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At least 883 records · Page 49Linked to original sources
PubMed1992

Improved efficiency in determining the titer of the Autographa californica baculovirus nonoccluded virus.

An economical and time-efficient method for titering the Autographa californica multiple-embedded nuclear polyhedrosis virus (AcMNPV) by the endpoint method is described. The method uses an electronic pipetting device to perform dilutions in the same 60-well microplate as is used for the assay, thus eliminating the need for test tubes or vials for making dilutions. Additionally, since small volumes are used for the dilutions, a substantial savings in medium is achieved. The effect of using three different lepidopteran cell lines in this assay for AcMNPV is also described. This test revealed that one line (the Trichoplusia ni IPLB-TN-R2 line) is at least 1.5 logs more sensitive to AcMNPV when using occlusion body formation as the measure of infection. The titer was about 6- to 12-fold higher in the IPLB-TN-R2 cell line than the other two lines when plaque assay procedures were used. The titer of a recombinant baculovirus with a bacterial beta-galactosidase gene was also measured in the three cell lines using X-gal as an indicator and showed the IPLB-TN-R2 line to be fourfold more sensitive to this virus.

Animals↗
PubMed2003

Filtration based DNA preparation for sexual assault cases.

Police departments in the United States currently have as many as 500,000 unprocessed swabs taken from rape victims. The standard method for purifying sperm from these swabs is to resuspend first all cells and to digest selectively the excess of the victim's epithelial cells. The intact sperm are then separated from the contaminating solubilized DNA by centrifugation, careful removal of supernatant, and extensive washing of the sperm pellet, all steps that are difficult to automate. Vacuum driven filtration is an alternative method for separating sperm from digested epithelial cells that requires only pipetting steps and can be readily automated in a 96 well format. Sperm DNA is enriched 45-fold using this process and the yield of PCR ready DNA is roughly 20% of the amount originally present on the swab.

Cell Separation↗
PubMed1998

99m Tc-ethylenedicysteine (99mTc-EC) renal clearance determination error for the multiple- and single-sample methods.

BACKGROUND: The aim of this work has been to assay determination error values for the clearance determined by single- and multi-sample method and to compare the error values obtained for both methods. METHODS: The assay has been conducted by two independent methods (Monte Carlo simulation, total differential). RESULTS: The results indicate that the relative root mean square error of the clearance determination by multisample method is practically constant above CI values 150 ml/min and amounts to ab +/-3-4%. For values below 150 ml/min the error increases and reaches +/-12% at CI=70 ml/min. The one sample method is less accurate and error increases from +/-(8-9)% 550 ml/min to +/- 25% at 70 ml/min. Moreover, for the multisample method the error estimated from total differential exceeds that obtained from Monte Carlo produced by factory of 3 to 4. For the one sample clearance determination both error estimates yield similar results. In both methods of clearance determination inaccurate pipetting makes a dominant contribution to the overall error. CONCLUSIONS: Form monitoring of patients' condition the multisample method should be employed. The use of one sample method should be limited to screening tests and for obtaining additional information, supplementing the dynamic scintigraphic studies

Journal Article↗
PubMed1992

Results of the multicenter evaluation of a novel homogeneous immunoassay for digoxin based on the cloned enzyme donor immunoassay technology.

A new CEDIA assay for the measurement of digoxin in serum on random access analyzers was evaluated by twelve laboratories in Europe and the United States. Studies on the analytical range, reproducibility, calibration stability, recovery in controls, interlaboratory comparability, comparability with routine methods, and the effect of various interfering factors have been performed and the results are presented in this paper. The analytical performance was comparable to that of routine methods provided the manual pipetting step for pre-incubation was performed with accurate pipettes. A major advantage of the CEDIA Digoxin assay in terms of convenience is the simple two-point calibration procedure. Moreover, digoxin can be determined within 15 minutes after receiving the samples on random access analyzers like Boehringer Mannheim/Hitachi analysis systems. Thus, the CEDIA Digoxin assay represents an attractive alternative to the measurement of digoxin on dedicated immunochemical assay systems.

Calibration↗
PubMed1992

Laboratory evaluation of five pesticides for control of Leptotrombidium fletcheri (Acari: Trombiculidae).

Five pesticides were evaluated against laboratory colonies of Leptotrombidium fletcheri (Womersly and Heaslip) by the Pasteur pipet technique. The pesticides were dieldrin (LC50 = 3.6 ppm, LC99 = 18.2 ppm), bromopropylate (LC50 = 9.2 ppm, LC99 = 239.6 ppm), dicofol (LC50 = 27.8 ppm, LC99 = 118.1 ppm), fenthion (LC50 = 15.4 ppm, LC99 = 29.7 ppm), and malathion (LC50 = 84.7 ppm, LC99 = 313.9 ppm). Dieldrin was the most toxic. Dicofol was recommended for further evaluation in field trials.

Animals↗
PubMedDate not supplied

Evaluation of the swab test on premises for detection of antimicrobial residues in bovine and porcine kidneys.

The effect of sample matrix on the sensitivity of the Swab Test On Premises (STOP) was evaluated for selected antimicrobials. Fluid was extracted from bovine and porcine kidneys, and fortified with known levels of drugs. Aqueous standards were also prepared at the same levels. An aliquot of the fortified fluid or water was pipeted onto a dry swab which was placed onto the surface of a STOP plate, and the plate was incubated as outlined in the test kit manual. Zones of bacterial growth inhibition were measured and recorded, and additional testing was performed with decreasing levels of drug until a minimum detectable level was determined. The effect of temperature on the sensitivity of the test was also evaluated by running samples in duplicate, one set at a nominal temperature of 28 degrees C, and the second set at a nominal temperature of 32 degrees C. Fortified bovine kidney fluid produced significantly larger zones than did porcine kidney fluid at both temperatures, but the mean zone sizes for fortified water were not significantly different from those of bovine or porcine kidney fluid at either temperature. For all 3 matrixes, zones of inhibition were significantly larger at 28 degrees than 32 degrees C.

Animals↗
PubMed2004

Development of automated high-throughput ecotoxicity and genotoxicity test systems and fields of application.

Bioassays like growth inhibition and genotoxicity assays are frequently used for the characterization of chemicals and contaminated environmental samples. In this work two standardized bioassays are automated completely using newly developed liquid handling stations and robotics. A high-throughput algal growth inhibition assay prototype is set up and optimized in cooperation with Hoelle & Huettner AG (Tuebingen, Germany) and Polygen GmbH (Langen, Germany). A software package for both the control of the test system and for data evaluation has been developed (Biolane Supervisor, BioLane Manager HTT). The applicability of the prototype is demonstrated by testing reference compounds with the automated assay in parallel with the Erlenmeyer flask assay. It is shown that EC50-values of chemicals do not differ significantly when incubation parameters like homogeneity of light intensity, temperature and evaporation during 72 hours of incubation are optimized. The umu-genotoxicity test is automated completely using a "RoboSeq 4204 SE" pipetting station (MWG AG, Ebersberg, Germany) equipped with two shakers, microplate photometer, four pipettes, stacker for ten microplates and incubation cabin with temperature regulation. As a result of miniaturization and automation large numbers of toxicants and far more treatments and parallels can be tested and, additionally, only low sample volumes are needed.

Automation↗
PubMed2004

[Determination of sodium monofluoroacetate in human blood and food samples by ion chromatography].

A method was developed for the determination of sodium monofluoroacetate in human blood by ion chromatography. The analysis was performed on a Dionex Ion Pac AS11 column (250 mm x 4 mm i.d.) with an AG11 guard column (50 mm x 4 mm i.d.). A conductivity detector, an SRS suppressor and a 25 microL sample loop were used. The eluent was 2.0 mmol/L Na2B4O7 with a flow rate of 1.0 mL/min. A blood sample of 0.2 mL was pipetted into a test tube followed by adding 1.0 mL of pure water. And then acetonitrile was used to dilute it to 3.0 mL. After the protein was settled down from the blood sample, the supernatant layer was filtered through a 0.45 microm filter before injected into the ion chromatographic system. Good linear relationship between the peak area and the concentration of sodium monofluoroacetate was found with correlation coefficient (r2) of 0.9978 within the range from 0.10 to 10.0 mg/L. The detection limit for blood was 2.5 mg/L. This method also can be used for the determination of sodium monofluoroacetate in food samples.

Animals↗
PubMed2005

[Clinical experience with a modified method of human embryo vitrification].

OBJECTIVE: To obtain clinical experience with a modified method of vitrification used to freeze supernumerary human embryos following in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). SETTING: Centre of Assisted Reproduction, 2nd gynaecological-obstetrical clinic of Faculty hospital of L. Pasteur and Medical Faculty of the University of P. J. Safáirik, Kosice. METHODS: A modified method of vitrification was used to freeze 215 human embryos after IVF-ICSI, obtained in 42 cycles. The embryos were frozen at 48 h after in vitro culturing. The modified method of vitrification consisted in the use of a series of solutions with increasing cryoprotectant concentrations (ethylene glycol and sucrose) up to the final concentration of 40% ethylene glycol and 1 M sucrose. A 100 microl pipetting "tip" was used as a freezing container. The embroys were thawed at room temperature and the cryoprotectant was washed out three consecutive steps with sucrose solutions of decreasing concentrations (1 M, 0.5 M, and the pure medium). The morphology of embryos was evaluated before and after thawing. After thawing, the embryos were cultured in vitro for 24 hours and only the embryos with at least one cleaved blastomere were used for transfer. RESULTS: The percentage of embryos which survived thawing and were capable of further development was 69.8% and 48%, respectively. The mean number of transferred and developing embryos was 2.9 +/- 1.4. The percentage of clinical pregnancies per thawing cycle and per transfer was 19.0% and 27.6%, respectively. Seven patients delivered 8 children. CONCLUSION: The results showed that our modification of vitrification procedure of human embryos is reliable and represents a simpler, shorter and cheaper alternative to slow-rate freezing.

Adult↗
PubMedDate not supplied

[Uncertainty of measurement as an integral part of the result and a tool of quality of work improvement for the chemical-toxicology laboratory].

An uncertainty in measurements is a numerical defined range that is supposed to find a true value with a given level of probability. The purpose of this study was to estimate a type A and B of uncertainty in the analysis of ethanol using the ADH method and prove it to be useful in lab practice. A large percentage of uncertainty was shown for pipetting of the ADH and perchloric acid solutions. This result formed the basis to make an effort in order to improve the procedure. The results of uncertainty of measurements given by method B are similar to the empiric method A results. The reduction of expenses and time saving of the analysis is an important advantage of the described way of uncertainty.

Alcohol Dehydrogenase↗
PubMedDate not supplied

[D.L.vo n. 626/94 - Musculoskeletal diseases caused by use of micropipette in laboratory].

The use of manual pipettes has been associated with a high prevalence of upper extremity and neck cumulative trauma disorders (CTD's) and work-related musculoskeletal disorders (WMSD's) among laboratory workers. The primary risk factor for these disorders are poor ergonomics in three specific areas: posture, repetition and force. Federal agencies have issued guidelines for pipetting practices to reduce the risk of injury and WMSD's. Pipette manufacturing have responded to the problem by improving the ergonomics and buttons and plungers that require less forces during operations. Another problem was still open with the traditional axial-design pipette: deviation of body or extremity from an ergonomically favourable neutral position. The new generation of micropipettes solves this problem.

Carpal Tunnel Syndrome↗
PubMed2005

The antimicrobial effect of nitric oxide on the bacteria that cause nosocomial pneumonia in mechanically ventilated patients in the intensive care unit.

BACKGROUND: Nosocomial pneumonia is the second most frequent nosocomial infection and the leading cause of death from hospital-acquired infection. Endogenously produced nitric oxide is an important component of the body's natural defense mechanism. Recent studies have demonstrated that exogenous gaseous nitric oxide (gNO) is bactericidal and that inhaled gNO is beneficial to bacterial clearance. OBJECTIVE: Determine the antimicrobial effect of exogenous gNO in vitro against organisms from culture collections and pathogens derived from tracheal aspirates of mechanically ventilated patients with pneumonia in an intensive care unit. METHODS: Using bacterial isolates in pure culture, a 0.5 McFarland standard (10(8) colony-forming-units [cfu] per mL) was prepared and further diluted to 1:1,000 with saline, to 10(5) cfu/mL. For each isolate tested, 3 mL was pipetted into each well of a 6-well plate, and placed in a specially designed incubator with compartments for both a treatment arm and a control arm. Both chambers received a continuous flow of heated, humidified gas. The treatment chamber had 200 ppm of gNO in the gas flow, which is higher than the clinically accepted concentration for gNO. Samples were drawn off at time intervals, plated onto Columbia agar base with 5% sheep blood, and placed in a traditional incubator at 35 degrees C for a minimum of 24 h. All tests were performed in duplicate. The colony-forming units were visually counted to determine percentage kill. RESULTS: There was total kill (100% of all colony-forming units) of each bacterial strain subjected to the test conditions at between 2 and 6 h of exposure to 200 ppm gNO. CONCLUSION: gNO is bactericidal against various strains of bacteria suspended in saline, including both Gram-positive and Gram-negative organisms, and those that commonly cause nosocomial pneumonia in mechanically ventilated patients. Future work should focus on developing strategies that maximize the antimicrobial effect while minimizing the effect of these same interventions on host cells.

Administration, Inhalation↗
PubMed2005

Automated template quantification for DNA sequencing facilities.

The quantification of plasmid DNA by the PicoGreen dye binding assay has been automated, and the effect of quantification of user-submitted templates on DNA sequence quality in a core laboratory has been assessed. The protocol pipets, mixes and reads standards, blanks and up to 88 unknowns, generates a standard curve, and calculates template concentrations. For pUC19 replicates at five concentrations, coefficients of variance were 0.1, and percent errors were from 1% to 7% (n=198). Standard curves with pUC19 DNA were nonlinear over the 1 to 1733 ng/microL concentration range required to assay the majority (98.7%) of user-submitted templates. Over 35,000 templates have been quantified using the protocol. For 1350 user-submitted plasmids, 87% deviated by >or=20% from the requested concentration (500 ng/microL). Based on data from 418 sequencing reactions, quantification of user-submitted templates was shown to significantly improve DNA sequence quality. The protocol is applicable to all types of double-stranded DNA, is unaffected by primer (1 pmol/microL), and is user modifiable. The protocol takes 30 min, saves 1 h of technical time, and costs approximately $0.20 per unknown.

Plasmids↗
PubMed1999

Serum dexamethasone levels after decadron phonophoresis.

OBJECTIVE: To determine serum levels of dexamethasone at several intervals after administration of Decadron (dexamethasone sodium phosphate) phonophoresis. DESIGN AND SETTING: This study was designed as a 2-factor analysis of variance with repeated measures on 1 factor (blood draws). Independent variables were group (gel/sham, gel/ ultrasound, dexamethasone/sham, dexamethasone/ultrasound) and blood draws (pretreatment, posttreatment, 15 minutes, and 30 minutes). The dependent variable was the serum level of dexamethasone. SUBJECTS: Forty healthy college students (21 males, 19 females; mean age = 22 +/- 1.3 years) with no known drug allergies or current medication use were randomly assigned to 1 of 4 treatment groups. The treatment site was the left forearm. MEASUREMENTS: After the pretreatment blood draw, a 10- minute ultrasound treatment was administered, followed by a posttreatment blood draw. Two additional blood draws followed at 15-minute intervals. A total of 4 serum samples (5 cc each) from each subject were centrifuged, and the pipetted serum was frozen for later analysis by double antibody radioimmunoassay. RESULTS: No significant amounts of serum dexamethasone were detected in 12 consecutive samples. Testing of additional samples was, therefore, discontinued. CONCLUSIONS: Decadron phonophoresis as used in this experiment did not result in detectable serum levels of dexamethasone. More study is needed to validate the efficacy of Decadron phonophoresis on serum dexamethasone levels.

Journal Article↗
PubMed1991

A dipstick ELISA for rapid detection of human blood meals in mosquitoes.

A dot-enzyme-linked immunosorbent assay, dot-ELISA, that allows for identification of human blood meals in mosquitoes in less than 2 h is presented. Strips of mylar-backed nitrocellulose paper, the dipstick, may be inoculated with anti-human capture antibody, blocked, dried and stored for at least 3 months before use. The Dipstick ELISA consistently detected human blood meals at 24 h post-blood meal in frozen Anopheles mosquitoes and at 32 h post-blood meal on samples eluted off filter-paper smears. This ELISA detects human IgG at dilutions of 1:25,600, and produces strong positive results at dilutions between 1:400 and 1:12,800. The Dipstick ELISA is highly specific, and no false positives were detected when tested against cow, horse, goat, dog, cat, pig, rabbit, mouse, rat, chicken, raccoon and opossum sera. All reagents for the assay are commercially available. The Dipstick ELISA meets requirements for a rapid and simple assay for the identification of human blood meals and should have particular application to short-term field studies and emergency epidemiological investigations. A modified protocol, the Trough ELISA, which treats dipsticks jointly in disposable pipet troughs during the conjugate and substrate steps, was developed and produced as a kit. The Trough ELISA produces 1-4% random false positives, and we recommend that the Trough ELISA not be used.

Animals↗
PubMed2006

[In vitro cytotoxicity of different self-etching dental adhesive systems].

The study evaluated the cytotoxicity of five self-etching dentin adhesive systems applied on dentin specimens of different thicknesses. The test materials (A: Adper Prompt-L-Pop, B: Xeno II, C: Clearfil SE Bond, D: One up Bond F, E: Resulcin Aqua Prime & Monobond) and a positive control (35% H202) were applied on 1.0, 1.5 and 2.5 mm thick bovine dentin specimens (each subgroup n = 5) in a dentin barrier test device. The experiments were performed with perfusion (2 ml/h) of the pulpal part of the chamber. The eluates were obtained before (baseline) and 15, 30, 45, 60 and 120 min after application of the adhesives and pipetted onto L-929 fibroblasts. Cytotoxicity of the materials was determined in relation to the baseline value using the MTT assay and statistical analysis was performed by ANOVA. After 15 min perfusion, test materials B-E applied on 1.0 mm and B and E applied on 1.5 mm dentin specimens exhibited cytotoxic potential. However, after 30 min perfusion none of the adhesives showed any toxicity. Cytotoxicity decreased with increasing thickness of the dentin slices and was lower for Adper Prompt-L-Pop compared to adhesives B-E. Self-etching adhesive systems might exhibit cytotoxic potential when applied on dentin of less than 1.5 mm thickness. However, cytotoxicity of the materials decreased with increasing dentin thickness and increasing duration of perfusion.

Acid Etching, Dental↗
PubMed2005

Isolation of Acanthamoeba sp. from conjunctival sac of healthy individuals using swab.

Is Acanthamoeba sp. normally found in the eyes? A study was carried out to establish the possibility of Acanthamoeba sp. as a part of the normal conjunctival flora. Conjunctiva swabbing were carried out in 286 healthy Orang Asli school children using sterile cotton swab. The swab was then inoculated onto non-nutrient agar (NN-A). Heat killed Escherichia coli that was used as food source for the growth of the amoebae was pipetted onto and away from the smear. The plates were incubated at 30 degrees C and examined daily using an inverted microscope for 14 days. Morphology of the trophozoites and cysts of the amoebae were used as the taxonomic criteria for identification. Positive-controls and negative-controls were done to check for the consistency of the technique used and monitoring of contamination respectively. None of the conjunctiva swab cultured was positive for Acanthamoeba sp. This finding may indicate that Acanthamoeba sp. is not part of normal conjunctival flora or conjunctiva swab is an insensitive technique to isolate the organism. However, a more extensive research is needed to investigate these possibilities.

Acanthamoeba↗
PubMed1991

Application of robotics and image processing to automated colony picking and arraying.

We describe a system that applies image processing and robotic techniques to automatically pick individual colonies from square petri dishes and array them in 96-well microtiter plates. Digital images of the colony distribution in the dishes are acquired using a video camera and frame buffer. Commercial image processing software is used to identify individual colonies and determine their locations. A Hewlett-Packard Microassay System robot reads the resulting coordinate file for each dish, picks cells from each identified colony and transfers the cells into a microtiter plate well. A disposable pipet tip is used as the sterile implement for colony picking. Custom holders position the dishes accurately and provide common coordinate systems for imaging and picking. The system is calibrated to account for the depth of agar in the dishes. The robot can process up to 10 dishes and 20 plates (1920 colonies) in a single run. It has successfully arrayed a cosmid library of the S. pombe genome consisting of approximately 6000 colonies in 30 petri dishes in about 40 hours of robot time. Future enhancements to the system are discussed.

Cells, Cultured↗