Storing microbiome samples properly ensures accurate analysis by preserving their original microbial composition. Missteps like delayed freezing, improper stabilization, or repeated freeze-thaw cycles can alter the samples, leading to skewed results. Here’s what you need to know:
- Immediate Action: Freeze at -80°C as soon as possible or use chemical stabilizers like RNAlater or OMNIgene.GUT if freezing isn’t feasible.
- Short-Term Storage: Refrigeration at 39°F (4°C) is an option for up to 72 hours but should only be temporary.
- Avoid Freeze-Thaw Cycles: Divide samples into smaller portions to minimize thawing.
- Use Proper Containers: Sterile, leak-proof containers with clear labeling are crucial.
- Quality Control: Regularly monitor storage conditions, inspect samples, and test DNA/RNA quality to prevent degradation.
These steps help maintain sample integrity, ensuring reliable results for microbiome analysis.
De-Risking Microbiome Research and Clinical Discoveries: Sample Collection, Storage and Transport
Preparing for Sample Collection
Getting everything in order before collecting microbiome samples is essential to ensure accurate results. Having the right tools and sticking to established procedures can significantly reduce the chances of contamination.
Materials You’ll Need
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Sterile collection containers
Use screw-cap tubes or vials made specifically for biological samples. These containers provide an airtight seal to prevent contamination and preserve the sample's integrity. Always opt for single-use, sterile containers to avoid cross-contamination. -
Disposable gloves
Wear disposable gloves during the entire collection process, and switch to a new pair between samples. Make sure to wash your hands before putting on gloves and after removing them. -
Waterproof labels
Use clear, waterproof labels along with a permanent marker to jot down essential details like the sample ID, collection date (MM/DD/YYYY), time, and type of sample. -
Preservatives or stabilization solutions
If you can’t process the sample immediately, use solutions like RNAlater, OMNIgene.GUT, or DNA/RNA Shield. These halt microbial activity and preserve nucleic acids, making them especially handy for fieldwork or home collections without access to refrigeration. -
Sample volume
Aim to collect about 0.2–2 grams of fecal material (or a similar volume for other sample types). This amount is ideal for multiple tests and mixes well with preservatives when needed.
Steps for Immediate Handling
Once you’ve gathered your materials, follow these steps to maintain the sample's integrity.
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Work quickly
Seal the container promptly and either add a preservative or cool the sample rapidly. This prevents microbial growth that could skew your results. -
Set up a clean workspace
Use a sterile, dedicated area with single-use tools. Avoid touching non-sterile surfaces, especially if multiple people are involved in the process. -
Keep detailed records
Document everything - collection time, any procedural deviations, and environmental conditions. These notes are crucial for later analysis and troubleshooting. -
Apply quality control
Include quality checks like negative controls (e.g., blank tubes) to monitor for contamination. Regularly review the collection process to ensure high standards are consistently met.
Sample Preservation Methods
Once you've collected your microbiome sample, time becomes a critical factor. The preservation method you choose directly affects the accuracy of your results, so it's important to understand how to keep your sample intact until analysis.
Freezing Samples for Long-Term Storage
Flash-freezing at -80°C is the gold standard for preserving microbiome samples. This method keeps the original microbial community structure intact and prevents overgrowth of fast-replicating bacteria, which could distort your findings.
Why is this method so effective? Research shows that freezing samples at -80°C minimizes changes in microbial diversity and composition compared to fresh samples. This makes it the most dependable option for accurate downstream analysis[2][6]. DNA of high quality - suitable for 16S rRNA analysis - can be extracted from samples stored at this temperature for over 2 years, and even up to 14 years, with only minimal shifts in microbial diversity[4].
Act quickly when freezing your samples. The sooner you can transfer your sample from collection to the freezer, the better. Leaving a sample at room temperature, even briefly, allows bacteria to grow and can skew your results. Long-term storage at -80°C has been shown to preserve microbiota composition for at least 4 weeks to 6 months, with only minor changes, such as slight increases in Lactobacillus and Staphylococcus[4].
One critical tip: avoid repeated freeze-thaw cycles. These cycles can damage microbial cell walls and degrade nucleic acids, leading to the loss of specific microbes and introducing bias into your analysis[1][3]. To prevent this, divide your sample into smaller portions before freezing. This way, you can thaw only what you need for each analysis, keeping the rest safely stored.
Short-Term Refrigeration Options
When immediate freezing isn't an option, refrigeration at 39°F (4°C) can serve as a temporary solution. This slows bacterial growth and fermentation, giving you extra time to transfer samples to long-term storage.
Studies show that refrigeration at 39°F (4°C) preserves microbiota composition for up to 72 hours without significant changes. However, for metagenomics analysis, it's best to transfer samples to -80°C storage within 24 hours[2][4]. Timing is key - some metabolites may begin to degrade if refrigeration exceeds 2 hours[4]. This makes refrigeration a stopgap measure rather than a final solution.
The 72-hour window is helpful for situations like weekend sample collections or limited lab access. But don’t stretch this timeframe - after 72 hours, the risk of introducing bias increases. Consistent temperature is also essential, so store samples in the main compartment of the refrigerator, not in the door, where frequent opening and closing can cause fluctuations.
Refrigeration has one clear advantage: accessibility. Most labs and even home settings have reliable refrigeration, making it a practical option for studies involving participant self-collection. Just remember, refrigeration is a short-term fix, and your ultimate goal should always be to move samples to -80°C storage as soon as possible.
Next, we'll look at chemical stabilization methods for preserving samples when freezing or refrigeration isn't immediately available.
Chemical Preservation Options
When freezing isn't feasible, chemical preservation becomes a reliable alternative for maintaining sample quality. This approach is particularly useful in field studies or remote settings where cold storage simply isn’t an option.
Chemical preservatives stabilize microbial DNA and RNA while preventing microbial community shifts. Timing is critical - adding the preservative immediately after sample collection helps preserve the original microbial composition[3]. Below, we’ll dive into some specific stabilization solutions tailored to different sample types.
Using Stabilization Solutions
One of the most commonly used solutions is RNAlater™, which works by inactivating nucleases to protect nucleic acids. This allows samples to remain stable at room temperature for several days without significant changes to the microbial community[2].
For gut microbiome studies, OMNIgene.GUT is a standout option. Studies show that samples stored in OMNIgene.GUT closely resemble those frozen at -80°C, making it ideal when cold storage isn’t available. It minimizes microbial composition changes during transport and can maintain sample integrity for several days to weeks at room temperature[2].
Another versatile option is DNA/RNA Shield™, which preserves both DNA and RNA while also inactivating pathogens. This makes it a great choice for handling diverse sample types, especially in field conditions where safety is a concern[3].
The process is straightforward: add the recommended volume of stabilizer immediately after collection, mix thoroughly, label the container, and store it as per the manufacturer’s guidelines[3].
For researchers seeking more budget-friendly or field-adapted solutions, ethanol or specialized buffers can also be effective.
Ethanol and DESS Buffer Methods
95% Ethanol is a cost-effective preservation method that can maintain microbial composition for up to eight weeks at room temperature[6]. It halts bacterial growth and metabolic activity immediately upon contact. However, ethanol may selectively affect certain bacterial taxa, so using it consistently across all samples is essential to ensure comparability[6].
Another option is the DESS Buffer, a mix of dimethyl sulfoxide, disodium EDTA, and saturated NaCl. This buffer effectively stabilizes DNA and prevents enzymatic degradation, making it well-suited for fieldwork where refrigeration isn’t available. It’s particularly popular in ecological studies conducted in remote locations[5].
| Preservation Method | Storage Duration | Key Advantages | Considerations |
|---|---|---|---|
| RNAlater™ | Several days | Protects DNA/RNA; widely validated | May alter some microbial profiles |
| OMNIgene.GUT | Several weeks | Closest to frozen samples; gut-specific | Higher cost than simpler solutions |
| 95% Ethanol | Up to 8 weeks | Affordable; long-term stability | May impact certain bacterial taxa |
| DESS Buffer | Extended periods | Easy for field use; prevents degradation | Requires precise preparation |
It’s important to note that chemical preservatives aren’t without limitations. If mismatched to the sample type, they can introduce biases in the microbial community. For instance, Tris-EDTA buffer, though sometimes used for DNA stabilization, has been shown to significantly alter microbial composition and is not recommended for microbiome studies[2].
Unstabilized samples pose even greater risks. Research has demonstrated that samples shipped without stabilization can experience microbial overgrowth, such as an overabundance of E. coli, which can completely distort the original community structure[3]. This highlights why immediate chemical stabilization is vital for accurate downstream analysis.
Selecting the right preservative depends on your sample type and research goals. Gut microbiome studies may benefit from OMNIgene.GUT or ethanol, while DNA/RNA Shield™ offers broader compatibility for diverse sample types. Always consult manufacturer guidelines and recent research to ensure the best choice for your study. Following these methods can help maintain sample integrity and produce reliable results for microbiome analysis.
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Step-by-Step Storage Guide
Now that you're familiar with different preservation techniques, let's dive into the practical steps for storing microbiome samples. These steps are designed to help you maintain the integrity of your samples.
Labeling and Sealing Samples
Start with clear and accurate labeling. Each sample should have a unique identifier that ties it to your study records. Include key details like the sample ID, collection date and time (formatted as MM/DD/YYYY with 12-hour AM/PM notation, e.g., 03/15/2024 2:30 PM), sample type, and initials of the collector. This ensures there’s no confusion later on [1][3].
Keep both physical and digital logs to reduce the risk of data loss. The physical log should travel with the samples, while the digital version stays secure in your lab’s information system. Record extra metadata like the collection location, environmental conditions, and preservation method to ensure traceability [1][3].
Seal your samples securely. Use sterile, leak-proof containers such as screw-cap tubes or cryovials. Tighten the caps firmly but avoid over-tightening. For extra protection, wrap the containers in parafilm or place them in a secondary sealed bag. This double-layered approach helps prevent contamination and leakage that could compromise your results [1][3].
Once your samples are labeled and sealed, maintaining the right temperature becomes the next critical step.
Temperature Control Guidelines
Start temperature control as soon as you collect the samples. For short-term storage (up to 72 hours), keep samples refrigerated at around 39°F (4°C). For long-term storage, freeze the samples at −112°F (−80°C) as quickly as possible. Delays in cooling can cause microbial communities to shift from their original state [2][6].
Avoid freeze-thaw cycles at all costs. Repeated freezing and thawing can degrade microbial DNA and RNA, leading to unpredictable changes in the sample's composition [2][6].
Use digital loggers to monitor storage conditions. These devices provide a continuous record, ensuring samples stay within the required temperature range. For transport, insulated coolers with ice packs work well for refrigerated samples, while dry ice is ideal for maintaining freezing temperatures during longer trips [2][3].
Document temperature readings regularly in your sample log. This not only helps troubleshoot unexpected results but also demonstrates compliance with storage protocols.
| Storage Duration | Temperature | Monitoring Method | Key Considerations |
|---|---|---|---|
| Up to 72 hours | 39°F (4°C) | Digital thermometer | Process samples promptly |
| Long-term | −112°F (−80°C) | Temperature logger | Avoid freeze-thaw cycles |
| Transport | Maintain cold chain | Insulated coolers | Use ice packs or dry ice |
For specialized products like Begin Rebirth RE‑1™, storage requirements are unique. These sachets use the Lyosublime™ delivery system, which keeps them stable without refrigeration. Store them in a cool, dry place, away from direct sunlight, and follow the manufacturer’s guidelines for temperature and humidity to maintain the viability of the prebiotics, probiotics, and postbiotics [7].
Consistency in storage practices is essential to prevent systematic bias.
Troubleshooting and Quality Control
After establishing proper storage practices, the next step is identifying and addressing errors to ensure your samples remain intact. Spotting problems early and applying the right quality control measures can safeguard your sample quality throughout the study.
Common Storage Mistakes
Delayed freezing can compromise your samples. To preserve microbial communities in their original state, freeze samples at −80°C (−112°F) within hours of collection [6][8].
Repeated freeze-thaw cycles are highly damaging. They can lead to cell lysis, DNA and RNA breakdown, and even the loss of key microbial species. To avoid this, divide your samples into smaller portions before freezing, so you only thaw what’s needed for each analysis [1][3].
Using the wrong chemical preservatives or incorrect ratios can disrupt the microbial profile. For example, Tris-EDTA buffer has been shown to significantly alter microbial composition [2]. Stick to validated preservatives like RNAlater or OMNIgene.GUT, and always follow the manufacturer’s recommended concentrations [2][3].
Poor sealing or labeling increases the risk of contamination and misidentification. Always check that containers are tightly sealed and properly labeled during each storage review.
Temperature fluctuations during storage or transport can cause major shifts in microbial composition, especially if stabilizers aren’t used [2].
By addressing these common pitfalls, you can maintain the integrity of your samples. Routine quality control checks, as outlined below, can help ensure these errors don’t go unnoticed.
Quality Control Measures
Effective quality control builds on the storage protocols you’ve already established.
Temperature monitoring is critical. Use data loggers or digital indicators to continuously track storage conditions. For transportation, insulated coolers with proper cooling agents help maintain the required temperature range. Any deviations should be recorded and assessed immediately [1][8].
Regular sample inspections are essential. Look for leaks, cracks, contamination, or damage to labels. Also, check for any changes in appearance or unusual odors. For short-term storage, inspect samples weekly; for long-term frozen storage, conduct monthly checks [1][8].
Comprehensive documentation of storage conditions and handling steps is a must. Record collection times, storage temperatures, preservation methods, and any incidents. Detailed metadata not only aids in troubleshooting but also ensures compliance with protocols [1][8].
Periodic testing of DNA and RNA quality is a proactive way to catch degradation before it affects your study. Testing a subset of samples or using control samples can help identify contamination or systemic issues within your storage setup [1][8].
Here’s a quick summary of recommended quality control checks:
| Quality Control Check | Frequency | What to Monitor | Action if Problems Found |
|---|---|---|---|
| Temperature logs | Daily | Storage temperature range | Document deviations and assess sample impact |
| Sample inspection | Weekly (short-term) / Monthly (long-term) | Container integrity and contamination signs | Isolate affected samples and investigate cause |
| DNA/RNA quality testing | Monthly or per batch | Sample degradation | Exclude compromised samples and review protocols |
If storage errors occur, document them immediately, noting the time, affected samples, and observed impact. Evaluate how these errors might affect sample quality, and consult with laboratory quality assurance personnel or microbiome experts. In some cases, you may need to exclude affected samples from key analyses to maintain the integrity of your results [8].
Specialized products, like Begin Rebirth RE‑1™, follow rigorous quality control protocols. This product undergoes over 50 QA/QC checkpoints during manufacturing, including lot-to-lot testing under both US and EU standards (GMP and HACCP). Additionally, seven of its nine probiotic strains are microencapsulated with a lipid coating, which protects them from stomach acids and digestive enzymes, eliminating the need for refrigeration [7].
Consistency across all samples is non-negotiable. Using the same storage containers, preservation methods, and temperature conditions for every sample helps ensure that any observed differences reflect true biological variation, not inconsistencies in storage.
Conclusion
Storing microbiome samples correctly is the backbone of reliable research and product development. When samples are preserved in a way that maintains their original microbial composition, researchers can confidently identify beneficial strains and assess therapeutic solutions.
Key storage practices include freezing samples immediately at −80°C (−112°F), which remains the most effective method for long-term preservation. This technique ensures microbial communities stay intact over extended periods [4].
A great example of these principles in action is Begin Rebirth RE-1™. This product not only follows proper storage protocols but also undergoes rigorous quality assurance, with over 50 QA/QC checkpoints and strict lot-to-lot testing aligned with US and EU standards. These measures guarantee consistency from production to consumption [7].
By adhering to these storage practices, researchers and developers can elevate the quality of their work. Properly stored samples are essential for advancing our understanding of the human microbiome and creating targeted interventions that support gut health, immune function, and overall well-being. As microbiome science progresses, the storage methods you use today will directly impact the reliability of future discoveries and the success of next-generation therapeutic products.
Investing in high-quality storage equipment, validated preservatives, and thorough quality control systems is not just a best practice - it’s a necessity for improving data accuracy and reproducibility. Whether you’re conducting academic studies or developing commercial products, following these guidelines is a step forward for microbiome science.
FAQs
What happens if microbiome samples aren’t stored correctly?
Improper storage of microbiome samples can seriously affect their quality, leading to unreliable analysis. Exposure to heat, moisture, or delays in freezing can cause the samples to break down, which alters the microbial composition and makes it harder to gather accurate data.
To preserve sample integrity and ensure dependable results, it’s crucial to stick to recommended storage practices. This includes maintaining the right temperature and preventing contamination. Careful handling is key to protecting the quality and credibility of microbiome research or testing.
What’s the best way to preserve microbiome samples: freezing or using chemical preservatives?
Freezing is often regarded as the best method for preserving microbiome samples because it effectively halts biological activity, keeping microbial structures intact. For optimal preservation, samples should ideally be stored at ultra-low temperatures, such as -80°F (-62°C), to reduce the risk of degradation over time.
When freezing isn't feasible, chemical preservatives offer a practical alternative. These solutions are formulated to stabilize microbial DNA and RNA, even at room temperature or under refrigeration. However, their reliability can differ based on the type of sample and the specific microbes being studied. To ensure accurate results, always adhere to the storage guidelines provided with your testing kit or by your laboratory.
Why should you avoid repeated freeze-thaw cycles when storing microbiome samples, and how can you prevent them?
Repeated freeze-thaw cycles can damage microbiome samples by breaking down DNA, RNA, and other biomolecules, which can lead to unreliable analysis results. To prevent this, it's crucial to store samples at a stable, suitable temperature - preferably in a freezer set to -80°F (-62°C) for long-term storage. When you need to access samples, thaw only the portion you plan to use right away and avoid refreezing any leftover material. A practical solution is to prepare single-use aliquots, which eliminates the need for repeated thawing and helps maintain the integrity of the samples.