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At least 1,063 records · Page 59Linked to original sources
PubMed1975

Immunological properties of Rickettsia rickettsii purified by zonal centrifugation.

The properties of Rickettsia rickettsii purified from infected chicken yolk sacs or mouse L cell cultures by sucrose density gradient centrifugation in a zonal rotor were examined in various ways. Rickettsiae derived from both L cells and yolk sacs had similar compositions: about 12% nitrogen, 1.5% phosphorus, 5% carbohydrate, and 30% fatty acids. On a dry-weight basis, purified rickettsiae were at least 2,000 times as effective as a commercial spotted fever vaccine in protecting guinea pigs against infection with spotted fever rickettsiae and mice against death from toxin of R. rickettsii. Gradient-purified rickettsiae (0.6 mug) induced a serological response in guinea pigs of the same magnitude as that stimulated by 1,600 mug of the commercial vaccine. Gradient-purified rickettsiae had little group reactivity in complement fixation tests but became anti-complementary upon storage. Microagglutination and hemagglutination tests with the purified antigen gave promise of usefulness in diagnosis of spotted fever. These results suggest that zonal centrifugation may be a valuable procedure for the preparation of R. rickettsii vaccine and diagnostic reagent.

Animals↗
PubMed1981

Interaction between a trachoma strain of Chlamydia trachomatis and mouse fibroblasts (McCoy cells) in the absence of centrifugation.

A system was devised for studying the interaction of a trachoma strain of Chlamydia trachomatis (G17) and mouse fibroblasts (McCoy cells) in the absence of centrifugation, which is usually employed to enhance the infection of cell cultures with non-lymphogranuloma venereum human strains of C. trachomatis. In this system, the conditions of infection more closely approached those encountered in natural infections, and the entry of G17 into host cells could be compared with the previously described entry of C. psittaci 6BC and a lymphogranuloma venereum strain (440L) of C. trachomatis. McCoy cells were infected by shaking at 37 degrees C with inocula suspended in 0.01 M phosphate buffer, pH 7.2, containing 0.2 M sucrose. The efficiency of infection (inclusion counts without centrifugation/inclusion counts with centrifugation) was 1.5% for monolayers and 7.5% for suspensions. When measured either by inclusion counts or by host cell-associated 14C-amino acid-labeled G17, association was proportional to G17 concentration and increased linearly for 60 min. Pretreatment of host cells with diethylaminoethyl-dextran (30 micrograms/ml, 30 min) raised the efficiency of infection to about 13% for both monolayers and suspensions. Host cells treated with cytochalasin B (2 x 10(-5) M, 90 min) or trypsin (50 micrograms/ml, 60 min) associated with G17 at undiminished rates. 14C-labeled G17 inactivated by heat (60 degrees C, 3 min) or ultraviolet light (1,800 ergs per mm2) associated with McCoy cells at the same rate as live G17. Comparison of these results with those previously reported for strains 6BC and 440L showed that strain G17 exhibited some, but not all, of the host cell association properties of the other two chlamydial strains.

Animals↗
PubMed1969

Size-separation of yeast mitochondria in the zonal centrifuge.

Mitochondria, released from yeast spheroplasts and subjected to rate separation through sorbitol gradients in the zonal centrifuge, migrated in a wide symmetrical zone. Electron micrographs showed that the mitochondria had been resolved within the zone according to size. The mean mitochondrial diameter at the leading edge was approximately twice that at the trailing edge of the particle zone. Activities of the enzymes cytochrome oxidase, malate dehydrogenase, and reduced nicotinamide adenine dinucleotide- and d-lactate cytochrome c reductases were essentially uniform throughout the mitochondrial zone. Mitochondria from a vegetative-petite mutant had almost the same size distribution as the isogenic wild type, but with somewhat larger mean diameter and either absent or markedly reduced enzyme activities. Mixtures of wild-type and petite mitochondria produced sedimentation profiles showing overlap of particle populations with respect to mean sedimentation rates and mitochondrial diameters, as well as intermediate levels of enzyme activities. Both cristate and noncristate organelles were present throughout the mitochondrial zone from these mixtures. Mitochondria centrifuged in sorbitol density gradients were well-preserved and yielded consistent sedimentation profiles, whereas particles in sucrose density gradients migrated more slowly, produced varied sedimentation profiles, and often showed spurious peaks, presumably due to particle aggregations.

Centrifugation↗
PubMed1970

Chromatography of microbial lipids by centrifugation through microparticulate gel.

An improved apparatus and a procedure are described by which the migration of sample components in column chromatography is accelerated by centrifugal force, thereby making it possible to use beds of densely packed gel prepared with ultrafine silica. This technique was used to resolve components of certain lipid mixtures where other methods have failed, and it has been found generally useful as an adjunct to other methods for the fractionation of lipids. Biologically active phosphoglycolipids from Mycobacterium tuberculosis and a phosphatidylglycerol-like substance from Mycoplasma pneumoniae which formed single spots on thin-layer chromatographic plates were each found to contain a major and several minor components by centrifugal chromatography. The method enabled us to isolate individual components of Wax D from M. tuberculosis rather than a spectrum of components. Minor components were resolved which, although present in insufficient quantity to influence results of chemical analyses, may be responsible for biological activity. The apparatus provides an essentially closed system which reduces highly volatile solvents to minimal evaporation during the chromatographic process. Samples are applied in solution and are not allowed to dry on the columns until after separation has been achieved. Consequently, polar, labile microbial lipids can be resolved without the use of harsh reagents which destroy some of their properties. Single components may be harvested by cutting and removing appropriate segments of the larger chromatograms or by eluting them from the columns.

Centrifugation↗
PubMed1965

Washing bacteria by centrifugation through a water-immiscible layer of silicones.

Hurwitz, Charles (Veterans Administration Hospital, Albany, N.Y.), Catherine B. Braun, and Richard A. Peabody. Washing bacteria by centrifugation through a water-immiscible layer of silicones. J. Bacteriol. 90:1692-1695. 1965.-A method is described which enables the separation of cells from aqueous suspension without altering the internal aqueous environment of the cells. The method consists of centrifuging the cells from the aqueous environment through a more dense, immiscible solvent consisting of a mixture of two silicones. A thin film of the aqueous environment equal to 7 x 10(-13) ml per cell remains with the bacteria during the separation procedure. The method by which this volume was determined is described. The procedure itself has no measurable effect on viability or permeability of the cells and permits recovery of about 90% of the cells from the aqueous environment. With this method, it has been found that the intracellular water volume of Escherichia coli ML 35 accessible to sucrose or inositol is 1.96 x 10(-12) ml +/- 6%, or 85% of the total volume of the cell determined by visual measurement.

Bacteriological Techniques↗
PubMed1982

Rapid detection of simulated bacteremia by centrifugation and filtration.

A centrifugation-filtration procedure was developed to expedite the recovery of microorganisms from blood. Fresh whole human blood was inoculated with various aerobic and facultatively anaerobic microorganisms (3 to 18 per ml). The seeded blood was carefully overlaid on a Ficoll-Hypaque gradient (density, 1.114 g/ml) and centrifuged (400 x g) for 45 min at ambient temperature. The entire gradient (plasma, leukocytes, and Ficoll-Hypaque) was removed and filtered through a 0.22-micrometer membrane filter. The filters were then placed on chocolate agar and incubated at 35 degrees C in humidified air containing 5% CO2. No statistically significant differences were detected between the numbers of microorganisms recovered by filtration and by direct culture of the original inoculum. Most microorganisms were detected within 18 h after filtration. This system has excellent sensitivity and negligible toxicity.

Blood↗
PubMed1984

Clinical correlations of serial quantitative blood cultures determined by lysis-centrifugation in patients with persistent septicemia.

The potential clinical value of colony counts determined by the lysis-centrifugation blood culture method was studied by reviewing the records of eight patients with persistent septicemia in whom colony counts were available on at least 3 days. Colony counts of the five patients who survived decreased steadily as the patients improved. One of the three patients who died had counts repeatedly below 1.0 CFU/ml while she was clinically stable and higher counts when her condition deteriorated. Two patients died despite decreasing colony counts. One was improving and died unexpectedly of an unrelated cause; the other died of candidiasis, but declining serial arabinitol/creatinine ratios suggested a partial response to therapy. In addition, septicemia related to infected intravenous catheters was documented by demonstrating large differences in colony counts determined simultaneously from two different sites in two patients and by demonstrating a precipitous drop in CFU per milliliter after removal of the infected catheter in one patient. Routine availability of colony counts appears to be an important advantage of the lysis-centrifugation method.

Adolescent↗
PubMed1985

Detection and serotyping of herpes simplex virus in MRC-5 cells by use of centrifugation and monoclonal antibodies 16 h postinoculation.

Monoclonal antibodies (Syva Co., Palo Alto, Calif.) were used for the detection and serotyping of herpes simplex virus (HSV) isolates by immunofluorescence 16 h after inoculation of MRC-5 monolayers in 3.7-ml shell vials and after low-speed centrifugation. A total of 119 specimens were inoculated into conventional tube cell cultures and shell vials. Of 98 specimens inoculated on the same day of receipt in the laboratory (fresh specimens), all 23 (23.5%) HSV-positive specimens were identified by serotype in 16 h in shell vials by immunofluorescence, whereas only 8 of 23 HSV-positive specimens (34.8%) produced cytopathic effects in conventional tube cell cultures in this time period. Similarly, of 21 original specimen extracts previously determined to be culture positive for HSV (stored specimens), all were detected and serotyped by the immunofluorescence test with monoclonal antibodies 16 h postinoculation compared with the recognition of only 8 of these isolates (38.1%) by cytopathic effect that soon. This technique of centrifugal inoculation of HSV in shell vials containing MRC-5 cells permitted detection of this virus in all positive specimens with serotype determination within 16 h postinoculation.

Antibodies, Monoclonal↗
PubMed1988

Comparison of primary rabbit kidney and MRC-5 cells and two stain procedures for herpes simplex virus detection by a shell vial centrifugation method.

By using a conventional tissue culture method as a standard, four shell vial centrifugation culture (SVC) formats were compared for herpes simplex virus (HSV) detection in 300 clinical samples. Both MRC-5 and primary rabbit kidney (PRK) cells were used in the conventional and SVC systems. In addition, both a direct monoclonal fluorescent antibody to HSV (MAb-FA; Syva Corporation, Palo Alto, Calif.) and an indirect HSV polyclonal antibody-horseradish peroxidase stain (poly-HRP; Difco Laboratories, Detroit, Mich.) were used to stain shell vials of both cell types. Conventional tubes were incubated for up to 7 days with daily examination for cytopathic effect, which was confirmed as HSV by staining with an MAb-FA. Shell vials were inoculated, centrifuged, incubated for 16 to 24 h, and stained directly with MAb-FA or indirectly with a poly-HRP stain. Of the 300 specimens examined, 82 (27%) were HSV positive by conventional tissue culture. PRK cells detected 81 (99%) positive specimens, compared with 74 (90%) specimens detected with MRC-5 cells. Of the 82 positive specimens by conventional culture, the SVC formats detected 68 by MRC-5 and MAb-FA, 74 by MRC-5 and poly-HRP, 64 by PRK and MAb-FA, and 77 by PRK and poly-HRP. Therefore, PRK stained by an indirect method with poly-HRP was the most sensitive of the SVC formats tested, detecting 94% of the positive specimens.

Animals↗
PubMed1988

Conventional tube cell culture compared with centrifugal inoculation of MRC-5 cells and staining with monoclonal antibodies for detection of herpes simplex virus in clinical specimens.

During a 15-month period, two methods for detection of herpes simplex virus (HSV) in 699 clinical specimens were compared: (i) 24-well-plate centrifugation (24WPC) with MRC-5 cells and staining with type-specific monoclonal antibodies (Syva Co., Palo Alto, Calif.) after incubation for 16 to 18 h and (ii) conventional tube cell culture with primary rabbit kidney and A549 cells. HSV was identified by conventional tube cell culture in 165 (24%) of 699 specimens and by the 24WPC method in 116 (17%) of 699 specimens. One specimen was positive for HSV by the 24WPC method alone, compared with 50 specimens positive only by conventional cell culture (P less than 0.0001). The sensitivity, specificity, and positive and negative predictive values of the 24WPC technique with MRC-5 cells for detection of HSV in clinical specimens were 70, 99.8, 99, and 91%, respectively. Centrifugal inoculation of MRC-5 cells in 24-well plates and staining with monoclonal antibodies after incubation for 16 to 18 h is an insensitive means of detecting HSV in clinical specimens and should not replace conventional tube cell culture with primary rabbit kidney cells.

Antibodies, Monoclonal↗
PubMed1989

Clinical experience with cytomegalovirus isolation using conventional cell cultures and early antigen detection in centrifugation-enhanced shell vial cultures.

A total of 1,915 clinical samples was inoculated by low-speed centrifugation into shell vials (Bartels Immunodiagnostics, Bellvue, Wash.) containing cover slip monolayers of MRC-5 fibroblasts. At 1 and 2 days postinoculation, one cover slip was stained by an indirect immunofluorescence technique using a monoclonal antibody (Biotech Research Laboratories for Dupont, Billerica, Mass.) to cytomegalovirus (CMV) early antigen (EA). Clinical samples were also inoculated into three MRC-5 or MRHF cell cultures which were observed for 30 days for the appearance of a cytopathic effect (CPE). Of 157 CMV-positive samples, 92 (59%) were identified by centrifugation-enhanced EA (CE-EA) and 131 (83%) produced a CPE. CE-EA was less sensitive than CPE for all types of samples, although 17% of CMV-positive samples were detected by CE-EA alone. Evaluation of the CMV status of patients with CE-EA-positive-CPE-negative samples indicated that these samples likely represented true CMV-positive results. The average elapsed time between culture inoculation and identification of CMV decreased as follows when both CE-EA and CPE, rather than CPE alone, were used: urines, 15 to 7 days; buffy coats, 18 to 9 days; lung samples, 13 to 8 days; throat samples, 18 to 7 days. Although CE-EA was less sensitive than 30-day cell culture, both CE-EA and CPE were identified as valuable in CMV detection, and neither could be discontinued without a decrease in the CMV isolation rate or an increase in the turnaround time.

Antibodies, Monoclonal↗
PubMed1991

Detection of bacteria in blood by centrifugation and filtration.

Culture of blood is the most frequent means of diagnosing bacteremia. However, conventional blood culturing methods are slow in isolating bacteria. We developed a method for isolation of bacteria by centrifugation and filtration. Fresh human whole blood was inoculated with facultatively anaerobic and aerobic microorganisms (3 to 172 microorganisms per 5 ml). Seeded blood was then mixed with Ficoll-Hypaque (density, 1.149 +/- 0.002 g/ml) and centrifuged (386 x g) for 30 min at ambient temperature. The entire gradient (plasma, leukocytes, and Ficoll-Hypaque) was removed and filtered through a 0.22-micron membrane filter (Millipore). The filters were then placed on chocolate agar plates and incubated at 35 degrees C in a humidified atmosphere containing 5% CO2. For each bacterium tested, approximately 35 to 100% of the viable microorganisms were recovered when compared with control cultures (pour plates of seeded blood). All bacteria produced isolated colonies on filters after overnight incubation (18 h). This procedure may prove to be a more rapid method for isolating bacteria from clinical blood samples than the blood culture bottle technique.

Bacteria↗
PubMed1992

Quantification of cytomegalovirus in bronchoalveolar lavage fluid after allogeneic marrow transplantation by centrifugation culture.

A technique to quantify cytomegalovirus (CMV) by centrifugation culture of bronchoalveolar lavage fluid from marrow transplant recipients was developed. This technique was used to assess the CMV response to antiviral treatment and the relationship between viral load, asymptomatic excretion versus symptomatic infection, and prognosis. Relative to tube cell culture, centrifugation culture of bronchoalveolar lavage fluid was more sensitive than direct fluorescent-antibody staining. It was also a rapid, replicable method for detecting and measuring the amount of CMV. There was no significant difference between viral load at diagnosis and after 9 days of treatment with ganciclovir and intravenous immunoglobulin. Viral load was not predictive of outcome, and there was no difference in amount of virus between patients with asymptomatic CMV excretion and those with CMV pneumonia. The amount of CMV may not be as important as other factors (e.g., host immune response) in the pathogenesis of CMV pneumonia.

Bone Marrow Transplantation↗
PubMed1998

Isolation of Francisella tularensis by centrifugation of shell vial cell culture from an inoculation eschar.

A 52-year-old man was admitted to the hospital following the development of an inoculation eschar and fever six days after being bitten by a tick. He was clinically diagnosed as suffering from rickettsiosis. Eschar biopsy cultures on standard bacteriological media remained sterile. However, inoculation of cells with the homogenized specimen by the centrifugation-shell vial technique (M. Marrero and D. Raoult, Am. J. Trop. Med. Hyg. 40:197-199, 1989) resulted in the recovery of a bacterium. Determination of the sequence of the 16S rRNA gene amplified from the organism and comparison of the sequence to other sequences identified it as a strain of Francisella tularensis, whereas the specific serology was still negative. Our findings demonstrate that the centrifugation-cell culture, which is a tool for investigation of tick-transmitted diseases, may have the potential to serve as a method for the cultural isolation of F. tularensis.

Animals↗
PubMed1999

Optimizing culture of Chlamydia pneumoniae by using multiple centrifugations.

Three methods for the recovery of Chlamydia pneumoniae from spiked nasopharyngeal and blood specimens, including extended culture and additional centrifugations, were compared. Additional centrifugations and a 7-day culture time resulted in a 500- to 5, 000-fold increase in the number of detectable inclusion-forming units.

Cells, Cultured↗
PubMed1977

Solid-phase radioimmunoassay of rubella virus immunoglobulin M antibodies: comparison with sucrose density gradient centrifugation test.

The solid-phase radioimmunoassay (RIA) method developed in our laboratory for demonstrating rubella virus-specific immunoglobulin G (IgG) antibodies (Kalimo et al., 1976) was further developed for demonstrating IgM antibodies. A total of 188 serum specimens were tested. The statistical probability of obtaining a false-positive IgM result, based on determinations of 100 rubella-negative sera, was below 0.001. Nonspecific inhibitors and IgM antibodies against other viruses tested did not interfere in the assay. In 2 out of 20 (10%) serum specimens with rheumatoid factor, a false-positive IgM result was obtained. The new RIA method was compared with sucrose density gradient centrifugation, followed by hemagglutination inhibition testing of the separated immunoglobulins with respect to demonstrating IgM antibodies. In patients with acute rubella infection, IgM antibodies were demonstrated by RIA in 9 out of 20 acute-phase sera and in all 20 early-convalescent-phase sera, compared with 7 out of 20 acute-phase sera and 19 out of 20 early-convalescent-phase sera by sucrose density gradient centrifugation. The results obtained indicate that the RIA method is reliable and sensitive and suitable for routine diagnostic use.

Antibodies, Viral↗
PubMed1967

Herpes simplex virus products in productive and abortive infection. I. Stabilization with formaldehyde and preliminary analyses by isopycnic centrifugation in CsCl.

Lysates of HEp-2 cells productively infected with herpes simplex virus yielded two bands on isopycnic centrifugation in CsCl gradients, ranging from 1.2 to 1.6 g/cm(3). One band, designated alpha, had a mean buoyant density of 1.27 g/cm(3) and contained herpes virions. Band beta had a mean density of 1.305 g/cm(3) and contained primarily complement-fixing viral antigens and little or no viral deoxyribonucleic acid (DNA). The products banding in the alpha and beta bands were unstable; fivefold or higher amounts were recovered by treating the cell extract with formaldehyde prior to centrifugation. Formaldehyde treatment increased the buoyant density of viral products in both the alpha and beta bands by about 0.015 g/cm(3). In addition, it stabilized hitherto inapparent products, forming a broad band gamma with a density range of 1.37 to 1.45 g/cm(3). The material in the gamma band was heterogeneous; it contained viral DNA, cellular DNA, and viral antigen. Formalinized lysates of DK cells abortively infected with herpes simplex virus yielded a beta band undifferentiated from that formed by extracts of productively infected cells. The gamma band was less dense and narrower. The alpha band was entirely missing.

Carcinoma↗
PubMed1967

Respiratory syncytial virus isolation by combined continuous flow-isopycnic banding centrifugation.

A new zonal centrifuge rotor (B-IX) which combines continuous sample flow centrifugation with isopycnic banding has been used to isolate and concentrate respiratory syncytial virus from liter volumes of culture fluid. This isolation technique utilizes a sucrose density gradient to trap and isopycnically band the virus particles, and permits recovery of the particles from the rotor in an unaggregated condition.

Carcinoma↗