A difficulty associated with collecting fecal samples, as opposed to other biological specimens like blood, urine or saliva, is that specimens often cannot be collected precisely on demand. This presents a noteworthy logistical challenge. If a fresh sample can not be processed immediately, then the gold standard for metagenomics, other applicable –omics techniques, and bacterial cultures is collection without a preservation agent and freezing immediately at -80°C. This protocol is not often feasible as most collections occur at home and need to be stored in the interim before being transported to the laboratory. Home freezers are not usually temperature-stable environments, which may affect the quality of the sample. A consideration with at-home fecal collections is the personal aversion or ‘yuck factor’ specific to this sample type, to the extent that donor recruitment and continued participation in multiple time-point collections or longitudinal studies can be an issue. There is an understandable aversion to storage of stool samples in a personal fridge or freezer beside the family ice cream.

Immediate processing of a fresh fecal sample is often the best scenario for all types of microbiome studies, including volatile short-chain fatty acid profiling. Without a stabilization agent, samples held at RT can experience major changes in taxa abundances after anywhere from 30 minutes to two days.

Below, both frozen and room temperature (RT) collection options are detailed.

Available collection methodologies, discussed in turn below, include:

• Short-term refrigeration
• Freezing or cryopreservation
• Tris-EDTA-buffer (RT)
• RNA later (RT)
• Commercial room temperature transport vials (RTTVs) (RT)
• Swabs with or without preservation medium (RT)
• 95% Ethanol (RT)
• Flinders Technology Association (FTA) cards (RT)
• Fecal occult blood test (FOBT) cards and Fecal Immunochemical Test (FIT) tubes

Low Temperature options

1) Refrigeration of a sample can bridge the gap between collection and processing by slowing the rate of bacterial growth. If processed within the first 24 hours, samples are suitable for metagenomics analysis. Although cooler temperatures do slow the fermentation reactions important for metabolomics analysis, even refrigeration conditions should not exceed 2 hours and some loss of volatile metabolites should be expected.

2) The gold standard for long-term storage of fecal samples is cryopreservation at -80°C. When compared to samples processed within 30 minutes of egestion via 16S rRNA gene sequencing, there was no significant change observed in microbiota composition at 4 weeks or 6 months. Furthermore, DNA of adequate quality for analysis has been recovered from samples stored for more than 14 years, albeit with some shearing. Aliquoting samples prior to freezing is advisable to avoid multiple freeze-thaw cycles, also known to shear genetic material and induce changes in taxa. Rapid freezing techniques, such as employing liquid nitrogen, provide higher quality samples for metatranscriptomics and proteomics analyses. A disadvantage to transporting frozen samples is the logistics of preventing thawing en route and the costs associated with cold-chain protocols.

Room Temperature options

3) Tris-EDTA buffer stabilizes genetic material at RT and can be used for 16S rRNA microbiota profiling and shotgun metagenomic sequencing, but there is some concern that microbial profiles may not reflect the original microbial community. This method is not well suited for any of the other –omics techniques, nor for bacterial cultures.

4) Although RNAlater was developed specifically to preserve RNA, genomic DNA can also be extracted. DNA quality and quantity are sufficient for metagenomics analysis, but typically yields are lower than other methods. Samples can be stored at RT for up to 6 days with minimal bias in the mRNA profile. Storage for longer periods ought to be avoided, since stability is impaired after 2 weeks. Neither protein-based techniques nor metabolomics are feasible.

5) There are a number of RTTV collection and stabilization devices on the market, designed to preserve the microbial DNA in fecal samples. These kits have user-friendly designs and can be sent in the mail, and are amiable to high-throughput technologies. The stabilization buffer lyses cells to protect the genetic material, rendering any proteomics, metabolomics or culturing techniques infeasible. Of note, studies have shown both Faecalibacterium and Bifidobacterium tend to be inadequately detected, which may bias results. These kits also tend to be more expensive than other methods.

6) Fecal swabs are user-friendly, cost effective, and mailable. They can be combined with other preservation methods or dried. Commercial swabbing kits, combined with media (i.e. Cary-Blair medium), are especially well-suited for culturing enteropathogens. Despite the low quantity of collected sample, they can be used for most –omics studies, although Gram-negative bacteria tend to be over-represented. Fecal swabs without preservative media often show an abundance of aerobic species and are not well-suited for other –omics studies, nor for culturing.

7) Fecal samples stored at RT in concentrations of 95-99% ethanol can yield DNA/RNA quality sufficient for metagenomics and metatranscriptomics analyses. Although cost-effective, there are a number of drawbacks. Ethanol quickly evaporates, and is both volatile and flammable; as such it is considered a hazardous good and is costly to send in the mail. Furthermore, ethanol is not agreeable to culturing methods.

8) FTA cards stabilize genetic material sufficient for a number of downstream analyses, but DNA quantity tends to be very low. In a comparative study by Rob Knight and colleagues, FTA cards demonstrated a high correlation of OTU abundance profiles when compared to fresh fecal samples. Curiously, FTA cards tend to collect increased bacterial taxa compared with other methods, whether by improved lyses of Firmicutes in the card matrix, or possibly contamination.

9) FOBT and FIT were designed to detect blood in stool, which is one of the early-onset markers for colon cancer. The dietary modifications prescribed prior to the FOBT test may affect results, since diet is known to influence gut microbiome composition. Although the use of these collection devices as a diagnostic test presents an opportunity to use a large cohort size, there are some drawbacks. Altered taxa abundances up to the phylum level have been observed, as well as issues regarding technical reproducibility and stability. Other studies have found the FOBT cards feasible for microbiome studies, especially large epidemiological studies requiring low-cost collection options. FTA, FOBT and FIT can be easily transported in the mail.

Making your choice

With the importance of studying the gut microbiome in health and disease, it is imperative to collect high-quality fecal samples capable of providing meaningful data. With the complexity associated with this research, there is often a necessity to interrogate larger cohorts. Studies employing the same collection device and study protocols can more readily be compared with each other or combined in a meta-analysis. Furthermore, collections of high-quality, properly stored samples can potentially be used in multiple studies or to interrogate different end-points.

The choice of fecal collection device is not a trivial one, and the team at Microbiome Insights would be glad to answer any of your questions. Our scientists and laboratory technicians have had the benefit of working with a diversity of collection devices and are able to offer advice on what might suit your research question and downstream analysis needs the best.

Note: Neither Microbiome Insights, Inc. nor this author has any financial interest in recommending a particular type of collection device.

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