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At least 19 recordsLinked to original sources
PubMed1979

Colloidal silica--aluminum modified--PVP density gradient centrifugation: centrifuge tube wall cell adherence, aggregation, separation properties and comparison to BSA and Ficoll.

A method is described for the inexpensive and easy preparation of colloidal silica--aluminum modified--polyvinylpyrrolidone (CS-AM-PVP) density gradient centrifugation medium. Using density gradient centrifugation, several cell separation and biochemical characteristics were studied: centrifuge tube wall cell adherence, mouse spleen cell density distribution, rebanding properties, mitogen response and cell aggregation. Cell adherence to the centrifuge tube wall using density gradients of CS-AM-PVP was compared with density gradients of bovine serum albumin and Ficoll. Few cells adhered to the centrifuge tube wall when CS-AM-PVP was used as a gradient medium; whereas, significant cell adherence to the centrifuge tube cell wall occurred when bovine serum albumin or Ficoll was used as a gradient medium. The CS-AM-PVP gradient medium did not inhibit the response to mitogens of mouse spleen cells which had been separated into density subpopulations in a discontinuous CS-AM-PVP density gradient, caused a minimum amount of cell aggregation, and was found to be non-toxic.

Aluminum↗
PubMed1979

Centrifugal elutriation (counterstreaming centrifugation) of cells.

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.

Animals↗
PubMed1975

Deterioration of rat-liver mitochondria during isopycnic centrifugation in an isoosmotic medium.

We have investigated the effect of the centrifugation speed on the behavior of rat-liver mitochondria during isopycnic centrifugation in an isoosmotic medium. The gradient was made with a macromolecular compound, glycogen dissolved in 0.25 M aqueous sucrose. The distribution curves of several mitochondrial enzymes change when the centrifugation reaches a certain speed: they are shifted toward regions of lower density. The results are plausibly explained by supposing that the inner mitochondrial membrane becomes permeable to sucrose at high centrifugation speeds, and that the granules swell. The main causal agent of the phenomenon is the hydrostatic pressure the mitochondria are subjected to during centrifugation. Morphological observations show that mitochondria are markedly deteriorated when centrifuged at high speed in the glycogen gradient; they are swollen and the outer membrane is broken; also frequently, a large electron-dense granule is seen in the matrix near the inner mambrane.

Animals↗
PubMed1977

Subcellular fractionation by centrifugation of homogenates of the neural lobe of the bovine pituitary gland: identification of different pools of hormone in the homogenate and isolation of neurosecretosomes.

1. Homogenates of bovine pituitary neural lobe tissue were subjected to differential centrifugation. Six subcellular fractions (I-VI) were obtained and the distribution among them of various cell organelles was studied by means of markers. Fraction II (800-3000 gav), the nerve-ending fraction (neurosecretosomes), contained sedimentable lactate dehydrogenase, hormone and a large proportion of the total Mg2 + +Na+ +K+-ATPase. 2. Lysis of the neurosecretosomes in hypotonic sucrose solutions led to loss of lactate dehydrogenase and vasopressin. 3. Centrifugation of the granule fraction (IV) on a sucrose gradient (1.3-2.0 M sucrose) gave a bimodal distribution of vasopressin. Purified neurosecretory granules were recovered from the denser band. Centrifugation on modified gradients (0.8-2.0 or 0.4-2.0 M sucrose) increased the yield of hormone in the denser band but the purity of the granules was decreased. 4. Considerable purification of the neurosecretosomes (fraction II) was achieved by "washing". Centrifugation of washed neurosecretosomes on sucrose density gradients led to the accumulation of all activities at a region between 1.4 and 1.5 M sucrose. 5. The distribution of Mg2+ +Na+ +K+-ATPase in centrifugal fractions indicated that the neurosecretosomes had been isolated in relatively high yield.

Adenosine Triphosphatases↗
PubMed1979

Improved blood culture technique based on centrifugation: clinical evaluation.

A total of 3,335 blood samples from 1,180 patients suspected of having bacteremia were analyzed concurrently by two methods: (i) supplemented peptone broth with sodium polyanethanol sulfonate and a CO2 atmosphere; and (ii) lysis centrifugation at 3,000 X g for 30 min onto a high-density, hydrophobic cushion. The centrifugation technique recovered 80% of the positive cultures as compared with 67% for the broth method. The centrifugation technique showed an apparent increase in the isolation of staphylococcus aureus, Pseudomonas, and yeasts. In almost every instance, the time required for detection of a positive culture was shortest for the centrifugation method. Contamination rates for both systems were comparable (1.4%). Quantitation, offered only by the centrifugation method, proved useful on several occasions in discriminating between an opportunistic infection versus a skin contaminant and in judging efficacy of antimicrobial therapy.

Bacteria↗
PubMed1977

[Studies on the manufacture of hematological and cytological specimens using the centrifugation technic].

Centrifugal preparation (Cp) represents a method of creating haematological parameters which, in addition to a qualitative improvement of representing single cells, is suitable to produce reproducible haematological smear preparations. In qualitative and quantitative respect centrifugal preparations will reflect the intravasal conditions better than the common blood smear technique. Further advantages are the concentration of cellular elements and in connection with it the opportunity of finding cells rarely present. With a small amount of work centrifugal preparations enable counting values to be established as well as cytological and cytochemical findings from the venous blood to be obtained. As a result of the test it turned out that the common glass slides cannot be replaced by plastic foils ones without certain disadvantages appearing. First investigations of attempting to represent cells in culture medium as well as analyzing liquors and processing other biological liquids with the help of the centrifugal preparation technique brought unsatisfactory results. Centrifugal preparation provides the possibility of performing pre-programmed haematological examinations beginning with blood collection and ending up in the evaluation by a computer.

Blood Specimen Collection↗
PubMed1975

The large-scale isolation of renin-containing granules from rabbit renal cortex by zonal centrifugation.

A new method for the isolation of renin-containing granules from rabbit renal cortex by sequential two-dimensional centrifugation is presented. A mitochondrial fraction produced by preparative differential centrifugation was subjected to isopycnic centrifugation on combined discontinuous and linear density gradients in the same SZ-14 reorienting gradient zonal centrifuge rotor. Renin activity, as assayed by radioimmunoassay for angiotensin I, was localized in a region of the density gradient with a corresponding density of 1.196 Gm. per cubic centimeter or 1.53 M sucrose. The renin-containing granules were enriched 5.0-fold over whole homogenate levels, while peroxisomes were enriched 17.4-fold, lysosomes 23.4-fold, and mitochondria 4.3-fold. Electron microscopic examination of the fraction with highest renin activity showed a granular fraction almost free of contamination from other subcellular material. The data supports the view that renin-containing granules are a distinct subcellular particle. This new method for the large-scale isolation of renin-containing granules makes it possible to obtain the quantity of material necessary to study the release renin at the subcellular level and permit the further biochemical purification and characterization of the enzyme.

Acid Phosphatase↗
PubMed1979

Isolation of alveolar type II cells by centrifugal elutriation.

Centrifugal elutriation (counterflow centrifugation) was used to develop a reproducible method for obtaining a nearly pure population of isolated alveolar type II cells. Lung was dissociated into individual cells with recrystallized trypsin, and the type II cells were partially purified by centrifugation on a discontinuous density gradient. The alveolar type II cells were finally purified by centrifugal elutriation. Cells were collected from the elutriator rotor by stepwise increases in flow rates. Cells obtained at flow rates of 7 and 14 ml per min were lymphocytes, other small cells, a few type II cells and cell debris; cells collected at flow rates of 18 and 22 ml per min were mainly type II cells; and cells collected at flow rates of 28, 34 and 43 ml per min were macrophages, some type II cells, other lung cells and cell aggregates. At flow rates of 18 and 22 ml per min, 1.9 +/- 1.0 x 10(6) cells per rat lung (mean +/- S.D., n=30) were recovered of which 86 +/- 6% were type II cells. At these flow rates, 94% of the cells excluded the vital dye erythrosin B from their cytoplasm. They consumed oxygen at a rate of 101 +/- 21 nmol per hr . 10(6) cells (mean +/- S.D., n=4), and their oxygen consumption increased only 10% after 10 mM sodium succinate was added. The cells incorporated [14C]leucine into protein and lipid for 4 hr. Electron micrographs of the cells collected at flow rates of 18 and 22 ml per min show a high percentage of morphologically intact alveolar type II cells. We conclude that centrifugral elutriation is a reproducible method for obtaining nearly pure, metabolically active alveolar type II cells.

Animals↗
PubMed1976

Hepatocyte perfusion within a centrifuge.

A centrifuge, as used in blood banks for washing erythrocytes, is shown to be a good perfusion chamber for hepatocyte suspensions being evaluated for potential extracorporeal liver support. The perfusate was cell-free oxygenated--pO2 300 millimeters of mercury--plasma, serum or tissue culture medium. A centrifuge that requires intermittent perfusion cannot deliver sufficient oxygen to maintain prolonged viability. A continuous flow unit can, at a flow of 800 milliliters per minute, support most functions of 100 grams of hepatocytes for six hours and some function for 12 hours. Metabolism of 2-14C tagged pyruvate to 14CO2 is the most useful sensitive measurement of liver function in this system. It is concluded that a centrifuge allowing a constant perfusion of oxygenated plasma into hepatocytes kept within the unit by centrifugal force is a suitable base for extracorporeal liver support.

Animals↗
PubMed1979

Centrifugation of stallion semen and its storage in large volume straws.

In a study of different methods of handling stallion semen for deep freezing, ejaculates were divided into 3 portions, the first of which was diluted 1:2 with lactose--egg yolk--glycerol diluent and frozen in pellet form. The second aliquot was centrifuged without any diluent and the third portion was initially diluted with an experimental diluent (Merck) and then centrifuged for 5 min at 1000 g. The second and third portions were frozen in large volume straws each of which contained one whole insemination dose of 1 or 2 X 10(8) progressively motile spermatozoa. The addition of a diluent to the semen before centrifugation and freezing (portion 3) resulted in an increase in sperm motility after thawing. Motility was further increased by the use of a recently developed diluent after centrifugation and before freezing. In one fertility trial, 12 of 19 mares (63%) conceived following a single insemination of frozen semen during one oestrous period.

Animals↗
PubMed1977

Adenine nucleotide metabolism of blood platelets. X. Formaldehyde stops centrifugation-induced secretion after A23187-stimulation and causes breakdown of metabolic ATP.

A23187 induced shape change, aggregation and secretion of platelets in plasma. When rapid cooling was used to stop secretion and centrifugation to separate the cells from the medium, maximal amounts of storage ATP plus ADP and preadsorbed [14C]serotonin were found in the supernatant immediately (less than 5 s) after A23187 addition. These results suggested that A23187 could cause shape change and aggregation through secreted ADP and not directly. When secretion was stopped with chilling and formaldehyde treatment before centrifugation, the secreted substances appeared after a lag of 60-120 s, i.e. after shape change was terminated and aggregation was well on its way. These two platelet responses thus seemed to be independent of secretion and induced directly by A23187. The absence of a lag period when secretion was stopped by chilling alone was thought to be due to centrifugation-induced secretion of platelets conditioned by A23187. Formaldehyde completely inhibited centrifugation-induced secretion. At 37 degrees C, formaldehyde caused rapid breakdown of metabolic ATP in platelets with a pattern dependent on the formaldehyde concentration: Below 50 mM, ATP was converted to inosine plus hypoxanthine via ADP, AMP and IMP and the adenylate energy charge was preserved. Above 100 mM, AMP was the end product with a drastic reduction in the adenylate energy charge. These changes were not due to lysis of the platelets, but were apparently caused by an formaldehyde-induced increase in cellular ATP consumption. Platelet secretion is usually associated with a conversion of metabolic ATP to hypoxanthine. Formaldehyde had to be used to stop secretion and since it caused breakdown of ATP, additional smaples were taken out for nucleotide determination during stirring of platelet-rich plasma with A23187. It was found that metabolic ATP was converted to inosine plus hypoxanthine only during the secretion step.

Adenosine Diphosphate↗
PubMed1978

Deformation of human erythrocytes in a centrifugal field.

A new method for altering red cell morphology by high-speed centrifugation of cells through a physiological medium is described. Cell shape is preserved for microscopic analysis by allowing the sedimenting cells to pass from the physiological medium into a glutaraldehyde fixative solution. Examination of the deformed, fixed cells indicates that the vast majority resemble spheres with a flat, triangular tail. Measurements of the overall length of deformed cells show a nearly linear relationship between cell length and centrifugal force; average cell length increased from 8 to 11 micrometer as the centrifugal field was increased from 2,000 to 15,000 g. These data suggest that this centrifugal technique may be useful for evaluating cellular deformability and, potentially, the material properties of red cells.

Erythrocyte Membrane↗
PubMed1979

Distribution of PNS myelin proteins and membrane enzymes in fractions isolated by continuous gradient zonal centrifugation.

Myelin was purified from adult rabbit sciatic nerve by two procedures: discontinuous gradient centrifugation and continuous gradient zonal centrifugation. Two fractions were obtained from the discontinuous gradient. The fraction floating on 0.32 M sucrose and the fraction recovered from the 0.32/0.85 M sucrose interface showed typical myelin membranes by electron microscopy and typical myelin proteins by gel electrophoresis. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) decreased from the top to the bottom of the discontinuous gradient. The myelin separated by zonal centrifugation on a continuous sucrose gradient showed three distinct peaks (on monitoring optical density) at 0.10, 0.30 and 0.57 M sucrose. The latter peak yielded 92% of the material applied. The two minor peaks of low density exhibited high CNP and acetylcholinesterase (AChE) activities but the specific activity of both enzymes increased markedly at the heavy end of the gradient. The zonal fractions showed typical myelin proteins in all fractions by polyacrylamide gel electrophoresis but with important quantitative differences. These results indicate that PNS myelin shows significant heterogeneity.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1978

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient.

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.

Acid Phosphatase↗
PubMed1978

New centrifugation blood culture device.

A single-tube blood culture device designed for centrifugation in a tabletop centrifuge is described. Reconstruction experiments using 21 different organisms and human donor blood indicate that excellent recovery can be obtained by centrifugation for 30 min at 3,000 X g.

Bacteria↗
PubMedDate not supplied

A comparative cytologic study of 100 urine specimens processed by the slide centrifuge and membrane filter techniques.

An evaluation of cytology preparations made by the slide centrifuge and membrane filter techniques was made on 100 consecutive urine specimens from 46 patients. The membrane filter technique gave better cellular recovery and produced slides qualitatively superior to those prepared by the slide centrifuge technique. The former, therefore, is the recommended method. The slide centrifuge might be of greater value with urine of low cellularity where the membrane filter has provided only a few to practically no cells. The diagnostic accuracy on clinical follow-up of the patients included in this study is 94% for both methods, with no false positive cases and a 6% false negative rate. This high degree of accuracy indicates that these concentrating methods may lead to clinically valuable information if used routinely in the cytology laboratory.

Adult↗
PubMedDate not supplied

[Homogenization of samples for the culture of mycobacteria. A comparative study of samples with and without centrifugation].

A homogenization method for sputum samples for the culture of acid-fast bacilli that omits the use of centrifugation, passage test tubes and balances is described. 10% sodium phosphate was used as decontaminating substance. This method facilitates the laboratory work since the samples so treated are incubated at 37 degrees C up to the next day when they are then cultured. 676 samples prepared according to this method were compared with samples prepared according to the standard method; a 6,21% positivity in the former compared to a 5,47% in the latter as well as a lower percentage of contamination were obtained. Contamination occurred in 9,91% of samples without centrifugation and in 14,35% of centrifuged samples.

Bacteriological Techniques↗