New Method for Preserving Gut Microbiome Samples

The gut Microbiome plays a crucial role in our health. It consists of various microorganisms living in our intestines, and they produce substances that impact our overall well-being. Issues like intestinal diseases, diabetes, and heart problems can be linked to the condition of our gut microbiome. Therefore, it is important to find effective ways to study it.

One key aspect of examining the gut microbiome is how we collect and store samples. The usual method involves freezing Stool Samples right after collection. This is because the makeup of the microbes in stool can change quickly if left at room temperature. However, freezing requires special equipment, which isn’t always convenient, especially for collecting samples in places like jungles. As a result, researchers have been looking into methods for storing stool samples without the need for freezing.

#Current Collection Methods

Various commercial kits have been developed to store stool samples at room temperature. Some have shown promise in keeping the microbiome stable, but there is no method that protects both the microbes and the substances they produce (Metabolites) at the same time. Many of the current kits also contain high amounts of salt, making them unsafe for use outside of a lab and complicating the measurement of metabolites.

One traditional method of Preservation is Drying. This method reduces water content, which can help stop the growth of harmful microbes. While some bacteria can survive in dry conditions, most cannot thrive, making drying a reliable method. In the past, field biologists have used drying for DNA samples, and studies have indicated that this method is effective.

However, there is concern that drying might lead to the loss of certain important volatile metabolites. These metabolites, including short-chain fatty acids produced by gut microbes, are essential for various health aspects. Yet, some research has indicated that the concentrations of these metabolites can remain stable even after freeze-drying, suggesting that drying may also work for stool samples.

#Study Purpose

This study aimed to examine a method of preserving stool samples using silica gel, a common drying agent. The goal was to see if this method could effectively maintain both the microbiome and metabolome profiles at room temperature.

#Methods

To test different methods of stool preservation, various storage techniques were applied. Here are the key methods used in the study:

1. Control Method: Stool samples were immediately frozen with liquid nitrogen after collection.
2. Room Temperature Control: Samples were stored at room temperature without any preservation.
3. FTA Card Method: Samples were placed on an FTA card to dry before storage at room temperature.
4. RNAlater Method: Samples were stored in a solution that stabilizes RNA at room temperature.
5. Silica Gel Method: Samples were placed between silica gel plates to absorb moisture and then stored.

Stools were collected from ten participants, ensuring they were healthy and not taking specific medications. The samples were stored for seven days, and some were tested after fourteen days for longer-term preservation.

#Sample Extraction

The next step involved extracting DNA and metabolites from the samples. The control and room temperature samples were dried using a special machine, and then the stool was mixed with solutions to help isolate DNA and metabolites. After extraction, DNA sequencing and metabolite measurement were performed to analyze the samples thoroughly.

#Results

#Qualitative Analysis

The first analysis focused on whether different methods could effectively detect bacteria and metabolites in stool samples. In total, 241 types of bacteria, 458 metabolic pathways, and 316 metabolites were identified. Comparing these findings with the control method showed that most techniques preserved a good number of microbes and pathways. However, the FTA card method did not retain many metabolites.

Among all methods, the silica gel technique had the highest count of preserved items, suggesting it was the most effective option.

#Quantitative Analysis

Next, the researchers looked at how accurate and consistent the results were across different methods. Precision indicates how close the measurements are to one another, while accuracy measures how close the results are to the true values.

For microbes and metabolic profiles, the silica gel method showed high precision and accuracy compared to other methods. The FTA card method had lower accuracy, particularly for metabolites, which indicated that it may not be suitable for preserving metabolomic data.

#Storage Method Comparisons

The researchers also examined how different methods affected the count of specific microbes, pathways, and metabolites. Although varying results were seen between the methods, some significant differences were observed, particularly with the FTA card and RNAlater methods, which did not perform as well.

When comparing rankings, the silica gel method maintained a strong correlation with the control method across various microbes and metabolites. This suggests that using silica gel for sample preservation could yield reliable and comparable results in future studies.

#Limitations

While this study highlighted the effectiveness of the silica gel method, it also had limitations. The analysis was complex, considering many different variables, which could lead to issues with multiple comparisons. Additionally, all participants were healthy, so more research is needed to see if these methods hold for sick individuals.

#Conclusion

Drying stool samples using silica gel can effectively preserve the gut microbiome and metabolites. This technique offers a practical alternative to freezing, making it more accessible for research. Further studies with larger groups will help confirm these findings and explore applications for other populations.