A method for cultivating Caenorhabditis elegans

Abstract

The invention discloses a method for cultivating a Caenorhabditis elegans, comprising the following steps: S1: taking a glass culture dish and treating it; S2: using a bacterial loop to pick a nitrogen-fixing bacteria strain and inoculating it into a 50 mL nitrogen-fixing bacteria liquid culture medium, performing constant temperature shaking culture at a culture temperature of 26-28° C. and shaking culture for 24 hours to obtain a nitrogen-fixing bacteria liquid; S3: introducing 15-20 mL of the nematode culture medium into the culture dish on a sterile clean bench and keeping it still for 48 hours; S4: using a pipette to take 200 uL of the nitrogen-fixing bacteria liquid and adding it to the nematode culture medium, then using a bacterial coating stick to evenly coat it, and culturing it at 25-28° C. for 24 hours; S5: adding sterilized Caenorhabditis elegans, and then culturing at 25-28° C. for 10-14 days to obtain a large number of cultured Caenorhabditis elegans. In the past, the most commonly used strain for culturing nematodes was Escherichia coli OP50. The number of nematodes obtained by culturing C. elegans with nitrogen-fixing strains was higher than that obtained by culturing nematodes with E. coli OP50 (0.4-1.8 times).

Description

Technical Field

[0001] The present invention relates to the technical field of Caenorhabditis elegans cultivation, and in particular to a method for cultivating Caenorhabditis elegans. Background Art

[0002] Soil nematodes are the most abundant multicellular animals in the soil and are indicator organisms of the soil ecological environment. Bacteria-eating nematodes generally account for more than 40% of soil nematodes, and can reach more than 90% in environments with strong microbial activity areas. Bacteria-eating nematodes are one of the indicator organisms of soil fertility. The genus Mesobranchus feeds on bacteria and is a common bacteria-eating nematode in farmland. This nematode has strong adaptability, wide distribution, short generation cycle, and responds quickly to changes in available substances in the soil. Therefore, it can be used to indicate changes in soil fertility. Through large-scale reproduction, it can be used to detect the impact of the addition of external aid substances on soil fertility.

[0003] At present, there have been reports on the cultivation and reproduction of other nematodes that feed on bacteria, but there have been no reports on the cultivation method of Caenorhabditis elegans. In the past, Escherichia coli was mostly used to propagate nematodes. The present invention uses a strain with nitrogen-fixing function to propagate nematodes. This strain is easy to cultivate, and Caenorhabditis elegans have a stronger reproduction ability after feeding. Summary of the invention

[0004] The present invention provides a method for cultivating Caenorhabditis elegans to solve the problem that the prior art proposed in the above background technology is only applicable to the propagation of nematodes on a small scale in a laboratory.

[0005] The present invention provides a method for cultivating a Caenorhabditis elegans, comprising the following steps:

[0006] S1: Take a glass petri dish and process it;

[0007] S2: Use an inoculation loop to pick up nitrogen-fixing bacteria strains and inoculate them into 50 mL of nitrogen-fixing bacteria liquid culture medium, perform constant temperature shaking culture at 26-28°C, shake culture for 24 hours, and obtain nitrogen-fixing bacteria liquid;

[0008] S3: In a sterile clean bench, add 15-20 mL of nematode culture medium into the culture dish and let it stand for 48 hours;

[0009] S4: Use a pipette to take 200uL of nitrogen-fixing bacteria liquid and add it to the nematode culture medium, then spread it evenly with a bacteria-spreading stick, and culture it at 25-28℃ for 24 hours;

[0010] S5: Add sterilized Caenorhabditis elegans and then culture at 25-28°C for 10-14 days to obtain a large amount of cultured Caenorhabditis elegans.

[0011] Preferably, in S1, the glass culture dish needs to be sterilized at 121° C. for 30 min.

[0012] Preferably, the nitrogen-fixing bacteria liquid culture medium is: 10 g mannitol, 0.2 g KH2PO4, 0.2 g MgSO4·7H2O, 0.2 g NaCl, 0.3 g K2SO4, 5 g CaCO3, and 1000 mL distilled water.

[0013] Preferably, the nematode culture medium is: 3 g NaCl, 2.5 g peptone, 20 g agar, 985 mL distilled water, 1 mL cholesterol stock solution (5 mg / mL), 1 mL 1 mol / L CaCl2, 1 mL 1 mol / L MgSO4, and 25 mL 1 mol / L phosphate buffer.

[0014] Preferably, in S5, 50-100 Caenorhabditis elegans are added.

[0015] Preferably, in S2, the shaking frequency of the constant temperature shaking culture is set at 170-180 rpm.

[0016] Preferably, in S3, the culture dish is sealed with a sealing film.

[0017] The present invention provides a method for cultivating Caenorhabditis elegans, which has the beneficial effect that the strain commonly used for cultivating nematodes in the past is Escherichia coli OP50. When Caenorhabditis elegans is cultivated with nitrogen-fixing strains, the number of nematodes obtained is higher than that of nematodes propagated with Escherichia coli OP50 (0.4-1.8 times). DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with specific embodiments.

[0019] Example 1

[0020] The present invention provides a method for cultivating a Caenorhabditis elegans, comprising the following steps:

[0021] S1: Take a glass culture dish and sterilize it at 121°C for 30 minutes;

[0022] S2: Use a bacterial loop to pick up the nitrogen-fixing bacteria strain and inoculate it into 50 mL of nitrogen-fixing bacteria liquid culture medium, perform constant temperature shaking culture, the culture temperature is 26°C, the shaking frequency is set at 170 rpm, and the shaking culture is performed for 24 hours to obtain nitrogen-fixing bacteria liquid;

[0023] S3: In a sterile clean bench, add 15-20 mL of nematode culture medium into a culture dish, seal the dish with a sealing film, and leave it alone for 48 hours;

[0024] S4: Use a pipette to take 200uL of nitrogen-fixing bacteria liquid and add it to the nematode culture medium, then spread it evenly with a bacteria-spreading stick, and culture it at 25-28℃ for 24 hours;

[0025] S5: Add 50-100 sterilized Caenorhabditis elegans, and then culture at 25°C for 10-14 days to obtain a large number of cultured Caenorhabditis elegans.

[0026] The nitrogen-fixing bacteria liquid culture medium comprises: 10 g of mannitol, 0.2 g of KH2PO4, 0.2 g of MgSO4·7H2O, 0.2 g of NaCl, 0.3 g of K2SO4, 5 g of CaCO3, and 1000 mL of distilled water.

[0027] The nematode culture medium is: 3g NaCl, 2.5g peptone, 20g agar, 985mL distilled water, 1mL cholesterol stock solution (5mg / mL), 1mL 1mol / L CaCL2, 1mL 1mol / L MgSO4, and 25mL 1mol / L phosphate buffer.

[0028] Example 2

[0029] The present invention provides a method for cultivating a Caenorhabditis elegans, comprising the following steps:

[0030] S1: Take a glass culture dish and sterilize it at 121°C for 30 minutes;

[0031] S2: Use a bacterial loop to pick up the nitrogen-fixing bacteria strain and inoculate it into 50 mL of nitrogen-fixing bacteria liquid culture medium, perform constant temperature shaking culture, the culture temperature is 27°C, the shaking frequency is set at 175 rpm, and the shaking culture is performed for 24 hours to obtain nitrogen-fixing bacteria liquid;

[0032] S3: In a sterile clean bench, add 15-20 mL of nematode culture medium into a culture dish, seal the dish with a sealing film, and leave it alone for 48 hours;

[0033] S4: Use a pipette to take 200uL of nitrogen-fixing bacteria liquid and add it to the nematode culture medium, then spread it evenly with a bacteria-spreading stick, and culture it at 25-28℃ for 24 hours;

[0034] S5: Add 50-100 sterilized Caenorhabditis elegans, and then culture at 26°C for 10-14 days to obtain a large number of cultured Caenorhabditis elegans.

[0035] The nitrogen-fixing bacteria liquid culture medium comprises: 10 g of mannitol, 0.2 g of KH2PO4, 0.2 g of MgSO4·7H2O, 0.2 g of NaCl, 0.3 g of K2SO4, 5 g of CaCO3, and 1000 mL of distilled water.

[0036] The nematode culture medium is: 3 g NaCl, 2.5 g peptone, 20 g agar, 985 mL distilled water, 1 mL cholesterol stock solution (5 mg / mL), 1 mL 1 mol / L CaCl2, 1 mL 1 mol / L MgSO4, and 25 mL 1 mol / L phosphate buffer.

[0037] Example 3

[0038] The present invention provides a method for cultivating a Caenorhabditis elegans, comprising the following steps:

[0039] S1: Take a glass culture dish and sterilize it at 121°C for 30 minutes;

[0040] S2: Use an inoculation loop to pick up the nitrogen-fixing bacteria strain and inoculate it into 50 mL of nitrogen-fixing bacteria liquid culture medium, perform constant temperature shaking culture, the culture temperature is 28°C, the shaking frequency is set at 180 rpm, and the shaking culture is performed for 24 hours to obtain nitrogen-fixing bacteria liquid;

[0041] S3: In a sterile clean bench, add 15-20 mL of nematode culture medium into a culture dish, seal the dish with a sealing film, and leave it alone for 48 hours;

[0042] S4: Use a pipette to take 200uL of nitrogen-fixing bacteria liquid and add it to the nematode culture medium, then spread it evenly with a bacteria-spreading stick, and culture it at 25-28℃ for 24 hours;

[0043] S5: Add 50-100 sterilized Caenorhabditis elegans, and then culture at 28°C for 10-14 days to obtain a large number of cultured Caenorhabditis elegans.

[0044] The nitrogen-fixing bacteria liquid culture medium comprises: 10 g of mannitol, 0.2 g of KH2PO4, 0.2 g of MgSO4·7H2O, 0.2 g of NaCl, 0.3 g of K2SO4, 5 g of CaCO3, and 1000 mL of distilled water.

[0045] The nematode culture medium is: 3 g NaCl, 2.5 g peptone, 20 g agar, 985 mL distilled water, 1 mL cholesterol stock solution (5 mg / mL), 1 mL 1 mol / L CaCL2, 1 mL 1 mol / L MgSO4, and 25 mL 1 mol / L phosphate buffer.

[0046] The nitrogen-fixing bacteria strain is specifically the nitrogen-fixing bacteria PhyLLobacterium brassicacearum strain, which is a Gram-positive bacterium, and has a transparent colony on the culture medium, a smooth surface, and a central bulge. The sequence number of the bacteria in NCBI GenBank is:

[0047]

[0048] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for cultivating a Caenorhabditis elegans, characterized in that: The following steps are involved: S1: Take a glass culture dish and process it. The glass culture dish needs to be sterilized at 121°C for 30 minutes; S2: Use an inoculation loop to pick up the nitrogen-fixing bacteria strain and inoculate it into 50 mL of nitrogen-fixing bacteria liquid culture medium, perform constant temperature shaking culture at 26-28°C, shake culture for 24 hours, and obtain nitrogen-fixing bacteria liquid; S3: In a sterile clean bench, add 15-20 mL of nematode culture medium into the culture dish and let it stand for 48 hours; S4: Use a pipette to take 200uL of nitrogen-fixing bacteria liquid and add it to the nematode culture medium, then spread it evenly with a bacteria-spreading stick, and culture it at 25-28℃ for 24 hours; S5: Add sterilized Caenorhabditis elegans and then culture at 25-28°C for 10-14 days to obtain a large number of cultured Caenorhabditis elegans; The nitrogen-fixing bacteria liquid culture medium is: mannitol 10g, KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, K2SO4 0.3g, CaCO3 5g, distilled water 1000mL; The nematode culture medium is: 3 g NaCl, 2.5 g peptone, 20 g agar, 985 mL distilled water, 1 mL 5 mg / mL cholesterol stock solution, 1 mL 1 mol / L CaCl2, 1 mL 1 mol / L MgSO4, and 25 mL 1 mol / L phosphate buffer.

2. The method for cultivating a Mesococcus nematode according to claim 1, characterized in that: In S5, 50-100 Caenorhabditis elegans should be added.

3. The method for cultivating a Mesococcus nematode according to claim 1, characterized in that: In S2, the shaking frequency of the constant temperature shaking culture was set at 170-180 rpm.

4. The method for cultivating a Mesococcus nematode according to claim 1, characterized in that: In S3, the culture dish was sealed with parafilm.