Continuous deformation and entry flow of single blood granulocytes into small caliber micropipets at various suction pressures have been studied to determine an apparent viscosity for the cell contents and to estimate the extent that dissipation in a cortical layer adjacent to the cell surface contributes to the total viscous flow resistance. Experiments were carried out with a wide range of pipet sizes (2.0-7.5 microns) and suction pressures (10(2)-10(4) dyn/cm2) to examine the details of the entry flow. The results show that the outer cortex of the cell maintains a small persistent tension of approximately 0.035 dyn/cm. The tension creates a threshold pressure below which the cell will not enter the pipet. The superficial plasma membrane of these cells appears to establish an upper limit to surface dilation which is reached after microscopic "ruffles" and "folds" have been pulled smooth. With aspiration of cells by small pipets (less than 2.7 microns), the limit to surface expansion was derived from the maximal extension of the cell into the pipet; final areas were measured to be 2.1 to 2.2 times the area of the initial spherical shape. For suctions in excess of a threshold, the response to constant pressure was continuous flow in proportion to excess pressure above the threshold with only a small nonlinearity over time until the cell completely entered the pipet (for pipet calibers greater than 2.7 microns). With a theoretical model introduced in a companion paper, (Yeung, A., and E. Evans., 1989, Biophys. J. 56:139-149) the entry flow response versus pipet size and suction pressure was analyzed to estimate the apparent viscosity of the cell interior and the ratio of cortical flow resistance to flow resistance from the cell interior. The apparent viscosity was found to depend strongly on temperature with values on the order of 2 x 10(3) poise at 23 degrees C, lower values of 1 x 10(3) poise at 37 degrees C, but extremely large values in excess of 10(4) poise below 10 degrees C. Because of scatter in cell response, it was not possible to accurately establish the characteristic ratio for flow resistance in the cortex to that inside the cell; however, the data showed that the cortex does not contribute significantly to the total flow resistance.

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Many nonadherent cells exist as spheres in suspension and when sucked into pipets, deform continuously like liquids within the fixed surface area limitation of a plasma membrane envelope. After release, these cells eventually recover their spherical form. Consequently, pipet aspiration test provides a useful method to assay the apparent viscosity of such cells. For this purpose, we have analyzed the inertialess flow of a liquid-like model cell into a tube at constant suction pressure. The cell is modeled as a uniform liquid core encapsulated by a distinct cortical shell. The method of analysis employs a variational approach that minimizes errors in boundary conditions defined by the equations of motion for the cortical shell where the trial functions are exact solutions for the flow field inside the liquid core. For the particular case of an anisotropic liquid cortex with persistent tension, we have determined universal predictions for flow rate scaled by the ratio of excess pressure (above the threshold established by the cortical tension) and core viscosity which is the reciprocal of the dynamic resistance to entry. The results depend on pipet to cell size ratio and a parameter that characterizes the ratio of viscous flow resistance in the cortex to that inside the cytoplasmic core. The rate of entry increases markedly as the pipet size approaches the outer segment diameter of the cell. Viscous dissipation in the cortex strongly influences the entry flow resistance for small tube sizes but has little effect for large tubes. This indicates that with sufficient experimental resolution, measurement of cell entry flow with different-size pipets could establish both the cortex to cell dissipation ratio as well as the apparent viscosity of the cytoplasmic core.

Formation of oil-water emulsions during bacterial growth on hydrocarbons is often attributed to biosurfactants. Here we report the ability of certain intact bacterial cells to stabilize oil-in-water and water-in-oil emulsions without changing the interfacial tension, by inhibition of droplet coalescence as observed in emulsion stabilization by solid particles like silica.Emulsions are commonly observed when liquid hydrocarbons and water are mixed during bioremediation or fermentation (2). These emulsions dramatically increase the area of the oil-water interface, thereby enhancing bioavailability. For a dispersion of one liquid in another to be stable enough to be classified as an emulsion, a third component, such as a surfactant, must be present to stabilize the system. Fine solid particles such as silica beads can also stabilize emulsions if they attach at the interface between the oil and the water to prevent droplets from coalescing (4,14,23). While bacteria can produce surfactants or emulsifiers that stabilize emulsions (1, 5, 18), some microorganisms can emulsify hydrocarbons even in the absence of cell growth or uptake of hydrocarbons (18). The latter observation suggests that emulsification may be associated with the surface properties of the cells, as a result of attachment to the oil-water interface by general hydrophobic interactions rather than specific recognition of the substrate (5, 21). Bacterial cells may, therefore, behave as fine solid particles at interfaces.Given that fine solid particles can stabilize oil-water emulsions, our hypothesis was that intact, stationary-phase bacteria can stabilize oil-water emulsions by adhering to the oil-water interface and that this property is related to cell surface hydrophobicity. We selected four hydrocarbon-degrading bacterial species and determined the surface properties of washed stationary-phase cells by cell adhesion to hydrocarbons, the contact angle, and the interfacial tension. The structure of the emulsions was observed by confocal microscopy, and the behavior of oil droplets in bacterial suspensions was measured with a micropipette apparatus. These results were used to make general inferences about the ability of intact, bacterial cells to stabilize oil-water emulsions.The n-alkane-degrading bacteria employed in this study were Acinetobacter venetianus 20), Rhodococcus erythropolis 20S-E1-c (9), and Rhizomonas suberifaciens EB2-1a (8). Pseudomonas fluorescens LP6a degrades a range of aromatic hydrocarbons but not n-alkanes (8). All bacterial cultures were grown in Trypticase soy broth (Difco, Sparks, Md.) with incubation at 28°C and gyratory shaking. Each culture was harvested at its stationary phase by centrifugation and washed twice with 100 mM phosphate buffer (pH 7).The harvested and washed cells were used to characterize the cell surface properties. Bacterial adhesion to hydrocarbons (BATH) was measured as described by Rosenberg et al. (17). Cells were resuspended in phosphate buffer to an optical density at 600 nm (OD 600 ) of about 0.6....

The remarkable stability of water-in-crude oil emulsions is due to the presence of a complex adsorbed layer at the surfaces of the dispersed droplets. Except for its role as a steric barrier, little is known about the in situ properties of this interfacial structure. In this study, new insights into the adsorbed layer are provided by direct micrometre-scale measurements. At low crude content in the bulk where, according to interfacial tension isotherms, there should be little or no surfactants on the droplet surface, the adsorbed layer displays pronounced rigidity and is capable of preventing coalescence and coagulation of the droplets. Such interfaces are highly dissipative and can be well described by the Boussinesq-Scriven model. As the supply of surface active materials in the bulk (i.e. the crude content) increases, the adsorbed layer transforms from a rigid structure to a fluid interface. This fluid layer continues to inhibit coalescence, although signs of weak interdroplet adhesion begin to appear. Under area compression, the fluid interface will discharge micrometre-sized emulsion droplets into the oil phase via a 'budding' mechanism.