Forest soil nematode separation method based on combination of Belman funnel method and centrifugal flotation method

Abstract

The invention discloses a forest soil nematode separation method based on combination of a Bellman funnel method and a centrifugal flotation method, and relates to the technical field of nematode extraction. The invention aims to solve the problem that the traditional single separation method is difficult to consider both efficiency and effect at the same time. Compared with a traditional single nematode separation method, the nematode separation method combining the Bellman funnel method and the centrifugal flotation method has the advantages that the separation efficiency is improved, loss and errors in the operation process are reduced, the nematode integrity can be better protected, the purity of a separated sample is ensured, and the separation efficiency is improved. Therefore, a more accurate and reliable basis is provided for subsequent nematode identification and counting. The forest soil nematode separation method based on the combination of the Bellman funnel method and the centrifugal flotation method can be obtained.

Description

Technical Field

[0001] The invention relates to the technical field of nematode extraction, and in particular to a method for separating forest soil nematodes based on a combination of a Baermann funnel method and a centrifugal flotation method. Background Art

[0002] In the context of global change, the structure and function, biodiversity and ecological balance of forest ecosystems are being disturbed by multiple factors such as climate warming, precipitation changes, biological invasions and human activities. These disturbance factors produce significant differences in different regions through direct and indirect feedback mechanisms, threatening the stability of forest ecosystems and may weaken their key role in global carbon cycle and climate regulation. Soil nematodes are the most abundant metazoans in soil ecosystems. They are widely distributed in the main trophic levels of the soil food web and are an important bridge connecting decomposers and high-trophic-level organisms. Changes in their trophic group structure not only affect the stability of soil ecosystem processes, but also can sensitively reflect environmental disturbances. Therefore, they are regarded as ideal environmental indicator organisms (LIU TM, 2020; Hou Linxiu, 2022). Therefore, accurate isolation and identification of soil nematodes is of great significance for revealing the dynamic changes of forest ecosystem processes under the background of climate change, evaluating the impact of environmental disturbances on soil ecological functions, and conducting ecological research. At the same time, this process provides a scientific basis for biodiversity conservation, soil health monitoring and the maintenance of ecosystem functions.

[0003] The separation of soil nematodes is a key step in community analysis. Improving the efficiency of nematode separation can more accurately reflect the number, structure and diversity of soil nematode communities, which is of great significance for the study of forest ecosystems. Domestic and foreign scholars have conducted extensive research on nematode separation technology. Currently, commonly used separation methods include wet selection, shallow plate method, sucrose centrifugation method, Carborundum sieving method and semi-automatic washing method. When choosing a suitable separation method, the specific characteristics of the soil and the research objectives should be comprehensively considered, and the separation steps and parameters should be reasonably adjusted to ensure the accuracy and reliability of the results.

[0004] Forest soil has a loose structure and is rich in organic matter, plant residues and roots. These components are easily mixed with nematodes during the separation process, increasing the difficulty of separation. At the same time, some forest soils contain a high proportion of clay. The strong adhesion of clay particles makes nematodes easily wrapped and difficult to completely separate. Therefore, a single separation method is often unable to cope with these complex characteristics, resulting in incomplete separation. Summary of the invention

[0005] The purpose of the present invention is to solve the problem that the traditional single separation method is difficult to take into account both efficiency and effect, and to provide a forest soil nematode separation method based on the combination of Baermann funnel method and centrifugal flotation method.

[0006] A method for separating forest soil nematodes based on a combination of the Baermann funnel method and the centrifugal flotation method is carried out in 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; place a layer of wire mesh in the funnel, and place two layers of gauze on the wire mesh, and then place a layer of nematode filter paper;

[0009] Step S2:

[0010] Evenly spread the fresh soil sample containing nematodes on the filter paper in step S1, and add water to immerse the soil sample; then separate at a temperature of 20 to 22° C. for 24 to 48 hours, open the clamp at the end of the rubber tube, and drain the water into a container for collection;

[0011] Step S3:

[0012] Add the remaining soil sample in the funnel and on the filter paper into the centrifuge tube, and pour the washing liquid of the funnel, gauze and filter paper into the centrifuge tube, stir evenly, and then place it in the centrifuge for the first centrifugation;

[0013] Step S4:

[0014] After the first centrifugation, the supernatant is discarded and sucrose solution is added; the mixture is stirred evenly and then placed in a centrifuge for a second centrifugation; after the centrifugation, the supernatant is collected and mixed with the water collected in step S2, and the mixture is passed through a sieve to collect the separated nematodes.

[0015] Beneficial effects of the present invention:

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

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

[0018] (3) The nematode separation method provided by the present invention, which combines the Baermann funnel method with the centrifugal flotation method, is easy to operate and solves the problem that a single separation method is difficult to achieve both efficiency and effect.

[0019] The invention can obtain a forest soil nematode separation method based on the combination of the Baermann funnel method and the centrifugal flotation method. DETAILED DESCRIPTION

[0020] Specific implementation method 1: This implementation method is a method for separating forest soil nematodes based on a combination of the Baermann funnel method and the centrifugal flotation method, and is carried out in 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; place a layer of wire mesh in the funnel, and place two layers of gauze on the wire mesh, and then place a layer of nematode filter paper;

[0023] Step S2:

[0024] Evenly spread the fresh soil sample containing nematodes on the filter paper in step S1, and add water to immerse the soil sample; then separate at a temperature of 20 to 22° C. for 24 to 48 hours, open the clamp at the end of the rubber tube, and drain the water into a container for collection;

[0025] Step S3:

[0026] Add the remaining soil sample in the funnel and on the filter paper into the centrifuge tube, and pour the washing liquid of the funnel, gauze and filter paper into the centrifuge tube, stir evenly, and then place it in the centrifuge for the first centrifugation;

[0027] Step S4:

[0028] After the first centrifugation, the supernatant is discarded and sucrose solution is added; the mixture is stirred evenly and then placed in a centrifuge for a second centrifugation; after the centrifugation, the supernatant is collected and mixed with the water collected in step S2, and the mixture is passed through a sieve to collect the separated nematodes.

[0029] The purpose of the first step of centrifugation in step S4 is to remove unnecessary impurities, suspended matter or liquid components dissolved in the supernatant, and optimize separation efficiency and purity. However, due to the effect of centrifugal force, some nematodes may also remain in the supernatant, but the proportion of these nematodes is relatively low, and from the perspective of experimental efficiency and target effect, its loss is acceptable. If there are special needs, the supernatant can be re-added to the centrifuge separately by improving the operation, and centrifuged again (lower centrifugation speed), and then only the nematodes therein are recovered, and then combined with subsequent steps to reduce nematode losses.

[0030] Specific implementation method 2: The difference between this implementation method and specific implementation method 1 is that the funnel described in step S1 is a plastic funnel with a diameter of 15 to 25 cm.

[0031] The other steps are the same as those in the first specific implementation.

[0032] Specific implementation method three: The difference between this implementation method and specific implementation method one or two is that the lower opening diameter of the plastic funnel is 3 to 4 cm and the height is 25 to 30 cm.

[0033] The other steps are the same as those in the first or second embodiment.

[0034] Specific implementation method 4: The difference between this implementation method and specific implementation methods 1 to 3 is that the fresh soil sample described in step S2 needs to be manually broken into small pieces before use 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 Embodiments 1 to 3.

[0036] Specific implementation method five: The difference between this implementation method and specific implementation methods one to four is that in step S3, a glass rod with an adsorption rubber head is used for stirring to remove roots and organic matter in the soil.

[0037] The other steps are the same as those in Specific Embodiments 1 to 4.

[0038] Specific implementation method 6: The difference between this implementation method and specific implementation methods 1 to 5 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 Embodiments 1 to 5.

[0040] Specific embodiment 7: The difference between this embodiment and specific embodiments 1 to 6 is that the concentration of the sucrose solution in step S4 is 790-810 g / L.

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

[0042] Specific embodiment eight: The present embodiment differs from specific embodiments one to seven in 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 Embodiments 1 to 7.

[0044] Specific embodiment 9: The difference between this embodiment and specific embodiments 1 to 8 is that after the second centrifugation in step S4 is completed, 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 ensure the activity and integrity of the nematodes.

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

[0046] Specific implementation method ten: The difference between this implementation method and specific implementation methods one to nine is that the set of sieves described 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 Embodiments 1 to 9.

[0048] The following examples are used to verify the beneficial effects of the present invention:

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

[0050] Step S1:

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

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

[0053] Step S2:

[0054] Weigh 100 g of fresh soil sample, break it into small pieces by hand, evenly spread it on the filter paper in step S1, and add water to immerse the soil sample; then separate it at a temperature of 20°C for 48 hours, 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 in the funnel and on the filter paper into a 250mL centrifuge tube, and pour the washing liquid of the funnel, gauze and filter paper into the centrifuge tube. Add water until the water-soil mixture weighs 200g, stir it evenly with a glass rod with an adsorption rubber head, and then place it in a centrifuge and centrifuge it at a speed of 2500r / min for 5min.

[0057] Step S4:

[0058] After the first centrifugation, the supernatant was discarded, and a 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 a speed of 1500 r / min for 5 minutes. After the centrifugation, the collected supernatant was immediately mixed with the water collected by the Baermann funnel method in step S2 to prevent the nematodes from losing water and deforming in the high-density sucrose solution. The separated nematodes were then collected through a nested sieve of 300 mesh, 400 mesh and 500 mesh sieves.

[0059] As shown in Table 1, the second time the rotation speed was reduced to 1500 r / min, the number of nematodes increased to 339.57 / 100 g, indicating that the low rotation speed protected the integrity of the nematodes, but the separation efficiency was slightly lower, and the impurities around the nematodes may not be completely removed, resulting in an increase in the number of nematodes, but the effect was not as good as that of Example 3 with a higher rotation speed.

[0060] Example 2: A method for separating forest soil nematodes based on a combination of the Baermann funnel method and the centrifugal flotation method, which is carried out in the following steps:

[0061] Step S1:

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

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

[0064] Step S2:

[0065] Weigh 100 g of fresh soil sample, break it into small pieces by hand, evenly spread it on the filter paper in step S1, and add water to immerse the soil sample; then separate it at a temperature of 20°C for 48 hours, 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 in the funnel and on the filter paper into a 250mL centrifuge tube, and pour the washing liquid of the funnel, gauze and filter paper into the centrifuge tube. Add water until the water-soil mixture weighs 200g, stir it evenly with a glass rod with an adsorption rubber head, and then place it in a centrifuge and centrifuge it at a speed of 2000r / min for 5min.

[0068] Step S4:

[0069] After the first centrifugation, the supernatant was discarded, and a 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 a speed of 2000 r / min for 5 minutes. After the centrifugation, the collected supernatant was immediately mixed with the water collected by the Baermann funnel method in step S2 to prevent the nematodes from losing water and deforming in the high-density sucrose solution. The separated nematodes were then collected through a nested sieve of 300 mesh, 400 mesh and 500 mesh sieves.

[0070] As shown in Table 1, the second rotation speed was 1900 r / min, and the number of nematodes was 359.36 / 100 g. The test results show that this rotation speed is close to efficient separation, and the mechanical damage is well controlled, and the effect is higher than the conditions of 1500 r / min and 2000 r / min.

[0071] Example 3: A method for separating forest soil nematodes based on a combination of the Baermann funnel method and the centrifugal flotation method, which is carried out in the following steps:

[0072] Step S1:

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

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

[0075] Step S2:

[0076] Weigh 100 g of fresh soil sample, break it into small pieces by hand, evenly spread it on the filter paper in step S1, and add water to immerse the soil sample; then separate it at a temperature of 20°C for 48 hours, 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 in the funnel and on the filter paper into a 250mL centrifuge tube, and pour the washing liquid of the funnel, gauze and filter paper into the centrifuge tube. Add water until the water-soil mixture weighs 200g, stir it evenly with a glass rod with an adsorption rubber head, and then place it in a centrifuge and centrifuge it at a speed of 2000r / min for 5min.

[0079] Step S4:

[0080] After the first centrifugation, the supernatant was discarded, and a 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 a speed of 1800 r / min for 5 minutes. After the centrifugation, the collected supernatant was immediately mixed with the water collected by the Baermann funnel method in step S2 to prevent the nematodes from losing water and deforming in the high-density sucrose solution. The separated nematodes were then collected through a nested sieve of 300 mesh, 400 mesh and 500 mesh sieves.

[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 / 100 g. This data verified that 1800 r / min was the best balance between efficiency and integrity.

[0082] Comparative Example 1: Baerman Funnel Method;

[0083] The separation time was long (48 hours) and the efficiency was low, with the number of nematodes being 240.42 / 100g.

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

[0085] Comparative Example 2: Centrifugal flotation method;

[0086] The two centrifugal speeds were selected as 2000r / min+2000r / min, and the test results showed that the number of nematodes increased to 302.73 / 100g, which was significantly higher than that of the Baermann funnel method. This shows the advantage of the centrifugal flotation method in separation efficiency, but the excessively high second speed may cause certain mechanical damage to the nematodes, resulting in the separation results being inferior to those of Examples 2 and 3.

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

[0088] Table 1

[0089]

[0090] The main purpose of the first centrifugation in the present invention is to preliminarily separate large particles of impurities (such as soil particles and organic debris) to lay the foundation for subsequent fine separation. Therefore, the rotation speed is uniformly set to 2000r / min, which not only ensures effective separation of impurities, but also avoids impurity residues due to too low a rotation speed or damage to nematodes due to too high a rotation speed.

[0091] The main purpose of the second centrifugation is to finely separate the target (nematodes) from the remaining impurities. The adjustment of the rotation speed will directly affect the separation efficiency and integrity of the nematodes. The specific effects are as follows:

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

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

[0094] In Example 1 (1500 r / min, 339.57 nematodes / 100 g), 1500 r / min is a relatively low speed, and the centrifugal force is relatively weak. A relatively small centrifugal force can not only effectively reduce mechanical damage, but also protect the integrity of the nematodes, but the ability to separate impurities is relatively weak, and some nematodes may still be wrapped by impurities and cannot be completely separated.

[0095] In Example 2 (1900 r / min, 359.36 nematodes / 100 g), 1900 r / min is a relatively high speed with a stronger centrifugal force, which can more efficiently extract nematodes from impurities, but the risk of mechanical damage increases. Compared with 1500 r / min, the number of nematodes increased slightly, indicating that the separation efficiency was improved. However, the increase in number may be small because some nematodes were mechanically damaged.

[0096] In Example 3 (1800 r / min, 367.51 nematodes / 100 g), 1800 r / min is between 1500 r / min and 1900 r / min, taking into account both separation efficiency and nematode integrity. Therefore, the number of nematodes in Example 3 (1800 r / min) is the highest, indicating that this speed can achieve the best separation effect. By comparing the above three examples, the effects of different speeds on the number and integrity of nematode separation can be clearly verified, thereby highlighting the excellent effect of Example 3.

[0097] The design of adjusting the rotation speed in the present invention is a scientific optimization based on the centrifugal separation principle, existing experimental data and practical application requirements, which ensures the comparability, consistency and scientificity of the experiment. At the same time, through verification and comparison, the rationality and feasibility of Example 3 as the optimal solution can be clearly highlighted.

Claims

1. A method for separating forest soil nematodes based on a combination of the Baermann funnel method and the centrifugal flotation method, characterized in that The separation method is carried out 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; place a layer of wire mesh in the funnel, and place two layers of gauze on the wire mesh, and then place a layer of nematode filter paper; Step S2: Evenly spread the fresh soil sample containing nematodes on the filter paper in step S1, and add water to immerse the soil sample; then separate at a temperature of 20 to 22° C. for 24 to 48 hours, 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 in the funnel and on the filter paper into the centrifuge tube, and pour the washing liquid of the funnel, gauze and filter paper into the centrifuge tube, stir evenly, and then place it in the centrifuge for the first centrifugation; Step S4: After the first centrifugation, the supernatant is discarded and sucrose solution is added; the mixture is stirred evenly and then placed in a centrifuge for a second centrifugation; after the centrifugation, the supernatant is collected and mixed with the water collected in step S2, and the mixture is passed through a sieve to collect the separated nematodes.

2. The method for separating forest soil nematodes based on the combination of Baermann funnel method and centrifugal flotation method according to claim 1, characterized in that The funnel described in step S1 is a plastic funnel with a diameter of 15 to 25 cm.

3. The method for separating forest soil nematodes based on the combination of Baermann funnel method and centrifugal flotation method according to claim 2, characterized in that The lower opening diameter of the plastic funnel is 3-4 cm, and the height is 25-30 cm.

4. The method for separating forest soil nematodes based on the combination of Baermann funnel method and centrifugal flotation method according to claim 1, characterized in that The fresh soil sample described in step S2 needs to be manually broken into small pieces before use.

5. The method for separating forest soil nematodes based on the combination of Baermann funnel method and centrifugal flotation method according to claim 1, characterized in that In step S3, a glass rod with an adsorbed rubber head is used for stirring.

6. The method for separating forest soil nematodes based on the combination of Baermann funnel method and centrifugal flotation method according to claim 1, characterized in that The rotation speed of the first centrifugation in step S3 is 2000-3000 r / min, and the centrifugation time is 4-6 min.

7. The method for separating forest soil nematodes based on the combination of Baermann funnel method and centrifugal flotation method according to claim 1, characterized in that The concentration of the sucrose solution described in step S4 is 790-810 g / L.

8. The method for separating forest soil nematodes based on the combination of Baermann funnel method and centrifugal flotation method according to claim 1, characterized in that The rotation speed of the second centrifugation in step S4 is 1500-2000 r / min, and the centrifugation time is 4-6 min.

9. A method for separating forest soil nematodes based on a combination of Baermann funnel method and centrifugal flotation method according to claim 1 or 8, characterized in that After the second centrifugation in step S4 is completed, the collected supernatant is immediately mixed with the water collected in step S2.

10. The method for separating forest soil nematodes based on the combination of Baermann funnel method and centrifugal flotation method according to claim 1, characterized in that The set of sieves described in step S4 is composed of 300 mesh, 400 mesh and 500 mesh sieves.

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