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PubMedDate not supplied

Blood cultures from Bangladeshi children with septicaemia: an evaluation of conventional, lysis-direct plating and lysis-centrifugation methods.

The use of a laboratory-made lysis-direct plating and lysis-centrifugation (LDP/LC) device for blood cultivation has been compared with the conventional broth culture method in respect of speed and sensitivity in detecting organism(s) and cost effectiveness. 400 blood cultures yielded 95 clinically significant isolates. Both methods recovered 73 organisms (76.8%); 20 (21%) were detected by LDP/LC methods only, and 2 (2.1%) were isolated by the conventional method only. All the 93 isolates (97.8%) recovered by LDP/LC were isolated within 48 h, whereas the broth culture method took 7 d to isolate a total of 75 organisms (78.9%). The LDP/LC method, with our laboratory-made device, costs one-fourth of the cost of the conventional broth culture system.

Bacteriological Techniques↗
PubMed1985

A fully automated method for clot based coagulation panels using an MCA Multistat Centrifugal Analyzer.

Small modifications of the commercially available software of the I.L. Multistat Centrifugal Analyzer (MCA) enabled us to evaluate clot based prothrombin times (PT), activated partial thromboplastin times (APTT) and thrombin times (TT) simultaneously within one run. This imparted a great deal of flexibility to this procedure in that batches and/or panels of clot based and other types of turbidimetric tests can be performed concurrently in any combination. The PT and TT by this turbidimetric procedure correlated very well with those of the Fibrometer in normal specimens as well as in a wide variety of coagulation defects. The turbidimetric APTT procedure, however, often produced clotting times longer than those achieved with the Fibrometer. The discrepancy between the Fibrometer and turbidimetric APTT procedures was shown to be the consequence of greater sensitivity of the latter to detect low concentrations of heparin, elevated fibrinogen split products and mild factor deficiencies. The available clot based procedures are suitable for use in detecting specific factor deficiencies and the presence of coagulation inhibitors.

Autoanalysis↗
PubMed1985

Development of a photometric assay for activated partial thromboplastin time and its application to the Cobas Bio centrifugal analyzer.

We describe a two-step procedure for APTT that can be performed on photometric devices. It includes preincubation of diluted plasma with ellagic acid and phospholipids and a starting reagent that contains calcium and a chromogenic peptide substrate for thrombin, Tos-Gly-Pro-Arg-pNA. Reaction time is recorded from addition of the starting reagent until thrombin formation occurs, and a prefixed amount of substrate is cleaved. The pattern of sensitivity to clotting factors and heparin was similar to clotting assays and the substrate used did not interfere with the activity of factor Xa. An application of the method was made for the Cobas(R) Bio centrifugal analyzer. Absorbance readings were sent to an external computer and were transformed into reaction times by a computer program. Although the results are independent on fibrinogen concentrations, from kinetic data of the reaction curve fibrinogen concentrations can be estimated. Correlation studies showed good correspondence to clotting methods (r = 0.92, n = 53) as well as an excellent precision (CV 3% for inter-assays, n = 15) and high throughput of samples (greater than 100/h) in the automated assay.

Apoproteins↗
PubMed1986

Centrifugal cytology: a new technique for urine cytology.

A new technique, centrifugal cytology, appears to have a high recovery rate of malignant cells from voided urine specimens resulting in a high probability of early disease detection and should be considered a good supplement to cytoscopic evaluation of high-risk populations.

Centrifugation↗
PubMedDate not supplied

Unilateral centrifugation of the otoliths as a new method to determine bilateral asymmetries of the otolith apparatus in man.

Ten healthy subjects were eccentrically rotated with constant speed on a Barany chair. Setting of a luminous line (LL) to the subjective vertical and ocular counter-roll (OCR) were evaluated. During eccentric position rotation subjects consistently reported illusory rotation and set the LL to an angle correlating to centrifugal force. At the same time an OCR of opposite direction was measured. In one patient, labyrinthectomized on the right side, only counterclockwise rotation of the luminous line was observed. Differences between "inner" and "outer" eye were evident for luminous line settings and OCR in some subjects. The results indicate that eccentric rotation is a valuable method to test for bilateral otolith asymmetries. The method can be applied to preflight tests of astronaut candidates for susceptibility to spacesickness. It is also offered for clinical evaluation of unilateral otolith impairments.

Adult↗
PubMed1994

Principle approaches to selection of the short-arm centrifuge regimens for extended space flight.

Eight +Gz regimens on the SAC varying in their values (within 0.8 to 1.6 G), exposures, schedules, etc. were analyzed. Some regimens were combined with water-salt supplements (WSS) or veloergometer training (VE). Weightlessness was simulated by 3- to 28-day water immersion. +3 Gz loads on the centrifuge with the radius of 7.5 m were applied prior to and post immersion. Regimens for human runs on the SAC as a novel, perspective countermeasure for interplanetary expeditions should be selected with due regard of the human tolerance, their efficiency, and subsequent verification and specification in orbital flights. These approaches showed that 3 days of exposure to 1.2 G combined with WSS and 6 days of exposure to G-loads from 0.8 to 1.6 G together with VE were most optimal.

Acceleration↗
PubMedDate not supplied

Human placental gonadotrophin-releasing hormone (GnRH) binding sites: II. Comparison of binding and inactivation of 125I-labelled GnRH agonist to subcellular fractions following density gradient centrifugation.

Human placental homogenates and membrane fractions were centrifuged on continuous sucrose density gradients, with or without buoyant density perturbation of plasma-membranes by digitonin, and aliquots of each gradient fraction were assayed for a range of plasma-membrane and intracellular organelle markers, and for specific binding and inactivation of radiolabelled GnRH agonist (GnRHA), Buserelin ([D-Ser(tBu)6] GnRH 1-9 ethylamide). GnRH agonist (GnRHA) binding equilibrated in the same regions of control gradients as the plasma-membrane markers, EGF-receptor and alkaline phosphatase. Moreover, binding of 125I-labelled GnRHA and 125I-labelled chicken GnRH II (ch GnRH II) was enriched in the same regions of the gradients, indicating that both bound to the same membrane fractions. Digitonin pretreatment increased the buoyant density of all three placental plasma-membrane markers to a similar degree. Intracellular organelle markers (and hCG content) equilibrated in different regions of the gradient to placental surface-membranes, and were not perturbed appreciably by digitonin. In contrast, inactivation of 125I-labelled GnRHA was associated largely with the soluble (cytosol) fraction which failed to enter the gradient, and little tracer inactivation was observed in fractions enriched in GnRHA binding activity. Similar results were obtained with fractionated rat pituitary membranes. We conclude that: (a) placental GnRH binding sites do not represent binding of radiolabelled ligand to GnRH-degrading enzymes, (b) degradation of radiolabelled ligand is associated largely with placental cytosol fractions, and (c) GnRH binding activity appears to be associated largely with placental plasma-membranes.

Animals↗
PubMed1982

Differentiation restriction in the neutrophil-granulocyte, macrophage, eosinophil-granulocyte pathway: analysis by equilibrium density centrifugation.

Bone marrow culture techniques and equilibrium density centrifugation of human bone marrow cells were used to analyse the neutrophil-granulocyte, macrophage and eosinophil-granulocyte progenitor hierarchy. The buoyant density of progenitor cells changes as cells differentiate down the granulocyte-macrophage pathway and this allows the construction of a density 'map' of the points at which differentiation decisions are made. Unipotent progenitors, neutrophil-granulocyte (G), monocyte-macrophage (M), eosinophil-granulocyte (Eo), are more dense than bi- and tripotent progenitors (GM and EoGM) and have a lower 7-day proliferative capacity (assessed as the clone size achieved in maximally stimulated agar cultures). Experiments in which marrow cells were separated on a basis of their density and either cultured in agar immediately or after an interval of 6 days in suspension culture, were performed to establish the density of the cells which give rise to each type of progenitor, i.e. to investigate parent-progeny relationships. In each case the parent cells were of lower density than the unipotent or bipotent progenitor in question. The ability to separate, at least partially, unipotent, bipotent and multipotent cells of closely related lineages is important since it facilitates studies of the intracellular events taking place as restriction of the cell's differentiation options takes place.

Bone Marrow Cells↗
PubMedDate not supplied

Separation of Lymnaea stagnalis hemocytes by density gradient centrifugation.

A chelating anti-clumping (alpha-C) buffer allowed blood cells (hemocytes) of a gastropod, Lymnaea stagnalis to be separated by discontinuous Percoll density gradient centrifugation. The hemocytes of L. stagnalis were separated into five fractions, having a density lower than 10, 20, 30, 40, and 50% Percoll, respectively. Trypan blue exclusion assays showed viability of separated hemocytes to be between 81 and 89%. Cytospin preparations of these hemocytes were examined. Small cells were mainly observed at high densities; at lower densities medium and large hemocytes were also present. No absolute separation was achieved. Some density fractions were enriched for hemocytes with regard to the distributions of two endogenous lysosomal enzymes (alpha-naphthyl acetate esterase and acid phosphatase).

Acid Phosphatase↗
PubMed1979

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.

Animals↗
PubMed1980

Centrifugal microfiltration: a simple way to enhance the sensitivity of the classical aluminium oxide adsorption method of fluorimetric catecholamine determination.

With the aid of a device utilizing filtration at high pressure produced by centrifugation and constructed from a disposable syringe, the process of solid--liquid separation was greatly simplified. The use of this device facilitated considerably the aluminium oxide adsorption method of catecholamine determination: practically 100% recovery from the adsorbent in as little as 100 microliter of eluent could be easily achieved, and the total amount of catecholamines could be transferred into the microcuvette of a fluorimeter. This resulted in an almost 10-fold increase of sensitivity.

Adsorption↗
PubMed1980

Separation of prostaglandins using the new horizontal flow-through coil planet centrifuge.

Prostaglandins and their metabolites have been separated using the new horizontal flow-through coil planet centrifuge to perform countercurrent chromatography. The system is a continuous-flow system which provides separations similar to column and thin-layer chromatography without the use of solid supports. Either phase of a two-phase solvent system can be used to elute the compounds with the low pressure produced by a metering pump. Separations were produced in PTFE tubing, 0.55 mm i.d. (wound in 0.68 cm helical diameter) with a total capacity of 24 ml. A solvent system of chloroform/acetic acid/water (2 : 2 : 1, v/v) was used at flow rates of 2.4 and 6 ml/h and at a rotation of 500 rev./min. Microgram quantities of prostaglandins E2, F2 alpha, A2, and B2 and thromboxane B2 can be separated. This method can be scaled up to 260-ml columns with comparable results.

Centrifugation↗
PubMed1980

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 s20,w f omega2 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. Experimental verification of the program was carried out.

Centrifugation, Density Gradient↗
PubMed1984

Assessment of the Mulder and Van Doorn kinetic procedure and rapid centrifugal analysis of UDP-glucuronosyltransferase activities.

The optimal experimental conditions of the enzyme assay described by Mulder and Van Doorn (1975, Biochem J. 151, 131-140) for the measurement of UDP-glucuronosyltransferase activities were tested towards structurally different aglycones. This assessment of this assay revealed that addition of Triton X-100 as enzyme activator was necessary because of its apparent inhibitory effects on interfering reactions. Under these conditions, accordance of the data with results published in the literature was obtained. We present for the first time an UDP-glucuronosyltransferase assay adapted on a fast analyser centrifuge which allows a rapid and sensitive measurement of enzyme activity that is very useful for kinetic constant determination, without consuming a large volume of reagents.

Animals↗
PubMed1986

Centrifugation or filtration in quantitative radioreceptor assays.

In quantitative radioreceptor assays the amount of a drug present in the medium to be assayed is inversely related to the amount of receptor-bound radiolabelled ligand. Usually, separation of the bound and free fractions of radiolabelled ligand is done by filtration, in which the bound fraction can easily be collected. However, the filtration disturbs the equilibrium between bound and free fractions, which may lead to erroneous results. Because the decrease in bound radiolabelled ligand is accompanied by an increase in free labelled ligand, we decided also to measure this free fraction after separation by centrifugation and to compare these data with the filtration data. In these experiments a radioreceptor assay for anticholinergics was employed. The results indicate that both methods are compatible in precision when appropriate conditions are used whereas each method has its specific features.

Animals↗
PubMed1989

A convenient procedure for the isolation of intact translatable mRNA by potassium iodide gradient centrifugation.

High concentrations of KI were found to efficiently protect RNA against degradation by RNases. When a sufficient amount of solid KI was added to cell lysates or subcellular fractions (9 g per 10 ml), the solutions could be stored at room temperature for several days without measurable degradation of mRNA. Ribonucleic acids were selectively sedimented when these KI-containing solutions were centrifuged at 72,000 x g for 24 h. The RNA pellets were found to be readily dissolved in bidistilled water and the redissolved RNA could be immediately submitted to oligo(dT)-cellulose chromatography to isolate the poly(A)-containing RNA. However, extraction with phenol/chloroform was found to be necessary, if total RNA or poly(A)-minus RNA was to be analysed. This procedure was found to be superior to other methods currently in use - especially with respect to the isolation of intact, translatable high-molecular-weight mRNA.

Blotting, Northern↗
PubMed1992

Management of sedimentation in centrifugal counter-current distribution of sperm cells in an aqueous 2-phase system.

It is generally assumed that centrifugal counter-current distribution (CCCD) in aqueous two-phase systems cannot be employed for analyzing or fractioning cell populations, due to large particles of sediment in the system caused by enhanced gravity. The present work was undertaken to find out whether addition of Percoll to a two-phase system would be a useful method to avoid this cell sedimentation. The results obtained show that bull spermatozoa partition as a unique peak in a CCCD using a Dextran T500-poly(ethylene glycol) 6000 system, and that sedimentation takes place significantly in the upper phase during the process. Addition of increasing concentrations of Percoll made this unique peak wider and two different populations of bull spermatozoa were finally obtained when Percoll concentration rose to 13.6%. This management of cell sedimentation in CCCD could be of great interest for analyzing cell heterogeneity, since the shortening of the time required for counter-current distribution should prevent the loss of cell viability during the separation process. Finally, the results obtained suggest that an increase of viscosity rather than of density is the phase feature which has greater influence on managing cell sedimentation in CCCD.

Animals↗
PubMed1980

Purification of adrenal medullary chromaffin cells by density gradient centrifugation.

A method has been developed for the isolation of chromaffin cells from guinea pig adrenal glands. Crude suspensions of adrenal cells are prepared by the digestion of adrenal with collagenase. Chromaffin cells are then purified from these crude suspensions by isopycnic centrifugation through a 5-25% (w/v) gradient of metrizamide. More than 90% of the cells in the preparation are viable chromaffin cells, as judged by trypan blue exclusion and fluorescence histochemistry. A more convenient method for monitoring the purity of chromaffin cell preparations, using neutral red staining, is also described. Chromaffin cells are the primary neutral red staining cell in the adrenal gland, and more than 90% of the purified chromaffin cells stain with this dye. Purified chromaffin cells contain 400 +/- 50 nmol of epinephrine/mg protein (133 +/- 16 nmol/10(6) cells), and secrete epinephrine in response to acetylcholine. At a concentration of 100 microM, acetylcholine causes a 10- to 20-fold increase in the secretion of epinephrine from the cells. The method described in this paper is a useful procedure for the preparation of pure, functional chromaffin cells.

Adrenal Medulla↗