Electron microscopy procedure influences detection of rotaviruses.
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Technical parameters of electron microscope staining procedures (type of stain, pH of stain, and time of staining) influence particle integrity for three groups of rotaviruses. Simian rotavirus SA11 (group A), Chinese adult diarrhea rotavirus and porcine rotavirus-like agent (group B), and porcine pararotavirus (group C) were tested. All rotavirus strains were quite stable in uranyl acetate and phosphotungstic acid at pH 4.5 and relatively stable in ammonium molybdate. However, staining with phosphotungstic acid at higher pH values with increased staining time yielded a reduction in the number of particles and particles that were broken or degraded to single-shelled particles or core particles. The different staining procedures were also tested in immunoelectron microscopy experiments. Antibody molecules bound to rotavirus particles were observed clearly only with phosphotungstic acid staining and not with uranyl acetate. We therefore recommend that uranyl acetate and phosphotungstic acid at pH 4.5 be used for negative staining of rotaviruses; phosphotungstic acid at pH 4.5 is optimal for immunoelectron microscopy. These technical points may be critical for rotavirus detection and are important for studies pertaining to the epidemiology and clinical importance of the non-group A rotaviruses. (+info)
A rapid method for staining large chylomicrons.
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In this report, we present a rapid method for producing high-quality micrographs suitable for determining the size distributions of particles in concentrated samples of postprandial chylomicrons and chylomicron remnants. The procedure consists of mixing particles with osmium tetroxide in water to stabilize the lipids of the particles. These fixed and positively stained particles are then negatively stained with phosphotungstate in the presence of dilute sucrose. This dual staining procedure prevents the fusion and clustering of chylomicrons during processing for electron microscopy and is effective with particles of different lipid compositions. In addition, this procedure is simple and rapid, adding only one mixing step and 5 min to the preparation time required for conventional negative stains. (+info)
Multilayered distribution of peptidoglycan in the periplasmic space of Escherichia coli.
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When a staining technique using phosphotungstic acid (PTA) in 10% (w/v) chromic acid was applied to cells of Escherichia coli, the periplasmic space was seen as a dark 15-nm-thick layer of uniform appearance and constant width. Our observations are consistent with peptidoglycan being the main material stained. Isolated sacculi as well as purified peptidoglycan (protein free) were also stained by the same procedure, the thickness of the peptidoglycan being 8.8 +/- 1.8 and 6.6 +/- 1.5 nm, respectively. The increased thickness of the PTA-stained layer in stationary phase cells correlated well with the increased thickness of isolated sacculi or purified peptidoglycan and with the increased amount of peptidoglycan in such cells. Thickness measurements on isolated peptidoglycan were compatible with a two to three layer structure for material from exponential phase cells and with a four to five layer structure for that from stationary phase cells. Furthermore, the results indicated an uneven distribution of peptidoglycan material in the periplasmic space, the peptidoglycan spanning the space from the inner to the outer membrane. (+info)
Comparison of improved precipitation methods for quantification of high-density lipoprotein cholesterol.
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We compared the standard Lipid Research Clinics heparin-Mn2+ (46 mmol/L) method and five improved precipitation methods for quantification of high-density lipoprotein (HDL) cholesterol. Three of these methods--a dextran sulfate-Mg2+ procedure, reported as a Selected Method, a modified heparin-Mn2+ (92 mmol/L) method, and a modified phosphotungstate-Mg2+ procedure--all gave similar results. Three other methods--the standard heparin-Mn2+ (46 mmol/L) method and two polyethylene glycol methods (75 g/L or pH 10 reagent at 100 g/L final concentrations)--gave slightly higher values for HDL cholesterol. Addition of NaCl or glucose to specimens did not significantly change protein precipitation. In terms of sedimentation effectiveness with hypertriglyceridemic specimens, the methods were ranked in the following order: polyethylene glycol (pH 10, 100 g/L) greater than dextran sulfate-Mg2+ greater than heparin-Mn2+ (92 mmol/L) = polyethylene glycol (75 g/L) greater than phosphotungstate-Mg2+ greater than heparin-Mn2+ (46 mmol/L). (+info)
Presence of basic proteins and ribonucleoproteins in the neck region of human spermatids and spermatozoa.
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The ultrastructural cytochemical study of the neck region of human spermatids and spermatozoa revealed that, besides the centrioles, the basal plate, the lateral cupshaped junction body and the annulus material showed affinity for both ethylene diamine tetra-acetic acid (EDTA) stain (a preferential staining method for ribonucleoproteins) and ethanolic phosphotungstic acid (PTA) stain (for cellular localisation of basic proteins). The capitulum and the surface of the striated columns were also stained with ethanolic PTA. The chromatoid body and annulate lamellae-which were frequently encountered in the neck region--stained with both EDTA and ethanolic PTA techniques. This suggests a possible participation of the chromatoid body and annulate lamellae in the formation of the neck region structures. (+info)
Interference of the chemotherapeutic agent etoposide with the direct phosphotungstic acid method for uric acid.
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A 62-year-old woman receiving chemotherapy with etoposide showed discrepant uric acid values as measured by a direct phosphotungstic acid (PTA) method (150 mg/L) compared with a uricase technique (40 mg/L). After ultrafiltration, the positive interference for the direct PTA method was retained in the protein fraction, but not in the filtrate. Adding exogenous etoposide to drug-free serum confirmed this interference for the direct PTA method, but not for the uricase procedure or a PTA technique preceded by dialysis. Decisions for aggressive patient management are often based on the magnitude of hyperuricemia. We do not recommend that the direct phosphotungstic acid method be used to measure uric acid in patients receiving etoposide. (+info)
Effect of reagent pH on determination of high-density lipoprotein cholesterol by precipitation with sodium phosphotungstate-magnesium.
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When determining high-density lipoprotein cholesterol by use of sodium phosphotungstate-magnesium precipitation method, I found that the pH of the sodium phosphotungstate reagent was a critical factor in the method. Unless the pH of the reagent was less than 7.6, the precipitation of low-density lipoprotein and very-low-density lipoprotein was incomplete. When the specimen pH was between 7.35 and 8.65, the pH of the serum of plasma did not influence the completeness of precipitation. Optimum concentrations of precipitation reagents, determined after the pH of the sodium phosphotungstate reagent was standardized to pH 6. 15, were 40 g/L for sodium phosphotungstate and 2 mol/L for MgCl2. The distribution of high-density lipoprotein cholesterol in a healthy adult population was skewed to the left for women (n = 34; mean = 660 mg/L) and bi-modal for men (n = 44; mean = 460 mg/mL). The central 95% reference interval was 280 to 880 mg/L for women and 250 to 750 mg/L for men. (+info)
Correlation between degradation and ultrastructure of peptidoglycan during autolysis of Escherichia coli.
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The kinetics of peptidoglycan degradation were examined under different conditions of autolysis of Escherichia coli. With cephaloridine- or moenomycin-induced autolysis, degradation did not exceed 25 to 35%, whereas in EDTA-induced autolysis it rapidly reached 65 to 70%. When nonautolyzing cells were fixed overnight with glutaraldehyde, followed by an osmium fixation, and thin sections were stained by the phosphotungstic acid method, a dark, 15-nm-thick layer of uniform appearance and constant width occupied the whole area between the inner and outer membranes of the envelope. The stained material was tentatively identified with peptidoglycan. Ultrastructural changes in this phosphotungstic acid-stained periplasmic space were investigated at different time intervals after induction of autolysis. In all cases, breakdown proceeded over the whole cell surface. During antibiotic-induced autolysis a progressive thinning down limited to the inner side of the layer was observed. During EDTA-induced autolysis, the rapid decrease in thickness correlated well with the important loss of material labeled with [3H]diaminopimelic acid. Considering these changes and the insufficient amounts of peptidoglycan (1.3 U/nm2) necessary to account for a regularly structured polymer occupying the whole 15-nm layer, it was speculated that peptidoglycan might be unevenly distributed throughout the periplasmic space. (+info)