An improved method for the separation of cells by sedimentation at unit gravity.

An apparatus is described for the separation of cells by sedimentation velocity at lg. The viscosity of the sample was raised by the addition of polyethylene oxide and subsequently the sample was layered on top of a density gradient via a sieve. With the aid of this procedure the various ploidy classes of rat-liver cells were enriched and murine leukemia cells were separated according to the phases of their life cycle.     

Two methods for electrophoretic elution of proteins from polyacrylamide gels.

An apparatus is described which improves an earlier technique for eluting proteins from polyacrylamide-gel slabs by electrophoresis against a sucrose gradient. Another elution method where the components are concentrated electrophoretically in a collodion bag by altering the current density is described. This method enables the elution of small amounts of sample, free from disturbing background material arising from the gel, and also permits subsequent dialysis and ultrafiltration without transfer losses. It can be used with alkaline and acidic buffers and has been applied in the purification of human pituitary thyrotropin (TSH).     

Exponential gradient maker using a disposable syringe

With a simple modification, any disposable syringe can become a reliable and easy to use exponential gradient maker. The modification consists of two notches, made with a razor blade, in the borders of the rubber sealing tip of the plunger. A clamp in the tube connected to the syringe allows control over solution flow. With the clamp prohibiting drainage, the body of the syringe is filled with the desired volume of starting solution I. A magnetic stir bar, small enough to spin inside the syringe is included. The notched plunger is introduced until no air space remains. This forms the fixed volume, closed mixing chamber, while the rest of the volume of the syringe forms the open chamber. The two chambers are connected through the notches in the plunger. The ending solution II is poured after the introduction of the plunger. Opening the clamp allows solution I in the closed chamber to flow out, and the solution II in the open chamber flows through the notches and mixes with solution I. This exponential gradient maker can be reused many times, but the low cost of the components makes it potentially disposable. This feature is especially useful when using toxic chemicals, or when pouring polyacrylamide gradient gels, since the apparatus may be disposed of after contamination or eventual polymerization.     

A universal gradient apparatus for multiple organic solvents. I. Principle, construction and gradient evaluation.

The mechanical and electrical construction and the evaluation of a gradient apparatus for multiple organic solvents and other liquids is described. Special attention is given to the problems of organic resistance, solvent density and evaporation. The apparatus is a solvent-resistant liquid pump with a positively controlled adjustable and reproducible flowrate.     

An Apparatus for Measuring Volumes of Small Objects

An apparatus for measuring volumes of small objects such as tissue blocks is described. The apparatus measures volumes by fluid displacement and consists of a micropipette adapted to fit the mouth of an Erleiuneyer flask, a Luer adaptor fused to the side of the flask, and a glass syringe. When assembled with fluid enclosed, the fluid rises to a low level in the micropipette. Withdrawal of fluid into the syringe lowers the fluid level below the mouth of the flask. The micropipette is raised, the object to be measured is placed in the flask, and the micropipette is joined to the flask again. Fluid returned to the flask from the syringe rises to a higher level in the micropipette. The difference between the two fluid levels equals the volume of the object measured.

This apparatus gives reproducible measurements and can be calibrated for absolute volume determination. It is inexpensive to construct and easy to use.     

Cerebrospinal fluid sampling technique and Astrup pH and PCO2 values.

The pH and PCO2 values measured by the Astrup technique were compared in cerebrospinal fluid (CSF) obtained using two different sampling techniques: 1) a direct or in vivo technique and 2) the widely accepted syringe sampling technique. In 65 pairs of measurements in 9 dogs it was found that the pH was always overestimated and the PCO2 always underestimated in the syringe sample when compared to the in vivo sample. The equations describing the relationships are as follows: 1) pH (syringe = 0.995 pH (in vivo) + 0.084 and 2) PCO2 (syringe) = 0.873 PCO2 (in vivo) + 0.2. The amount by which the syringe sample underestimated the true PCO2 value increased with the absolute PCO2 value, consistent with the possibility of there being a diffusional loss of CO2 during the transfer of CSF from the syringe to the pH electrode (PCO2 (in vivo)- PCO2 (syringe) = 2.4, 4.9, 7.5, and 10.0 mmHg at in vivo PCO2's of 20, 40, 60, and 80 mmHg). This study indicates that the technique used for sampling CSF is crucial to the expected accuracy of the results and that the number of transfers of CSF during the sampling and measurement procedures should be minimized in order to obtain reliable results.     

A simple microtechnique for isoelectric focusing on a density gradient.

Isoelectric focusing in a density gradient can be performed on chromatographic columns of 10–15-ml volume. A polyacrylamide salt bridge was used to make electrical contact between the density gradient tube and the electrode compartment. An appropriate gradient mixer can easily be made from two 10-ml syringes. The small column permits a resolution of isoenzymes which is comparable to the separation with a commercial 110-ml column.With the described apparatus one uses ten times less Ampholine and biological sample. The time of isoelectric focusing is reduced three to four times.     

Mobility measurements by photometric analysis of zone electrophoresis in a sucrose gradient column

A density gradient zone electrophoresis apparatus has been designed for measuring electrophoretic mobilities on small amounts (about 10 μg) of viruses or other ultraviolet-absorbing materials. Dialysis membranes separate the gradient column from electrode chambers and eliminate the need to maintain hydrostatic equilibrium between the buffer chambers. The virus zone was located by pumping the gradient column through an ultraviolet flow cell, a procedure which did not disturb the virus zone. The apparatus was tested by measuring the mobilities of tobacco mosaic and brome mosaic viruses in different buffers, in sucrose, glucose, and glycerol gradients, at 0° and 15°, and during ascending and descending electrophoresis.     

Syringe pumped high speed flow cytometry of oceanic phytoplankton.

BACKGROUND: Nanophytoplankton (2-20 microm) are less numerous than picophytoplankton (<2 microm) in the oceans but their biomass and production are comparable and sometimes higher. The accuracy of cytometry-based enumeration of phytoplankton ultimately depends on cell abundance and sample flow rate. Commercial flow cytometers in which sheath and core streams are driven by air pressure cannot produce sufficiently high, stable sample flow rate. The present study demonstrates the applicability of a syringe pump for flow cytometric enumeration of oceanic nanophytoplankton on two meridional transects across the Atlantic Ocean. METHODS: Commercially available syringe pumps were used to deliver live phytoplankton samples into a flow cell of standard flow cytometers (FACSort, FACSCalibur, BD) with increased flow rate of > 1.0 ml min(-) (1) compared to the normal air pressure sample delivery of < 0.1 ml min(-) (1). An auxiliary application of syringe pump flow cytometry for calibrating 0.5 microm bead concentration standards is also discussed. RESULTS: The results demonstrated that flow cytometry of samples injected at rates above 0.1 ml min(-) (1) is achievable and worthwhile. Counts of phytoplankton in air and syringe pumped samples agreed closely. Syringe pumping of samples offered a broader range of flow rates up to 0.8-1.0 ml min(-) (1) without detrimental effect on flow cytometric enumeration of cells. The increased number of coincidences at high flow rates led to an approximate 10% decrease of Cyanobacteria counts when the acquisition rate approached 1,000 particles s(-) (1), but seemed to have a lesser effect on counting rarer phytoplankton. The syringe pump flow cytometry allowed enumeration of phytoplankton groups at concentrations of 5-100 cells ml(-) (1), cell concentrations equivalent to those of Cyanobacteria in the twilight deep ocean. CONCLUSION: The proposed syringe pump modification of a FACS instrument represents a significant improvement for accurate enumeration of the less abundant phytoplankton and so gives better estimations of phytoplankton distribution and standing stocks.     

A separation chamber to sort cells and cell organelles by weak physical forces: III. Preparative electrophoresis of cells in stationary density gradients at low electric field strength

An apparatus was designed for preparative density gradient electrophoresis of mammalian cells. In a low conductivity isotonic Ficoll density gradient of 1.5 cm length, human erythrocytes treated with neuraminidase were separated from untreated erythrocytes at an electric field strength of approximately 2.7 v/cm. Within 5 min two bands of erythrocytes were visible. Electrophoretic separation was completed within 25 min. The fractionation is performed in a design consisting of three Perspex circular plates, bottom and top plates of which can be displaced simultaneously relative to the stationary middle plate by a worm-gear mechanism. The middle plate contains a cylindrical separation chamber of 50 cm² and 1.5 cm high. Top and bottom plates contain cones and flow deflectors for the undisturbed thin layering of cell suspensions and for introduction of the density gradient. Also present in top and bottom plates are electrode compartments containing a large platinum electrode and a cellophane membrane that isolates the separation chamber hydrodynamically but not electrically from the electrode compartment. The electrode compartments were flushed with electrophoresis buffer to remove products of electrophoresis as well as the (low) generated Joule heat.     

A simplified high-resolution gradient analysis system

A simplified high-performance, semiautomatic apparatus for the analysis and fractionation of density gradient preparations is described. In this system the gradient, mounted directly under a standard recording spectrophotometer, is displaced upwards through a vertical straight-path flow cell, then downward to a fraction collector.The resolution attainable with this apparatus is greater than that required for the high fidelity analysis of small volume centrifuged density gradients. The system is easily adaptable to cesium gradients, zonal rotors, and to chromatographic columns where its high-resolution, bubble-free performance makes practical the analysis of both small and large volumes. It is significantly simpler and less costly to construct than other apparatus of comparable performance.     

A semiautomated procedure for rapid intrarectal instillation of fluids in the guinea pig.

A penumatic operated semiautomatic system was developed for the intrarectal instillation of solutions in the guinea pig. The apparatus permitted one person to inject large numbers of animals at considerable savings in time compared to manually operated syringe techniques. The procedure was shown to be both rapid and safe for administering solutions intrarectally in the guinea pig.     

Cell electrophoresis research directed toward clinical cytodiagnosis.

The application of cell electrophoresis to cytodiagnosis requires that a scientifically established basis exists for identifying abnormal cells electrophoretically, that research to detect such differences in the cytodiagnostic setting is possible and that a rapid and simple method of cell electrophoresis is adaptable to the clinical setting. Data are presented indicating modifications of electrophoretic mobility due to herpes simplex virus type 1 infection and Rous sarcoma virus transformation of culture cells. A simple apparatus for electrophoretically separating cells on a density gradient and collecting them for subsequent analysis is described, and results of experiments with this apparatus are consistent with those obtained by microscopic electrophoresis. Laser-doppler spectroscopic electrophoresis is suggested as a rapid method adaptable to clinical application.     

Transverse agarose pore gradient gel electrophoresis of DNA.

Transverse agarose pore gradient gels were prepared on GelBond in the concentration range of nominally 0.2-1.5% SeaKem GTG agarose, using density stabilization by glycerol and incorporation of a dye to define the gel concentration at each point on the pore gradient gel. The distribution of the dye was evaluated by photography, video-acquisition and digitization of the gradient mixture and by densitometry of the gel. The gel was applied to the electrophoresis of a 1-kb standard ladder of DNA fragments, using standard submarine apparatus. The method extends to agarose gel electrophoresis the benefits of semi-automated analysis of 'Ferguson curves' described in application to polyacrylamide gel by Wheeler et al. (J. Biochem. Biophys. Methods 24, 171-180).     

Rapid, multisample isoelectric focusing in sucrose density gradients using conventional polyacrylamide electrophoresis equpiment: a two-peak transient in the approach-to-equilibrium.

Using a semiporous plug of agar gel to support a sucrose density gradient column without restricting electrical conductivity, Massey and Deal [J. Biol. Chem.248, 56 (1973)] were able to use a conventional polyacrylamide gel electrophoresis apparatus to carry out single tube isoelectric focusing experiments in density gradients in only 2 hr using minute amounts (50 μg) of sample and very little ampholyte (0.18 ml); no cooling apparatus was required. In this work we report that 1) polyacrylamide provides a superior gel plug and 2) that ten isoelectric focusing tubes can easily be run simultaneously in a conventional polyacrylamide gel electrophoresis apparatus. In addition, the isoelectric points of eight proteins, with pI values ranging from 5.1 to 8.8 have been determined and the kinetics of the approach-to-isoelectric-focusing-equilibrium have been analyzed. Of special interest is the discovery that in the initial stages of focusing, in these sucrose density gradients, a major peak is formed at each end of the column; these two peaks migrate toward each other and finally coalesce into a single peak. Similar, although less pronounced, effects were previously observed by Catsimpoolas and Wang [Anal. Biochem.39, 141 (1971)] in focusing experiments in polyacrylamide gels. With all other conditions constant, the time required to reach equilibrium is 1) less in broad range (e.g., 3–10) pH gradients than it is in narrow range (e.g., 5–8) pH gradients and 2) generally greater with higher molecular weight substances than with lower molecular weight substances. Explanations are given for all of these kinetic phenomena.     

Automated kinetic analysis of pyruvate kinase

An apparatus for the automatic determination of enzyme kinetics of pyruvate kinase is described. A continuous plit of velocity versus substrate concentration is obtained using quantities of enzyme and substrates comparable to manual determinations. The automated procedure offers a number of advantages over manual methods including elimination of repetitive pipetting, simpler reaction temperature regulation, reduced analysis time, and possible on-line computer analysis. The apparatus utilizes a commercially available column uv flow monitor to measure NADH/NAD changes in the coupled lactic dehydrogenase reaction at 340 nm in a continuous flow system. The optical density changes are directly related to the velocity of the enzyme-catalyzed reaction. A linear substrate gradient is generated from a density gradient maker to provide the required relationship between velocity and substrate concentration. The system is calibrated by forming a gradient from a hemoglobin solution of known concentration. The procedure has been evaluated by determination of the kinetic parameters of three of the isozymes of pyruvate kinase. Values obtained by the continuous flow method are in close agreement with those obtained by individual point determination in a recording spectrophotometer.     

Free-flow electrophoresis. I. Theoretical and experimental investigations of the influence of mechanical and electrokinetic variables on the efficiency of the method.]

In the present paper, various types of band-broadening effects in free-flow electrophoresis were investigated. They resulted from the velocity profiles of the liquid curtain and electroosmosis, temperature gradient, thermal diffusion and sample inlet geometry. An analytical free-flow electrophoresis apparatus permitted easy observation of these parameters. The experiments showed that the influence of the temperature gradient was negligible, whereas the effect of the velocity profiles on band broadening was higher than theoretically expected. In preparative work this is of utmost importance, since overlap of bands is not desirable. In this case, the zeta potential of the walls can be adjusted to that of the material to be separated, resulting in a considerable reduction of band broadening. Various approaches are indicated. Further attention was given to the influence of the relaxation effect on the separation. Conditions are shown where particles are separated either according to their surface charge density or to their size. The practical relevance of the results is discussed.     

A capillary-based microfluidic instrument suitable for immunoaffinity chromatography

The analysis of biological samples to produce clinical or research data often requires measurement of analytes from complex biological matrices and limited volumes. Miniaturized analytical systems capable of minimal sample consumption and reduced analysis times have been employed to meet this need. The small footprint of this technology offers the potential for portability and patient point-of-care testing. A prototype microfluidic system has been developed and is presented for potential rapid assessment of clinical samples. The system has been designed for immunoaffinity chromatography as a means of separating analytes of interest from biological matrices. The instrument is capable of sub-microliter sample injection and detection of labeled antigens by long wavelength laser-induced fluorescence (LIF). The laboratory-constructed device is assembled from an array of components including two syringe pumps, a nano-gradient mixing chip, a micro-injector, a diode laser, and a separation capillary column made from a polymer/silica (PEEKsil) tube. An in-house program written with LabVIEW software controls the syringe pumps to perform step gradient elution and collects the LIF signal as a chromatogram. Initial columns were packed with silica beads to evaluate the system. Optimization of the device has been achieved by measuring flow accuracy with respect to column length and particle size. Syringe size and pressure effects have also been used to characterize the capability of the pumps. Based on test results, a 200-microm x 25-mm column packed with 1-microm silica beads was chosen for use with a 500-microL syringe. The system was tested for mixer proportioning by pumping different compositions of buffer and fluorescent dye solutions in a stepwise fashion. A linear response was achieved for increasing concentrations of fluorescent dye by online mixing (R2=0.9998). The effectiveness of an acidic gradient was confirmed by monitoring pH post-column and measuring premixed solutions offline. Finally, assessment of detectability was achieved by injecting fluorescent dye solutions and measuring the signal from the LIF detector. The limit of detection for the system with these solutions was 10.0 pM or 10.0 amol on-column. As proof-of-principle, immunoaffinity chromatography was demonstrated with immobilized rabbit anti-goat IgG and a fluorescent dye-goat IgG conjugate as a model antigen.     

西藏东达山3种嵩草属植物气孔特征沿海拔的变化

嵩草属(Kobresia)植物是藏东南高山草甸的优势种和建群种,对该区畜牧业发展和维持生态系统平衡起着重要作用。选择西藏左贡县东达山为研究地点,从林线开始,海拔每升高约100m设置1个样带直至高山草甸分布边缘,共8个样带,调查各样带中物种的组成及盖度,并依据相对盖度和相对频度计算3种嵩草植物矮生嵩草(K.humilis)、线叶嵩草(K.capillifolia)和大花嵩草(K.macrantha)在群落中的重要值,同时取样观察它们叶片远、近轴面表皮细胞形态,测量气孔长度及保卫细胞宽度,计算气孔密度,探讨嵩草属植物对海拔梯度的适应性。结果表明:(1)3种嵩草属植物叶表皮细胞均呈波浪状,气孔器仅分布于远轴面,近轴面无气孔器分布。(2)3种嵩草属植物气孔密度沿海拔梯度的变化均呈单峰曲线分布格局,且在海拔4 537m样带处达到最大值,并表现为矮生嵩草(777.6个/mm2)线叶嵩草(476.4个/mm2)大花嵩草(414.3个/mm2)。(3)随海拔的增加,矮生嵩草和线叶嵩草气孔长度显著增大(P0.05),而保卫细胞宽度显著减小;但大花嵩草气孔长度随海拔的升高而显著减小,保卫细胞宽度基本保持不变。(4)矮生嵩草和线叶嵩草气孔密度、长度和保卫细胞宽度与海拔梯度均显著相关,气孔特征对海拔梯度变化的敏感程度高,与其在群落中重要值高的分布特征一致;而大花嵩草仅气孔密度和长度与海拔梯度显著相关,气孔特征对海拔梯度变化的敏感性低,与其在群落中重要值低的分布特征一致;嵩草属植物气孔密度、长度和保护细胞宽度与海拔梯度之间的相关性,反映出它们在海拔梯度上对生境的适应程度。可见,3种嵩草属植物气孔特征对海拔梯度上生境变化的适应性不同,从而影响它们在群落中的分布范围和物种优势度,其中矮生嵩草和线叶嵩草对环境变化敏感,而大花嵩草对环境变化相对不敏感;保卫细胞宽度与气孔长度同样对植物适应环境变化起重要作用。     

A self-refilling syringe for dispensing anaerobic media

Summary A standard Luer-lock self-refilling syringe was modified to dispense anaerobic medium at pre-set volumes. It is suitable for transferring anaerobic medium for serial dilutions, where a consistent volume delivered is necessary. Sulfate reducing and methanogenic bacteria have been cultivated routinely on media dispensed with this apparatus.