Electroacoustic fusion of millilitre volumes of cells in physiological medium

A technique is described in which erythrocytes suspended in 1.1 ml of 145 mM NaCl, have been fused by electrofusion. The cells in suspension were brought into close contact by setting up a 3 MHz ultrasonic standing wave in a cylindrical cell container. The aluminium foil base of the container served both to transmit ultrasound and as an electrode for electrofusion. The electric pulse was generated by a capacitor discharge system. The electric field strength required to fuse cells increased as the ionic strength of the cell suspending phase increased. Cells in physiological saline fused at an electric field strength of 7.3 kV/cm with a 50 microseconds pulse.     

Clarification of small volume microbial suspensions in an ultrasonic standing wave

The removal of Saccharomyces cerevisiae and Escherichia coli from 2·5 ml suspensions in ultrasonic standing wave formed at 1 or 3 MHz has been characterized. The standing wave was set up by a plane transducer and reflector mounted in the vertical plane. Cells in the ultrasonic field first concentrated in vertical planes at half wavelength separations. The ultrasound was then pulsed to allow clumps of concentrated cells to sediment in a controlled way during the short 'off' intervals. Yeast removal from suspension at a concentration of 3 × 10⁹ ml⁻¹ (14% volume v/v) was 99·5% in a total time of 4·5 min. Almost total (99·5%) clarification of prokaryote ( E. coli ) suspension was achieved here for the first time in a standing wave field. The clarification of a 1·3 × 10¹¹ ml⁻¹ (16% v/v) E. coli suspension occurred over 11·5 min. The period decreased to 7 min in the presence of a polycationic flocculant, polyethyleneimine. The implications of the results for design of systems to further reduce clarification times are discussed. Removal efficiency for both S. cerevisiae and E. coli decreased with decrease in cell concentration. This concentration dependence is shown not to be simply a consequence of acoustic interaction between single cells. Flow cytometry of stained cells detected no loss of cell viability arising from the ultrasonic procedure.     

Disruption of Brewers' yeast by hydrodynamic cavitation: Process variables and their influence on selective release

Intracellular products, not secreted from the microbial cell, are released by breaking the cell envelope consisting of cytoplasmic membrane and an outer cell wall. Hydrodynamic cavitation has been reported to cause microbial cell disruption. By manipulating the operating variables involved, a wide range of intensity of cavitation can be achieved resulting in a varying extent of disruption. The effect of the process variables including cavitation number, initial cell concentration of the suspension and the number of passes across the cavitation zone on the release of enzymes from various locations of the Brewers' yeast was studied. The release profile of the enzymes studied include alpha-glucosidase (periplasmic), invertase (cell wall bound), alcohol dehydrogenase (ADH; cytoplasmic) and glucose-6-phosphate dehydrogenase (G6PDH; cytoplasmic). An optimum cavitation number Cv of 0.13 for maximum disruption was observed across the range Cv 0.09-0.99. The optimum cell concentration was found to be 0.5% (w/v, wet wt) when varying over the range 0.1%-5%. The sustained effect of cavitation on the yeast cell wall when re-circulating the suspension across the cavitation zone was found to release the cell wall bound enzyme invertase (86%) to a greater extent than the enzymes from other locations of the cell (e.g. periplasmic alpha-glucosidase at 17%). Localised damage to the cell wall could be observed using transmission electron microscopy (TEM) of cells subjected to less intense cavitation conditions. Absence of the release of cytoplasmic enzymes to a significant extent, absence of micronisation as observed by TEM and presence of a lower number of proteins bands in the culture supernatant on SDS-PAGE analysis following hydrodynamic cavitation compared to disruption by high-pressure homogenisation confirmed the selective release offered by hydrodynamic cavitation.     

Observation of yeast cell movement and aggregation in a small-scale MHz-ultrasonic standing wave field

Aggregation of suspended yeast cells in a small-scale ultrasonic standing wave field has been monitored and quantified. The aggregation effect is based on the acoustic radiation force, which concentrates the cells in clumps. The ultrasonic chamber employed (1.9 MHz, one wavelength pathlength) had a sonication volume of 60 l. The aggregation process was observed from above the transducer through a transparent glass reflector. A distinct, reproducible, pattern of clumps formed rapidly in the sound field. The sound pressure was estimated experimentally to be of the order of 1 MPa. Microscopic observations of the formation of a single clump were recorded onto a PC. The time dependent movement patterns and travelling velocities of the cells during the aggregation process were extracted by particle image velocimetry analysis. A time dependent change was seen in the particle motion pattern during approach to its completion of clump formation after 45 s. Streaming eddies were set-up during the first couple of seconds. The scale of the eddies was consistent with Rayleigh micro-streaming theory. An increase in the travelling velocity of the cells was observed after 30 s from initially about 400 m s^–1 to about 1 mm s^–1. The influence of a number of mechanisms on particle behaviour (e.g. micro-streaming, particle interactions and convective flow) is considered. The experimental set-up introduced here is a powerful tool for aggregation studies in ultrasonic standing waves and lays the foundation for future quantitative experiments on the individual contributions of the different mechanisms.     

Filtration of bacteria and yeast by ultrasound-enhanced sedimentation

Continuous flow filtration of suspensions of eukaryotic cells by ultrasonic standing wave enhanced sedimentation has recently been reported. The filtration efficiency for Escherichia coli in such a filter has been characterized at frequencies of 1 and 3 MHz in the present work and compared with results for Saccharomyces cerevisiae. The yeast can be filtered at greater than 99% efficiency at a flow rate of 5 ml min^-1 at either frequency. The filtration efficiency of the smaller E. coli at 3 MHz is in excess of 80% at concentrations in the region of 10¹⁰ mI^-1 but decreased at lower concentrations. However, E. coli in a mixed suspension with yeast were, because of inter-particle interactions, removed with the filtrate at an efficiency ranging from 80 to 50% over the eight orders of bacterial concentrations tested (down to 10³ mI^-1) at 3 MHz. Quantitative considerations show that poor filtration of pure suspensions of the smaller cells at the lower frequency arises because, at reasonable flow rates, the residence time is not sufficient for the cells to reach the pressure nodal cell concentration regions. The filtration efficiencies of both cell types are comparable at 3 MHz. It is suggested that the more comparable efficiencies arise because concentration regions are narrower at the high frequency and Stokes drag by the filter bulk flow inhibits sedimentation of the concentrated cells.     

A study of the spatial organisation of microbial cells in a gel matrix subjected to treatment with ultrasound standing waves

Retention and manipulation of microbial cells through exploitation of ultrasonic forces has been reported as a novel cell immobilisation technique. The spatial ordering of yeast cells, within suspensions subjected to an ultrasonic standing wave field, was analysed for the first time. A technique, based on `freezing' the spatial arrangement using polymer gelation was developed. The resultant gel was then sectioned and examined using microscopic techniques. Light Microscopy confirmed the presence of specific regions in the ultrasonic field, where the cells are organised into bands corresponding to the standing waves' pressure nodal planes. Computer Image Analysis measurement of several physical parameters associated with this cell distribution matched the values derived from the theoretical model. The spatial cell-cell re-arrangement within each band and uneven distribution along the nodal planes have been analysed by Scanning Electron Microscopy. These results complement the ongoing study of the process of immobilisation of microbial cells by ultrasound standing waves. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spatially periodic discrete contact regions in polylysine-induced erythrocyte—Yeast adhesion

Cell-cell adhesion occurs when human erythrocytes and yeast cells are suspended together in suprathreshold concentrations of polylysine in saline. The threshold polycation concentration for adhesion depends on cell concentration and decreases with increasing polycation molecular weight. The threshold concentration was similar for erythrocyte-erythrocyte adhesion and for yeast-erythrocyte adhesion. Transmission electron micrographs show that the erythrocytes adhere to yeast as if to engulf the cell. The regions of close contact between the erythrocyte membrane and the yeast cell walls are spatially discrete. The contact separation distance for the asymmetric erythrocyte-yeast adhesion is very similar to that (0.83 μm) observed when polylysine-induced adhesion occurs in the symmetrical erythrocyte-erythrocyte system. The spacing is attributed to the growth of a squeezing wave as an interfacial instability, on the intercellular aqueous layer. Freeze-fracture electron microscopy of cells that were not fixed during preparation for microscopy confirms the discrete nature of contacts between polylysine treated erythrocytes.     

Spatially periodic discrete contact regions in polylysine-induced erythrocyte-yeast adhesion

Cell-cell adhesion occurs when human erythrocytes and yeast cells are suspended together in suprathreshold concentrations of polylysine in saline. The threshold polycation concentration for adhesion depends on cell concentration and decreases with increasing polycation molecular weight. The threshold concentration was similar for erythrocyte-erythrocyte adhesion and for yeast-erythrocyte adhesion. Transmission electron micrographs show that the erythrocytes adhere to yeast as if to engulf the cell. The regions of close contact between the erythrocyte membrane and the yeast cell walls are spatially discrete. The contact separation distance for the asymmetric erythrocyte-yeast adhesion is very similar to that (0.83 μm) observed when polylysine-induced adhesion occurs in the symmetrical erythrocyte-erythrocyte system. The spacing is attributed to the growth of a squeezing wave as an interfacial instability, on the intercellular aqueous layer. Freeze-fracture electron microscopy of cells that were not fixed during preparation for microscopy confirms the discrete nature of contacts between polylysine treated erythrocytes.     

Spatially periodic discrete contact regions in polylysine-induced erythrocyte-yeast adhesion

Cell-cell adhesion occurs when human erythrocytes and yeast cells are suspended together in suprathreshold concentrations of polylysine in saline. The threshold polycation concentration for adhesion depends on cell concentration and decreases with increasing polycation molecular weight. The threshold concentration was similar for erythrocyte-erythrocyte adhesion and for yeast-erythrocyte adhesion. Transmission electron micrographs show that the erythrocytes adhere to yeast as if to engulf the cell. The regions of close contact between the erythrocyte membrane and the yeast cell walls are spatially discrete. The contact separation distance for the asymmetric erythrocyte-yeast adhesion is very similar to that (0.83 micron) observed when polylysine-induced adhesion occurs in the symmetrical erythrocyte-erythrocyte system. The spacing is attributed to the growth of a squeezing wave as an interfacial instability, on the intercellular aqueous layer. Freeze-fracture electron microscopy of cells that were not fixed during preparation for microscopy confirms the discrete nature of contacts between polylysine treated erythrocytes.     

Proton pumping and the internal pH of yeast cells, measured with pyranine introduced by electroporation.

The internal pH of yeast cells was determined by measuring the fluorescence changes of pyranine (8-hydroxy-1,3,6-pyrene-trisulfonic acid), which was introduced into the cells by electroporation. This may be a suitable procedure for the following reasons. (i) Only minor changes in the physiological status of the cells seemed to be produced. (ii) The dye did not seem to leak at a significant rate from the cells. (iii) Different incubation conditions produced large fluorescence changes in the dye, which in general agree with present knowledge of the proton movements of the yeast cell under different conditions. (iv) Pyranine introduced by electroporation seemed to be located in the cytoplasm and to avoid the vacuole, and therefore it probably measured actual cytoplasmic pH. (v) Correction factors to obtain a more precise estimation of the internal pH are not difficult to apply, and the procedure may be useful for other yeasts and microorganisms, as well as for the introduction of other substances into cells. Values for the cytoplasmic pHs of yeast cells that were higher than those reported previously were obtained, probably because this fluorescent indicator did not seem to penetrate into the cell vacuole.     

Mechanism of disintegration of biological cells in ultrasonic cavitation

On the basis of elastic waves released by imploding cavitation bubbles, a mechanism for biological cell disintegration in high intensity ultrasounds has been proposed. Comparison of this mechanism with the published results on yeast cells shows many points of agreement suggesting that yeast cell disintegration in ultrasonic cavitation occurs by shear stresses developed by viscous dissipative eddies arising from shock waves.     

Applications of ultrasound streaming and radiation force in biosensors

Direct radiation force (DRF) and acoustic streaming provide the main influences on the behaviour of particles in aqueous suspension in an ultrasound standing wave (USW). The direct radiation force, which drives suspended particles towards and concentrates them in acoustic pressure node planes, has been applied to rapidly transfer cells in small scale analytical separators. The DRF also significantly increased the sensitivity of latex agglutination test (LAT) by concentrating the particles of an analytical sample in the pressure node positions and hence greatly increasing the antibody-antigen encounter rate. Capture of biotinylated particles and spores on a coated acoustic reflector in a quarter wavelength USW resonator was DRF-enhanced by 70- and 100-fold, respectively compared to the situation in the absence of ultrasound. Acoustic streaming has been successfully employed for mixing small analytical samples. Cavitation micro-streaming substantially enhanced, through mixing, DNA hybridization and the capture efficiency of Escherichia coli K12 on the surface of immunomagnetic beads. Acoustic streaming induced in longitudinal standing wave and flexural plate wave immuno-sensors increased the detection of antigens by a factor of five and three times, respectively. Combined DRF and acoustic streaming effects enhanced the rate of the reaction between suspended mixture of cells and retroviruses. The examples of a biochip and an ultrasonic immuno-sensor implementing the DRF and acoustic streaming effects are also described in the review.     

Investigation of enhancement of two processes, sedimentation and conjugation, when bacteria are concentrated in ultrasonic standing waves

Cells aggregate and can be recovered from suspension when exposed to an ultrasonic standing wave field. The acoustic force on individual cells in a standing wave decreases with particle volume. A plane ultrasonic field generated by a transducer driven at 3.3 MHz was used here to investigate the removal of Escherischia coli, cells with dimensions of the order of 1.0 m, from batch suspension by sedimentation over a range of concentrations (10³ to 10¹⁰ cells ml^–1). Cell removal efficiencies greater than 90% were achieved at initial concentrations of 10¹⁰ cells ml^–1. Removal efficiencies decreased gradually to zero, as initial bacterial concentration was reduced to 10⁷ cells ml^–1. It was found that, when low concentrations of E. coli (10³ to 10⁵ cells ml^–1) were added to suspensions of larger particles (i.e. yeast cells) that were of sufficient concentration to form aggregates in the sound field, E. coli could be harvested to an efficiency of 40%. The results imply that the E. coli became trapped and sediment with aggregates of larger particles. Some strains of bacteria are capable of DNA transfer by conjugation. The transfer rate of E. coli RP4 plasmid is order of magnitudes greater when conjugation occurs on solid medium rather than in liquid suspension. We have investigated whether the conjugation rate would also be higher in ultrasonically induced E. coli clumps than in free suspension. The donor strain was mixed with a recipient strain of E. coli, then sonicated in a capillary at 4.6 MHz in a tubular transducer for 5 min. The bacteria aggregated successfully. Results showed a three-fold increase in the rate of conjugation compared to a liquid mating control.     

Sub-micron particle manipulation in an ultrasonic standing wave: Applications in detection of clinically important biomolecules

Separation of particles from the suspending phase is of interest, among others, to clinical analysts. A system that enables manipulation of sub-micron sized particles in suspensions of analytical scale volume (10–50 l) using a non-cavitating ultrasonic standing wave is described. Particle suspensions, contained in glass capillary tubes of 1–2 mm internal dimension, are treated on the axis of a tubular transducer generating a radial standing wave field at 4.5 MHz. Microparticles (of average diameter range 0.3–10 m) suspended in buffer are concentrated within seconds at preferred regions separated by submillimetre distances. Concentration of suspended latex particles was inhibited in solutions containing protein at levels similar to those occurring in clinical specimens when the suspensions were sonicated in capillaries of circular cross-section. This effect was associated with acoustic streaming of the suspending fluid. Silica microparticles (more dense and less compressible than latex) could be concentrated in the presence of streaming. Latex particles concentrated readily in square cross-section capillaries where no streaming was observed. With sub-micron particles, the geometry of the sample chamber, the suspending phase composition and the size, density and compressibility of the microparticles all influence particle manipulation. The radial standing wave system has been used to enhance agglutination of antibody-coated latex microparticles in the presence of antigen allowing rapid and highly sensitive detection of clinically important biomolecules. The sensitivity of conventional diagnostic tests for microbial antigen has been improved by application of ultrasound and clinical utility has been demonstrated, in particular, for detection of meningitis-causing bacteria.     

Effect of ultrasound on the membrane potential and embryonic development of sea urchin eggs

Eggs of sea urchin Strongylocentrotus intermedius were exposed to continuous wave ultrasonic energy at a frequency of 2 MHz for 3 min. Ultrasonic treatment results in the appearance of monstrous embryos that die at the latest stages of their development. The number of dead embryos sharply grows with the increase in the spatial average intensity from 0.4 W/cm2 up to 1.6 W/cm2, when it comprises 100%. Dramatic decrease from 99% to 17% in the absolute value of transmembrane potential for both fertilized and unfertilized eggs was observed after ultrasonic treatment. These results seem to be caused by the action of stable cavitation. The given estimations show that for ultrasonic intensities more than 0.2 W/cm2 and providing the presence of gaseous bubbles in the vicinity of eggs shell the value of shear stress in an egg shell becomes great enough to cause the cytoplasmic gel fragmentation.     

腺苷甲硫氨酸合成酶的提取纯化研究

腺苷蛋氨酸具有转甲基、转硫和转氨丙基等重要生理作用,已成为治疗疾病的重要药物。目的：为腺苷蛋氨酸合酶的基因克隆做准备。方法：研究了腺苷甲硫氨酸合成酶的提取和纯化。腺苷蛋氨酸合酶为胞内酶,其提取需先进行细胞破碎,然后进行盐析和离子交换层析等方法来纯化。酵母的破壁试验考察了研磨、加入有机溶剂和超声波等不同的破碎方法。结果：超声波破碎法最好,得到粗酶液的酶活力为0．934U／ml;经过硫酸铵盐析后,利用离子交换层析法纯化腺苷甲硫氨酸合成酶,作出了腺苷甲硫氨酸合成酶的穿透曲线和洗脱曲线。     

The disruption ofSaccharomyces cerevisiae cells and release of glucose 6-phosphate dehydrogenase (G6PDH) in a horizontal dyno bead mill operated in continuous recycling mode

Baker’s yeast was disrupted in a 1.4-L stainless steel horizontal bead mill under a continuous recycle mode using 0.3 mm diameter zirconia beads as abrasive. A single pass in continuous mode bead mill operation liberates half of the maximally released protein. The maximum total protein release can only be achieved after passaging the cells 5 times through the disruption chamber. The degree of cell disruption was increased with the increase in feeding rate, but the total protein release was highest at the middle range of feeding rate (45 L/h). The total protein release was increased with an increase in biomass concentration from 10 to 50% (w/v). However, higher heat dissipation as a result of high viscosity of concentrated biomass led to the denaturation of labile protein such as glucose 6-phosphate dehydrogenase (G6PDH). As a result the highest specific activity of G6PDH was achieved at biomass concentration of 20% (ww/v). Generally, the degree of cell disruption and total protein released were increased with an increase in impeller tip speed, but the specific activity of G6PDH was decreased substantially at higher impeller tip speed (14 m/s). Both the degree of cell disruption and total protein release increased, as the bead loading increased from 75 to 85% (v/v). Hence, in order to obtain a higher yield of labile protein such as G6PDH, the yeast cell should not be disrupted at biomass concentration and impeller tip speed higher than 20% (w/v) and 10 m/s, respectively.     

The influence of bakers’ yeast cells on protein adsorption in anion exchange expanded bed chromatography

Baker’s yeast was disrupted in a 1.4-L stainless steel horizontal bead mill under a continuous recycle mode using 0.3 mm diameter zirconia beads as abrasive. A single pass in continuous mode bead mill operation liberates half of the maximally released protein. The maximum total protein release can only be achieved after passaging the cells 5 times through the disruption chamber. The degree of cell disruption was increased with the increase in feeding rate, but the total protein release was highest at the middle range of feeding rate (45 L/h). The total protein release was increased with an increase in biomass concentration from 10 to 50% (w/v). However, higher heat dissipation as a result of high viscosity of concentrated biomass led to the denaturation of labile protein such as glucose 6-phosphate dehydrogenase (G6PDH). As a result the highest specific activity of G6PDH was achieved at biomass concentration of 20% (ww/v). Generally, the degree of cell disruption and total protein released were increased with an increase in impeller tip speed, but the specific activity of G6PDH was decreased substantially at higher impeller tip speed (14 m/s). Both the degree of cell disruption and total protein release increased, as the bead loading increased from 75 to 85% (v/v). Hence, in order to obtain a higher yield of labile protein such as G6PDH, the yeast cell should not be disrupted at biomass concentration and impeller tip speed higher than 20% (w/v) and 10 m/s, respectively.     

Ultrasonic cell separator as a cell retaining device for high density cultures of plant cell

A system of ultrasonic filter device consisted of an ultrasonic generator, ultrasonic cell separation chamber (resonator) and a guide column, which was developed for suspension cultures of a plant cell. The key operation parameters affecting the efficiency of separation of cells from medium fluid were found to be the voltage of ultrasonic generator, the convective flow rate, and the distance between transducer and reflector. In the high density cultures ofAloe saponaria (>17 g DCW/L), the ultrasonic filter was so efficient that the cell holding time in the separation chamber was 10-fold higher than the case without ultrasonic wave at a convective flow rate of 0.24 cm/min. Furthermore, in perfusion type of high cell density cultures, cell aggregates were observed to be densely held in the ultrasonic chamber by ultrasonic force overcoming both gravitational and drag forces by pump. The accumulated cells were finally overflowed after the holding capacity of the chamber was reached. Back pressure was applied periodically to the resonator to flush cells back to bioreactor. The ultrasonic cell separator could operate over 75 min at a convective flow rate of 0.1 cm/min and at a cell concentration of 17 g DCW/L.     

Novel electrode structures for large scale dielectrophoretic separations based on textile technology

The use of dielectrophoresis (DEP) to date has mainly been limited to processing small volumes due to difficulties in the fabrication of microelectrodes over large surface areas. To overcome this problem a novel approach to the construction of micro-electrode arrays has been developed based on weaving. A plain weave cloth was made from 100 microm diameter stainless steel wires and 75 decitex polyester yarns. The stainless steel wires formed the weft, and were kept parallel and apart by a warp of flexible polyester yarns, with a gap of around 150 microm between the metal wires. The metal wires were alternately connected to earth and signal of an AC power source, and it was shown that it was possible to collect yeast cells suspended in deionised water at the metal wire surfaces by dielectrophoresis. The polyester yarn was also found to distort the electric field, creating further areas of electric field non-uniformity around the polyester yarns, further enhancing the capability of the system to attract cells. A 14 ml separation chamber was built from the cloth by alternately sandwiching perspex slabs and cloth together. The DEP chamber was able to effectively collect life yeast from a flow of suspended cells through the cloth using an applied field of 1 MHz at flow rates up to 5 ml min-1. However, some loss occurred due to sedimentation. Also, the chamber was able to separate dead and live yeast cells at 30 Vpk-pk, 2 MHz, with some cell loss due to sedimentation.