Cell Surface Analysis Techniques: What Do Cell Preparation Protocols Do to Cell Surface Properties?

Abstract

Cell surface analysis often requires manipulation of cells prior to examination. The most commonly employed procedures are centrifugation at different speeds, changes of media during washing or final resuspension, desiccation (either air drying for contact angle measurements or freeze-drying for sensitive spectroscopic analysis, such as X-ray photoelectron spectroscopy), and contact with hydrocarbon (hydrophobicity assays). The effects of these procedures on electrophoretic mobility, adhesion to solid substrata, affinity to a number of Sepharose columns, structural integrity, and cell viability were systematically investigated for a range of model organisms, including carbon- and nitrogen-limited Psychrobacter sp. strain SW8 (glycocalyx-bearing cells), Escherichia coli(gram-negative cells without a glycocalyx), and Staphylococcus epidermidis (gram-positive cells without a glycocalyx). All of the cell manipulation procedures severely modified the physicochemical properties of cells, but with each procedure some organisms were more susceptible than others. Considerable disruption of cell surfaces occurred when organisms were placed in contact with a hydrocarbon (hexadecane). The majority of cells became nonculturable after air drying and freeze-drying. Centrifugation at a high speed (15,000 × g) modified many cell surface parameters significantly, although cell viability was considerably affected only in E. coli. The type of washing or resuspension medium had a strong influence on the values of cell surface parameters, particularly when high-salt solutions were compared with low-salt buffers. The values for parameters obtained with different methods that allegedly measure similar cell surface properties did not correlate for most cells. These results demonstrate that the methods used to prepare cells for cell surface analysis need to be critically investigated for each microorganism so that the final results obtained reflect the nature of the in situ microbial cell surface as closely as possible. There is an urgent need for new, reliable, nondestructive, minimally manipulative cell surface analysis techniques that can be used in situ.

Role of cranberry juice on molecular‐scale surface characteristics and adhesion behavior of Escherichia coli

“…Bacterial cells were centrifuged for 15 min at 190g and resuspended in the desired media for the force measurements (described below). This low centrifugal force was chosen because previous results from our laboratory have shown that it is sufficient to pellet E. coli, but does so without causing any artifacts in cell surface characteristics and adhesion behavior (Bell and Camesano, 2005), as can be found when high forces are applied to bacteria (Pembrey et al, 1999).…”

Escherichia coli strains harvested from springs in Kampala, Uganda: cell characterization and transport in saturated porous media

“…Hydrophobicity was determined with the microbial adhesion to hydrocarbons method (Pembrey et al, 1999;Walker et al, 2005), where percentage partitioning of cells into dodecane was measured as cell hydrophobicity. To do this, 4 ml of bacteria suspension of known optical density (OD) and 1 ml of dodecane were vigorously mixed in a test tube for 2 min and left to stand for 15 min to allow phase separation.…”

Cell Preparation Methods Influence Escherichia coli D21g Surface Chemistry and Transport in Saturated Sand

“…Th e zeta-potential measurements are in agreement with previous studies in which it was demonstrated that centrifugation can alter the surface charge of bacteria. For example, Pembrey et al (1999) reported that harvesting by high-speed centrifugation (15,000 × g) generally reduced the net surface charge of E. coli. Th is eff ect was attributed to removal of materials from the cell surface and thus generating a new and very diff erent microenvironment interface.…”