Thymol turbidity and thymol flocculation tests in Nigerian control subjects.
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Twelve laboratories in the north Rhine-Westphalia participated in comparing TPHA and FTA-ABS tests with the so-called classical syphilis reactions (VDRL test, cardiolipin and pallida complement-fixation reaction, as well as Meinicke clearing reaction II). TPHA and FTA-ABS tests proved to be superior to the conventional methods both as regards specificity and sensitivity. Present-day serological tests of syphilis can thus be limited to the TPHA test to exclude syphilis, the FTA-ABS test as a confirmatory reaction and the VDRL test to judge the need or effectiveness of antisyphilitic treatment. These reactions which supersede the conventional syphilis tests serve for rational, reliable and cheap serological diagnosis of syphilis.
The efficiency of poliovirus elution from fiber glass cartridge filters (K27), epoxy-fiber glass-asbestos filters (M780), and pleated cartridge filters was assessed by using 3% beef extract (pH 9.0) or 0.1 M glycine (pH 11.5). Poliovirus type I, strain LSc, was seeded into 20- to 25-gallon (ca. 75.6- to 95.6-liter) samples of treated sewage effluent and concentrated by using a filter adsorption-elution technique. Virus elution was accomplished by using either two 600-ml portions of 3% beef extract (pH 9.0), or two 1-liter portions of 0.1 M glycine (pH 11.5). In all experiments, beef extract elution followed by organic flocculation was found to be superior, yielding a mean recovery efficiency of 85%, with recoveries ranging from 68 to 100%. Elution with 0.1 M glycine (pH 11.5) followed by inorganic flocculation resulted in a mean recovery efficiency of 36%. The variable range of recoveries with beef extract could not be significantly improved by varying the type of beef extract or by extending the elution time to 30 min. Second-step reconcentration of 1-liter seeded sewage effluent and renovated wastewater samples indicated that organic flocculation was a more efficient method for virus recovery than inorganic flocculation. Beef extract concentrations of less than 3% were found to be efficient in the recovery of poliovirus from renovated wastewater.
Effects of treatments with proteolytic enzymes and protein-modifying reagents on flocculation of brewer's yeast IFO 2018 were investigated. The floc-forming ability of the yeast cells was irreversibly eliminated by treatment with papain, trypsin, chymotrypsin or pepsin, indicating that certain proteins on the cell surface participate in the yeast flocculation. Chemical modification with reagents, known to act on disulfide bridges, carboxyl and/or phosphate groups, phenolic groups, amino groups, and imidazole groups, also destroyed the ability to flocculate, although in some cases a high concentration (8 M) of urea was necessary in addition to protein-modifying reagents. Thus, it is suggested strongly that these functional groups of amino acid residues of the proteins are essential for the floc-forming ability of brewer's yeast cells.
Spectrophotometric characteristics of bilirubin at low concentrations (0.005-2.500 mg/100 ml) have been studied under various physical conditions in order to gain a better understanding of the state of bilirubin when preparing "solutions" for laboratory use. Standing, minimal shaking, or stirring of the bilirubin preparations at pH 7.4 progressively reduced and altered the maximal spectral absorption of bilirubin (440 nm) in aqueous buffered media. The shift to 415-420 nm is attributed to oxidation of the pigment whereas shoulder formation is attributed to the formation of large size particles (flocculants). In the presence of antixidants (L-ascorbic acid and nitrogen gas) and EDTA the maximal absorption peak remained at 440 nm but decreased in magnitude concomitant with development of progressively increasing shoulder at 480-560 nm. In the absence of antioxidants and EDTA maximal absorption shifted to 415-420 nm and the magnitude of 480-560 nm shoulder formation was less. At the higher concentrations of bilirubin and with reduction in pH of the buffer in the absence of antioxidants, the shift to lower wave lengths was reduced and 450-560 nm shoulder formation was increased. In the absence of antioxidants and EDTA at the lower concentrations of bilirubin and in more alkaline media, the reduction at 440 nm and the shift of maximal absorption to the shorter wave lengths was enhanced. At pH 12, stirring of antioxidant-EDTA-containing solutions of bilirubin resulted in neither a shift of maximal absorption to the shorter wave lengths nor the formation of 480-560 nm shoulder. The formation of 480-560 nm shoulder was accompanied by the visual appearance of turbidity. The formation of flocculants when a "solution" is agitated indicates that significant portions of the pigment were in fact, not in solution and must have existed previously as a finely dispersed colloidal sol or supersaturated solution which progressed to a colloidal sol. Spectral curves of bilirubin, therefore, may represent a composite resulting from four physical states of bilirubin: (1) bilirubin truly in solution with the spectral peak at 440 nm; (2) bilirubin in the fine colloidal dispersion with spectral characteristics similar to those of bilirubin in solution; (3) bilirubin flocculant giving 480-560 nm shoulder; and (4) oxidation products of bilirubin with the spectral peaks lower than 440 nm. Increasing the pH of the aqueous media containing bilirubin (0.05 mg/100 ml) from 7.4 to 12.0 increased the molar extinction coefficient of bilirubin, E1M/440 1cm, progressively to a maximum at pH 12 of 6.35 X 10(4). Very dilute bilirubin preparations (0.005-0.050 mg/100 ml) in aqueous media, pH 7.4, exhibited spectral evidence of rapid oxidation (more so at higher pH), but spectral shoulder formation was still observed after mechanical agitation. Thus, the solubility of bilirubin in 0.1 M phosphate buffer at pH 7.4 appears to be less than 0.005 mg/100 ml.
A major problem in the development of a particulate hepatolienographic agent is the prevention of particle aggregation upon contact with blood. When iothalamate ethyl ester, a particulate suspension for potential use as an hepatolienographic agent, was mixed with human or animal plasmas, it resulted in moderate to severe flocculation. In a study conducted to define the binding of plasma proteins to the iothalamate ethyl ester particles, plasma was reversibly adsorbed to various particle preparations. Fractions obtained by incubation of the particle-plasma complexes in buffers of increased ionic strength and decreased pH were subjected to polyacrylamide disc gel electrophoresis. The fraction containing the specifically bound proteins produced three distinct bands corresponding to prealbumin, albumin, and fibrinogen. Since the particles flocculated only when mixed with plasma, it is postulated that the fibrinogen-particle interaction is a major factor leading to particle agglutination.
Five two-year-old heifers were each inoculated intravenously with 0.02 mg M. bovis strain AN5. Clinical, haematological and microbiological observations were made during the course of the experiment and antibody levels were measured before and after infection by means of the indirect immunofluorescent antibody (IFA) and bentonite flocculation tests. All cattle developed M. bovis infection varying in severity from peracute tuberculous pneumonia resulting in death within 33 days to chronic progressive generalised tuberculosis. Only cattle developing peracute or acute forms of tuberculosis showed marked haematological changes characterised by leucopenia with lymphopenia. Bacteraemia was detected in the two cattle with peracute tuberculosis 22 days after infection. Anti-mycobacterial antibody was detected after infection in all cattle but fluctuated markedly during the course of the disease. Of a total of 61 serum samples examined from all cattle after inoculation with M. bovis, only 38 were positive to the IFA test and 30 to the bentonite flocculation test. Only 18 were positive to both tests at any one time. IgM was the predominant type of anti-mycobacterial antibody detected by the IFA test and this was found to cross-react with M. avium in almost every sample.
The atheroscleroit lesions, associated with the celiac intimal smooth muscle cushions, of four and five year old White Carneau pigeons were studied with the light and electron microscopes. Light microscopic examination of the spontaneous lesions demonstrated large intimal cushions composed of smooth muscle abundant collagen, clusters of foam cells and cholesterol crystal clefts. Ultrastructural examination of the intimal atheroma revealed dilatations between apposing endothelial cells which contained a flocculent material, similar to that seen in the subendothelial space. The subendothelial compartment contained abundant collagen, extracellular lipid, vesiculated material and cell processes which contained a flocculent matrix and tubular-like elements. In addition, fibroblast-like interlaminar cells were often observed. Numerous intimal smooth muscle cells were seen which displayed varied morphology. Abundant foam cells were also present within the intimal atheromas. The presence of atherosclerotic lesions in preexisting intimal smooth muscle cushions suggests that hemodynamic factors may be important in the progression of these spontaneous lesions. Endothelial cell dilatations may provide an important route of transport for circulating elements which may accumulate withing the subendothelial space. Morphologically, it appears that the smooth muscle cells undergo modification and may represent the precursors of foam cells in this species.
Barium x-ray patterns of ketonuric diabetic Chinese hamsters displayed marked dilatation of the stomach, small and large intestine. Hypomotility was manifested by flocculation of barium in the small and large bowel. Impaired transit time was further characterized by prolonged emptying of the stomach (mean 570 min diabetics; 200 min controls) and delayed stool formation (mean 230 min diabetics; and 100 min controls) and passage (mean 457 min diabetics; 210 min controls). Ultrastructural analysis of Auerbach's myenteric plexuses of the small intestine indicated acute degeneration in certain distal, unmyelinated axons. Swelling, deposition of glycogen, aggregation of neurofilaments and dense accumulation of lamellar bodies were observed. The severity and frequency of barium flocculation, glycogen deposition, aggregation of neurofilaments and lamellar inclusion bodies in axons were directly related to duration of ketonuria. The data strongly suggest that autonomic neuropathology in the plexuses of Auerbach may be a critical factor underlying gastrointestinal dysfunction in the ketonuric diabetic Chinese hamster.
Extracellular acidosis (ph 5.9 to 6.5) was found to be significantly retard the subcellular response of Ehrlich ascites tumor cells (EATC) to anoxic injury. The cells initially showed a reversible stage of cell injury with diffuse mitrochondrial condensation and swelling of endoplasmic reticulum (ER) at pH 7.9 by 1 h, while by 3 h at this pH all cells showed high amplitude and intramatrical flocculent densities of mitochondria, and fragmentation of membrane systems. At pH 7.4 cells were normal at 15 min., the mitochondria were condensed as above by 1 h, some cells still showed condensed mitochondria and dilated ER while others showed high amplitude swelling of mitochondria by 3 h and all cells showed irreversible changes by 4 h. At pH 5.9 to pH 6.5 mitochondria remained condensed until 3 h and only by 6 h did some cells show high amplitude swelling, whereas most cells showed persistent mitrochondrial condensation. Not until 8 h did all cells show high amplitude swelling of mitochondria. Furthermore, ultrastructural differences in the pattern of necrosis were seen at the lower pH values consisting of greater density of the cell sap, irregularly dispersed flocculent densities in apposition to fragmented cytoplasmic membranes and in mitochondria, and more prominent persistent clumping of nuclear chromatin.
Intact nerve myelin compacts to a dehydrated structure of closely apposed membranes when exposed to isotonic solutions at least 10 mM in calcium or tetracaine. The repeat period of the membrane pair in the compacted structure measured by X-ray diffraction is about 126 A in both central and peripheral mammalian nerve myelins whereas the normal periods are about 158 and 178 A, respectively. The electron density profile of compacted myelin shows an asymmetric membrane unit with thickness similar to that of the symmetric bilayer of flocculated myelin lipids. The centrosymmetrically averaged myelin membrane profile is similar to that of the lipid bilayer except at the surface where residual protein is concentrated. Dispersions of extracted total myelin lipids flocculate under similar conditions to those causing myelin compaction, indicating that similar forces act in both processes. Compaction is always accompanied by lateral segregation of intramembrane particles out of the close-packed domains. Lateral displacement of intramembrane proteins form compacted domains can be driven by the attraction of the lipid surfaces for each other. Rates of compaction vary with compacting reagent, concentration, tissue, and temperature, and probably reflect the permeability of the tissue. Extensive compaction by calcium or tetracaine leads to disruption and vesiculation of the spirally wrapped myelin membranes.
Cellular aggregates or nodules which formed in Galleria mellonella larvae in response to injections of killed Bacillus cereus were examined during their first 24 hr of development. The initial aggregation stage was very rapid, occurring within 1 min, and was brought about by the release of an adhesive flocculent material by granular haemocytes in contact with the bacteria. This material, which subsequently became increasingly electron dense owing to melanin deposition, consolidated and by 5 min ramified all the intercellular spaces. The granular cells degenerated, releasing portions of cytoplasm form their peripheries, and eventurally appeared in the matrix as voids containing loosely flocculent material. No bacterial breakdown was seen, however. From 2-4 hr, plasmatocytes began to encapsulate the necrotic mass, and by 24 hr a complete sheath, composed of three discrete regions of cells, was formed. These findings are discussed in relation to the various proposed mechanisms of nodule and capsule formation.
Coated vesicles (CVs), plain synaptic vesicles (PSVs), and nonvesicular flocculent material were isolated from synaptosomes and examined with goniometry and high-resolution electron microscopy after either negative staining or various biochemical procedures. The flocculent material (i.e. the presynaptic matrix material except CV shells) is largely composed of particulate or elongated (chainlike) structures; some of this material (here referred to as particle/chain material) is attached to PSVs. The results obtained were: (a) the proteinaceous properties of the CV coat (also referred to as CV shell) and the particle/chain material were demonstrated with chymotrypsin; (b) the CV shell, studied with various negative-staining techniques, differs from the particle/chain material since it has no 3-4-nm globular subunits and reacts differently to alkaline pH; (c) the particle/chain material consists of aggregates of 3-4-nm globular subunits, four of which yield 8-10-nm fine particles; and these particles can be further aggregated into chains 8-10 nm wide and up to 30-60 nm long showing a "hollow" core; (d) vinblastine sulfate induced ringlike or helical crystalloid precipitates closely resembling the vinblastine-induced microtubule crystals reported in the literature, but vinblastine had no effect on either the CV shell material or the particle/chain material.
In a comparative study, the enzyme-linked immunosorbent assay, using peroxidase labeled anti-rat immunoglobulin M and immunoglobulin G, and the passive hemagglutination test were applied to determine the primary and secondary antibody response to lipopolysaccharide and tetanus toxoid in rats. In the enzyme-linked immunosorbent assay, the antigens were bound to the wells of polystyrene microplates, tetanus toxoid directly, and lipopolysaccharide after complexing it with methylated bovine serum albumin. After incubation with dilutions of the rat sera, the amount of antibody bound to the solid phase was quantified by means of peroxidase-labeled anti-immunoglobulin. The specificity of the enzyme immunoassay was tested by absorption of the sera with their respective antigens. The enzyme-linked immunosorbent assay proved to be more sensitive than the hemagglutination reaction, except when titers were determined during the secondary response to tetanus toxoid. Besides its specificity and sensitivity, the enzyme-linked immunosorbent assay is a convenient method for measuring both immunoglobulin M and immunoglobulin G antibodies. At low serum dilutions of lipopolysaccharide antisera, inhibition of the reaction in the enzyme-linked immunosorbent assay occurred. This phenomenon could be prevented by heating the sera at 56 degrees C for 30 min. Lipopolysaccharide was immunogenic in rats over an extremely wide dose range (from 10 pg to 1 mg); the optimal immunogenic dose of lipopolysaccharide for young adult rats was 0.1 to 1,000 mug when administered intravenously, and that of tetanus toxoid was 5 to 10 lines of flocculation, as determined by the Ramon flocculation test.
Six monkeys (Macaca fascicularis) were used for the present study. In animals which survived for 2-6 days after section of C5 to T1 dorsal roots, at least four types of degenerating afferent terminal were observed - electron-dense, electron-lucent, neurofilamentous and flocculent. The electron-dense degeneration was the most common and was seen as early as 2 days after rhizotomy. The neurofilamentous type was the second commonest and was found predominantly in the 3 days' survival material. The electron-lucent and flocculent types were less commonly encountered. Since the profiles exhibiting neurofilamentous hyperplasia showed varying degrees of electron density it is suggested that this type of degeneration progresses to the electron-dense type with time. The present study also showed that the primary afferent terminals in the cuneate nucleus of the monkey are mostly large and that they contain round vesicles. They are commonly found within synaptic complexes in which they are presynaptic to dendrites of various sizes, and are themselves postsynaptic to smaller axon terminals containing flattened vesicles. Degenerating terminals forming isolated synapses were less commonly seen. No dorsal root axon terminals formed axosomatic synapses.