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  • Thumbnail for Toward imaging live, competent Acinetobacter baylyi with Liquid Cell Atomic Force Microscopy
    Toward imaging live, competent Acinetobacter baylyi with Liquid Cell Atomic Force Microscopy by Tenente, Ricardo

    Imaging live bacteria with an atomic force microscope (AFM) is a challenging process, taking into account the lateral forces exerted by the probe on cells that must be immobilized but still immersed in liquid. The Lang-Lostroh labs use AFM to examine Acinetobacter baylyi cells that are competent (able to take up DNA from the environment). In order to image cells while they are competent, it is necessary to find an effective combination of liquid media and sample preparation method while maintaining the cells alive, competent, and attached to the AFM pucks with porcine gelatin. Many combinations were tested using different washing and gelatin immobilization media (distilled deionized water, and phosphate-buffer saline), as well as using different media (distilled deionized water, phosphate-buffer saline, and diverse dilutions of Luria-Bertani broth with and without sodium chloride) during imaging itself. The combinations that presented the most satisfying images of immobilized cells with AFM were submitted to membrane integrity and competence assays. Washing and immobilizing the cells with distilled deionized water and imaging them with 50% Luria-Bertani broth without sodium chloride was the most successful combination. Even though the cells did not show detectable competence ability, they were alive yet inactive since after the imaging procedure they were still able to form colonies on plain Luria-Bertani agar plates. It provides insight on a viability spectrum that must be taken into account when distinguishing cell viability. Further research should focus on testing smoother media transitions with access to food source to avoid osmotic pressure stress and starvation.