These experiments were performed using a Nanowizard III AFM (Bruker, Billerica, MA, USA) equipped with an inverted light microscope (Zeiss, Jena, Germany). NP-O10 probes (Bruker, Billerica, MA, USA) with a nominal spring constant of 0.06 N/m for experiments with S. aureus and of 0.35 N/m for experiments with C. glabrata were used to measure the interactions between single-cells and the different material surfaces. For this, given their respective sizes S. aureus (~1 µm of diameter), cells were first immobilized on colloidal probes [43], while C. glabrata cells (~4–5 µm in diameter) were immobilized directly on the tipless cantilevers. All cantilevers were cleaned using oxygen plasma before use. For all conditions, force maps of 10 × 10 pixels were recorded on 1 × 1 µm² areas on the surfaces. In the case of S. aureus, colloidal probes were obtained by attaching a single silica microsphere (5 μm diameter, Bangs Laboratories, Fishers, Indiana, IN, USA) with a thin layer of UV-curable glue (NOA 63, Norland Edmund Optics, Barrington, NY, USA) on the cantilevers using the AFM. Afterwards these were immersed for 1 h in tris buffer saline (TBS, pH 8.5) containing 4 mg/mL of dopamine hydrochloride (Sigma-Aldrich Chemie GmbH, Munich, Germany), rinsed in PBS 1X, and used directly for cell probe preparation. The spring constant of the colloidal probes, ranging from 0.0554 to 0.0932 N/m was determined prior to cell immobilization using the thermal noise method [49]. Post preparation, the colloidal probe was brought into contact with an isolated bacterium and subsequently retracted with the attached bacterial cell. The attachment of the cell on the silica microsphere was confirmed using optical microscopy and by performing a force distance curve on the Petri dish. The maximum force applied equaled 250 pN and a constant approach and retraction velocity of 1 µm/s was applied. For each condition, experiments were repeated for seven cells originating from at least three different cultures, and 400 force curves were recorded for each cell.

In the case of C. glabrata cells, the cantilevers were incubated in a solution of Concanavalin A (ConA, Sigma-Aldrich, 0.1 mg/mL) overnight and rinsed in acetate buffer. The spring constant of the ConA functionalized cantilevers, ranging from 0.213 to 0.361 N/m, was determined prior to cell attachment using the thermal noise method [49]. The ConA cantilever was then brought into contact with an isolated yeast cell and retracted with the attached yeast cell. In this case also the proper attachment of the cell on the cantilever was confirmed using an inverted light microscope (Zeiss, Jena, Germany). In contrast to experiments with S. aureus, tipless cantilever can be used without a sphere because of the larger size of the C. glabrata cells. The maximum force applied equaled 1000 pN and a constant approach and retraction velocity of 5 µm/s was applied. For each condition, experiments were repeated for seven cells, originating from at least three different cultures, and 400 force curves were recorded for each cell.

For both types of cell, probes were finally used to measure cell–surface interaction forces in PBS 1X for S. aureus, and in acetate buffer for C. glabrata, both at room temperature. Data were analyzed using the JPK Data Processing software (Bruker, Billerica, MA, USA). Adhesion force and rupture distance histograms were obtained by calculating the maximum adhesion force and the rupture distance of the last peak for each obtained curve. The mean values and standard deviations were calculated and the significance was tested by performing a two sample t-test by using OriginLab (OriginLab Corporation, Northampton, MA, USA). The force distance curves were finally analyzed by using the JPK Data Processing software (JPK Instruments, Berlin, Germany). The adhesion force and the rupture distance were obtained and summarized in histograms with a bin interval of 50 pN and 20 nm for S. aureus and 100 pN and 50 nm for C. glabrata, respectively. The mean values and standard deviations were calculated using OriginLab (OriginLab Corporation, Northampton, MA, USA).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.