Integrated and high-throughput method to collect, store, recover, and manage microbial isolates in mini-arrays

Clinical studies of vaccines generally require collections of microbial isolates obtained from various body sites over multiple years. Further, large microbe collections are needed for research due to increasing appreciation for the phenotypic and genotypic diversity among a single microbial species. However, large collections are not generally available due to method limitations. We show a new way to create, recover, and manage microbe collections in 96- or 384-well plates using 50% glycerol at -20°C. Fifty percent glycerol remains liquid at -20°C and permits only the chosen isolates to be reliably sampled without first thawing all other isolates in the plate. Consequently, the glycerol sampling allows integration of microbe collection, labeling, recovery, and storage steps. Creating a microbe collection as an array in microplates reduces physical storage space by 6- or 23-fold with 96-well or 384-well plates, respectively. The array permits direct analysis of the collection with high-throughput assay systems. Further, we show that Streptococcus pneumoniae could be stored for 11 years as a microplate array. Standardized microbe arrays created in microplates with the new method could be easily distributed for studies of microbial structures, genetic diversity, antibiotics, and microbe-host interactions. This integrated method suggests how automated microbe management systems can be created.IMPORTANCEEpidemiologic and microbiology studies require large microbial collections, and the use of microplates could facilitate the creation and management of these collections. However, recovering individual isolates from microplates is manual and tedious. In this study, we demonstrate a simple method for recovering a selected individual isolate from a microplate at -20°C using 50% glycerol. Additionally, we found that Streptococcus pneumoniae could be revived for more than 10 years in microplates. This new method of recovering microbes from frozen microplates could greatly streamline many large-scale epidemiologic studies, particularly those related to pneumococcal vaccine studies. This new method may ultimately automate the collection, management, and storage of microbial isolates.