Methods to preserve individual bacteria and microbiome samples for nucleic acid analyses without altering cellular structure or integrity.

One of the major challenges of the microbiome studies is unavailability of a reliable preservative media for long term storage of microbes that maintains the cellular structure and integrity without causing partial lysis or cellular leakage. Such a preservative would allow for many downstream analyses without compromising DNA and RNA quality. To alleviate these technical challenges, we designed a proof of principle study which included ABRF MMRG (Metagenomics and Microbiome Research Group) Members, representing multi-institutional sites. We formulated a preservative media that was suitable for long term storage of samples, maintained cellular structure of microorganisms, exhibited no detectable DNA or RNA leakage in the solution, preserved DNA and RNA integrity, preserved Gram stain and other stainability (SYBR Green, DAPI, etc) features, and maintained enzymatic cell wall digestion by Metapolyzyme. In this proof of principle study, we preserved three Gram positive (Bacillus subtilis ATCC23857, Enterococcus faecalis OG1RF, and Staphylococcus epidermidis PCI 1200), and one Gram negative (Escherichia coli K-12 substr. MG1655) bacterial strains and evaluated for the preservation competence. For long term storage and to evaluate the features of the preservative solution, microbial reference cultures were grown to early log phase. After storage (2 ys at 4 °C), cells were evaluated using microscopy, centrifuged to check for leakage of RNA and DNA, extracted nucleic acids using various extraction kits, and evaluated on a Bioanalyzer and Fragment Analyzer. This study suggests that the MMRG preservation approach can be used for long-term storage of microbial cells without compromising the morphological and genetic integrity and can be used to enable DNA and RNA extraction efficiency studies. However, further studies are required to evaluate additional microbial strains as well as to verify any genetic changes using an in-depth comparative sequencing approach.