A new generation of microbiological research methods has been revealed that single cell level biotechnology "shows its skill"

Microorganisms play a very important role in the earth ecosystem They are the main driving force of the global biogeochemical cycle and are related to almost all multicellular life forms (including plants, animals and humans), so it is very important to understand and study microorganisms However, the traditional research methods of microbiology are facing many challenges Studies have shown that even in the same population of microbial cells, there may be significant differences in gene transcription and translation, protein activity, and metabolite abundance, indicating that there are multiple levels of heterogeneity between microbial cells At the same time, the traditional microbiological research methods need to re culture the studied microbial objects in the laboratory, and then study the pure cultured microbial population, which often results in the laboratory research results can not truly reflect the original state of microbial cells in nature, so it is urgent to develop new in-situ research methods In addition, there are only a few microorganisms in nature that can be cultured in the laboratory, and there are still a large number of microorganisms that can not be explored and studied by traditional methods Traditional research methods of microbial physiology (picture source: Reference 1) Under such a background, the emergence of single cell scale Microbiology provides a new strategy and technical thinking for solving these important challenges in microbiology research, which is expected to help researchers more intuitively and deeply understand the internal state of each cell and its physiological and ecological functions in nature In order to distinguish these novel methods from the traditional methods, the concept of next-generation physiology approaches emerged, which is independent of the prior knowledge demand of microbial community genetic composition and focuses on cell function They do not need to be cultured in the laboratory and are non-destructive, so microbiologists can bridge the gap between the separation fields in the history of microbiological research https://doi.org/10.1038/s41579-020-0323-1 On February 13, nature's Nature Reviews Microbiology published a review entitled "next generation physics approvals to study microbiology function at single cell level" In this review, the researchers first discussed the general concept of the new generation of microbial physiological research methods, and then described in detail the existing technologies to study cell phenotype without destroying the research cells, and discussed how to combine these methods with cell classification technology and a series of powerful downstream applications Research methods of new generation microbial physiology (picture source: Reference 1) It is worth noting that this review describes the existing technology of non-destructive cell phenotype from three aspects: unlabeled method, stable isotope detection method with matrix or heavy water and base simulation detection method Non-invasive optical microscopy and Raman microscopy are used to characterize the morphology of single cells and observe the chemical composition of cells, mainly to observe the information of temporary characteristics, but the time-resolved analysis of living cells also provides the observation of cell dynamic process The phenotype observation of light microscope includes the formation of spores, the storage and distribution of compounds, the process of cell division, the behavioral response of cells to external stimuli (such as, oxygen, chemotaxis, magnetic or phototaxis, etc.) or the occurrence of intrinsic autofluorescence from cofactors, pigments or vitamins Through database comparison, compounds with known Raman bands can be identified in cell Raman spectra Those cells with specific characteristics can be separated according to their optical properties or chemical composition Raman spectra of cells (image source: Reference 1) Isotopic detection involves incubation of microbial samples with isotopic labeled reactants (matrix or water), tracking their binding to cell components, and identifying anabolically active microorganisms Incubation of substrate 13C glucose or 15NH4 + with microorganisms can make it incorporated into biomass and track the flow of intermediates in the system, while incubation of heavy water 2H2O or H218O with microorganisms can provide a labeling strategy Base simulation detection (SAP) uses the infrastructure that most laboratories can use, namely standard fluorescence microscope and FACS instrument Sap is relatively easy to implement compared to sip SAP mainly includes fluorescence analogues, nonstandard substrates and protein analysis based on activity and affinity Work flow of new generation microbial physiology research (picture source: Reference 1) The non-destructive nature of the new generation of microbial physiological research methods makes it possible to analyze the key downstream of a single cell expressing a specific phenotype The authors encourage microbiologists to work with researchers other than microbiologists For those non microbiologists who are willing to go out of their comfort zone and enter the field of living system, there are great opportunities, and they may affect microbiomics in many ways At the end of the paper, the author said that once the methods mentioned in this paper are widely used, they will greatly help microbial research to shift from related research to the causal understanding of microbial activity and function.