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1. Preparation before sampling

Before sampling, it is necessary to determine the experimental purpose, sampling site, sample quantity, sampling method, sampling tool, transportation, route and weather conditions. It is usually necessary to prepare a sampling report to record in detail the specific conditions of the sampling, such as sampling date, time, detailed location of the sampling point (GPS positioning), weather conditions (temperature, precipitation, humidity, clouds, etc.) and other information.
After determining the sampling point, you can collect and understand the sample type, weather data, hydrological data, etc. of the place in advance. Before actual sampling, conduct site survey based on the collected data, and finally determine the exact sampling point.
Sampling tools and soil samples must be sterilized in advance (wrapped in tin foil, after autoclaving, oven at 120°C overnight) to avoid interference from foreign substances; avoid the use of substances that absorb water, release solvents or plasticizers, etc. Use utensils to hold soil samples.
Container washing: After cleaning the container with water and detergent to remove dust and oil, rinse it with tap water, soak it in 10% nitric acid or hydrochloric acid for 8 hours, take it out and rinse it with tap water 3 times, and finally rinse it with distilled water.
Container sterilization: ①High temperature and high pressure sterilization, and dry the container in time; ②If it cannot be sterilized, you can use 75% alcohol and sterile water to rinse repeatedly.
Remarks: ①The treated container should be used within 2 weeks; ②Before changing the sample site for sample collection, use alcohol to disinfect and sterilize the equipment, or use the sample site water to rinse the equipment.

2 Soil sample

2.1 Location selection

① The soil type of the sampling site has clear characteristics, the terrain is relatively flat and stable, and the vegetation is good;
② Sampling points should not be set in places with subordinate landscape features such as slope foot and depression;
③ Man-made disturbances in towns, houses, fences, roads, ditches, ridges, manure pits, composting sites, graves, etc. are large, which may cause disorder of soil characteristics, make the soil lose its representativeness, and it is not easy to set up sampling points;
④ The sampling point is at least 300m away from the railway and highway;
⑤ Sampling points shall be based on complete profile development, clear layers, and no intrusive bodies. No sampling points shall be set up where the soil erosion is serious or the topsoil is damaged.
⑥ Sampling points should not be arranged in marginal areas with multiple soil types and multiple parent materials staggered and small in area. Note: After determining the sampling point, you can use the accurate map or GPS to locate the sampling point, or mark the sampling point for future re-sampling or comparison experiments.

2.2 Operation method of soil sampling

Ordinary soil:

A. Remove the mulch on the soil, including plants, moss, visible roots, litter, and visible soil animals.
B. Wipe the sampler with alcohol cotton. After the alcohol has evaporated completely, the sampler can be infiltrated with the soil at the sampling square. This step needs to be repeated each time the sample is replaced.
C. Multi-point mixed sampling with the same method (3 to 5 points are recommended to be mixed in equal amounts), remove plants, visible animals, and stone particles in the sample, and then choose whether to sieve according to the soil conditions, ① 2mm sieve is recommended; ②Some organic matter (such as coarse humus or peat) cannot pass through a 2mm sieve, you can choose to pass a 4-5mm sieve at this time; ③If the soil viscosity is too high or the water content is too high, you can choose not to pass the sieve.
D. Divide the mixed soil samples into multiple portions, each 3-5g, quick-frozen with liquid nitrogen (if conditions do not permit, you can choose to put it in an ice box and transport it back to the laboratory as soon as possible), and then transfer to -80°C Refrigerator storage is used to determine soil microorganisms; another part of the soil is reserved for determination of soil physical and chemical indicators (such as organic matter, total nitrogen, mineral elements, etc.).

Rhizosphere soil of crops (including dwarf plants such as grassland):

A. Collect plant plants, remove large pieces of soil from the roots, and transport them to the laboratory on ice;
B. After shaking the roots to remove the loose soil from the roots, use a sterile brush to collect residual soil from the roots;
C. Sampling soil samples at multiple points with the same method and mixing them evenly, then quick-freeze in liquid nitrogen and store them in a refrigerator at -80°C.

Forest rhizosphere soil:

A. Collect the rhizosphere soil 10-20cm below the ground and transport it to the laboratory on ice;
B. Pass a 2-5mm aseptic sieve;
C. Sampling soil samples at multiple points with the same method and mixing them evenly, then quick-freeze in liquid nitrogen and store them in a refrigerator at -80°C.

3 Water sample

3.1 Sampling collection

The sampling depth and range of the water body can be determined and selected according to the purpose of the experiment, and the corresponding sampler is used for sample collection.

3.2 Operation method of water sampling

According to the experimental settings, select a filter membrane with appropriate pore size, and use a water suction filter to filter 1-100 L of water (different water quality varies greatly), or filter until a visible covering is visible on the filter membrane. Collect the filter membrane for DNA extraction or store in a refrigerator at -80℃.
Remarks: According to the microbial content in the water body and the project experience, the amount of water with rich flora such as sewage is generally 200-1000ml, natural water such as lakes and rivers is generally 1-2 L, and water with low flora content such as tap water is normal. For 1-5 L, or even more.

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