: A method for isolating nematodes from forest soil based on a combination of the Bellman funnel method and centrifugal flotation, comprising the following steps:

[0072] Step S1:

[0073] Connect a rubber tube to the end of a 20cm diameter plastic funnel and clamp the end of the rubber tube with a spring clip; place a layer of wire mesh inside the funnel, then place two layers of gauze on the wire mesh, and finally place a layer of nematode filter paper on the gauze.

[0074] The plastic funnel has a lower opening diameter of 3.5cm and a height of 27cm;

[0075] Step S2:

[0076] Weigh 100g of fresh soil sample, break it into small pieces by hand, spread it evenly on the filter paper in step S1, and add water until the soil sample is submerged; then, after separating at 20℃ for 48h, open the spring clamp at the end of the rubber tube and drain the water into a beaker for collection.

[0077] Step S3:

[0078] Add the remaining soil sample from the funnel and filter paper to a 250mL centrifuge tube, and pour the washing liquid from the funnel, gauze and filter paper into the centrifuge tube as well. Add water until the water-soil mixture weighs 200g. First, stir it evenly with a glass rod with an absorbent tip, and then place it in a centrifuge and centrifuge at 2000r / min for 5min.

[0079] Step S4:

[0080] After the first centrifugation, the supernatant was discarded, and sucrose solution (concentration of 800 g / L) was added until the soil suspension weighed 200 g. The soil suspension was first stirred evenly with a glass rod, and then placed in a centrifuge and centrifuged at 1800 r / min for 5 min. After centrifugation, the collected supernatant was immediately mixed with the water collected in step S2 using the Bellman funnel method to prevent the nematodes from losing water and deforming in the high-density sucrose solution. The separated nematodes were then collected by passing them through a nested sieve with 300 mesh, 400 mesh and 500 mesh screens.

[0081] As shown in Table 1, the second rotation speed was 1800 r / min, and the number of nematodes was the highest, reaching 367.51 nematodes / 100g. This data verifies that 1800 r / min is the optimal balance between efficiency and integrity.

[0082] Comparative Example 1: Bellman funnel method;

[0083] The separation time was long (48 hours) and the efficiency was low, with a nematode count of 240.42 per 100g.

[0084] The above experimental results are consistent with the characteristics of the traditional Bellman funnel method, which mainly relies on gravity, has low separation efficiency, and may have a large amount of residual impurities.

[0085] Comparative Example 2: Centrifugal flotation;

[0086] The centrifugation speeds were set at 2000 r / min and 2000 r / min respectively. The results showed that the number of nematodes increased to 302.73 per 100g, which was significantly higher than that of the Bellman funnel method. This demonstrates the advantage of centrifugal flotation in separation efficiency. However, the excessively high second speed may have caused some mechanical damage to the nematodes, resulting in separation results that were not as good as those in Examples 2 and 3.

[0087] Table 1 shows the number of soil nematodes isolated under different treatment conditions;

[0088] Table 1

[0089]

[0090] The primary purpose of the first centrifugation in this invention is to preliminarily separate large particulate impurities (such as soil particles and organic debris), laying the foundation for subsequent fine separation. Therefore, the rotation speed is uniformly set to 2000 r / min, which ensures effective separation of impurities while avoiding impurity residue due to excessively low rotation speed or damage to nematodes due to excessively high rotation speed.

[0091] The main purpose of the second centrifugation is to finely separate the target (nematodes) from the remaining impurities. Adjusting the rotation speed directly affects the separation efficiency and integrity of the nematodes, specifically as follows:

[0092] Low speed (1500-1600 r / min): It is beneficial to protect the integrity of nematodes, but may reduce separation efficiency; medium speed (1700-1800 r / min): It maximizes separation efficiency while protecting the integrity of nematodes and is the optimal speed range; high speed (>2000 r / min): It may cause mechanical damage to nematodes and is not suitable as the speed for the second centrifugation.

[0093] As shown in Table 1, in all three embodiments, the first centrifugation speed was 2000 r / min. This speed provided sufficient centrifugal force to effectively remove impurities while avoiding indirect damage to the nematodes caused by squeezing or colliding with impurities due to excessive speed. Since the first centrifugation speed was consistent, the difference in the number of nematodes was mainly related to the difference in the second centrifugation speed.

[0094] In Example 1 (1500 r / min, 339.57 nematodes / 100g), 1500 r / min is a relatively low speed, resulting in weak centrifugal force. Lower centrifugal force not only effectively reduces mechanical damage but also protects the integrity of the nematodes. However, it has a weaker ability to separate impurities, and some nematodes may still be encapsulated by impurities and not be completely separated.

[0095] In Example 2 (1900 r / min, 359.36 nematodes / 100g), 1900 r / min is a higher rotation speed, providing stronger centrifugal force and enabling more efficient extraction of nematodes from impurities, but the risk of mechanical damage also increases. Compared to 1500 r / min, the number of nematodes increased slightly, indicating improved separation efficiency. However, the increase in number may be limited due to mechanical damage to some nematodes.

[0096] In Example 3 (1800 r / min, 367.51 nematodes / 100g), the speed of 1800 r / min falls between 1500 r / min and 1900 r / min, balancing separation efficiency and nematode integrity. Therefore, Example 3 (1800 r / min) showed the highest number of nematodes, indicating that this speed achieves the best separation effect. The comparison of the three examples clearly verifies the impact of different speeds on the number and integrity of separated nematodes, thus highlighting the superior performance of Example 3.

[0097] The design for adjusting the rotation speed in this invention is a scientific optimization based on the principle of centrifugal separation, existing experimental data, and practical application requirements, ensuring the comparability, consistency, and scientific rigor of the experiments. Furthermore, through verification and comparison, the rationality and feasibility of Example 3 as the optimal solution are clearly demonstrated.

Claims

1. A method for isolating nematodes from forest soil based on a combination of the Bellman funnel method and centrifugal flotation, characterized in that... The separation method is performed according to the following steps: Step S1: Connect a rubber tube to the end of the funnel and clamp the end of the rubber tube tightly; place a layer of wire mesh inside the funnel, then place two layers of gauze on the wire mesh, and then place a layer of nematode filter paper on the gauze. The funnel mentioned in step S1 is a plastic funnel with a diameter of 15-25cm, a lower opening diameter of 3-4cm, and a height of 25-30cm. Step S2: Spread the fresh soil sample containing nematodes evenly on the filter paper in step S1, and add water until the soil sample is submerged; then separate at a temperature of 20~22℃ for 24~48h, open the clamp at the end of the rubber tube, and drain the water into a container for collection; Step S3: Add the remaining soil sample from the funnel and filter paper to the centrifuge tube, and also pour the washing liquid from the funnel, gauze and filter paper into the centrifuge tube. Stir well first, and then place it in the centrifuge for the first centrifugation. In step S3, a glass rod with an absorbent tip is used for stirring; In step S3, the first centrifugation is performed at a speed of 2000~3000 r / min for 4~6 min. Step S4: After the first centrifugation, discard the supernatant and add sucrose solution; stir well and then place in a centrifuge for a second centrifugation; after centrifugation, collect the supernatant and mix it with the water collected in step S2, pass it through a sieve, and collect the separated nematodes; The concentration of the sucrose solution mentioned in step S4 is 790~810 g / L; In step S4, the second centrifugation speed is 1500~2000 r / min, and the centrifugation time is 4~6 min; After the second centrifugation in step S4, the collected supernatant is immediately mixed with the water collected in step S2; The sieve set mentioned in step S4 consists of 300-mesh, 400-mesh, and 500-mesh sieves.

2. The method for isolating nematodes from forest soil based on a combination of the Bellman funnel method and centrifugal flotation as described in claim 1, characterized in that... Before using the fresh soil sample mentioned in step S2, it needs to be manually broken into small pieces.

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