A method for recovery of enteroviruses from milk]

A method of detection of enteric viruses in milk was studied. The high protein content of milk and the protein nature of enterovirus allowed the detection of these viruses using the organic acid flocculation method. The poliovirus type 1 (Mahoney strains) and the E.C.H.O.1 isolated from the environment were used as virus model and were inoculated to creamed, half-creamed and whole UHT commercialized milk. The method consists on a milk sample clarification with acid precipitation and centrifugation. The clarified extract is reduced to a final volume of 10 to 15 ml after addition of beef extract powder and protein precipitation. This technique allows the recovery of 26 to 36% of poliovirus type 1 and 10 to 46% of E.C.H.O.1 viruses. In this work, the ferric chloride (FeCl3), added in 0.5 to 1 mM final concentration, was used as an adjuvant for the organic acid precipitation.     

Collecting Human Chromosomes for Whole-Mount Electron Microscopy

Harvesting chromosomes for the whole-mount technique and subsequent electron microscopy observation is a difficult step which may be overcome by the following method: direct centrifugation on formvar-coated grids of a lymphocyte suspension by means of a slide centrifuge, grids being then processed in the usual way.     

Continuous fractionation of human plasma proteins by precipitation from the suspension of the recycling stream

The conventional cold-ethanol batch fractionation method of human plasma is converted to an automatically controlled continuous fractionation process. The selected protein fractions are precipitated by mixing in the recycled product stream of the suspension. Compared to the batch process, the continuous fractionation process generates less coprecipitation and less spontaneous nucleation, allowing efficient centrifugation of precipitates, and the yield and purity of albumin in the final fraction is significantly increased.     

Observations on the separation of Theileria sporozoites from ticks

Hyalomma anatolicum anatolicum ticks infected with Theileria annulata were partially fed on rabbits and then ground up with tissue culture medium. The ground up ticks were treated by centrifugation at 100 g, filtration through membranes of 8 μm pore diameter and centrifugation on a discontinuous density gradient of Percoll. Counts of sporozoites and tick debris were made from Giemsa stained slides of samples at each stage of the separation. Debris was removed during light centrifugation and filtration at a greater rate than sporozoites. After filtration approximately 41% of the original sporozoites remained in the suspension. After density gradient centrifugation most sporozoites were found in a distinct zone, at approx. 1·08 g/cm³ density, separate from most dense debris and light debris and soluble contaminants. After this final centrifugation approximately 24% of the original sporozoites remained in the recovered suspension.     

A Centrifugation Technique for Preparing Medium-Free Resinous Mounts of Feulgen-Stained Pollen Tubes

Air-dried pollen of Tradescantia paludosa and a colchicine doubled Allium ascalonium-fistulosum interspecific hybrid was sown in culture tubes on an inorganic salt-lactose liquid medium containing 0.02% colchicine. After a 16-20 hr incubation at 22 C, pollen tubes were harvested by centrifugation for 3-5 min at 1100-1400 rev/min and fixed with acetic-alcohol (1:3). Feulgen staining was carried out in the culture tubes with fluid changes made after the centrifugation following each step. Single drops of the final pollen-45% acetic acid suspension were flattened under silicone-treated coverglasses which were removed by the quick freeze technique prior to counterstaining with fast green, dehydration, and mounting in Diaphane or Canada balsam. Medium-free, Feulgen-stained, resin-mounted preparations of well-dispersed pollen tubes with arrested metaphases were obtained.     

The Use of Density Gradient Centrifugation for the Separation of Germinated from Nongerminated Spores

S ummary : The density gradient centrifugation of a suspension of spores of B. subtilis 8057 on both sucrose and renografin gradients gave 2 distinct fractions. Germination evidence suggested that the heavier fraction consisted of dormant spores and the less dense fraction, germinated spores. It is concluded that density gradient centrifugation may provide a useful technique for the separation of germinated from nongerminated spores.     

A new dimension in suspension fusion techniques with polyethylene glycol.

Polyethylene glycol (PEG) induces the hybridization of mammalian cells at a much higher frequency when the cells are attached to a substrate during treatment than when the cells are treated in suspension. Since many cell types, e.g., lymphocytes, cannot attach to a substrate, a new technique for the PEG-induced fusion of cells in suspension was developed. This technique, referred to as "pancake fusion," is based on the centrifugation of suspended cells onto a coverslip and the PEG treatment of the cells on the coverslip as if they were attached to a substrate. With this technique, the frequency of hybridization of human white blood cells, which are incapable of attaching to a substrate, can be greatly increased.     

Purification of nuclei from a flagellate protozoan, Trypanosoma brucei

A simple and rapid technique is described for the isolation of nuclei from the flagellate protozoan Trypanosoma brucei. Cells were disrupted by nitrogen cavitation in the presence of hexylene glycol which enhances nuclear stability. Isolated nuclei were separated from nuclei trapped with cytoskeletal fragments and flagellae by Percoll density gradient centrifugation. Centrifugation in a medium with increased sucrose concentration greatly improved the separation of free nuclei from those trapped in cell debris. The isolation procedure resulted in the recovery of 34% of the nuclei present in the whole cell suspension. Electron microscopic and chemical analysis indicated that the recovered nuclei were in good condition and were highly purified.     

Attempt to use monocytes for HLA-DR typing

Two methods of isolating monocytes from peripheral defibrinized blood are described and compared with each other: 1. Adherence of monocytes on the surface of Petri-dishes and 2. Two-phase technique with centrifugation over a gradient of Percoll. The technique with Percoll has advantages as compared with the method of adherence: 1. Greater purity of monocyte suspension and higher percentage of monocytes (85% of monocytes on an average compared with 60% in adherence). 2. A damage of membrane can sometimes be observed in those monocytes gained by adherence and as a consequence of that there is a tendency to unspecifically positive cytotoxic reactions. In monocytes isolated by means of these two methods HLA-DR antigens could be identified. There was no complete correspondence between the cytotoxical reactions with monocytes and B-lymphocytes.     

Fractionation of a population ofSaccharomyces cerevisiae yeasts by centrifugation in a dextran gradient

A method of the fractionation of aSaccharomyces cerevisiae yeast population in dextran gradients is described. The elaboration of this method was based on the finding of a correlation between the size of individual cells and the number of bud scars on their surface and rapid indication of the scars by fluorescence microscopy. The basic conditions for fractionation (determined experimentally) were as follows: 2 ml. yeast suspension (100 mg. dry weight) was applied to the surface of a continuous dextran gradient of 9–16% concentration and was centrifuged at a relative centrifugal force of 200 G for 15 minutes. In fractionation of a whole population, the best fractionation was obtained in a linear gradient. Repeated separation of fractions obtained by centrifugation in a linear gradient in a concave gradient further separated cells without bud scars and accumulated cells with five scars and over. Three fractions were obtained by this technique. The first contained 90–98% cells without bud scars, the second 55–65% cells with 1–4 bud scars and the third 50% cells with five bud scars and over.     

Characteristics of mechanically disrupted bakers' yeast in relation to its separation in industrial centrifuges

The viscosity, density, and sedimentation characteristics of suspensions of whole and mechanically disrupted yeast cells were measured. Mechanical disruption increases the suspension viscosity and its non-Newtonian behavior. Experiments showed a good correlation between laboratory- and industrial-scale centrifugation results.     

Differential Centrifugation in Culture and Differentiation of Rat Neural Stem Cells

(1) The study of neural stem cells (NSC) has attracted much attention in recent years because of their therapeutic potential. However, the problem in culture and differentiation of NSC was how to obtain single cell suspension that preserves the function of NSC, and remove the debris caused by mechanical dissociation. In the present study, we try to find a simple and effective way to address the problem, i.e. differential centrifugation. (2) After a gentle mechanical dissociation using Pasteur pipette, the suspension was first centrifuged at 100 g for 5 min, and then recentrifuged at 400 g for 6 min. Finally, the two deposits were resuspended and seeded into culture flask respectively. The suspension from the second deposit was allowed for further culture and differentiation. Immunofluorescence technique was used to identify neural stem cell, neuron, astrocyte, and oligodendrocyte. (3) After the second differential centrifugation, single cell suspension was obtained with 2–3 cell clusters, and the cells not only grew to form neurospheres, but also differentiated into neurons, astrocytes, and oligodendrocytes. (4) Differential centrifugation is a simple and effective way to obtain single cell suspension, which will help make large-scale production of neurodifferentiated cells more effective.     

Synchronization of somatic embryogenesis from carrot cells at high frequency as a basis for the mass production of embryos

Synchronization of somatic embryogenesis at high frequency is a useful system for the mass production of embryos. Many attempts have been carried out, however, it was difficult to obtain the system in which most of the initial embryogenic cells or cell clusters synchronously differentiate to embryos. In carrot suspension cultures, high frequency, synchronous embryogenesis systems (following three systems) have been established.(1) Small spherical single cells from suspension cultures obtained by sieving and density gradient centrifugation in Percoll solutions differentiated to embryogenic cell clusters at high frequency when they were cultured in a medium containing 2,4-dichlorophenoxyacetic acid (0.05 micromolar), zeatin (1 micromolar) and mannitol (0.2 molar). (2) Embryogenic cell clusters from suspension cultures obtained by sieving, density gradient centrifugation in Ficoll solutions, and subsequent centrifugation at a low speed for a short time synchronously differentiated to embryos, especially globular embryos at high frequency, when they were cultured in a medium containing zeatin (0.1 micromolar) but no auxin. (3) Embryogenic cell clusters obtained by above method are cultured at cell densities of 2×10³ cell clusters ml^-1. Globular embryos which were sieved from embryos induced synchronously differentiated to torpedo-shaped embryos at high frequency when they were cultured at densities below 150 globular embryos ml^-1.Using these systems, the whole process of embryogenesis from single cells to whole plants could be synchronously induced at high frequency.Abbreviations ABA abscissic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellin A₃ - IAA indoleacetic acid - NAA naphthylacetic acid     

Filtration and agar embedding applied to fine needle aspirates for tumor diagnosis by electron microscopy. A combined technique.

OBJECTIVE: To develop a novel method for processing of fine needle aspirates subjected to electron microscopic (EM) study. STUDY DESIGN: Included 70 cases of poorly differentiated malignant tumors in which a definitive diagnosis was not possible on light microscopic (LM) examination and that thus required application of an ancillary technique such as FNA/EM, for diagnosis. We have established a novel method of processing, a technique of filtration through nylon mesh filters to eliminate red blood cells (RBCs) and necrotic debris, followed by agar well embedding to avoid loss of diagnostic material during processing without centrifugation at later steps after agar embedding, thus minimizing the time required for processing. It was successfully carried out in 70 cases. RESULTS: The combined technique was extremely effective in eliminating RBCs and necrotic debris. It also avoided further loss of valuable diagnostic material. An accurate diagnosis was rendered in 70 cases; that was not possible by LM alone. The whole procedure saves two to three hours of processing as centrifugation is not required after the agar embedding step. CONCLUSION: This technique was found to be cost- and time-effective, particularly suitable for developing countries, where financial resources are limited.     

Isolation of rat liver microsomes by gel filtration

A method was developed for the rapid isolation of liver microsomes by means of gel filtration. Such microsomes were compared to microsomes prepared by conventional centrifugation techniques. Both preparations were of similar composition as regards concentrations and activities of certain microsomal enzymes as well as contamination by other liver cell organelles. The main advantages over conventional techniques are the considerable decrease of time required for isolation of the microsomes, the improved removal of solutes like hemoglobin, the improved suspension stability of the preparation, and that the technique does not require facilities for ultracentrifugation.     

Fractionation of cells and subcellular particles with Percoll

At present, centrifugation is the most common method for separation and isolation of cells and subcellular particles. The technique can be used for a wide range of applications. During latter years it has become obvious what a powerful method density gradient centrifugation is, especially when used in conjunction with sensitive assays or clinical treatments. The most active areas for use of density gradient centrifugation include purification for in vitro fertilization of sperm of both human and bovine origin, isolation of cells for cell therapy of patients receiving chemo- and radiation therapy and basic research both on cellular and subcellular levels. These treatments and investigations require homogeneous populations of cells and cell organelles, which are undamaged after the separation procedure. Percoll, once introduced to reduce convection during centrifugation, has proved to be the density gradient medium of choice since it fulfills almost all criteria of an ideal density gradient medium. Recently good results have also been obtained after silanization of colloidal silica particles, e.g. BactXtractor. The latter medium has proved to be useful in recovery of microorganisms from food samples free of inhibitors to the Polymer Chain Reaction (PCR). The separation procedures described for Percoll in this review seem to be applicable to any cells or organelles in suspension for which differences in size or bouyant density exist. Furthermore, since Percoll media are inert, they are well suited for the separation of fragile elements like enveloped viruses.     

Packing of human erythrocytes in a centrifugal field

The rate of packing of human erythrocytes in whole blood and washed ones in aqueous suspension was investigated in a centrifugal field of 250 g. The Voigt-Kelvin rheological model was found to be well suited to describe the packing process. The ratio of the elastic modulus to viscosity was evaluated from this model. Its value suggests that the flexibility of the cell plays a minor role compared to other viscosity factors. Also the model suggests that the rate of packing is a complicated function of various viscoelastic factors. Empirical parameters describing the rate of packing are sensitive to drastic changes in cell flexibility, such as caused by formaldehyde treatment, whereas no fluidizing effect of procaine on cell membrane was detected. The rate of packing is not affected by decreasing the pH from 7.4 to 6.5. The method of mild centrifugation could be of some use for rapid evaluation of substantial flexibility changes in washed blood cells.     

SERUM ‘ANTI-MYELIN ANTIBODIES’: A SPECTROFLUOROMETRIC ASSAY PROCEDURE1

A simple spectrofluorometric procedure has been devised to determine serum antibodies, directed to constituents of the myelin sheath. It is an adaptation of the indirect immunofluorescent technique. A suspension of highly purified bovine myelin is incubated successively with a test rabbit serum and fluoresceinisothiocyanate-conjugated anti-rabbit gamma-globulin. Intensity of fluorescence in the final myelin suspension is determined spectrofluorometrically. Sera from rabbits with experimental allergic encephalomyelitis, induced by whole bovine spinal cord, generally gave fluorescence at least 10 times that of normal rabbit serum. Fluorescence of sera with high demyelinating activity was more intense than that of sera with equivocal demyelinating activity. The assay is specific for immunoglobulins directed to myelin constituents, organ-specific and species-independent. Rabbit anti-galactosylceramide serum with known demyelinating activity gave high fluorescence similar to that in sera of rabbits inoculated with whole spinal cord. Galactosylceramide could absorb a substantial portion of‘anti-myelin antibodies’of the anti-galactosylceramide serum but it did not absorb‘anti-myelin antibodies’of serum of rabbits with whole tissue-induced experimental allergic encephalomyelitis. This assay system may be useful for further studies of ‘anti-myelin antibodies’.     

An integrated approach for increasing the survival of autologous fat grafts in the treatment of contour defects

Autologous fat grafting as a technique to correct soft-tissue defects is a controversial subject. The high percentage of fat resorption and the resulting need for additional grafting considerably reduce the value of this method. The purpose of this study was to evaluate the clinical application of tissue-culturing methodology in the handling of the lipocyte aspirate in an endeavor to improve the survival rate and therefore the take of the grafted lipocytes. The method consists of syringe aspiration of the lipocytes from the donor site, isolation of intact lipocytes by gentle centrifugation, suspension of the aspirate in an enriched cell culture medium, and injection of the cell suspension into preformed subdermal tunnels. A number of media were tested and shown to prolong the survival of lipocytes ex vivo using fluorescent acridine orange stain. Implementing the integrated cell culture techniques increased the lipocytes' viability, as indicated in clinical evaluation in which the amount of graft take ranged between 50 and 90 percent. The results of 15 patients with varied types of cases who were operated on using this new methodology show that the tissue defect was filled and remained so in postoperative follow-ups of 6 to 24 months. A three-dimensional CAT scan-aided evaluation method was developed and used in one of four case histories presented herein.     

Preparation of isolated liver endothelial cells and Kupffer cells in high yield by means of an enterotoxin

A new method for preparing non-parenchymal rat liver cells (NPC) is described. The liver cell suspension, prepared by perfusing the liver with collagenase, was treated with enterotoxin from Clostridium perfringens for 15 min. The enterotoxin made the parenchymal cells leaky, and these cells could be separated from the NPC by centrifugation in a solution containing Nycodenz (20%, w/v). During the centrifugation, the NPC floated, while the parenchymal cells sedimented. The yield of NPC per liver (200 g rat) was about 250 X 10(6) cells. The NPC were further separated into endothelial cells, Kupffer cells and stellate cells by centrifugal elutriation. This method was particularly useful for preparing endothelial cells in high yield (100 X 10(6) cells per liver). Intravenously injected formaldehyde-treated albumin was selectively taken up by the endothelial cells. Isolated endothelial cells in suspension as well as in surface culture maintained their ability to endocytose this ligand.