Preservation of Microbial Pure Cultures and Mixed Communities

Abstract

Microorganisms are valuable and irreplaceable resources for scientific research and biotechnological innovation and should be safeguarded. Therefore, systematic preservation of isolated pure cultures, enriched mixed cultures, or environmental samples should become an integral part of good research practice. Cryopreservation of biological material is a low-tech, widely applicable way of long-term and stable storage. Its success is mostly dependent on the cryoprotective agent, used to protect cells from mechanical injuries due to ice formation, the stability of the freezing temperature, and the correct manipulations before and after storage. Although cryopreservation success can be organism dependent, the protocol described here proved successful for various fastidious pure and mixed cultures when frozen at À80 C using 5% (v/v) dimethyl sulfoxide as cryoprotective agent. Numerous parameters of the protocol can be changed or optimized, and guidelines are given to develop a custom-made cryopreservation protocol.

Development of Microbiome Biobanks – Challenges and Opportunities

“…Kerckhof et al [11] evaluated a cryopreservation protocol for a methanotrophic coculture, an oxygen-limited autotrophic nitrification/denitrification biofilm, and fecal material from a human donor, and succeeded in preserving both community structure (composition and abundance of taxa) and functionality of microbiomes. Vekeman and Heylen [12] described methods for the cryopreservation of mixed communities but only at -80°C and not at ultra-low temperatures.…”

Cryopreservation and Resuscitation of Natural Aquatic Prokaryotic Communities

“…We have adapted the cryopreservation procedure developed by Vekeman and Heylen (2017) for the cryopreservation of single strains or mixed cultures to cryopreserve natural aquatic communities. This previously published protocol had been applied by the authors exclusively to laboratory cell cultures grown in high cell densities (>10 8 cells/mL, Vekeman and Heylen, 2017 ) while cell densities in marine bacterial communities range typically from 10 5 to 10 6 cells/mL ( Kirchman, 2008 ). For this reason, but also to minimize the carryover of substrates to new culture media, a step to concentrate the cells present in aquatic samples before cryopreservation was added.…”

“…This volume was subsequently transferred into previously prepared 2 mL tubes with 0.5 mL cryoprotectant containing 10% (v/v) sterile-filtered DMSO (ACS reagent, 99.9%, Sigma-Aldrich, St. Louis, MO, United States) in inorganic ASW, equilibrated at 4 • C (DMSO final concentration: 5%). Next, the filters were completely immersed into the medium with cryoprotectant and resuspend cells and the tubes were kept at 4 • C for 15 min for equilibration (Vekeman and Heylen, 2017). The tubes were then flash-frozen in liquid nitrogen and transferred to −80 • C for long-term storage.…”

Dissimilatory nitrogen reduction in intertidal sediments of a temperate estuary: small scale heterogeneity and novel nitrate-to-ammonium reducers

“…All isolates were preserved at −80°C as described previously (Vekeman and Heylen, 2015 ). In short, isolates obtained from defined media were preserved in 10% DMSO prepared with NSW.…”