The accuracy and reproducibility of microbiological research are heavily dependent on the proper collection, storage, and handling of samples. Inadequate procedures can introduce biases and compromise data stability. Therefore, a comprehensive understanding of the sample collection process is paramount in microbiological research.
Consistency: To ensure uniformity and reliability, samples must be collected consistently across different collection points, employing standardized sampling steps and using uniform consumables (reagents and supplies) from the same brand and batch.
Mimicking Natural Conditions: Collected and stored samples should closely replicate the conditions within the organism, preserving the integrity of the microbiome.
Practicality: During the collection process, practicality is essential to facilitate the collection of an adequate quantity of samples with ease, ensuring the experiment's success.
1) Prepare a bedpan and feces container, wear gloves, and wash hands before collecting fresh fecal samples.
2) Immediately place the collected fecal sample in a laboratory freezer for sub-packaging and labeling.
3) Use a sterile tool to collect a sample from the middle part of the feces to avoid surface contaminants.
4) Subpackage the samples in the required quantity for extraction, preferably in anaerobic bags.
5) Store long-term samples in liquid nitrogen and then at -80°C to prevent freezing and thawing.
6) During shipping, we need to protect samples with larger tubes or self-sealing bags and maintain low temperatures with dry ice.
1) Collect samples within 2 hours if possible.
2) If samples need to be collected on different days, do so at the same time to maintain consistency.
1) Remove the entire gut with a sterile scalpel and extract the required gut segment's contents.
Place the contents in the freezer for sub-packaging and labeling.
2) Subpackage in sterile centrifuge tubes (200-500 mg per tube) and store at 4°C. For long-term storage, use liquid nitrogen, followed by -80°C.
Notes: Maintain consistency in the sampling site for samples from the same experiment.
Wash tissue samples in sterile phosphate-buffered saline, use a sterile microscope slide to scrape adhering tissue bacteria, and store at -80°C.
Notes: Ensure consistency in the sampling site.
For throat swab: swab tonsils and throat, dip in sampling solution, and cap.
For nasal swab: insert into nasal passage, rotate, dip in sampling solution, and cap.
Notes: Maintain sterile sampling conditions, and collect within 72 hours and preferably in the morning. Both nasal and throat samples can be collected and mixed.
Wash hands, use a labeled container, collect 5ml, avoid contamination, seal, record the collection time, and store at -80°C.
Notes: Do not store at 4°C due to potential changes.
For mouse samples, use aseptic techniques for collection. Generally, the neck and chest are dissected, the trachea and one side of the lung lobe are ligated. A puncture needle is inserted into the upper end of the trachea, and physiological saline or PBS is repeatedly flushed for 3-5 times. The specific operation is as follows:
1) Equipment required for the operation (except for disposable syringes, the tracheal tube is already sterilized; other equipment needs high-pressure sterilization).
2) Anesthetize and secure the mouse.
3) Remove the hair from the neck and disinfect.
4) Make a midline incision on the neck.
5) Expose and dissect the trachea, and tie a knot near the trachea.
6) Cut half of the trachea at the far end of the knot and insert a tracheal tube. After insertion, tie the tube.
7) Extract 10ml of sterile physiological saline using a 10ml syringe, and inject into the trachea through the tracheal tube. Repeat the aspiration for three times, and place the liquid in a sterile centrifuge tube. Repeat the same operation twice. Each time, about 7 ml can be aspirated. Place it in a sterile centrifuge tube.
8) The collected lavage fluid is filtered through a 0.22um filter membrane and then stored at -80℃. Alternatively, DNA can be directly extracted.
For human samples, use a bronchoscope. Maintain sterility, store at -80°C, and ensure consistent sampling volume.
1) Administer local anesthesia to the oropharynx using 1% lidocaine, followed by an intravenous injection of 5mg of midazolam.
2) Position the patient supine, provide oxygen support, and monitor electrocardiography and blood pressure.
3) Perform bronchoalveolar lavage with a bronchoscope, injecting 1% lidocaine for local anesthesia through the bronchoscope biopsy channel. Insert the bronchoscope into a subsegment of the right middle lobe or the upper lobe of the left lung. Inject 120mL of physiologic saline heated to 37°C, and recover the fluid using a 50mL syringe. Transport the sample to the laboratory promptly at 4°C.
4) After packaging, subject the sample to high-speed centrifugation at 15,600×g at 4°C for 30 minutes. Discard the supernatant and freeze the cell pellet at -80°C for storage. Ensure consistent sample volumes throughout the process.
For EDTA anticoagulation tubes, collect 3-5ml of whole blood, ensuring thorough mixing with EDTA for proper anticoagulation. Store at -80°C (-20°C) and avoid using heparin anticoagulant, which may affect the experiment. During transport, use dry ice. For Separation Gel Vacuum Collection Tubes, place the separation gel at the tube's bottom. After the blood sample coagulates, centrifuge it, creating an isolation layer between serum and blood clots. The serum remains unchanged. Store at -80°C (-20°C).
Avoid subjecting blood samples to repeated freeze-thaw cycles. If samples have been stored for an extended period or have undergone multiple freeze-thaw cycles, communicate in advance to ensure nucleic acid extraction quality. Prolonged storage or multiple freeze-thaw cycles can lead to nuclear rupture, resulting in free nucleic acids in the plasma. Fresh, anticoagulated blood can typically be stored at 4°C for 12-24 hours, but rapidly frozen samples must be stored at low temperatures. Be cautious of repeated freeze-thaw cycles.
Use plastic blood collection tubes to prevent freezing cracks, avoiding glass tubes or overfilling.
During ERCP surgery, collect bile from the common bile duct. Use a strictly sterile side-view endoscope, avoiding any suction operations before inserting the endoscope from the mouth into the duodenum and then into the common bile duct. Prior to injecting the contrast agent, use a sterile sphincterotomy catheter through the endoscope's operating channel to extract 3-5ml of bile from the common bile duct. Immediately place the collected sample in a sterile sputum cup and store it at -80°C.
For Common Soil Samples, determine the sampling range according to the research purpose. Sterilize and disinfect sampling tools in advance. Remove surface debris and use an ethanol-flamed shovel to dig 5-20cm underground. After eliminating visible impurities, pass the soil through a 2mm sieve. Collect and combine samples from three or more points, with each tube holding 3g-5g. Store in sterile centrifuge tubes and transport to the laboratory at 0°C or below for DNA extraction. If immediate experimentation isn't possible, store at -80°C. Using 5 ml EP or 15ml, 50ml centrifuge tubes is strongly recommended to load soil samples. Seal with sealing film to prevent sample cross-contamination.
For Rhizosphere Soil Samples, Sterilize and disinfect tools in advance. Collect rhizosphere soil 20cm deep in a 50mL sterile tube. Immediately store it in liquid nitrogen, sieve out plant roots, animal debris, and other impurities through a 20-mesh sieve. Package in sterile centrifuge tubes, with 3-5g per tube. Seal and store at -80°C for future use.
Place the studied object in a pre-sterilized container, add PBS buffer, and agitate to detach microorganisms from the object's surface. Then, extract microorganisms directly from the PBS buffer or filter the buffer through a filter membrane before DNA extraction.
In summary, key elements in 16S analysis for research articles include proper collection, storage, and handling of samples, which are crucial for accurate and reproducible microbiological research. If you have any questions about sample collection, we are here to help. In the next blog, we will discuss 16S analysis. At our Boston lab, we offer comprehensive multi-omics services, including 16S, metagenome, and Microbiome+Metabolome, to support your research.