Isolation of anucleated yeast protoplasts by means of density gradient centrifugation

An improved method for the preparation in high yield of anucleated Saccharomyces cerevisiae has been developed. This method is based on a two-stage centrifugation of the original protoplast mixture in linear density gradients (1–10%, w/v) of Ficoll 400. The yield of anucleated protoplasts was 5–9%, its value depended on the frequency of the nucleus-free protoplasts in the original mixture.The anucleated protoplasts were characterized by RNA, DNA and protein content, and by light and electron microscopy. The protoplasts lacking nuclei had about one third the diameter of the nucleated ones, and reduced of DNA, RNA and protein in comparison to normal protoplasts. Electron microscopy showed a typical yeast ultrastructure in anucleated protoplasts except that they lacked nuclei and exhibited a higher frequency of lipid granules and exocytotic electron-dense vesicles located close to the plasmalemma.     

Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll

Step gradients of polyvinylpyrolidone-coated colloidal silica particles (Percoll) were used to isolate and purify early development stages of Schistosoma mansoni (cercariae, skin stage, and 5-day-old schistosomula). With this method, mechanically transformed schistosomula can be isolated in higher purity and yield than that obtained with conventional procedures. In addition, use of the method revealed that schistosomula undergo a dramatic change in density during the first hours after transformation from cercariae. In other experiments, 5-day-old schistosomula were effectively purified from contaminating lung tissue by means of the Percoll gradient procedure. After purification on Percoll, schistosomula display no evidence of damage when examined by light microscopy and no loss in viability as judged by recovery of adult worms from mice.     

Fractionation of plant protoplast types by iso-osmotic density gradient centrifugation

Summary A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H₂O, the gradients provide a density range from 1.017 to 1.069 g/cm³ at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm³, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.     

Separation of oyster hemocytes by density gradient centrifugation and identification of their surface receptors

Glutaraldehyde-fixed hemocytes of Crassostrea virginica were subjected to differential centrifugation on a 5, 10, 15, and 25% discontinous sucrose gradient. Five subpopulations of cells were separated by this technique. Subpopulation 1 coincides with the small granulocytes, subpopulation 2 is comprised of hyalinocytes, subpopulation 3 of medium-sized granulocytes, subpopulation 4 of large granulocytes, and subpopulation 5 of a mixture of very large granulocytes and aggregates of small cells. By using several plant and animal lectins, it was ascertained that cells of subpopulations 1, 3, and 4 were agglutinated with Con A and extracts of the albumin glands of Helix pomatia and Cepaea nemoralis while those of subpopulation 2 were agglutinated by the same three lectins as well as wheat germ agglutinin. By applying the Con A-peroxidase cytochemical technique, it was determined that approximately 20% of the granulocytes of subpopulations 1 and 3 do not possess Con A-binding sites and only 18% of the large cells comprising subpopulation 5 possess such sites. These results suggest that the subpopulations of C. virginica granulocytes distinguishable by their dimensions and densities may be further subdivided by differences in specific surface binding sites.     

Enrichment of heterokaryocytes between mesophyll and epidermis protoplasts by density gradient centrifugation after electric fusion

Summary Mesophyll protoplasts from Nicotiana glauca were fused with epidermal protoplasts from N. langsdorffii by an electric pulse. After the fusion products were centrifuged on stepwise density gradient centrifugation using Percoll and sea water, somatic hybrids were observed at 70%–80% in the fraction recovered from the intermediate specific gravity fraction between epidermis and mesophyll protoplasts. From offsprings of these somatic hybrids, teratomatous plants were regenerated. Since the difference of specific gravity between mesophyll and epidermis protoplasts is inherent, this procedure can be essentially applied to obtain somatic hybrids between any combination of plants. The significance of this study is discussed in relation to obtaining somatic hybrids between plant materials without any appropriate genetic markers.     

Isolation of pea nodule bacteroids utilizing silica sol (Percoll) density gradient centrifugation

Isolation of bacteroids from effective (Fix⁺) and ineffective (Fix^–) pea nodules, inoculated withRhizobium leguminosarum K, were performed by a density gradient centrifugation method using silica sol (Percoll). Only one zone (=1.064–1.072; n-zone) was recognized in the Fix⁺ nodule which contained typical Y-shaped bacteroids while two zones (n-zone and =1.125–1.145; n'-zone) were obtained from the Fix^– nodule. The cells in the n'-zone, which are long rods differed morphologically from free-living cells at any growth phase (=1.108–1.125; f-zone and =1.074–1.078; f'-zone), and differed from Y-shaped bacteroids by cell density. The esterase isozyme pattern of bacteroids in the n-and n'-zones also showed clear differences from that of f-and f'-zone of free-living cells.     

The morphology of erythroid cells separated by density gradient centrifugation through ficoll

Summary The regenerating blood of geese injected with phenylhydrazine was subjected to large scale, zonal centrifugation through density gradients of Ficoll. In this way, erythroid cells were fractionated according to their respective stages of development. Highly enriched fractions were obtained, containing cells that were well preserved as assessed by both light and electron microscopy. The separated cells exhibited ribosome density and nucleic acid and protein staining patterns typically associated with erythrocyte differentiation. Morphometric analysis of nuclei indicated that despite an apparent net increase in the amount of compact chromatin during development, comparatively little difference existed between the volumes of condensed chromatin present in immature and mature cells. Instead, there was a three fold decrease in nuclear volume between young erythroblasts and reticulocytes, coupled with a concomitant decrease in the volume occupied by dispersed chromatin, RNP and nucleoli. These observations are discussed in relation to molecular changes associated with nuclear differentiation in erythroid cells.Supported by grants from the National Research Council of CanadaWe thank Dr. G. Setterfield for assistance with the EM data and we are grateful to the N.R.C. for use of centrifuges and the zonal rotor     

Evaluation of centrifugation parameters for density gradient experiments by means of a programmable pocket calculator

A calculation program is proposed suitable for programmable pocket calculators (e.g. HP series) to estimate s_20,w ∫ ω² dt values from density gradient centrifugation data. The program can be applied to linear or exponential density gradients prepared from sucrose or glycerol solutions spun in zonal rotors or swinging bucket rotors. A wide solute concentration range and temperature range is accounted for. Constants for empirical density calculation of glycerol and sucrose solutions concentrated in % (w/v) are estimated. Experiment verification of the program was carried out.     

Differentiation under the control of insulin of rat preadipocytes in primary culture: Isolation of homogeneous cellular fractions by gradient centrifugation

Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats. In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10^?9 M). During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid:CoA ligase. When VLDL and heparin were added with insulin to the medium, this effect was not potentiated. On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells. With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture. The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions. It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development.     

Efficacy of four density gradient separation media to remove erythrocytes and nonviable sperm from canine semen

The objective was to compare four commercially available density gradient centrifugation (DGC) media (ISolate [Irvine Scientific; Santa Ana, CA, USA], Percoll [Pharmacia; Uppsala, Sweden], PureCeption [SAGE In-Vitro Fertilization, Inc.; Trumbull, CT, USA], PureSperm 100 [Nidacon International AB; Molndal, Sweden]) for their ability to separate viable, motile sperm from contaminant nonviable (immotile and/or dead) sperm and red blood cells (RBC). Pooled sperm-rich fractions from four healthy dogs were assessed using Spermvison SAR (Minitube of America). For this, 1 mL of the blood/sperm admixture was pipetted over 4 mL of DGC media: 50%/90% ISolate (Irvine Scientific), 45%/90% Percoll (Pharmacia), 40%/80% PureCeption (SAGE In-Vitro Fertilization, Inc.), and 40%/80% PureSperm 100 (Nidacon International AB). After centrifugation, five 1-mL fractions (A, B, C, D, and E) and the sperm pellet (bottom fraction F) were separated. Sperm morphology and red blood cell/sperm ratio (RBC/S) per fraction were determined on stained slides. All DGC media separated RBC from sperm; the highest red blood cell/sperm ratio was present in ISolate (Irvine Scientific) and Percoll (Pharmacia) fraction A (29.4 ± 29.7 and 28.2 ± 20.8, respectively), and in fractions A and B of both PureCeption (SAGE In-Vitro Fertilization, Inc.) (37.0 ± 22.8 and 39.6 ± 24.3, respectively) and PureSperm 100 (Nidacon International AB) (25.2 ± 5.9 and 23.0 ± 3.9, respectively). The fractions with the highest total sperm recovery, motile sperm recovery, as well as overall motility were ISolate (Irvine Scientific) and Percoll (Pharmacia) fraction D (33.9 ± 29.4%; 40.99 ± 27.9%; 71.2 ± 21.8% and 36.4 ± 14.5%; 39.3 ± 15.8%; 88.6 ± 2.3%, respectively), and for PureCeption (SAGE In-Vitro Fertilization, Inc.) and PureSperm 100 (Nidacon International AB), the sperm pellet, fraction F (78.8 ± 28.3%; 88.0 ± 17.4%; 70.2 ± 11.1% and 73.1 ± 21.0%; 75.4 ± 24.6%; 80.6 ± 17.1%, respectively). In the pellet for PureCeption (SAGE In-Vitro Fertilization, Inc.), more sperm and motile sperm were recovered than in ISolate (Irvine Scientific) and Percoll (Pharmacia) fractions D (P < 0.0163). Therefore, DGC media should be considered for canine semen purification when contaminated with blood or when separation of motile versus immotile sperm is needed.     

Separation of components of the biomass from high rate algal ponds using PercollR density gradient centrifugation

The biomass of a High Rate Algal Pond was separated into individual components of algae, bacteria and detritus. The two stage technique involved mechanical and chemical disaggregation of concentrated pond samples, followed by separation on preformed Percoll^R/sucrose density gradients. Throughout a diurnal cycle, monitored in September 1990, between 85 and 90% of the total chlorophylla was recovered in the algal fraction. The greatest loss of chlorophyll from the sample occurred during the concentration stage; no further losses were encountered during physical and chemical disaggregation. The technique enabled the direct gravimetric determination of the separated algal biomass. The potential applications of the technique are discussed.     

Determination of small differences in buoyant density of macromolecules by analytical density gradient equilibrium centrifugation.

A sensitive method is proposed for the determination of small differences between the buoyant densities of different species of monodisperse macromolecules by analytical density gradient equilibrium centrifugation. The procedure involves the measurement at sedimentation equilibrium of the bandwidths of the concentration distribution of the separate macromolecules and of a mixture of the different species. The difference in buoyant densities can then be estimated from the difference between the bandwidths.     

蔗糖梯度离心法纯化Vero细胞乙脑疫苗的纯度分析及与层析法的比较

通过对蔗糖梯度离心法纯化Vero细胞乙脑疫苗的纯化工艺进行分析,发现现行工艺中收取的病毒组分中,不同蔗糖浓度中病毒的纯度是不同的,而呈峰型且与病毒效力峰及蛋白浓度峰不重合。实验结果对今后工艺的改进具有指导意义。另外应用Sephacryl-s-1000凝胶层析法对乙脑病毒进行了纯化研究和分析,发现蔗糖梯度离心法和层析法纯化的病毒的纯度及回收率分别为291．46u／mg、96．01％和309．41 u／mg 65．08％,Sephacryl-s-1000凝胶层析法同样可以获得较高的纯度和收率。     

Concentrating Toxoplasma gondii and Cyclospora cayetanensis from surface water and drinking water by continuous separation channel centrifugation

Aims:  To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters.
Methods and Results:  Ready-to-seed vials with known quantities of T. gondii and C. cayetanensis oocysts were prepared by flow cytometry. Oocysts were seeded at densities ranging from 1 to 1000 oocysts l⁻¹ into 10 to 100 l test volumes of finished drinking water, water with manipulated turbidity, and the source waters from nine drinking water utilities. Oocysts were recovered using continuous separation channel centrifugation and counted on membrane filters using epifluorescent microscopy. Recovery efficiencies of both parasites were ≥84% in 10 l volumes of drinking water. In source waters, recoveries ranged from 64% to 100%, with the lowest recoveries in the most turbid waters. Method precision was between 10% and 20% coefficient of variation.
Conclusion:  Toxoplasma gondii and C. cayetanensis are effectively concentrated from various water matrices by continuous separation channel centrifugation.
Significance and Impact of the Study:  Waterborne transmission of T. gondii and C. cayetanensis presents another challenge in producing clean drinking water and protecting public health. Detection of these parasites relies on effectively concentrating oocysts from ambient water, otherwise false negatives may result. Validation data specific to T. gondii and C. cayetanensis concentration methods are limited. Continuous separation channel centrifugation recovers oocysts with high efficiency and precision, the method attributes required to accurately assess the risk of waterborne transmission.     

Plasmodium yoelii and Plasmodium berghei: Isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation

Percoll (colloidal silica coated with polyvinylpyrrolidone) and Ficoll (MW 400,000) were used to separate erythrocytes infected with Plasmodium yoelii and Plasmodium berghei from uninfected red blood cells. Samples of blood collected from mice in different phases of malarial infection were overlaid on cushions of 55% Percoll, 20% Ficoll, or 28% Ficoll, respectively, centrifuged, and the interphase layers compared. The best yield of parasitized erythrocytes (PE) was achieved using Percoll when about 95% of the erythrocytes infected by the late developmental forms of the parasites (late trophozoites, schizonts, and gametocytes) were recovered from the gradient interphase, irrespective of the phase of the infection and the number of young erythrocytes in the sample. No alteration of antigenicity (assessed by immunofluorescence) or of osmotic fragility (over the range of 160–460 mOsm) could be detected in PE separated by Percoll or by Ficoll. In addition, parasites separated on Percoll gradients showed no significant ultrastructural changes and retained their normal infectivity to mice. Although both gradient media could be used for the separation of Plasmodium-infected erythrocytes, Percoll presented some advantages over Ficoll. Apart from the better reproducibility of the separation of high yields of very pure PE obtained with Percoll, its lower viscosity allowed easier handling, and lower centrifugal forces were needed to enable the cells to reach their isopycnic positions. Thus, Percoll fulfilled many of the criteria for an ideal density gradient medium. Parasitized erythrocytes were isolated by an easy, reproducible, and inexpensive procedure, and separated cells retained their normal structure, antigenicity, and infectivity.     

Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa

The aim of the present study was to evaluate the effect of selecting a sperm subpopulation by means of a discontinuous density gradient centrifugation (DGC) on the quality of ram thawed semen, and the relationships between sperm parameters assessed in unselected and in selected sperm samples with in vivo fertility after intrauterine artificial insemination (IUI) using unselected sperm samples. Semen samples from twenty males were collected by artificial vagina and cryopreserved following a standard protocol. After thawing, unselected sperm samples were used in an in vivo fertility trial and sperm motility (subjective and objective, assessed by means of CASA) and membrane and acrosomal integrities (microscopy) were evaluated on unselected and selected sperm samples. In addition, plasmalemma integrity (YO-PRO-1/PI), membrane fluidity (Merocyanine 540/YO-PRO-1), mitochondrial activity (Mitotracker Deep Red/YO-PRO-1), and DNA fragmentation index (%DFI) assessed by Sperm Chromatin Structure Assay (SCSA^®) were evaluated by flow cytometry before and after sperm processing using DGC. Results showed that DGC improved all sperm parameters significantly, except the %DFI, which increased after the selection procedure. No relationships were found between sperm parameters evaluated in unselected sperm samples and in vivo fertility. However, we found a positive correlation between spermatozoa with high membrane fluidity within the viable sperm population (VIABMerocyanine+) evaluated in selected sperm samples and in vivo fertility (r = 0.370, P = 0.019). In conclusion, our results suggest that selected spermatozoa represent a sperm subpopulation different to the unselected one that could be related with the in vivo fertility.     

Immobilization of immunoglobulins on polystyrene latex beads: characterization by density gradient centrifugation.

Isopycnic banding by density gradient centrifugation was used to measure density changes in complexes formed by the immobilization of each of four different immunoglobulins (IgG) (bovine, dog, rabbit, and sheep) on polystyrene latex beads (0.109 +/- 0.0025 micrometer diameter). Subtractive measurements of density changes allowed calculation of the mass of immobilized IgG under varying experimental conditions. The immobilization data were correlated with adsorption isotherms which incorporated charge repulsion forces. The effects of pH and NaCl concentration on the immobilization were studied for the latex-bovine IgG system. It was found that the mass of immobilized immunoglobulins was increased from 10 to 20% by removing the IgG from its isoelectric range.     

Separation of basolateral plasma membranes from smooth endoplasmic reticulum of the rat enterocyte by zonal electrophoresis on density gradients

Basolateral plasma membranes of rat small intestinal epithelium were purified by density gradient centrifugation followed by zonal electrophoresis on density gradients. Crude basolateral membranes were obtained by centrifugation in which the marker enzyme, (Na⁺ + K⁺)-ATPase, was enriched 10-fold with respect to the initial homogenate. The major contaminant was a membrane fraction derived from smooth endoplasmic reticulum, rich in NADPH-cytochrome c reductase activity. The crude basolateral membrane preparation could be resolved into the two major components by subjecting it to zonal electrophoresis on density gradients. The result was that (Na⁺ + K⁺)-ATPase was purified 22-fold with respect to the initial homogenate. Purification with respect to mitochondria and brush border membranes was 35- and 42-fold, respectively. Resolution of (Na⁺ + K⁺)-ATPase from NADPH-cytochrome c reductase by electrophoresis was best with membrane material from adult rats between 180 and 250 g. No resolution between the two marker enzymes occurred with material from young rats of 125 to 140 g. These results demonstrate that zonal electrophoresis on density gradients, a simple and inexpensive technique, has a similar potential to free-flow electrophoresis.