Identifying effective cryoprotection agents for non-model bacterial species

ABSTRACT Host-associated bacteria live amongst eukaryotes within varied niches and form relationships ranging from facultative to obligate. With advancement in the studies of such symbiotic associations, fastidious bacteria are increasingly becoming targets for genetic manipulation. However, there are limited resources for screening possible agents, enabling in vitro culturing and storage of these microbes. In this study, we present a simple protocol for optimizing cryopreservation of non-model organisms in laboratory settings using conventional chemicals. Our initial motivation for this observation was to discover a cryoprotection agent for independently cultured Mycetohabitans spp., fungal endosymbionts. We tested several common bacterial cryoprotection agents like glycerol, bovine serum albumin (BSA), and dimethyl sulfoxide (DMSO) over an ultralow freeze-thaw cycle to determine an adequate method of cryoprotection for assorted bacteria. We observed different recovery rates across bacterial species and cryopreservation methods, and identified cryoprotectants that reliably resulted in viable bacteria for each of the strains tested. We present this as a resource for those working with other fastidious and host-associated bacteria that may be missing effective cryopreservation methods. IMPORTANCE The ability to cryopreserve bacteria is important for optimizing laboratory procedures, preserving strains that have been genetically manipulated, and growing fresh cultures of microorganisms without in vitro evolution from serial subculturing. There are several known cryoprotection agents of bacteria, but there are limited accessible studies that collect these together and screen them for effectiveness with new bacteria studied in laboratory settings. With several fastidious and host-associated microorganisms emerging as new model systems, we aim to generate a resource for determining long-term storage solutions for novel organisms of interest.