Single cell bacterial cloning, used to isolate and propagate individual bacterial cells, is an important research tool across a wide range of scientific disciplines. In microbiome research, the isolation and cultivation of new bacterial species could help uncover new metabolic pathways or regulatory mechanisms that can aid in the development of new therapeutics. In genetics, studying bacterial mutants or strains enables researchers to understand the evolutionary significance of certain genetic modifications. For instance, it can be used to determine resistance mechanisms of antibiotic-resistant bacteria, which can be useful in drug development. Additionally, in biotechnology and industrial sectors, bacterial isolation can be used in strain development to create engineered strains that have desirable traits (i.e., enhanced protein production) for applications like biofuel production, biopharmaceuticals, and bioremediation.

Traditional methods for single cell bacterial isolation involve manual techniques and selection based on dilution-to-extinction or streaking on agar plates but quickly reach their limits in terms of clonality, throughput, contamination risk, and automation capability. And even with automated colony picking, the method contains a lot of time consuming steps., Cells must first be streaked onto a selective agar medium, and after waiting for growth, they can then be picked and transferred into liquid culture.As a result, these challenges have prompted the development of more advanced and efficient technologies.

Introducing CYTENA’s Advanced B.SIGHT Microbial Isolation Platform

The B.SIGHT is an advanced microbial isolation platform that overcomes the challenges by automating the process and accomplishing the isolation of microbes in one step. The platform utilizes state-of-the-art optical microscopy to perform cell isolation directly from liquid media and CYTENA’s Single-Cell Dispensing technology to deposit cells into liquid media in multi-well plates, making the process more efficient and less labor-intensive. This second-generation platform enables rapid and efficient isolation of a wide range of microorganisms such as E. coli and yeast strains, including Pichia. The instrument has the flexibility to accommodate a wide range of multiwell plates (96-, 384- and single-well plates) with heights up to 27 mm, enabling the use of low-profile deep well plates.

Simply pipette the sample into the reservoir of a disposable, single-use EASY.ON cartridge, which have been precisely engineered by microfluidic experts to ensure cell viability with the gentlest handling. The EASY.ON cartridge is then mounted and secured with a magnetic flap for quick and easy loading. The ability to dispose of the single-use cartridges eliminates the risk of cross contamination between samples. The platform includes a dispenser chip that rapidly generates 50 pL microdroplets from the sample inside a disposable cartridge. High-resolution optical microscopy and real-time image analysis effectively isolates single cells according to set parameters such as size, roundness and even fluorescence intensity. This advanced functionality significantly improves the selection rate of droplets containing individual cells, to ensure purity.

The B.SIGHT workflow offers a rapid and dependable method for single-cell isolation following transformation. It achieves this with fewer plates compared to traditional colony picking robots while generating the same number of clones. This streamlined approach can be applied to various applications, including the generation of mutant libraries and the development of cell factories with new pathways.

The key features of the instrument include:

• Dual-channel high resolution microscopy: The device is equipped with an innovative and dual-camera system that can efficiently and rapidly isolate cultures of both fluorescent and label-free bacteria, either directly into liquid culture or agar.
• High-speed isolation of cells: Process a 96-well plate in 3 minutes and a 384- plate in under 8 minutes.
• High cell viability: Fast, gentle, label-free isolation directly into liquid culture or agar maintains high cell viability.
• Documented isolation: Nozzle images confirm the clonality of dispensed single cells. Images of the dispensed single cellare stored on the hard drive, providing data for documentation or downstream analysis.
• Intuitive and easy-to-use software: The software enables users to easily design their plate setup and see in real time how each cell is sorted through the nozzle of the cartridge and dispensed.
• Automation ready: The lid can be programmed to open and close automatically for a seamless integration with a robotic arm plate carrier.

The B.SIGHT device, with its compact design, can be operated within an anaerobic chamber, facilitating the isolation of obligate anaerobic bacteria. It offers the convenience of control from outside the chamber by connecting a monitor, keyboard, and mouse, or through a remote-control terminal. In microbiome research, specifically for the cultivation of human gut bacteria, the B.SIGHT’s Single-Cell Dispensing workflow substantially reduces the time required to obtain pure cultures compared to traditional agar plate methods. Importantly, this accelerated workflow maintains comparable isolate diversity and relative abundance coverage, ensuring reliable and efficient microbiome analysis. Additionally, the B.SIGHT has demonstrated the ability to capture microbial species not detected through traditional methods, allowing researchers to uncover previously uncharacterized or overlooked microbial species to achieve a deeper understanding of microbial diversity.

Researchers can also easily harness the power of single-cell genomics for uncultivated bacteria with the B.SIGHT platform. The advanced Automated Offset Compensation (AOC) system can accurately dispense single cells in picolitre droplets directly into the center of the well in 96- or 384-well PCR plates, circumventing the need for manual droplet alignment at target substrate positions. This enables downstream miniaturization with lysis buffer volumes of less than 1 μL for single-cell whole genome sequencing, thereby reducing costs.

With its seamless integration into automation workflows, the B.SIGHT workflow can be easily scaled up, delivering cost-effective and high-throughput capabilities to obtain reliable and reproducible results. Researchers can streamline and expedite their workflows, effectively saving valuable time and resources. The B.SIGHT can open new possibilities for microbiological research, empowering researchers to gain deeper insights and accelerating microbial product discovery and development projects.

For more information on how the B.SIGHT Microbial Isolation Platform can transform your workflow, please visit:

About the Author

Dr. Hussein Hamzeh, Global Product Manager for single-cell dispensers at Cytena

Dr. Hussein Hamzeh combines scientific expertise with a strong commitment to innovation and product development. He holds a Ph.D. from the University of Bonn and conducted his research at The Max Planck Institute for Neurobiology of Behavior – Caesar, specializing in biophysics and biomedicine. Dr. Hamzeh’s scientific curiosity led him to participate in captivating summer excursions in Woods Hole, USA, where he joined Prof. U.B. Kaupp in unraveling cell signaling during chemotaxis. Before this, he served as a research engineer at CNRS, France, driven by his passion for innovation, after obtaining his M.Sc. in Applied Physics from the Lebanese University in Beirut.