Proper collection of human stool samples is critical to the success of microbiome research. Recent technological advances have enabled scientists to explore the microbiome in ways never before possible. Initial findings show significant promise for improving human health through microbiome science. While the microbiome may hold secrets to a host of human conditions and ailments, without properly collected samples this potential won't be realized. Because the gut is a particularly rich source of microbes, stool samples are excellent biospecimens for answering research questions.

To obtain high quality data, proper preservation of biospecimens is critical. Preserving samples by freezing them prior to shipment is currently the most validated approach for microbiome research and Second Genome Solutions can provide comprehensive instruction and materials for collection of frozen stool.

Frozen collection and transportation can be challenging for some research teams. Second Genome Solutions has also validated a chemical preservative for collection and stabilization of nucleic acids for microbiome research. The benefit of this method is that the samples are collected and can be stored at room temperature. The elimination of freezing simplifies collection for the study participant and minimizes the variation that can be associated with handling of frozen specimens. If you need assistance with stool sample collection please contact our team.

Samples that remain at ambient or warm temperatures during storage and transport are at risk of continued growth of bacteria within the sample. This growth can significantly impact community analysis. The research team at Second Genome Solutions continues to test a variety of methods for preserving biospecimen quality.

Mice and other rodents share many similarities with the human gut microbiome at high taxonomic levels, making them useful model systems for evaluating host microbiota interactions applicable to human biology. Obtaining a clean, uncontaminated sample should be a best practice for all researchers engaging in animal microbiome studies. There are special cages that can be purchased to collect the feces away from the cage floor, however, these solutions can be expensive and may be overkill for initial studies of this type.

We describe two different approaches for collecting animal feces from research animals. In order to maximize the study group replicates, efforts should be made to collect individual animal samples. If your animals are small or specimens for multiple assays are needed, it may be necessary to conduct a series of collections from the animals throughout the day. To maintain consistent sampling across all replicates, a daily collection schedule is recommended. At the time of DNA/RNA isolation, daily samples can be pooled for processing and analysis.

Transfer the animal to a disinfected cage. If space is a constraint, a disinfected or sterile tray or pan with walls high enough to contain the animal can be used instead. Properly disinfect the cage or tray by using a sodium hypochlorite solution, or other oxidative disinfectant, at a concentration of 5000-6000 parts per million. Once placed in the fresh cage, the animal will typically produce fecal pellets within a few minutes. Label a sterile collection tube, such as BD Falcon or Eppendorf tube, with corresponding animal ID. To ensure sufficient DNA yield, it is recommended that approximately 200mg of feces is collected from each animal; roughly 6 to 10 pellets from an average sized mouse. Once the animal has produced sufficient fecal material for the study, open the labeled collection tube and transfer the fecal pellets via sterile forceps or sterile gloved hands to the tube. Snap freeze, place on ice or inside freezer immediately. After completing, place the animal back in its original cage. Between each animal's collection, disinfect collection cage, as well as any forceps or other instrument used. Prior to taking the next animal from its cage, gloves should be changed.

With fresh, sterile gloves, remove one animal at a time from its cage. Position the animal with a sterile, labeled Eppendorf tube directly under its tail. You may also place a sterile petri dish on the bench just below where the clean catch is being performed. Gently squeeze the animal. Fecal pellets are typically excreted promptly. The fecal pellets can be collected directly into the Eppendorf tube, or allowed to fall into the petri dish. If pellets fall to petri dish collect with sterile forceps and add to the collection tube. The amount of feces an animal can comfortably produce at any given time is limited, so it might be a good idea to plan multiple collections in a day and pool the different collection points into a single sample. After collection is complete, return animal to its original cage. Snap freeze, place on ice or inside freezer immediately. Please be sure to change gloves after each animal to avoid any cross transfer of the microbiota. This method is can be useful when studying animals that may have loose stool and collection from a cage or tray is challenging.

Both of these methods are fast, non-invasive, and easy to carry out. However, the clean catch method offers the least risk of contamination and/or cross contamination and minimizes the need for repeatedly disinfecting or sterilizing a large area such as a cage. Regardless of method selected, it is important to always follow proper aseptic practice, including frequent changing of gloves and lab coat, wearing a mask or hair tie if needed, and routinely disinfecting your bench top.

Swab Method

Moisten sample collection swab, such as the Sterile Catch-All Sample Collection Swabs, in sterile SCF-1 solution or lysis buffer. Stretch skin area to be swabbed, approximately 4 cm2, with one hand. In the other hand hold the swab so that the shaft is parallel to the skin surface. Rub the swab back and forth approximately 50 times, applying firm pressure to skin. Immediately after swabbing, swirl each swab in a 2 ml labeled collection tube containing 300 ul lysis buffer with beads, such as MOBIO Laboratories MicroBead Solution. Press the swab sponge against the tube wall multiple times for 20 seconds to ensure transfer of bacteria from swab to solution. Freeze samples until ready to process. To minimize sample cross-contamination, the person sampling the individuals should wear a fresh pair of sterile gloves for each collection.

Cup Scrub or Cylinder Sampling Method

Label sample collection tube. Place a sterile cylindrical object open at both ends on the skin collection area. Holding the cylinder in place, dispense the neutralizing sample solution into the cylinder using a repeat pipette with a sterile tip. Place a sterile rubber policeman or spatula in the cylinder and rub the skin for 1 minute. This rubbing motion will suspend the skin microorganisms into the neutralizing solution. Transfer the solution to the sterile sample collection tube using a pipette. Using fresh neutralizing solution, repeat this process at the same site twice more and pool samples for processing. Freeze samples until ready to process. Label sample collection tube. Place a sterile cylindrical object open at both ends on the skin collection area. Holding the cylinder in place, dispense the neutralizing sample solution into the cylinder using a repeat pipette with a sterile tip. Place a sterile rubber policeman or spatula in the cylinder and rub the skin for 1 minute. This rubbing motion will suspend the skin microorganisms into the neutralizing solution. Transfer the solution to the sterile sample collection tube using a pipette. Using fresh neutralizing solution, repeat this process at the same site twice more and pool samples for processing. Freeze samples until ready to process.

Tape Stripping Method

This method involves the repeated application and stripping of adhesive tape to the skin surface. Begin with a clean tape specifically designed for skin sampling, such as D-SQUAME Skin Sampling Discs. Identify an area of skin for sampling approximately 2cm2. The most common area selected for tape stripping experiments is the upper buttock skin just under the waistband. Using a new disc each time, tape-strip the area at least 15 times until the skin has a glistening surface. A superficial wound may result. The study participant should heal from this within one week. Place all collected tapes on a laminated storage card labeled with patient ID and freeze samples until ready to process. This method involves the repeated application and stripping of adhesive tape to the skin surface. Begin with a clean tape specifically designed for skin sampling, such as D-SQUAME Skin Sampling Discs. Identify an area of skin for sampling approximately 2cm2. The most common area selected for tape stripping experiments is the upper buttock skin just under the waistband. Using a new disc each time, tape-strip the area at least 15 times until the skin has a glistening surface. A superficial wound may result. The study participant should heal from this within one week. Place all collected tapes on a laminated storage card labeled with patient ID and freeze samples until ready to process.

Microbiota collection from Mice using Swab Method

Shave mice on the dorsum, 24 h prior to the collection of skin microbiota. At time of collection, moisten a collection swab, such as the Sterile Catch-AllTM Sample Collection Swabs, in sterile SCF-1 solution or lysis buffer. Gently press the swab against the exposed skin of the mouse and rubbed back and forth. Immediately after swabbing, swirl each swab in a 2 ml labeled collection tube containing 300 ul lysis buffer with beads, such as MOBIO Laboratories MicroBead Solution. Press the swab sponge against the tube wall multiple times for 20 seconds to ensure transfer of bacteria from swab to solution. Freeze samples until ready to process. To minimize sample cross-contamination, the person sampling the individuals should wear a fresh pair of sterile gloves for each collection.