Basic Principles of Sampling
Representativeness and consistency: the experimental group and control group samples are kept as consistent as possible in terms of sampling site and time processing, thus ensuring the credibility of the experiment.
Swiftness: the operation process is rapid to minimize the time from sample collection to experiment.
Hypothermia: After all samples have been isolated, the separation step should be performed at 4°C or on ice. The separated samples are immediately stored at -80°C to avoid further metabolic activity of the samples.
Blood Sample Collection Guidelines
Serum
1) Collect blood in a procoagulant tube (or centrifuge tube if no procoagulant tube is available). Place the procoagulant tube/centrifuge tube upright and stationary on a test tube rack to avoid shaking
2) Avoid direct sunlight and room temperature.
3) After checking for complete clotting, centrifuge at 2500 ~ 3000 rpm for 5 min at 4°C. Aspirate 200 µL of supernatant into a 1.5 mL centrifuge tube. If more supernatant is available, divide into multiple tubes of 200 µL each. Freeze at -80 °C until use.
4) Repeated freezing and thawing is strictly prohibited. If the sample fails due to hemolysis, the sample should be prepared again. Qualified samples are transported on dry ice.
Plasma
1) Blood collection with an anticoagulation tube. Immediately after blood collection, gently invert up and down 8 times to evenly mix the anticoagulant with the blood (gentle movements to avoid hemolysis).
2) Centrifuge the blood at 2500~3000 rpm for 5 min at 4 ℃. 200 µL of supernatant should be divided into 1.5 mL centrifuge tubes and stored at -80 ℃ for freezing.
3) Plasma should be separated from whole blood immediately after blood sample collection, and no later than 1 h after blood collection. Qualified samples are transported on dry ice.
Fecal Sample Collection Guidelines
Human feces
1) Defecate directly into a clean container to avoid contamination of the stool sample by urine, toilet walls, etc. as much as possible. Take samples from the same time period or mix all samples before sampling.
2) Scoop the stool sample with a sterile spoon. Put the appropriate amount of stool sample into a sterile preservation tube, dispense the sample 200 mg/tube and quickly put it into liquid nitrogen for more than 15 min for quenching.
3) The collected samples are immediately put into -80 ℃ for storage until use.
Mouse/rat feces
1）Put the mice/rats to be collected into a clean cage lined with sterilized filter paper. Transfer to lyophilization tubes with sterile forceps immediately after defecation.
2) 6-8 pellets of mouse feces and 2-3 pellets of rat feces.
3) If samples need to be repeatedly tested, use sterile sticks to mix and split in multiple tubes to avoid repeated freeze-thawing. The samples should be stored at -80℃ immediately after collection for use.
Notes:
Feces samples should be kept away from urine to avoid cross contamination. Urine contains high levels of urea, which can interfere with subsequent stool sample testing.
After collecting fecal samples, it is recommended to quench them in liquid nitrogen for 15 min and then freeze them at -80 ℃.
Uniform form of stool sample is recommended (e.g. solid, liquid, freeze-dried stool, etc.)
Human fecal sample providers should not receive antibiotic treatment within the last three months of sampling.
Urine Sample Collection Guidelines
Urine
1) Take fasting morning urine, intermediate urine (clinical) or 1 h morning urine (animal) in a 50 mL centrifuge tube.
2) Centrifuge the supernatant at 10,000 rpm for 10 min at 4 ℃, dispense in 1.5 mL centrifuge tubes, and store at -80 ℃.
3) If the 1-h urine volume of the animal is not enough, the urine can be collected in several times. If 24 h urine is to be collected, a metabolic cage with a cryogenic device needs to be used for collection, and the sample should be frozen in time.
4) After collecting urine samples, it is recommended to quench them with liquid nitrogen for 15 min, and then freeze them at -80 ℃ in multiple tubes.
Animal Tissue Sample Collection Guidelines
Animal tissues: including brain, heart, liver, spleen, lung, kidney, muscle and skin; mollusks such as Schistosoma haematobium.
1) Take an appropriate amount of tissues and organs and wash them well with pre-chilled PBS buffer or physiological saline.
2) Quickly peel off non-essential fat and connective tissue, and then rinse well with PBS buffer or physiological saline.
3) Absorb the water with clean dust-free blotting paper and divide the tissue into 50-100 mg/tubes to be used.
4) Mark the tubes well and quickly put them into liquid nitrogen for freezing for at least 15 min and freeze at -80 ℃. Transport on dry ice.
Notes:
For animal tissue samples that cannot meet the 50 mg/sample (e.g. hippocampal tissue, brain tissue, etc.), consider mixing samples in the same group to meet the 50 mg/sample requirement.
Body Fluid Sample Collection Guidelines
Body fluid samples: such as joint fluid, saliva, bile, cerebrospinal fluid, rumen fluid, etc.
1) Collect the body fluid samples.
2) Centrifuge at 3000 rpm for 15 min at 4 ℃. The supernatant is taken and divided into 1.5mL centrifuge tubes every 0.5mL. Refrigerated at -80 ℃.
Plant Sample Collection Guidelines
Leaf, stem, flower
1) Take a whole leaf/section of stem/one flower, put it into a centrifuge tube or tin foil and label it. Quickly place in liquid nitrogen and freeze for at least 15 min.
2) Place the centrifuge tubes containing the samples quickly into self-sealing bags (one bag per group). Place a label in the self-sealing bag with the sample information.
3) Quickly place in -80 °C freezer for freezing. Send a sufficient amount of dry ice.
Notes:
When collecting leaf samples, it is best to collect them at noon when the light is good.
Keep samples consistent when collecting samples, especially the same group of samples (color, senescence, percentage of veins, light, position, etc.).
Roots
1) Take the roots of the whole plant and quickly rinse off any soil, medium or nutrient solution on the roots with PBS buffer or ultrapure water.
2) Absorb the water on blotting paper and divide into 500 mg/tube.
3) After labeling, quickly put into liquid nitrogen for freezing treatment for at least 15 min, freeze at -80 ℃ and send on dry ice.
Fruits and seeds
(1) For fruits with high water content and large volume (tomato, watermelon, apple, etc.), the sample should be divided into small uniform pieces (≈ 200 mg/sample) and packed into 2 mL centrifuge tubes. After labeling, the sample is quickly frozen in liquid nitrogen for at least 15 min.
(2) For fine-grained seeds (Arabidopsis seeds, grain seeds, etc.), mix the seeds of the same plant before dispensing (≈ 200 mg/sample) into centrifuge tubes. After labeling, quickly place in liquid nitrogen and freeze for at least 15 min.
3) For those requiring extraction of the whole fruit (whole grapes, etc.), the fruit needs to be packed in 50 mL centrifuge tubes/self-sealing bags. After labeling, quickly put into liquid nitrogen for freezing for at least 15 min, freeze at -80 ℃ and send on dry ice.
Root secretions
1) Take the roots of the whole plant and quickly rinse off the soil on the roots with 1×PBS.
2) Place the whole roots to a large container with ultrapure water and incubate for a period of time.
3) Take a certain volume of liquid sample of root secretion, centrifuge at 1200rpm for 15min, remove impurities and freeze into dry powder using a freeze dryer.
4) Store at -80 degrees.
Rhizosphere Soils Sample Collection Guidelines
1) Removal (shaking off) of loose soil around the roots: the intact root tissue should be ensured as much as possible and excess soil should be removed.
2) Buffer shaking to wash down inter-root soil (tightly adhered): To prevent damage to microorganisms and root tissue cells during shaking, direct treatment with sterile water is recommended. The shaking can be done manually or with a shaker etc. The time and intensity vary according to the ease of washing and the root tissue.
3) High-speed centrifugation to collect precipitation: After treatment with sterile water flushing, soil precipitation can be collected after high-speed centrifugation. If the precipitation is small, the soil and microorganisms can also be collected by filtering through 0.22μm membrane or by direct vacuum lyophilization (without lyophilization conditions, the turbid solution can be directly lyophilized and sent).
4) Storage: Dry ice is sent and stored at -80°C.
Cell Sample Collection Guidelines
Suspension cultured cells
1) Transfer to a 1.5 mL inlet centrifuge tube together with culture medium.
2) Centrifuge at low speed for 5 min (less than 3000 rpm) to allow cells to settle at the bottom of the tube (1*107 cells is appropriate).
3) Pour off the culture medium (as cleanly as possible). Rinse 2 ~ 3 times repeatedly with pre-chilled PBS. Centrifuge at low speed for 5 min and discard the supernatant.
4) Mark the centrifuge tube. Insert the tip of the centrifuge tube into liquid nitrogen and quench the cell precipitate for 1 min. Freeze at -80 ℃ and send on dry ice.
Adherent cells
1) Carefully discard the culture fluid and place the culture dish upside down on blotting paper to absorb as much of the culture fluid as possible.
2) Add pre-chilled PBS at 4 ℃ and shake gently for 1 min to wash the cells (if added with a pipette gun, add against the wall of the culture dish to avoid washing up the cells). Discard the PBS and aspirate the residual PBS several times with a micropipette.
3) Quench the cells by exposing the bottom (outer wall) of the culture dish to liquid nitrogen (1*107 cells is appropriate). Add 500 μL of pre-cooled methanol-water (4:1, V/V). Scrape off the cells with a clean cell scraper. Transfer to a 1.5 mL centrifuge tube with a pipette.
4) Continue adding 500 μL of methanol-water (4:1, V/V) and transfer as many of the remaining cells as possible to the centrifuge tube. Seal the centrifuge tubes using sealing film and store at -80 °C and send on dry ice.
Microbial Sample Collection Guidelines
Microorganisms: Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Pseudomonas malodorosa, yeast, Bacillus spp., Aeromonas spp., Aeromonas motileis, Bacillus glucuronoxidans, etc.
1) Transfer to an imported 15 ml centrifuge tube together with the medium. centrifuge at low speed below 3000 rpm and let the bacteria settle at the bottom of the tube (1*108 bacteria is appropriate).
2) After pouring off the medium (as clean as possible), carefully resuspend and wash the sediment once with pre-chilled PBS solution.
3) Insert the tip of the centrifuge tube into liquid nitrogen and quench the bacteria for 1 min. Freeze at -80 ℃, preferably prepare two tubes for each sample for backup.
Microbial culture broth (detection of extracellular metabolism)
Method 1: After mixing the cultured bacterial broth, take 10 mL of bacterial broth. Remove the bacterium by rapid filtration with a 0.2 μm pore size filter membrane. The filtrate should be dispensed by 1 mL/tube, frozen at -80 ℃ and sent on dry ice.
Method 2: After the culture solution is well mixed, take 1 mL of bacterial solution and centrifuge at 3000 rpm, 4 ℃ for 10 min. 800 μL of supernatant is taken, freeze at -80 ℃, and send on dry ice.
Microbial-extracellular metabolites (cell supernatant)
1) Use a suitable container to collect cell culture fluid or other biological fluids.
2) Centrifuge at 3000g, 4°C, for 15min. Transfer the supernatant carefully to a new sterile centrifuge tube.
3) Centrifuge the isolated cell supernatant at 16000g, 4°C, for 10min (the tip of the gun should not touch the impurities on the bottom side of the tube). Transfer the supernatant carefully to a new sterile centrifuge tube and store at -80°C.
