Separation of Marine Bacteria according to Buoyant Density by Use of the Density-Dependent Cell Sorting Method

The purpose of this study was to test whether some phylogenetic groups of natural marine bacteria have unique buoyant densities that allow them to be separated by the density-dependent cell sorting (DDCS) method. We first concentrated a natural bacterial assemblage to collect sufficient numbers of cells. They were separated into three fractions by DDCS, and the community structure in each was clarified by fluorescence in situ hybridization. The cells of Archaea tended to appear in the high-density fraction, whereas those of Cytophaga-Flavobacterium-Bacteroides were in the low-density fraction. We also calculated the sedimentation velocities of three typical marine bacteria (low density, middle density, and high density) using their buoyant density. The sedimentation velocities were approximately 10, 20, and 30 μm h⁻¹; these velocities have ecological implications when the heterogeneity of bacteria is considered at a microscale. To our knowledge, this is the first report on the buoyant density of natural marine bacteria.     

Separation of active and inactive fractions from starved culture of Vibrio parahaemolyticus by density dependent cell sorting

The co-existence of physiologically different cells in bacterial cultures is a general phenomenon. We have examined the applicability of the density dependent cell sorting (DDCS) method to separate subpopulations from a long-term starvation culture of Vibrio parahaemolyticus. The cells were subjected to Percoll density gradient and separated into 12 fractions of different buoyant densities, followed by measuring the cell numbers, culturability, respiratory activity and leucine incorporation activity. While more than 78% of cells were in lighter fractions, about 95% of culturable cells were present in heavier fractions. The high-density subpopulations also had high proportion of cells capable of forming formazan granules. Although this was accompanied by the cell specific INT-reduction rate, both leucine incorporation rates and INT-reduction rates per cell had a peak at mid-density fraction. The present results indicated that DDCS could be used to separate subpopulations of different physiological conditions.     

Factors affecting sedimentational separation of bacteria from blood

Rapid diagnosis of blood infections requires fast and efficient separation of bacteria from blood. We have developed spinning hollow disks that separate bacteria from blood cells via the differences in sedimentation velocities of these particles. Factors affecting separation included the spinning speed and duration, and disk size. These factors were varied in dozens of experiments for which the volume of separated plasma, and the concentration of bacteria and red blood cells (RBCs) in separated plasma were measured. Data were correlated by a parameter of characteristic sedimentation length, which is the distance that an idealized RBC would travel during the entire spin. Results show that characteristic sedimentation length of 20 to 25 mm produces an optimal separation and collection of bacteria in plasma. This corresponds to spinning a 12-cm-diameter disk at 3,000 rpm for 13 s. Following the spin, a careful deceleration preserves the separation of cells from plasma and provides a bacterial recovery of about 61 ± 5%.     

Spectrum of Physical Properties Among the Virions of a Whole Population of Vaccinia Virus Particles

Populations of virions released from cultures of L cells infected with vaccinia virus are composed of particles which differ substantially from each other in sedimentation rate and buoyant density. Clumps of two and three virions sediment enough faster than single particles so that fractions containing only singles and others with predominantly pairs can be isolated. The observed velocity range for single particles is much greater than that attributable to diffusion and convection in the centrifuge. Plaquing efficiency is three times higher in a small fraction of the slowest-moving virions than in the major part of the population, even though no size difference can be seen in the electron microscope. Isopycnic densities in potassium tartrate range from 1.15 to 1.23, enough to account for the observed range in velocities. Centrifugation was done in the BXIV zonal rotor at very low virion concentration (less than 10⁸ per ml at any point in the spectrum). Virus count and state of aggregation were determined by electron microscopy.     

Constancy of cell buoyant density for cultured murine cells

The relationship between cell cycle and cell density was determined for three different lines of mouse cells by equilibrium centrifugation of suspension cultures. The mean cell densities of the three lines differed significantly, with values of 1.0622, 1.0678, 1.0540 gm/ml for 70Z/3, S 107, and ABE 8, respectively. However, the density distributions within each of the three lines were indistinguishable, with an average coefficient of variation about 5% of the mean reduced density (i.e., density minus one). Quantitative DNA analysis of the cells separated by density showed that the proportion of cells in G1, S, and G2 + M phase of the cell cycle changed very little or not at all with cell density. In addition, cells separated by size (and therefore by phase of the cell cycle) using velocity sedimentation had the same means and distributions of densities. These results indicate that there is little or no change in cell density as the cells traverse the life cycle and that buoyant density appears to be a constant property of a cell type.     

Isolation of Cell Surface Membranes from Cultured C6 Glioblastoma Cells

Abstract: Plasma membranes were isolated from C6 glioblastoma cells by two methods. In the first method cells were treated with concanavalin A and lysed in hypotonic medium. After partial separation of plasma membranes from other cell material, the lectin was displaced with a-methyl-D-mannoside. In the second method untreated cells or cells iodinated in a lactoperoxidase-catalyzed reaction were homogenized in isotonic medium. Membrane fractions obtaincd by either homogenization procedure were further purified by rate zonal and equilibrium centrifugations into linear density gradients. Disruption of the glioblastoma cell membrane gives rise to heterogeneous assemblies of mem- brane fragments. Two populations of plasma membranes were isolated from untreated and from iodinated cells: a "lighter")membrane fraction characterized by relatively lower sedimentation velocity and buoyant density, and a "heavier" membrane fraction of relatively faster sedimentation velocity and higher buoyant density. Both fractions showed electrophoretic patterns similar to those of ¹²⁵I-labeled cell surface proteins. Their specific (Na⁺+ K⁺)-ATPase activity was seven- to eightfold the homogenate activity (recovery, 13.1%). Both fractions were, however, still contaminated by smooth endo- plasmic reticulum, as judged from the activity 0: NADPH-dependent cytochrome c reductase (recovery, 2.4%). It is suggested that plasma membrane fragments present in the two fractions might differ in the organization of their structures, e.g., membrane vesicle intactness and membrane orientation.     

DNA strand specificity of temporal RNA classes produced during infection of Bacillus subtilis by SP82.

The DNA of the Bacillus subtilis bacteriophage SP82 has been separated into heavy (H) and light (L) fractions by centrifugation in buoyant density gradients in the presence of polyguanylic acid. Competition-hybridization experiments were performed with these separated fractions using RNAs isolated from cells labeled at intervals which account for 80% of the lytic cycle and unlabeled competitor RNAs isolated from phage-infected cells at 2-min intervals throughout infection. The analysis of temporal RNA classes were facilitated by use of a double reciprocal plot of the data. Five temporal classes binding to the H fraction and three binding to the L fraction were detected; the possible existence of an additional class transcribed from the H fraction is discussed. RNA synthesized in the presence of chloramphenicol contains two of the three classes produced from L-DNA and two of the five classes transcribed from H-DNA.     

Separation and Purification of Bacteria from Soil

Bacteria were released and separated from soil by a simple blending-centrifugation procedure. The percent yield of bacterial cells (microscopic counts) in the supernatants varied over a wide range depending on the soil type. The superantants contained large amounts of noncellular organic material and clay particles. Further purification of the bacterial cells was obtained by centrifugation in density gradients, whereby the clay particles and part of the organic materials sedimented. A large proportion of the bacteria also sedimented through the density gradient, showing that they had a buoyant density above 1.2 g/ml. Attachment to clay minerals and humic material may account for this apparently high buoyant density. The percent yield of cells was negatively correlated with the clay content of the soils, whereas the purity was positively correlated with it. The cell size distribution and the relative frequency of colony-forming cells were similar in the soil homogenate, the supernatants after blending-centrifugation, and the purified bacterial fraction. In purified bacterial fraction from a clay loam, the microscopically measured biomass could account for 20 to 25% of the total C and 30 to 40% of the total N as cellular C and N. The amount of cellular C and N may be higher, however, owing to an underestimation of the cell diameter during fluorescence. A part of the contamination could be ascribed to extracellular structures as well as partly decayed cells, which were not revealed by fluorescence microscopy.     

Effect of the growth state on protein turnover in two lines of cultured BHK cells

The adherence, phagocytic activity and buoyant density of mouse peritoneal exudate colony forming units (CFU-PE) were investigated. There was a significant enrichment in the proportion of CFU-PE in the adherent cells population, defined as cells adhering to a plastic surface within 30 minutes of incubation. The phagocytic activity of CFU-PE was studied by incubating exudate cells with iron particles for 45 minutes. The cells were then separated into phagocytic and non-phagocytic cell fractions by passing the incubation mixture through a magnetic field. A significant enrichment of CFU-PE was seen in the phagocytic cell fraction. When exudate cells were fractionated in a Ficoll discontinuous density gradient, more than 88% of CFU-PE were recovered at the 16/18% and 18/20% interfaces. It is concluded that CFU-PE are adherent cells, have strong phagocytic activity and have a buoyant density between 1.0562 and 1.0703. When bone marrow cells were studied by these techniques, the committed stem cells for both granulocytes and macrophages (CFU-C) were enriched in both non-adherent cell and non-phagocytic cells populations. In the Ficoll density gradient, CFU-C banded at a heavier density region than CFU-PE.     

Post-irradiation thymocyte regeneration after bone marrow transplantation. II. Physical characteristics of thymocyte progenitor cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H x AKR)F1 bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, the thymocyte progenitor cells show the same distribution as CFUs. The relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

Buoyant density variation during the cell cycle of Saccharomyces cerevisiae

Cell buoyant densities of the budding yeast Saccharomyces cerevisiae were determined for rapidly growing asynchronous and synchronous cultures by equilibrium sedimentation in Percoll gradients. The average cell density in exponentially growing cultures was 1.1126 g/ml, with a range of density variation of 0.010 g/ml. Densities were highest for cells with buds about one-fourth the diameter of their mother cells and lowest when bud diameters were about the same as their mother cells. In synchronous cultures inoculated from the least-dense cells, there was no observable perturbation of cell growth: cell numbers increased without lag, and the doubling time (66 min) was the same as that for the parent culture. Starting from a low value at the beginning of the cycle, cell buoyant density oscillated between a maximum density near midcycle (0.4 generations) and a minimum near the end of the cycle (0.9 generations). The pattern of cyclic variation of buoyant density was quantitatively determined from density measurements for five cell classes, which were categorized by bud diameter. The observed variation in buoyant density during the cell cycle of S. cerevisiae contrasts sharply with the constancy in buoyant density observed for cells of Escherichia coli, Chinese hamster cells, and three murine cell lines.     

Selective aggregation of proteoglycans with hyaluronic acid.

Conditions were established to separate proteoglycan aggregate (AH1) from a bovine nasal septum extract by associative rate zonal sedimentation on a NaCl gradient. The AH1 has a higher protein content than the mixed aggregate-monomer (A1) isolated by conventional associative CsCl density gradient centrifugation from a portion of the same extract. The same associative rate zonal conditions separated the A1 fraction into aggregated AH1 containing hyaluronic acid and nonaggregated proteoglycan monomer (N1) essentially free of hyaluronic acid. The AH1 fraction is richer in protein and keratin sulfate than is N1. Dissociative rate zonal sedimentation of A1 under conditions which totally sedimented most of the disaggregated monomer (AH1-D1) and the nonaggregated monomer N1 separated a less sedimentable protein and keratan sulfate-rich proteoglycan monomer (AH1-D2). Chromatography on Sepharose 2B under dissociative conditions demonstrated that the nonaggregated N1 monomer is intermediate in size between the disaggregated monomers AH1-D1 and AH1-D2. N1 has a buoyant density higher than AH1 and is practically equivalent to AH1-D1. All are dense fractions so that separation by CsCl density gradient equilibration is not feasible.     

Cytoplasmic and outer membranes separation in Rhodopseudomonas sphaeroides

A cell envelope fraction has been prepared after mechanical disruption of lysozyme-EDTA spheroplasts from depigmented Rhodopseudomonas sphaeroides (aerobically grown in the light). On linear sucrose gradients this fraction can be separated in a cytoplasmic membrane fraction and an outer membrane fraction. The cytoplasmic fraction (buoyant density: 1.18 g/cm³) has been characterized by its succinic dehydrogenase activity and by its composition. The outer membrane fraction (buoyant density: 1.21 g/cm³) does not contain any respiratory activity nor hemoproteins. The same fractionation has been done on cells repigmented in the dark by lowering the O₂ pressure. In that case the same two fractions have been detected in addition to the chromatophore fraction (buoyant density: 1.14 g/cm³). However both, and specially the outer membrane fraction, were contaminated by chromatophore material.     

Isolation and characterization of outer and inner envelope membranes of cyanelles from a glaucocystophyte, Cyanophora paradoxa

Envelope membranes were isolated by sucrose density gradient floatation centrifugation from the homogenate of cyanelles prepared from Cyanophora paradoxa. Two yellow bands were separated after 40 h of centrifugation. The buoyant density of one of the two fractions (fraction Y2) coincided with that of inner envelope membranes of spinach or plasma membranes of cyanobacteria. The other yellow fraction (fraction Y1) migrated to top of sucrose-gradient even at 0% sucrose. Pigment analysis revealed that the heavy yellow fraction was rich in zeaxanthin while the light fraction was rich in β-carotene, and the both fractions contained practically no chlorophylls. Another yellow fraction (fraction Y3) was isolated from the phycobiliprotein fraction, which was the position where the sample was placed for gradient centrifugation. Its buoyant density and absorption spectra were similar to outer membranes of cyanobacteria. We have assigned fractions Y2 and Y3 as inner and outer envelope membrane fractions of cyanelles, respectively. Protein compositions were rather different between the two envelope membranes indicating little cross-contamination among the fractions. H. Koike and Y. Ikeda contributed equally.     

Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation Ii. Physical Characteristics of Thymocyte Progenitor Cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H × AKR)F₁ bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, thymocyte progenitor cells show the same distribution as CFUs. the relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

Wet and dry bacterial spore densities determined by buoyant sedimentation

The wet densities of various types of dormant bacterial spores and reference particles were determined by centrifugal buoyant sedimentation in density gradient solutions of three commercial media of high chemical density. With Metrizamide or Renografin, the wet density values for the spores and permeable Sephadex beads were higher than those obtained by a reference direct mass method, and some spore populations were separated into several density bands. With Percoll, all of the wet density values were about the same as those obtained by the direct mass method, and only single density bands resulted. The differences were due to the partial permeation of Metrizamide and Renografin, but not Percoll, into the spores and the permeable Sephadex beads. Consequently, the wet density of the entire spore was accurately represented only by the values obtained with the Percoll gradient and the direct mass method. The dry densities of the spores and particles were determined by gravity buoyant sedimentation in a gradient of two organic solvents, one of high and the other of low chemical density. All of the dry density values obtained by this method were about the same as those obtained by the direct mass method.     

Wet and dry bacterial spore densities determined by buoyant sedimentation.

The wet densities of various types of dormant bacterial spores and reference particles were determined by centrifugal buoyant sedimentation in density gradient solutions of three commercial media of high chemical density. With Metrizamide or Renografin, the wet density values for the spores and permeable Sephadex beads were higher than those obtained by a reference direct mass method, and some spore populations were separated into several density bands. With Percoll, all of the wet density values were about the same as those obtained by the direct mass method, and only single density bands resulted. The differences were due to the partial permeation of Metrizamide and Renografin, but not Percoll, into the spores and the permeable Sephadex beads. Consequently, the wet density of the entire spore was accurately represented only by the values obtained with the Percoll gradient and the direct mass method. The dry densities of the spores and particles were determined by gravity buoyant sedimentation in a gradient of two organic solvents, one of high and the other of low chemical density. All of the dry density values obtained by this method were about the same as those obtained by the direct mass method.     

Evaluation of methods for extraction of bacteria from soil

Abstract Several methods for dispersion of soil were tested for possible use in procedures for extraction of bacteria. Physical cell damage on cells and efficiency in extraction of indigenous cells from soil, were investigated. Cell damage by the dispersion methods was investigated by measuring the physical cell integrity and viability of pure cultures of Escherichia coli and Bacillus subtilis , as well as soil bacteria extracted from soil, when dispersed in slurries of γ-sterilized soil. Separation of bacteria and soil particles on the basis of buoyant density was conducted with the nonionic density gradient medium Nycodenz. When slurries of γ-sterilized soil with added pure cultured cells were centrifuged (10000 × g ) over cushions of Nycodenz (1.3 g ml⁻¹), practically all the added cells were recovered in a layer on top of the cushion. This proves that a reversible attachment and cosedimentation is not an important phenomenon in this procedure. The efficiency of the different dispersion methods for the extraction of indigenous soil bacteria, was assessed after separation of dislodged and attached soil bacteria. This separation was done either on the basis of sedimentation rate by low speed centrifugation, or buoyant density by Nycodenz density gradient centrifugation. The physical dispersion by ultrasonic treatment and chemical dispersion by the use of a chelating agent together with a detergent, were inferior to physical dispersion either by Waring blender (for large volumes) or a rotating rubber pestle treatment (for smaller volumes). The physical dispersion did not appear to be destructive to the cells tested.     

Targeted cytotoxic cells in human peripheral blood lymphocytes.

We have isolated subsets of cells from human PBL and have investigated their abilities to mediate lysis targeted by bispecific antibodies. Targeted cytotoxic cells were divided into two distinct types based on buoyant density. The low buoyant density fraction contained all of the targetable cytotoxic activity in unstimulated PBL, including both T and K cells targeted with anti-CD3 and anti-Fc gamma RIII (CD16) containing bispecific antibodies, respectively. Both types of targetable cytotoxic cells required IL-2 for maintenance of cytotoxic activity, expressed the CD56 (NKH1) marker, and mediated MHC-unrestricted lysis. The targetable T cells in low density PBL were exclusively CD8+ and represented only about 2% of the total PBL. The high buoyant density lymphocytes, depleted of NK cells, had no targetable activity, but were able to generate over several days, targetable T cell activity in the presence of a TCR cross-linking signal plus IL-2. Unlike the low-density cells, the activated high buoyant density effector T cells did not express CD56, consisted of both CD4+ and CD8+ cells, and did not mediate MHC-unrestricted lysis. These cells proliferated more rapidly and generated more total lytic activity than the low-density fraction. Our studies show that targetable cytotoxic activity in human PBL is mediated by several subsets of cells with different activation requirements. Presumably all of these activities could be directed against unwanted cells in clinical or preclinical studies involving targeted cytotoxic cells.     

Physical heterogeneity of mouse thymus lymphocytes.

Mouse thymocytes have been separated by velocity sedimentation in a density gradient. The resulting fractions have been analyzed using electrophoretic light scattering. The electrophoretic distributions of the individual sedimentation fractions reveal the presence of physically distinct subpopulations. Comparison of the mean mobilities of each fraction indicates that the faster-sedimenting cells tend to have a higher electrophoretic mobility.