Donald F. Gerson

Cell surface energy, contact angles and phase partition III. Adhesion of bacterial cells to hydrophobic surfaces

The densities of adhesion of Staphylococcus epidermidis, Staphylococcus aureus and Serratia marcescens to five types of plastics were studied in relation to interfacial free energies at the aqueous interfaces of both the bacteria and the plastics. The free energy of adhesion of bacteria to plastic in an aqueous medium is a linear function of partititon of the bacteria between the solid surface and the liquid phase. These results show that the thermodynamics of the partitioning of a suspended particle between two immiscible liquid phases also apply to partitioning between a liquid and a solid phase.

Determination of cell/medium interfacial tensions from contact angles in aqueous polymer systems

The contact angles on cell layers of a series of polymeric droplets from aqueous two-phase systems of dextran and poly(ethylene glycol) have been used to determine the critical or limiting interfacial tension for spreading on the cell layers. Test droplets of the denser dextran-rich phase were formed in the lighter poly(ethylene glycol)-rich phase. The interfacial tensions, gamma, between the phases were determined with the pendant drop method, and a linear relationship was found between gamma-1/2 and the cosine of the angle the droplets made with the cell layers (Good-Girifalco plot). We were thus able to determine the limiting or critical interfacial tension, gamma c, for spreading on the cell layers. The value of gamma c is a measure of the interfacial energy of the cell/bathing medium interface. Values of gamma c obtained by this method include the following: 0.65 and 0.84 microN . m-1 for human erythrocytes and neutrophils, respectively; 0.93 microN . m-1 for porcine pulmonary macrophages; 0.75--3.60 microM . m-1 for various transformed murine lymphoid cell lines, and 2.53 microN . m-1 for Balb/c murine spleen lymphocytes. Exposure to various agents has differing effects on gamma c. Concanavalin A reduces gamma c, and bacterial lipopolysaccharide increases gamma c of murine spleen lymphocytes. The calcium ionophore, A23187, increases gamma c of both porcine pulmonary macrophages and murine spleen lymphocytes. This new method provides a quantitative approach to the cell surface energy and hydrophobicity which are thought to play an important role in membrane-mediated phenomena and in cell adhesion.

Cell surface energy, contact angles and phase partition. II. Bacterial cells in biphasic aqueous mixtures.

Partition coefficients in biphasic mixtures of poly(ethylene glycol) and Dextran are compared to cell surface energies obtained from contact angles of each liquid phase on cell layers. Linear relationships are observed between these two independent measurements for a variety of bacterial cells. The results demonstrate the importance of interfacial phenomena and contact angles in the phase-partition process.

Production of Clostridium difficile toxin in a medium totally free of both animal and dairy proteins or digests

In the hope of developing a vaccine against Clostridium difficile based on its toxin(s), we have developed a fermentation medium for the bacterium that results in the formation of Toxin A and contains no meat or dairy products, thus obviating the problem of possible prion diseases. Particular preparations of hydrolyzed soy proteins, especially Soy Peptone A3, have been found to replace both the meat/dairy product tryptone in the preparation of working cell banks and seed media, and NZ-Soy BL4 does the same in the fermentation medium. These replacements yield even higher toxin titers.

Adhesion, phagocytosis and cell surface energy. The binding of fixed human erythrocytes to rat macrophages and polymethylpentene

Fixed human erythrocytes were used as model particles for the study of adhesion and phagocytosis by rat peritoneal macrophages. Erythrocytes were fixed with various concentrations of glutaraldehyde or tannic acid, or were treated with neuraminidase. Adhesion and phagocytosis of these cells were measured. In addition, the surface energy of these erythrocytes and macrophages was estimated by the contact angle technique. Free energies of adhesion, based on the cell surface energies, were correlated with both adhesion and phagocytosis.

Interfacial Free Energies of Cells and Polymers in Aqueous Media

The interfacial free energy at the surface of cells in aqueous media, γCM determines the free energy of adhesion and the equilibrium constant for adhesion not only between different cells but also between cells and polymer surfaces. Previously described methods for measuring contact angles on cells in polymer containing, biphasic aqueous media (Biochim. Biophys. Acta. 602, 269, 1980; 640, 557, 1980) have been extended to provide values of γCM for cells in physiological media. To do this, an empirical equation of state was developed to describe the relations between contact angles and interfacial free energies in 3 component systems consisting of 2 fluids (gas or liquid) and one solid. In such systems, the bulk phase (gas or liquid) is designated “B”, the test droplet (liquid) is designated “D” and the solid phase is designated “S”. The contact angle between the drop and the solid when both are immersed in the bulk fluid is given by θ. The equation which was developed thus relates measured contact angles and bulk fluid-drop interfacial free energies, γBD to both of the solid-liquid interfacial free energies γSB, and γSD. This was used to determine cell-medium interfacial free energies from contact angle data. Cell-medium interfacial free energies in polyethyleneglycol-dextran biphasic media, and the polymer concentrations in these media were extrapolated to give γCM in polymer-free media.

Cell surface energy and membrane associated actin in lymphocytes

We have shown previously that membrane associated actin correlates with the migratory abilities of lymphocytes during recirculation, and that cell surface energy correlates with the adhesiveness of lymphocytes to other cells. In this study, measurements of actin content and cell surface energy have been made for various lymphocyte subpopulations to examine the possibility that recirculation ability may be related to nonspecific adhesiveness. We have found that: both cell surface energy and actin content combine to provide a consistent explanation for the relative rates of recirculation of various lymphocyte subpopulations, and cell surface energies and actin contents vary independently in these lymphocyte subpopulations. Comparison of the actin contents and cell surface energies of metastatic and nonmetastatic lymphoma cell lines indicated that the differences in metastatic potential were more likely attributable to specific receptor-ligand interactions than to nonspecific adhesiveness. Cell surface energy and actin content are consistent with the greater adhesiveness of B cells than T cells to nylon wool, providing a physical basis for this common cell separation technique.

Ammonium Concentration Control in Fed-Batch Fermentations for the Production of Biomass and Enzymes

A control system has been devised to maintain stable ammonium concentrations throughout a fed-batch fermentation. The control system is based on an ammonium gas-sensing electrode that monitors a pH adjusted effluent stream from the fermentor. The ammonium electrode (Orion 95–12) was stable throughout the fermentation period. This control system was used to study the growth of Escherichia coli and Saccharomyces cerevisiae at controlled ammonium concentrations. Apparent specific growth rates, biomass and protein production, and glucose and ammonium yield were determined.

The pressure fluctuation spectrum as a measure of mixing and emulsification in a biochemical reactor

SummaryPressure fluctuation spectra were measured inside a biochemical reactor at various mixing speeds and aeration rates. These spectra change considerably depending on the mixing conditions and appear to provide a detailed picture of the turbulent conditions within the reactor. Droplet size distributions were measured for 4% (v/v) hydrocarbon in the reactor. It was found that these droplet size distributions could be qualitatively predicted by analysis of the Rayleigh-Taylor instabilities at the oil-water interface in conjunction with the observed pressure fluctuation spectra.