Public Health in Wartime

Author Institution: Chairman, Department of Bacteriology, Ohio State Universit

To gate or not to gate: revisiting drinking water microbial assessment through flow cytometry fingerprinting

Flow cytometry has been utilized for over a decade as a rapid and reproducible approach to assessing microbial quality of drinking water. However, the need for specialized expertise in gating—a fundamental strategy for distinguishing cell populations—introduces the potential for human error and obstructs the standardization of methods. This work conducts a comprehensive analysis of various gating approaches applied to flow cytometric scatter plots, using a dataset spanning a year. A sensitivity analysis is carried out to examine the impact of different gating strategies on final cell count results. The findings show that dynamic gating, which requires user intervention, is essential for the analysis of highly variable raw waters and distributed water. In contrast, static gating proved suitable for more stable water sources, interstage sample locations, and water presenting a particularly low cell count. Our conclusions suggest that cell count analysis should be supplemented with fluorescence fingerprinting to gain a more complete understanding of the variability in microbial populations within drinking water supplies. Establishing dynamic baselines for each water type in FCM monitoring studies is essential for choosing the correct gating strategy. FCM fingerprinting offers a dynamic approach to quantify treatment processes, enabling options for much better monitoring and control. This study offers new insights into the vagaries of various flow cytometry gating strategies, thereby substantially contributing to best practices in the water industry. The findings foster more efficient and reliable water analysis, improving of standardizing methods in microbial water quality assessment using FCM

Streptavidin-Binding Peptide (SBP)-tagged SMC2 allows single-step affinity fluorescence, blotting or purification of the condensin complex

<p>Abstract</p> <p>Background</p> <p>Cell biologists face the need to rapidly analyse their proteins of interest in order to gain insight into their function. Often protein purification, cellular localisation and Western blot analyses can be multi-step processes, where protein is lost, activity is destroyed or effective antibodies have not yet been generated.</p> <p>Aim</p> <p>To develop a method that simplifies the critical protein analytical steps of the laboratory researcher, leading to easy, efficient and rapid protein purification, cellular localisation and quantification.</p> <p>Results</p> <p>We have tagged the SMC2 subunit of the condensin complex with the Streptavidin-Binding Peptide (SBP), optimising and demonstrating the efficacious use of this tag for performing these protein analytical steps. Based on silver staining, and Western analysis, SBP delivered an outstanding specificity and purity of the condensin complex. We also developed a rapid and highly specific procedure to localise SBP-tagged proteins in cells in a single step procedure thus bypassing the need for using antibodies. Furthermore we have shown that the SBP tag can be used for isolating tagged proteins from chemically cross-linked cell populations for capturing DNA-protein interactions.</p> <p>Conclusions</p> <p>The small 38-amino acid synthetic SBP offers the potential to successfully perform all four critical analytical procedures as a single step and should have a general utility for the study of many proteins and protein complexes.</p