Washing Medium Research Articles

Removal of Particles from Substrates by Electro-Osmotic Flow

Abstract The detergent process or detergency is defined as the removal of soil, or matter out of place, from a substrate immersed in a medium through the application of a mechanical force in the presence of a detergent which lowers the adhesion of the soil to the substrate1). Regarding the mechanical force in the detergent process for porous solids such as fabrics, detergency is considered to be governed by flow behavior of the washing medium inside the porous solids. For example, the fabric can be considered as a system of capillaries; the space between fibers is a capillary and the fiber surface is the capillary wall. In the washing process, soil particles adhering to the fiber surface can be detached by the tangential flow of washing liquid in the capillaries.

A possible role for potassium and pyruvate in the modulation of sperm motility in the rabbit oviducal isthmus.

Spermatozoa were recovered from the isthmus of the rabbit oviduct at 4 and 11 h post coitum using several defined flushing media. The motility of spermatozoa in the isthmic flushings was subsequently analysed from video recordings. There was little sperm movement in the native isthmic fluid, but vigorous flagellar activity and hyperactivated movement were induced by flushing the isthmus with 0.25 M-sucrose, apparently an effect of dilution. Flushing with a complex culture medium resulted in similar stimulation of sperm movement. When pyruvate was present in the medium, hyperactivated flagellar bending was stimulated, whereas these movements were virtually absent when glucose alone was present. The stimulating effect of dilution was less pronounced when the flushing medium contained 50 mM-potassium. When the isthmus was flushed with media containing 50 mM-K+ and the K+ concentration was lowered to 5 mM during a washing procedure, large-amplitude flagellar movements were sequentially suppressed and restored. The restoration of large-amplitude movements was enhanced when pyruvate was present in the 5 mM-K+ washing medium. These results suggest that alterations in the concentration of both K+ and pyruvate may have a role in regulating the motility of rabbit spermatozoa in the oviducal isthmus, K+ being inhibitory and pyruvate stimulatory.

Tubing bomb coal liquefaction technique

An experimental coal liquefaction test procedure is described in which coal, solvent, and hydrogen are reacted in a small, agitated batch reactor (tubing bomb) that is rapidly heated in a sand bath. After completion of the desired reaction time, the reactor is quench-cooled, and solid and liquid reaction products are removed using cyclohexane as a wash medium. Conversion of coal to cyclohexane soluble material is reproducible and correlates well with the conversion to 538/sup 0/C (1000/sup 0/F) minus boiling point liquids obtained by larger scale process units. The principal data obtained with the tubing bomb technique are: hydrogen consumption (molecular hydrogen and hydrogen transferred from the solvent), gas make (H/sub 2/S, CO /SUB x/, and hydrocarbon gases), water make, liquid yields, solid residue (cyclohexane insolubles), and conversion. Details of the apparatus, experimental procedure, sample analysis procedures, and data workup are described. Experimental results for a bituminous, a subbituminous coal and a lignite are shown to illustrate the technique.

Cryoprotectant removal temperature as a factor in the survival of frozen rice and sugarcane cells

Removal of cryoprotective additives through use of a room temperature (22 °C) washing step, instead of 0 °C, was found to improve the recovery of sugarcane suspension culture and rice callus tissues. Cultured cells were cryoprotected by gradual addition of a mixture of polyethylene glycol, glucose, and DMSO (PGD) to a final concentration of 10%-8%-10%, w/v, respectively, added at either 0 or 22 °C. After a programmed slow freezing of the cells, they were thawed rapidly and the cryoprotectants were gradually diluted and washed out using a 22 or 0 °C washing medium. Viability of suspension cultured sugarcane cells protected with PGD was greatly diminished when a cold washing solution was used, whether the cells had been frozen (−23 °C) or not. Two mutant lines of rice callus when frozen to −196 °C in PGD and thawed showed less growth than unfrozen cells, but their growth was improved by washing the thawed cells with a 22 °C solution. With all cultures tested, the addition of PGD at 0 °C and post-thaw washing out at 22 °C gave improved survival. Particularly with the rice lines, optimizing the addition and washing procedures allowed culture survival of liquid nitrogen freezing not otherwise attained.

Association of type 1 pili with the ability of livers to clear Salmonella typhimurium.

The role of type 1 pili in the adherence of Salmonella typhimurium strain SR-11 to hepatic sinusoidal cells was investigated. An average of 66.7% of piliated organisms was cleared by perfused livers on a single pass. Mannose and alpha-methyl-D-mannoside inhibited such trapping in a dose-dependent manner. Preincubation of the bacteria, but not the liver, with either sugar also inhibited trapping, suggesting that the sugar binds to bacterial, not hepatic, receptors. Significant numbers of previously trapped bacteria could be eluted by adding mannose to the wash medium. Bacteria with reduced piliation, obtained either by growing bacteria on agar or by using a nonpiliated variant of the parent strain, were trapped to a significantly lesser extent than the parent strain. The liver appears to selectively trap heavily piliated organisms since reperfusion of bacteria through a second liver results in significantly less trapping than occurs with the first perfusion. In vivo, the nonpiliated variant strain was cleared much more slowly than the piliated parent strain. Mannose and alpha-methyl-D-mannoside, but not glucose, decreased clearance rates of piliated organisms. Cumulatively, the data suggest that type 1 pili are a major factor in hepatic clearance of S. typhimurium.

Adenine Nucleotide Content of Corn Roots as Affected by Injury and Subsequent Washing

The adenine nucleotide content of the 2-centimeter segments excised from tray-grown corn (Zea mays L., WF9 x Mo17) roots declines for the first hour after excision. Concomitant with the loss of adenine nucleotides is a decline in respiration and a leakage of K(+). With continued washing, these parameters partially or completely recover and increased phosphate influx develops. Increasing the wound effect by cutting 0.5-centimeter segments gives a more rapid and pronounced degradation of adenine nucleotides and slower recovery. Conversely, the mild injury caused by submerging intact roots induces less degradation and produces greater net adenine nucleotide synthesis during recovery; adding auxin to the washing medium produces a similar result. With all treatments, there is stabilization of energy charge at about 0.85.Brief submersion or rubbing of intact roots, as well as recutting washed and recovered root segments, will initiate the transient loss of adenine nucleotides but will not induce increased phosphate influx.It is suggested that the loss in adenine nucleotides may reflect homeostasis in energy charge via catabolism arising from membrane permeability changes.

Vacuole/Extravacuole Distribution of Soluble Protease in Hippeastrum Petal and Triticum Leaf Protoplasts

The subcellular distribution of soluble protease in anthesis-stage, anthocyanin-containing Hippeastrum cv. Dutch Red Hybrid petal protoplasts has been reevaluated and that of Triticum aestivum L. var. Red Coat leaf protoplasts determined using (125)I-fibrin as a protease substrate and improved methods for protoplast and vacuole volume estimation. Results indicate that about 20% of the Hippeastrum petal-soluble protease and about 90% of the wheat leaf-soluble protease can be assigned to the vacuole. Protoplast isolation enzyme labeled with (125)I has been used to assess the efficiency of removing isolation enzyme from protoplasts by repeated washing and by separation of protoplasts from debris using density centrifugation. Results of these studies suggest that protoplasts prepared by both methods retain low levels of isolation enzyme. However, when protoplasts prepared by either method were lysed with washing medium lacking osmoticum, little isolation enzyme contaminated the lysates.

66] Preparation and use of protoplasts for studies of lipid metabolism

Abstract Spinach protoplasts are useful in lipid metabolism studies because they provide a homogeneous, differentiated plant tissue that can take up precursors directly from the medium. they are the material of choice for subcellular localization because they can be easily lysed and the intact organelles separated on density gradients. Crude fungal enzyme preparations are used to degrade the cell wall of the spinach leaves, which are stripped of their epidermal cells by brushing and sliced finely to increase the surface for enzyme action. Protoplasts are harvested from the digested leaf slices by filtration and washed and purified by repeated centrifugation. If protoplasts are to be used in metabolism studies, they are stabilized with a salt solution containing divalent cations and incubated in strong light with lipid or fatty acid precursors. For subcellular localization studies, salts are not present in the wash medium to reduce chloroplast aggregation in the gradients and protoplasts are lysed by shearing in a microemulsifying needle or passage through 20- and 10-μm mesh before sucrose density gradient centrifugation. Comparison with marker enzymes for organelles provides a method for the complete subcellular localization of an enzyme activity.

The Rapid Isolation of Vacuoles from Leaves of Crassulacean Acid Metabolism Plants

A technique is presented for the isolation of vacuoles from Sedum telephium L. leaves. Leaf material is digested enzymically to produce protoplasts rapidly which are partially lysed by gentle osmotic shock and the inclusion of 5 millimolar ethyleneglycol-bis (beta-aminoethyl ether)N,N'-tetraacetic acid in the wash medium. Vacuoles are isolated from the partially lysed protoplasts by brief centrifugation on a three-step Ficoll-400 gradient consisting of 5, 10, and 15% (w/v) Ficoll-400. A majority of the vacuoles accumulate at the 5 to 10% Ficoll interface, whereas a smaller proportion sediments at the 10 to 15% Ficoll-400 interface. The total time required for vacuole isolation is 2 to 2.5 hours, beginning from leaf harvest.The yield of vacuoles is approximately 44%. The major vacuole layer is < 7% contaminated by marker enzymes from the cytoplasm and other organelles but shows no contamination by chloroplasts. Isolated vacuoles were stable for >15 hours when left in Ficoll; however, dispersion into media of various osmotic concentrations resulted in decreased stability. Addition of mercaptobenzothiazole, CaCl(2), MgCl(2), bovine serum albumin, ethylenediaminetetraacetic acid, polyethylene glycol 600, and KH(2)PO(4) to the vacuole isolation media did not increase the stability of the isolated vacuoles.THIS TECHNIQUE WITH ONLY SLIGHT MODIFICATIONS HAS BEEN USED TO ISOLATE LEAF CELL VACUOLES FROM THE FOLLOWING CRASSULACEAN ACID METABOLISM PLANTS: pineapple, Kalanchoë fedtschenkoi, and Echeveria elegans. Spinach leaves also were used successfully.

Effect of Polymer Deposition and Washing Medium on the Felting Shrinkage of Chlorinated Wool Fabrics

The felting shrinkage of chlorinated wool fabrics treated with a group of preformed polymers has been investigated. The results suggest that after polymer deposition on the fabric, interfiber bridges and transient adhesive junctions formed between the fibers prevent relative fiber movements when the fabric is subjected to bending deformations in a washing medium. In the case of Hercosett-treated fabrics the shrinkproofing mechanism seems to include the elimination of directional bias in the movement of fibers by fiber lubrication. This latter mechanism is probably prevalent in fabrics made from Hercosett-treated top. These results are supported by the frictional and mechanical behavior of the polymers. The effect of surfactants in the washing medium on felting shrinkage depends on their ionic nature. Anionic and nonionic surfactants seem to reduce shrinkage by interfiber repulsions and limited fiber lubrication. Although they are operating by the same mechanisms, wettability of the fibers seems to be a key factor in the case of cationic surfactants. Differences in the concentration dependence of the surface tension of the three surfactants support this hypothesis.

Ouabain potentiation of Ca release from fragmented cardiac sarcoplasmic reticulum.

The present study was performed to determine the effect of ouabain on Ca release from fragmented sarcoplasmic reticulum (FSR) isolated from cardiac muscles by changing the ionic environment and by caffeine. FSR was isolated from cat, guinea pig and frog cardiac muscles as microsomal fractions obtained biochemically, namely, by means of ultra-centrifugation at 8000 × g-35000 × g. After sufficient Ca 2+ -uptake by FSR in a potassium methan-sulfonate (KMS) medium, one ml of the suspension was passed through a Millipore filter. By this treatment, almost all FSR adhered to the filter. Three ml of potassium chloride washing medium was added to alter the ionic environment. One min later the washing medium was passed through the filter. A KMS washing medium containing 30 mM caffeine was used to induce the Ca release by caffeine. In the case of ouabain, the medium containing ouabain (2 × 10 -6 M) was used. The time of ouabain treatment was 1 min. The grade of Ca release from FSR was almost the same among cat, guinea pig and frog cardiac FSR. Ouabain markedly potentiated Ca release from FSR by altering the ionic environment. These results suggest that the positive inotropic action of ouabain is dependent upon the potentiation of Ca release in SR vesicles of cardiac muscle.

Ouabain potentiation of ca release from fragmented cardiac sarcoplasmic reticulum

The present study was performed to determine the effect of ouabain on Ca release from fragmented sarcoplasmic reticulum (FSR) isolated from cardiac muscles by changing the ionic environment and by caffeine. FSR was isolated from cat, guinea pig and frog cardiac muscles as microsomal fractions obtained biochemically, namely, by means of ultra-centrifugation at 8000 × g-35000 × g. After sufficient Ca2+-uptake by FSR in a potassium methan-sulfonate (KMS) medium, one ml of the suspension was passed through a Millipore filter. By this treatment, almost all FSR adhered to the filter. Three ml of potassium chloride washing medium was added to alter the ionic environment. One min later the washing medium was passed through the filter. A KMS washing medium containing 30 mM caffeine was used to induce the Ca release by caffeine. In the case of ouabain, the medium containing ouabain (2 × 10-6 M) was used. The time of ouabain treatment was 1 min. The grade of Ca release from FSR was almost the same among cat, guinea pig and frog cardiac FSR. Ouabain markedly potentiated Ca release from FSR by altering the ionic environment. These results suggest that the positive inotropic action of ouabain is dependent upon the potentiation of Ca release in SR vesicles of cardiac muscle.

Ultrastructural demonstration of glucose-6-phosphate dehydrogenase activity in steroid-secreting cells.

The ultrastructural localization of glucose-6-phosphate dehydrogenase (NADP-linked) has been attempted in steroid-secreting cells. Rat adrenocortical cells and newt testicular glandular cells were fixed in an ice-cold mixture of 1% methanol-free formaldehyde and 0.25% glutaraldehyde. Potassium ferricyanide was used as the final electron acceptor. After incubation, the final copper ferrocyanide precipitate is exclusively observed in the hyaloplasm of these cells, provided that an electron carrier (1.0 mM PMS) has been added to the medium in order to by-pass the tissue "diaphorase" (NADPH-ferricyanide reductase) reaction. No precipitate appears in the absence of glucose-6-phosphate (substrate). Incubation in a medium devoid of PMS results in an exclusively mitochondrial reaction; the latter is that of the "diaphorase", which in these cells is mitochondrial. These results prove the importance of utilizing exogenous electron carriers (such as PMS) in coenzyme-linked dehydrogenase cytochemistry. Although polyvinyl alcohol was included in the washing and incubation media, in order to increase their viscosity, problems still exist concerning ultracytochemical localization of this "soluble" enzyme; these problems are discussed in the paper.

Calcium and pancreatic β-cell function: 4. Evidence that glucose and phosphate stimulate calcium-45 incorporation into different intracellular pools

β-Cell-rich pancreatic islets were microdissected from ob/ob-mice and used for studies of 45Ca uptake and washout. Irrespective of whether the experiments were performed at 21 or 37°C both glucose and phosphate stimulated the net uptake of lanthanum-nondisplaceable 45Ca. The stimulatory effect of phosphate was additive to that produced by glucose. 45Ca incorporated in response to phosphate differed from that taken up in the presence of 20 mM glucose in being easily washed out although it was not affected by the glucose concentration of the washing medium. The efflux of 45Ca was reduced after introducing phosphate into a medium used to perifuse islets which had accumulated 45Ca in response to 20 mM glucose. This suggests that the outward calcium transport can be influenced also by intracellular trapping of the cation. The glucose-stimulated insulin release was inhibited by phosphate; an effect reversed by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. It is concluded that a common effect of glucose and phosphate is to trap calcium in the pancreatic β-cells but that there are fundamental differences between their effects on intracellular distribution of calcium and on insulin release.

Calcium and pancreatic β-cell function: 3. Validity of the La 3+-wash technique for discriminating between superficial and intracellular 45Ca

Exposure to La 3+ has been proposed as a tool for discriminating between superficially and intracellularly located 45Ca. Two different pools of glucose-stimulated 45Ca were identified in β-cell-rich pancreatic islets microdissected from ob/ob mice using a standard procedure of washing with 2 mM La 3+ at 37°C. The results were not critically dependent on the concentration of La 3+ within a range of 1–20 mM. However, the distribution of 45Ca was markedly influenced by the temperature of the washing medium. Reduction of the temperature to 1°C abolished the glucose effect on the La 3+-displaceable 45Ca and considerably increased the amounts of 45Ca not displaceable with La 3+. Separate studies in a perifusion system revealed that La 3+ in itself was less effective than temperature reduction in depressing the efflux of 45Ca from the isolated islets. The data indicate that washing in cold La 3+ solution gives a better estimate of the intracellular islet calcium than the original procedure. It is suggested that the glucose-stimulated part of the 45Ca, which is displaced by washing with La 2+ at 37°C, is derived from the interior of the β-cells.

Cleaning of surgical instruments: a preliminary assessment.

An assessment has been made of some techniques used for cleaning surgical instruments at the Central Sterile Supply Departments (C.S.S.D.) at Dudley Road Hospital, Birmingham and the Walsgrave Hospital, Coventry. Tests were carried out using radioisotope methods, instruments being soiled with human serum albumin (h.s.a.) labelled with Technetium 99m (99mTc). It was found that cleaning efficiency varies with the kind of instrument: polypropylene instruments are cleaned more easily than stainless-steel ones: instruments with serrated surfaces, joints, or hinges are particularly difficult to clean. As would be expected, the use of detergents improves the degree of cleaning; the cleaning effect seems to depend on the alkalinity of the washing medium.

Storage of human platelets by freezing.

Prolonged, probably indefinite storage of viable and functional human platelets is now possible by freezing with dimethylsulfoxide (DMSO). These platelets have a nearly normal survival upon reinfusion and are capable of sustained hemostatic effectiveness in thrombocytopenic patients. Adaptation of the freezing technique for large-scale usage has more recently been achieved. The method is mainly based on the following principles: (1) use of plasma for suspension of the platelet concentrate; (2) gradual addition (0.5% every 2 min) of DMSO to a final concentration of 5% and its gradual removal; (3) a slow cooling rate of about 1 degree C per min and rapid thawing (in 1 min); (4) use of a polyolefin plastic bag for freezing; (5) a washing medium of 20% plasma in Hanks' balanced salt solution; (6) final resuspension of the platelets in 50% plasma in Hanks' solution.

連続遠沈法による解凍赤血球の検討

1. Objective and ProcedureFresh red blood cells are to be frozen either slowly or rapidly with glycerol for preservation as viable cells in a frozen medium. From the viewpoint of deglycerolizing such preserved viable erythrocytes there have been introduced three different techniques, viz., the reversible agglomeration (Huggins' method), the batch washing and the continuous centrifuge procedure.We conducted a study attempting the evaluation of the continuous centrifuge technique for deglycerolizing preserved red cells, using a Hemonetics Model 15 apparatus, in comparison with Huggins' method as to effects on osmotic pressure, free Hg concentration, erythrocytic resistance, electrolyte balance, 2, 3-DPG and ATP levels and recovery rate of viable cells.2. ResultsAs compared with Huggins' procedure, the continuous centrifuge method produced rather inconsistent results for which the rates of red cell and washing medium injection appear to have played an important role. In case of the washing medium being introduced into the apparatus before erythrocytes it would be probable that sufficient washing of red cells could hardly be accomplished. The failure in obtaining constantly optimal osmotic pressure, erythrocyte resistance and electrolyte balance with the subject procedure has thus been conjectured to be due to insufficient washing of preserved cells.3. ConclusionThe continuous centrifuge seems to be valuable method provided it be performed attentively insomuch as the glycerol concentration employed is fairly low and as electrolyte solutions are used for washing the cells.

Use of phospho-cellulose paper disks for the assay of histone acetyltransferase

A modified method for the assay of histone acetyltransferase is presented. Previously reported methods depended upon the determination of the incorporation of radioactivity from [ 14C]acetyl coenzyme A into trichloroacetic acid (TCA)-precipitable material. However, as shown in this paper, [ 14C]acetylated histone cannot be precipitated quantitatively by TCA. In the method described in this paper, phospho-cellulose (P-cellulose) paper disks are used as an adsorbent for [ 14C]acetylated histone and 0.05 m carbonate buffer, pH 9.2, is used as a washing medium. This P-cellulose disk method allows more quantitative determination of [ 14C]acetylated histone than the TCA-precipitation methods.

Effect of washing the hepatic microsomal fraction in sucrose solutions and in sucrose solution containing edta upon the metabolism of foreign compounds

Washing the hepatic microsomal fraction of the rat in 0·25 M sucrose containing 0·05 M Tris buffer (pH 7·4) resulted in the metabolism and binding to cytochrome P-450 of Type I substrates, although not of a Type II substrate. The levels of microsomal cytochrome b 5, cytochrome P-450 and NADPH-cytochrome c reductase was unchanged, whilst the activity of NADPH-cytochrome P-450 reductase was reduced. The inhibitors of lipid peroxidation EDTA and Mn 2+ added to the washing medium prevented the decrease in the metabolism of Type I substrates. It is suggested that the effects of washing may be related to the increase in the level of peroxidised microsomal lipid which could lead to a selective destruction of the Type I binding site. EDTA added to the washing medium also produced an increase above control values, in the metabolism and binding of both Type I and Type II substrates, which may be related to the apparent increase in the amount of microsomal cytochrome P-450. Washing the microsomal fraction almost completely abolished the ability of acetone to enhance aniline hydroxylation. It is concluded that the effects of acetone are not due to the displacement of endogenous substrates bound to the microsomal fraction.