Accelerating microbiome research

Cultivation using the classical agar plate method is used to study microbial communities. However, this method is time consuming and adds up to 21 days to go from microbiota to pure isolates. Single-cell isolation using the B.SIGHT allows high-throughput, label-free sorting of the smallest bacteria and accelerates the workflow by more than 5-fold.1 This device can efficiently separate cultures of both fluorescent and unlabeled bacteria, including complex samples.2

A microbiota sample, such as stool, can be suspended in anaerobic PBS supplemented with peptone (0.05% wt./vol.), l-cysteine (0.05% wt./vol.) and dithiothreitol (DTT) (0.02% wt./vol.) to obtain a 10-fold dilution (wt./vol.). The bottle containing the suspension must be flushed with an anaerobic gas mixture (89.3% N2, 6% CO2, 4.7 H2) prior to closing the lid. After vigorous manual shaking to re-suspend the sample and sterilizing the rubber stopper by flaming twice with alcohol, the faecal slurry can be diluted another 10-fold by rapidly transferred the mixture into another Schott bottle containing the same anaerobic medium using a syringe that has been flushed previously with the anaerobic gas mixture. The diluted anaerobic faecal dilutions can then be brought into the anaerobic workstation and further diluted with filter-sterilized anoxic PBS to a dilution of 10-8. The final dilution is filtered (0.2 μm) to remove any debris that may clog the nozzle of the B.SIGHT cartridge.2

Thanks to the instrument’s ability to generate extremely small 50 pL droplets, the precise dispensing of single bacteria into PCR plates can be combined with the I.DOT, our contact-free low-volume liquid handling platform, to enable cell lysis in only 350 nL. This miniaturization scales down the amplification reaction by 20 folds, resulting in significant cost savings. Downstream sequencing of the 16S rRNA gene enables taxonomic classification of the isolated bacterial cells. Since all steps are performed in standard microwell plates, this workflow can easily be scaled up to analyze thousands of individual organisms to gain insight on the composition and heterogeneity of complex microbial samples without the need for cultivation.