Shear viscoelasticity of suspensions of biological cells with viscoelastic membrane II

To discuss the relaxation phenomena of biological cell suspensions, we calculate the complex intrinsic viscosity of dispersions of spherical cells with viscoelastic membrane as a function of the frequency taking account of interfacial tension at both the interfaces of the membrane. The Maxwell model and two kinds of the three-parameter models are used to describe the viscoelasticity of the cell membrane. The results are computed mainly for the Maxwell model similarly in case of the Voigt Model (Abe, K., Takano, Y. and Sakanishi, A. Biorheology 21 405-414, 1984). The computed results of the four models, the Voigt, the Maxwell and the two kinds of the three-parameter viscoelastic models, are compared with the experimental data.     

Red cell extensional recovery and the determination of membrane viscosity.

A theory of membrane viscoelasticity developed by Evans and Hochmuth in 1976 is used to analyze the time-dependent recovery of an elongated cell. Before release, the elongated cell is the static equilibrium where external forces are balanced by membrane elastic force resultants. Upon release, the cell recovers its initial shape with a time-dependent exponential behavior characteristic of the viscoelastic solid model. It is shown that the model describes the time-dependent recovery process very well for a time constant in the range of 0.1-0.13 s. The time constant is the ratio membrane surface viscosity eta:membrane surface elasticity mu. Measurements for the shear modulus mu of 0.006 dyne/cm give a value for the surface viscosity of red cell membrane as a viscoelastic solid material of eta = mu tc = (6-8) X 10(-4) poise . cm.     

Red cell and ghost viscoelasticity. Effects of hemoglobin concentration and in vivo aging.

To assess the influence of intracellular hemoglobin concentration on red cell viscoelasticity and to better understand changes related to in vivo aging, membrane shear elastic moduli (mu) and time constants for cell shape recovery (tc) were measured for age-fractionated human erythrocytes and derived ghosts. Time constants were also measured for osmotically shrunk cell fractions. Young and old cells had equal mu, but tc was longer for older cells. When young cells were shrunk to equal the volume (and hence hemoglobin concentration and internal viscosity) of old cells, tc increased only slightly. Thus membrane viscosity (eta = mu . tc) increases during aging, regardless of increased internal viscosity. However, further shrinkage of young cells, or slight shrinkage of old cells, caused a sharp increase in tc. Because this increased tc is not explainable by elevated internal viscosity, eta increased, possibly due to a concentration-dependent hemoglobin-membrane interaction. Ghosts had a greater mu than intact cells, with proportionally faster tc; their membrane viscosity was therefore similar to intact cells. However, the ratio of old/young membrane viscosity was less for ghosts than for intact cells, indicating that differences between young and old cell eta may be partly explained by altered hemoglobin-membrane interaction during aging. It is postulated that these changes in viscoelastic behavior influence in vivo survival of senescent cells.     

Local measurements of viscoelastic parameters of adherent cell surfaces by magnetic bead microrheometry.

A magnetic bead microrheometer has been designed which allows the generation of forces up to 10(4) pN on 4.5 micron paramagnetic beads. It is applied to measure local viscoelastic properties of the surface of adhering fibroblasts. Creep response and relaxation curves evoked by tangential force pulses of 500-2500 pN (and approximately 1 s duration) on the magnetic beads fixed to the integrin receptors of the cell membrane are recorded by particle tracking. Linear three-phasic creep responses consisting of an elastic deflection, a stress relaxation, and a viscous flow are established. The viscoelastic response curves are analyzed in terms of a series arrangement of a dashpot and a Voigt body, which allows characterization of the viscoelastic behavior of the adhering cell surface in terms of three parameters: an effective elastic constant, a viscosity, and a relaxation time. The displacement field generated by the local tangential forces on the cell surface is visualized by observing the induced motion of assemblies of nonmagnetic colloidal probes fixed to the membrane. It is found that the displacement field decays rapidly with the distance from the magnetic bead. A cutoff radius of Rc approximately 7 micron of the screened elastic field is established. Partial penetration of the shear field into the cytoplasm is established by observing the induced deflection of intracellular compartments. The cell membrane was modeled as a thin elastic plate of shear modulus mu * coupled to a viscoelastic layer, which is fixed to a solid support on the opposite side; the former accounts for the membrane/actin cortex, and the latter for the contribution of the cytoskeleton to the deformation of the cell envelope. It is characterized by the coupling constant chi characterizing the elasticity of the cytoskeleton. The coupling constant chi and the surface shear modulus mu * are obtained from the measured displacements of the magnetic and nonmagnetic beads. By analyzing the experimental data in terms of this model a surface shear modulus of mu * approximately 2 . 10(-3) Pa m to 4 . 10(-3) Pa m is found. By assuming an approximate plate thickness of 0.1 micron one estimates an average bulk shear modulus of mu approximately (2 / 4) . 10(-4) Pa, which is in reasonable agreement with data obtained by atomic force microscopy. The viscosity of the dashpot is related to the apparent viscosity of the cytoplasm, which is obtained by assuming that the top membrane is coupled to the bottom (fixed) membrane by a viscous medium. By application of the theory of diffusion of membrane proteins in supported membranes we find a coefficient of friction of bc approximately 2 . 10(9) Pa s/m corresponding to a cytoplasmic viscosity of 2 . 10(3) Pa s.     

Viscoelastic properties of the oxygenated sickle erythrocyte membrane

Although most apparent in permanently misshapen irreversibly sickled erythrocytes (ISC), biochemical and structural alterations are present in the majority of sickle cell membranes. The relationship of membrane rigidity to cell shape and its dependence upon the internal hemoglobin cytosol are not clarified. We therefore examined the frequency dependent viscoelasticity of oxygenated, packed sickle red cell and ghost suspensions and hemoglobin solutions prepared from density gradient separated ISC and reversibly sickled cell (RSC) fractions. Low amplitude, oscillatory shear was applied in a Weissenberg cone and plate viscometer and the resultant viscoelastic signals provided a dynamic viscosity (eta') and elastic storage modulus (G') which varied with frequency of deformation. The viscoelastic response of the cell and ghost suspensions reflected the material properties of the membrane over most of the frequency range tested. Sickle erythrocyte, red ghost, and white ghost suspensions demonstrated greater viscocoelasticity than comparable normal suspensions. The viscoelastic magnitude of ISC was several-fold greater than normal, with little variation of viscoelasticity with frequency. RSC samples which were characterized by normal shape, size, and internal hemoglobin concentration were also significantly harder than normal, although similar in frequency dependence. Red ghosts prepared from ISC manifested 80% of the viscoelasticity of intact ISC despite diminution of the internal hemoglobin concentration by 90%. Under conditions of low amplitude shear, the behavior of the RSC membrane is compatible with a cytoskeleton possessing an increased number of molecular associations. The mechanical stability of the ISC membrane is related to a substantial, intrinsic reorganization of the cytoskeleton.     

Influence of preparative procedures on the membrane viscoelasticity of human red cell ghosts

The effects of systematic variations in the preparative procedures on the membrane viscoelastic properties of resealed human red blood cell ghosts have been investigated. Ghosts, prepared by hypotonic lysis at 0 degrees C and resealing at 37 degrees C, were subjected to: measurement of the time constant for extensional recovery (tc); measurement of the membrane shear elastic modulus (mu) via three separate techniques; determination of the membrane viscosity (eta m) via a cone-plate Rheoscope. Membrane viscosity was also determined as eta m = mu X tc. Compared to intact cells, ghosts had shorter tc, regardless of their residual hemoglobin concentration (up to 21.6 g/dl). However, prolonged exposure to hypotonic media did increase their recovery time toward the intact cell value. The shear elastic modulus, as judged by micropipette aspiration of membrane tongues (mu p), was similar for all ghosts and intact cells. This result, taken with the tc data, indicates that ghosts have reduced membrane viscosity. Rheoscopic analysis also showed that eta m was reduced for ghosts, with the degree of reduction (approx. 50%) agreeing well with that estimated by the product mu p X tc. However, flow channel and pipette elongation estimates indicated that the ghost membrane elastic modulus was somewhat elevated compared to intact cells. We conclude that: ghosts have reduced membrane viscosity; ghosts have membrane rigidities close to intact cells, except possibly when the membrane is subjected to very large strains; the reduction in eta m is not directly related to the loss of hemoglobin; prolonged exposure of ghosts to low-ionic strength media increases the membrane viscosity toward its initial cellular level. These data indicate that the mechanical characteristics of ghost membranes can be varied by changing the methods of preparation and thus have potential application to further studies of the structural determinants of red cell membrane viscoelasticity.     

Rheological properties of mammalian cell culture suspensions: Hybridoma and HeLa cell lines

Data on viscous (eta') and elastic (eta') components of the complex viscosity versus oscillatory angular frequency (0.01 to 4.0 rad/s) with increasing strains were obtained for hybridoma cell (62'D3) and HeLa cell (S3) suspensions in PBS at 0.9 (mL/mL) cell volume fraction using a Weissenberg rheogoniometer equipped with two parallel plate geometry at ambient temperature. Both cell suspensions exhibited shear thinning behavior. From the measured viscoelastic properties, the yield stress was calculated. Hybridoma cell suspension (15 mum as the mean diameter of cells) showed the yield stress at 550 dyne/cm(2) that was 1.8 times higher than the value of HeLa cell suspension (22 mum mean diameter) as measured at the oscillatory angular frequency, 4.0 rad/s. The apparent viscosities of HeLa cell suspension at four concentrations and varying steady shear rate were also determined using the Brookfield rotational viscometer. The yield stress to steady shear test was about 130 dyne/cm(2) for HeLa cell suspension at 0.9 (mL/mL) cell volume fraction. The apparent viscosity was in the range about 1 approximately 1000 Poise depending on the cell concentration and shear rate applied. A modified semiempirical Mooney equation, \documentclass{article}\pagestyle{empty}\begin{document}$ \eta = \eta _0 \exp [K\dot \gamma ;{ - \beta } \phi /(1 - K'\sigma \phi _c /D)] $\end{document} was derived based on the cell concentration, the cell morphology, and the steady shear rate. The beta, shear rate index, was estimated as 0.159 in the range of shear rate, 0.16 to 22.1 s(-1), for the cell volume fractions from 0.6 to 0.9 (mL/mL). In this study, the methods of determining the shear sensitivity and the viscous and the elastic components of mammalian cell suspensions are described under the steady shear field. (c) 1993 John Wiley & Sons, Inc.     

Intrinsic viscoelasticity of blood cell suspensions: effects of erythrocyte deformability

The intrinsic viscoelasticity of erythrocyte suspensions holds great potential for specifying the deformability of the individual, noninteracting cells in an oscillatory shear flow field. In order to extrapolate to zero cell concentration, the complex viscoelastic modulus was measured as a function of hematocrit using 2 Hertz oscillatory flow and a shear rate of 10/sec. This was done for both normal cells and cells with severely reduced deformability when hardened with glutaraldehyde. Suspension media were blood plasma, isotonic saline, and Dextran solutions. The real parts of the complex intrinsic visco-elasticities were obtained by an extrapolation using a regression fit to Huggins' equation. For normal cells in native plasma the values ranged from 1.7 to 2, increasing to the range 2.4 to 3.1 when the plasma was diluted with isotonic saline solution. For hardened cells the value obtained was near 3.5. These results are compared with theories for suspensions of both rigid and deformable particles. Several theories for deformable particles predict an increase in intrinsic viscoelasticity with increases in the ratio of the viscosity of the interior of the particle to that of the suspending medium. This ratio controls the balance between rotational and deformational response of the cell in the flow field. The trends of these theories were observed in the measurements.     

Effects of cell lysis on the rheological behavior of red blood cell suspensions

Rheological studies of lysed cell suspensions are performed with a magneto acoustic ball microrheometer. Two methods for lysing the cells are developed in order to provide cell volume concentrations identical to control intact cell suspensions. The first uses a freeze-thaw technique and the second uses sonication. It is found that cell suspensions disrupted by sonication have a lower viscosity than intact suspensions, whereas cell suspensions lysed by the freeze-thaw method exhibit a higher viscosity. Sonication is discovered to have a detrimental impact on the cell membrane, and to cause complete destruction of the cell membrane structure. Measurements of the steady state viscosity show that indeed the presence of the membrane is not detected, and that what is measured is mainly the viscosity of the hemoglobin solution. On the other hand, freeze-thaw results indicate that at least two phenomena occur. The first phenomenon, occurring during the first freeze-thaw cycle, produces an increase in viscosity and in viscoelasticity. The second one, taking place after subsequent freeze-thaw cycles, induces a decrease in the bulk rheological properties. Several possible mechanisms are presented to explain the observed phenomena.     

A cellular tensegrity model to analyse the structural viscoelasticity of the cytoskeleton

This study describes the viscoelastic properties of a refined cellular-tensegrity model composed of six rigid bars connected to a continuous network of 24 viscoelastic pre-stretched cables (Voigt bodies) in order to analyse the role of the cytoskeleton spatial rearrangement on the viscoelastic response of living adherent cells. This structural contribution was determined from the relationships between the global viscoelastic properties of the tensegrity model, i.e., normalized viscosity modulus (eta(*)), normalized elasticity modulus (E(*)), and the physical properties of the constitutive elements, i.e., their normalized length (L(*)) and normalized initial internal tension (T(*)). We used a numerical method to simulate the deformation of the structure in response to different types of loading, while varying by several orders of magnitude L(*) and T(*). The numerical results obtained reveal that eta(*) remains almost independent of changes in T(*) (eta(*) proportional, variant T(*+0.1)), whereas E(*) increases with approximately the square root of the internal tension T(*) (from E(*) proportional, variant T(*+0.3) to E(*) proportional, variant T(*+0.7)). Moreover, structural viscosity eta(*) and elasticity E(*) are both inversely proportional to the square of the size of the structure (eta(*) proportional, variant L(*-2) and E(*) proportional, variant L(*-2)). These structural properties appear consistent with cytoskeleton (CSK) mechanical properties measured experimentally by various methods which are specific to the CSK micromanipulation in living adherent cells. Present results suggest, for the first time, that the effect of structural rearrangement of CSK elements on global CSK behavior is characterized by a faster cellular mechanical response relatively to the CSK element response, which thus contributes to the solidification process observed in adherent cells. In extending to the viscoelastic properties the analysis of the mechanical response of the cellular 30-element tensegrity model, the present study contributes to the understanding of recent results on the cellular-dynamic response and allows to reunify the scattered data reported for the viscoelastic properties of living adherent cells.     

Viscoelastic properties for kappa- and iota-carrageenan in aqueous NaI from the liquid-like to the solid-like behaviour

The dynamic mechanical behaviour of a series of concentrations of kappa-carrageenan (KC; 0.35-1.6% w/w) and iota-carrageenan (IC; 0. 2-1% w/w) in 0.2 M NaI has been investigated. The flow behaviour of KC within the concentration range 0.004-0.8% (w/v) was also described. The high intrinsic viscosity of KC in 0.2 M NaI (23.4 dl g(-1)) and the great increase in viscosity with increasing concentration, in comparison with linear flexible polysaccharides, is well consistent with the stiffness of KC helices in NaI. The variation of the 'zero-shear' specific viscosity of KC in 0.2 M NaI with the degree of space-occupancy (c[eta]) displays two critical concentrations at c* approximately 0.09% w/v (c*[eta] approximately 2) and c** approximately 0.4% w/v (c**[eta] approximately 10). Different viscoelastic behaviours were exhibited from the liquid-like to the solid-like depending upon the type of carrageenan. From the application of the time-temperature superposition, classical frequency-temperature master curves could be obtained for KC, but not for IC. Moreover, for KC, a concentration-frequency master curve could be constructed for the concentrations below 1.5%, indicating a 'solution-like' behaviour in this entire concentration range, although systems above 0.8% were visually gel-like. It is proposed that the rigidity of the KC helices is responsible for the slow relaxation rates of the gel-like samples. At higher concentrations (beyond 1.6%) a frequency-temperature superposition was no longer possible. In contrast to KC, IC behaved as a typical viscoelastic gel with a very weak frequency dependence of the storage modulus at all temperatures. This indicates the existence of associations beyond simple entanglements for IC. Creep experiments performed at higher carrageenan concentrations in 0.1 M NaI further corroborated the differences in the viscoelastic behaviour between KC and IC.     

Complex viscosity of bovine red blood cells in suspensions

The complex viscosity eta* has been measured of bovine red blood cells suspended in a medium of isotonic NaCl solutions including dextran and buffered with potassium phosphate at pH 7.0. A multiple lumped resonator apparatus was used at the frequencies of 144, 572, 1491, 3742, and 8026 Hz at 20.0 degrees C. Due to the high molecular weight of dextran the medium also exhibited some visco-elasticity eta s*. So we adopted the complex specific viscosity eta sp* = (eta*-eta s*)/[eta s*]. At 20.0 degrees C eta sp* decreased with the frequency where the hematocrit was 0.233 and eta s 0.34 poise. The measurements were made for the medium with different viscosity at 5.0 degrees C and 25.0 degrees C. The results are compared with the theory of elastic shells.     

Effects of cellular morphology on the viscoelastic behavior of high hematocrit RBC suspensions

Using a constant-amplitude (+/- 1 degree) oscillatory Couette viscometer (f = 0.01-1.0 Hz), we have measured the viscous (eta') and elastic (eta") components of the complex viscosity at 25 degrees C for shape-transformed human RBC suspended in isotonic buffer at 80% hematocrit. Morphology-altering drugs employed were: ECHINOCYTIC AGENT 2,4-dinitrophenol (DNP, 0.1-5 mM); STOMATOCYTIC AGENT chlorpromazine hydrochloride (CPZ, 0.01-0.1 mM). All suspensions exhibited decreasing eta' and eta" with increasing frequency. Compared to biconcave, control RBC suspensions, salient effects of shape transformation included: 1) for DNP, a dose-related elevation of both eta' and eta", with a 850% increase in eta' and a 2500% increase in eta" at 5 mM and the lowest frequency; 2) for CPZ, a dose-related elevation of both eta' and eta", with a 170% increase in eta' and a 280% increase in eta" at 0.1 mM and the lowest frequency; 3) for both DNP and CPZ, the elevations of eta' and eta" were inversely related to frequency. Using 2 mM DNP and various concentrations of CPZ, both eta' and eta" could be returned to control with 0.08 mM CPZ; further increases of CPZ at constant DNP led to elevations of both components. Comparisons of eta' and eta" to steady shear viscometric data indicated that neither a nominal shear rate approach nor a RMS complex viscosity technique was able to completely reconcile these data; a modified Kelvin-Voigt model proved useful in evaluating cellular versus membrane contributions to eta". These results indicate that RBC morphology is an important determinant of the oscillatory behavior of RBC suspensions and suggest the usefulness of the technique for studies of drug-membrane interactions.     

A computational model for the transit of a cancer cell through a constricted microchannel

We propose a three-dimensional computational model to simulate the transient deformation of suspended cancer cells flowing through a constricted microchannel. We model the cell as a liquid droplet enclosed by a viscoelastic membrane, and its nucleus as a smaller stiffer capsule. The cell deformation and its interaction with the suspending fluid are solved through a well-tested immersed boundary lattice Boltzmann method. To identify a minimal mechanical model that can quantitatively predict the transient cell deformation in a constricted channel, we conduct extensive parametric studies of the effects of the rheology of the cell membrane, cytoplasm and nucleus and compare the results with a recent experiment conducted on human leukaemia cells. We find that excellent agreement with the experiment can be achieved by employing a viscoelastic cell membrane model with the membrane viscosity depending on its mode of deformation (shear versus elongation). The cell nucleus limits the overall deformation of the whole cell, and its effect increases with the nucleus size. The present computational model may be used to guide the design of microfluidic devices to sort cancer cells, or to inversely infer cell mechanical properties from their flow-induced deformation.

     

Tubulin-specific antibody and the expression of microtubules in 3T3 cells after attachment to a substratum : Further evidence for the polar growth of cytoplasmic microtubules in vivo

Mouse 3T3 cells were allowed to attach to and spread on glass. The expression of cytoplasmic microtubules during the respreading process was monitored by immunofluorescence microscopy using monospecific antibody against tubulin. During radial attachment of the cells a ring of flattened cytoplasm is seen around the nucleus. Cytoplasmic microtubules then enter this spreading ring from the perinuclear region and elongate toward the plasma membrane. At later times microtubules appear perpendicular to the plasma membrane and seem to be in intimate contact with it giving the impression that they “stretch” the cytoplasm. When the cells assume their typical fibroblastic shape numerous microtubules are seen. They traverse the cytoplasm. Some come close to the plasma membrane and some bend to conform to the shape of the cell. Changes in microtubular organization correlate well with changes in cell shape. These results together with our previous observations on the assembly of cytoplasmic microtubules upon recovery from colcemid treatment suggest that microtubules may grow as polar structures from a microtubular organizing center towards the plasma membrane. The hypothesis that cytoplasmic microtubules might confer polarity on the cell is discussed.     

The superposition of steady on oscillatory shear and its effect on the viscoelasticity of human blood and a blood-like model fluid

To understand the pulsatility of human blood flow in vivo, it is necessary to separately investigate (1) steady shear and oscillatory flow, and (2) the superposition of steady shear flow on oscillatory flow performed under in vitro conditions. In this study a variable steady shear rate was superimposed in parallel on oscillatory shear at a constant frequency (0.5 Hz) for human blood (45% hematocrit), and an aqueous polyacrylamide polymer solution (AP 30E, concentration 300 ppm). The effect of superposition of the above two shear flows on the viscoelasticity of blood was more pronounced for the elastic (η′') than for the viscous (η′) component of viscoelasticity. With increasing superimposed shear rate, both η′ and η′' decreased, especially at the low shear region. This behavior can be explained by the viscoelastic properties of blood and the phenomena of blood aggregation and disaggregation. Quantitatively, the dependence of the viscous component of complex viscosity on superimposed shear for both blood and polymer solution is described by a modified Carreau equation. The elastic component of complex viscosity decreased exponentially with increasing superimposed shear, and it is described by an exponential model. © 1997 Elsevier Science Ltd     

Viscoelastic properties of an exopolysaccharide: Aeromonas gum, produced by Aeromonas nichidenii 5797

The viscoelastic properties of Aeromonas (A) gum in water were investigated by using the Rheometric Scientific ARES controlled strain rheometer. An intrinsic viscosity of 8336 ml/g was obtained according to the Fuoss-Straus equation. The effect of salt concentration on intrinsic viscosity revealed that the A gum exists as semiflexible chain. Typical shear-thinning (pseudoplastic) behavior was observed at concentrations higher than 0.52%. The zero shear viscosity (eta(0)) increased with increasing polysaccharide concentration (c) showing a gradient of approximately 1.0, 2.9 and 4.8 in different concentration domains. The critical concentrations c* and c**, at which the transitions from a dilute solution of independently moving chains to semidilute and then concentrated domains occurred, were determined roughly to be 1.2% and 3.5%. The results from dynamic experiments revealed that the A gum solution shows characteristics of polymer solutions without any evidence of gel-like character. All the results from steady and dynamic tests suggest that the A gum is a non-gelling polysaccharide. The temperature dependence of apparent viscosity was described by Arrhenius equation and the flow activation energy was estimated to be 45.2 kJ/mol, which is independent on polymer concentration.     

A novel two-layer, coupled finite element approach for modeling the nonlinear elastic and viscoelastic behavior of human erythrocytes

A novel finite element approach is presented to simulate the mechanical behavior of human red blood cells (RBC, erythrocytes). As the RBC membrane comprises a phospholipid bilayer with an intervening protein network, we propose to model the membrane with two distinct layers. The fairly complex characteristics of the very thin lipid bilayer are represented by special incompressible solid shell elements and an anisotropic viscoelastic constitutive model. Properties of the protein network are modeled with an isotropic hyperelastic third-order material. The elastic behavior of the model is validated with existing optical tweezers studies with quasi-static deformations. Employing material parameters consistent with literature, simulation results are in excellent agreement with experimental data. Available models in literature neglect either the surface area conservation of the RBC membrane or realistic loading conditions of the optical tweezers experiments. The importance of these modeling assumptions, that are both included in this study, are discussed and their influence quantified. For the simulation of the dynamic motion of RBC, the model is extended to incorporate the cytoplasm. This is realized with a monolithic fully coupled fluid-structure interaction simulation, where the fluid is described by the incompressible Navier–Stokes equations in an arbitrary Lagrangian Eulerian framework. It is shown that both membrane viscosity and cytoplasm viscosity have significant influence on simulation results. Characteristic recovery times and energy dissipation for varying strain rates in dynamic laser trap experiments are calculated for the first time and are found to be comparable with experimental data.     

Relative Contribution of the Cell Wall, Cytoplasmic Membrane, and Cytoplasm to the Gram-Positive Characteristic of Bacillus megaterium.

Bartholomew, J. W. (University of Southern California, Los Angeles), and Thomas Cromwell. Relative contribution of the cell wall, cytoplasmic membrane, and cytoplasm to the gram-positive characteristic of Bacillus megaterium. J. Bacteriol. 90:643-647. 1965.-A comparison of the roles of the cell wall, cytoplasmic membrane, and cytoplasmic components revealed that the intact cell wall was the dominant contributor to the gram-positive state. Protoplasts of Bacillus megaterium were confirmed as being gram-negative, as reported by Gerhardt et al. The "gram-positive protoplast" report of Amano et al. was shown to be a laboratory-produced artifact, resulting from the comparison of smears made from saline suspensions of Escherichia coli cells with smears made from formalin-sucrose suspensions of B. megaterium protoplasts.     

Red blood cell orientation in orbit C = 0.

Two modes of behavior of single human red cells in a shear field have been described. It is known that in low viscosity media and at shear rates less than 20 s-1, the cells rotate with a periodically varying angular velocity, in accord with the theory of Jeffery (1922) for oblate spheroids. In media of viscosity greater than approximately 5 mPa s and sufficiently high shear rates, the cells align themselves at a constant angle to the direction of flow with the membrane undergoing tank-tread motion. Also, in low viscosity media, as the shear rate is increased, more and more cells lie in the plane of shear, undergoing spin with their axes of symmetry aligned with the vorticity axis of the shear field in an orbit "C = 0" (Goldsmith and Marlow, 1972). We have explored this latter phenomenon using two experimental methods. First, the erythrocytes were observed in the rheoscope and their diameters measured. Forward light scattering patterns were correlated with the red cell orientation mode. Light flux variations after flow onset or stop were measured, and the characteristic times of erythrocyte orientation and disorientation were assessed. The characteristic time of erythrocyte orientation in Orbit C = 0 is proportional to the inverse of the shear rate. The corresponding coefficient of proportionality depends on the suspending medium viscosity eta o. The disorientation time tau D, after flow has been stopped, is such that the ratio tau D/eta o is independent of the initial applied shear stress. However, tau D is much shorter than one would expect if pure Brownian motion were involved. The proportion of erythrocytes in orbit C = 0 was also measured. It was found that this proportion is a function of both the shear rate and eta o. At low values of eta o, the proportion increases with increasing shear rate and then reaches a plateau. For higher values of eta o (5 to 10 mPa s), the proportion of RBC in orbit C = 0 is a decreasing function of the shear stress. A critical transition between orbit C = 0 and parallel alignment was observed at high values of eta o, when the shear stress is on the order of 1 N/m2. Finally, the effect of altering membrane viscoelastic properties (by heat or diamide treatment) was tested. The proportion of oriented cells is a steep decreasing function of red cell rigidity.