Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 μl of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.     

Anaerobic column chromatography in the presence of detergents and its application to a bacterial HCN-producing enzyme

An easy method to deoxygenize protein or detergent containing buffer solutions by stirring and sparge flushing with an inert gas is described. The deoxygenation of buffers and the establishment of anaerobic conditions in a column chromatography system was followed continuously with an oxygen electrode at the outlet. All polyethylene tubes were placed inside stainless steel tubes and the interspace flushed with nitrogen. A peristalic pump with silicone rubber tubing, mounted in a small plastic box, was likewise flushed with nitrogen. By this method a very unstable cyanide-producing enzyme preparation, solubilized with Triton X-100 from a Pseudomonas species, could be fractionated on a phenyl Sepharose CL-4B column without loss of activity.     

Errors in measuring water potentials of small samples resulting from water adsorption by thermocouple psychrometer chambers

The adsorption of water by thermocouple psychrometer assemblies is known to cause errors in the determination of water potential. Experiments were conducted to evaluate the effect of sample size and psychrometer chamber volume on measured water potentials of leaf discs, leaf segments, and sodium chloride solutions. Reasonable agreement was found between soybean (Glycine max L. Merr.) leaf water potentials measured on 5-millimeter radius leaf discs and large leaf segments. Results indicated that while errors due to adsorption may be significant when using small volumes of tissue, if sufficient tissue is used the errors are negligible. Because of the relationship between water potential and volume in plant tissue, the errors due to adsorption were larger with turgid tissue. Large psychrometers which were sealed into the sample chamber with latex tubing appeared to adsorb more water than those sealed with flexible plastic tubing. Estimates are provided of the amounts of water adsorbed by two different psychrometer assemblies and the amount of tissue sufficient for accurate measurements of leaf water potential with these assemblies. It is also demonstrated that water adsorption problems may have generated low water potential values which in prior studies have been attributed to large cut surface area to volume ratios.     

A plant cell bioreactor with medium-perfusion for control of somatic embryogenesis in liquid cell suspensions

The Braun Biostat BF2 bioreactor system employs a novel aeration and agitation system, designed to enhance gaseous exchange and reduce shear stresses on submerged cell suspension cultures. The Biostat BF2 bioreactor employs a central pivoting spindle, around which the aeration tubing is wound forming a large paddle-type structure suspended from the top-plate and swung in a circle by a solid-state magnetic stirrer.The aeration tubing is a polypropylene capillary membrane, which has a unique microporous structure and is ideal for aeration, permitting two-way, bubble-free, gaseous exchange of the medium. This tubing can be rendered porous and can be used in the perfusion of aqueous solutions, enabling cell-free media exchange to be conducted. Thin-walled silicone rubber tubing, although gas permeable to a degree, cannot be made porous to aqueous solutions.The bioreactor was inoculated with a suspension culture of Sitka spruce (Picea sitchensis [Bong.] Carr.) known to be embryogenic and capable of maturing to plantlets on solidified medium. The perfusion capability of the bioreactor was employed to replace the inital proliferation medium with maturation medium in order to induce the development of the somatic embryos in submerged cell culture. The size ratio of the somatic embryo heads was monitored over 7 weeks. This cell line was found to mirror just the initial elongation, previously observed in shake-flask culture.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - SSPM Selby Sitka proliferation medium - SSMM Selby Sitka maturation medium The following was presented at the NERC TBLG '95 Meeting as the Bioreactor Workshop     

Toxicity of Tissue Culture Media exposed to Polyvinyl Chloride Plastic

IT has become clear that some plastics contaminate materials or fluids with toxic substances^1–4,8. I report here evidence that serum-containing solutions perfused through polyvinyl chloride administration tubing, of the type used routinely for blood transfusions, become highly toxic to heart cells isolated in tissue culture.     

Ethane exhalation as an index of in vivo lipid peroxidation: Concentrating ethane from a breath collection chamber

A simple method is described for concentrating ethane from large volumes of air; the sensitivity limits for detecting exhaled ethane are increased by 100-fold or more. The method is intended for monitoring low-level ethane exhalation resulting from peroxidative damage to tissue lipids in vivo. Various adsorbents for concentrating ethane and ethylene were tested; in addition, the permeability characteristics of various types of tubing were studied in order to construct a suitable, inexpensive breath collection chamber for laboratory animals. Recommended adsorbents are activated charcoal and molecular sleve; activated alumina is a poor adsorbent. Polyvinyl and Teflon tubing are impermeable, whereas latex and silicone tubing are permeable to ethane. When ethane and ethylene are injected intraperitoneally into mice, the exhalation of these volatile hydrocarbons is readily monitored in the breath collection chamber. Whereas ethylene and the C₃–C₅ alkanes probably are metabolized by mice, ethane apparently is not; however, a portion of the ethane appears to be trapped by absorption within body tissues.     

Improved immunomagnetic enrichment of CD34(+) cells from umbilical cord blood using the CliniMACS cell separation system

Background aimsCD34+ enrichment from cord blood units (CBU) is used increasingly in clinical applications involving ex vivo expansion. The CliniMACS instrument from Miltenyi Biotec is a current good manufacturing practice (cGMP) immunomagnetic selection system primarily designed for processing larger numbers of cells: a standard tubing set (TS) can process a maximum of 60 billion cells, while the larger capacity tubing set (LS) will handle 120 billion cells. In comparison, most CBU contain only 1–2 billion cells, raising a question regarding the optimal tubing set for CBU CD34+ enrichment. We compared CD34+ cell recovery and overall viability after CliniMACS processing of fresh CBU with either TS or LS.MethodsForty-six freshly collected CBU (≤ 36 h) were processed for CD34+ enrichment; 22 consecutive units were selected using TS and a subsequent 24 processed with LS. Cell counts and immunophenotyping were performed pre- and post-selection to assess total nucleated cells (TNC), viability and CD34+ cell content.ResultsTwo-sample t-tests of mean CD34+ recovery and viability revealed significant differences in favor of LS (CD34+ recovery, LS = 56%, TS = 45%, P = 0.003; viability, LS = 74%, TS = 59%, P = 0.011). Stepwise linear regression, considering pre-processing unit age, viability, TNC and CD34+ purity, demonstrated statistically significant correlations only with the tubing set used and age of unit.ConclusionsFor CD34+ enrichment from fresh CBU, LS provided higher post-selection viability and more efficient recovery. In this case, a lower maximum TNC specification of TS was not predictive of better performance. The same may hold for smaller scale enrichment of other cell types with the CliniMACS instrument.     

Mass production of Beauveria bassiana for regulation of Leptinotarsa decemlineata populations

A simple liquid medium which enhanced the production of conidiospores by an isolate of the entomophagous fungus Beauveria bassiana is described. Spore production was attained using cultures floating in inflated sections of plastic tubing (“polyethylene cushions”) and glass bottles. A method is described for determining lethal doses and lethal times for second- and third-instar Leptinotarsa decemlineata larvae.     

Axenic storage of small volumes of Tetrahymena cultures under liquid nitrogen: A miniaturized procedure.

A method for freezing and storing samples of Tetrahymena thermophila in short lengths of Teflon tubing is described. Survival rates of the organism frozen with this technique are sufficient to permit routine storage of many strains in one quarter of the LN₂ refrigerator space heretofore required.     

Effects of estradiol-17β on cholesterol metabolism in the rat: A study using a deuterium label and mass spectrometry

The effect of estradiol-17β (E₂) on several important aspects of cholesterol metabolism were examined in the rat. Ovariectomized rats were implanted subcutaneously with 1 or 4 cm. of silastic tubing packed with E2, and were also given 2% D₂O in their drinking water. The E2 diffused slowly out of the implants and the two different lengths of tubing resulted in constant E2 blood concentrations of either high (4.0 cm) or physiological (1.0 cm) levels. By measuring the rate of incorporation of deuterium into plasma cholesterol by mass spectrometry over a period of 42 days, we determined the rate constant of cholesterol synthesis and cholesterol turnover time and rate under two E2 dosage conditions. E2 treatment did not affect the rate constant of cholesterol synthesis or the cholesterol turn-over time. However, cholesterol turnover rate (mg synthesized/day) showed a dose dependent reduction with increasing doses of E2. This may be secondarily caused by E2's suppression of both food Intake and subsequent weight gain; E2 treated animals are smaller and, therefore, synthesize less cholesterol per day. Additionally, E2 treated animals showed a rise in plasma cholesterol levels and in the fraction of labeled cholesterol appearing in the plasma.     

Immobilized electric eel acetylcholinesterasemii. II. Flow kinetics of acetylcholinesterase chemically attached to nylon tubing.

Acetylcholinesterase has been attached covalently to the inner surface of nylon tubing. An experimental study has been carried out on the flow kinetics; solutions of acetylthiocholine at various concentrations were passed through tubing at various flow rates, and measurements made of the rates of formation of product. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, four different methods of analysis being employed. It is found that at lower substrate concentrations and flow rates the reactions are largely diffusion controlled. The Km(app) values are substantially higher than the Km value for diffusion-free conditions, but approach it as the flow rate is increased, when the diffusion layer becomes less important. The results are entirely consistent with the Kobayaski-Laidler theory, and provide guidelines for the design of open tubular heterogeneous enzyme reactors, both for industrial and analytic purposes.     

A flow-through amperometric sensor based on dialysis tubing and free enzyme reactors.

A generic flow-through amperometric microenzyme sensor is described, which is based on semi-permeable dialysis tubing carrying the sample to be analyzed. This tubing (300 microm OD) is led through a small cavity, containing the working and reference electrode. By filling this cavity with a few microl of an appropriate enzyme solution, an amperometric enzyme sensor results. As the dialysis tubing is impermeable for large molecular species such as enzymes, this approach does not require any immobilization chemistry, and as a consequence the enzyme is present in its natural free form. Based on this principle, amperometric sensors for lactate, glucose, and glutamate were formed by filling cavities, precision machined in Perspex, with buffered solutions containing respectively, lactate-, glucose-, and glutamate-oxidase. All sensors showed a large linear range (0-35 mM for glucose, 0-3 mM for lactate, and 0-5 mM for glutamate) covering the complete physiological range. The lower detection limit was in the order of 15-50 microM. Applicability in flow injection analysis systems is demonstrated.     

Evaluation of silicone tubing toxicity using tobacco BY2 culture

Summary Silicone tubing is frequently used for gas exchange in cell culture systems, due to its biocompatibility and high permeability to CO₂ and O₂. In cell culture chambers, medium pH and oxygen levels are often maintained by gas exchange through a coil of silicone tubing. Culture medium is recirculated between the gas exchanger and the culture chamber which contains a suspension of cells. We report that the type of agent used for silicone vulcanization (peroxide or platinum) can markedly affect its biocompatibility, and that tobacco cell culture represents a particularly sensitive indicator of tubing cytotoxicity. Under the conditions studied (cell suspension maintained with forward-reverse flow and stirring), peroxide-cured silicone tubing was toxic to the tobacco BY2 cell culture, in contrast to the platinum-cured silicone tubing that was completely biocompatible. Upon further investigation by mass spectrometry, it was determined that a component with a molecular mass of 288 Da, possibly a tetrachlorinated biphenyl, was present in culture medium in contact with peroxide-cured tubing but not in medium in contact with platinum-cured tubing. Additional curing of peroxide-cured tubing resulted in cell morphology and viability comparable to controls. These data suggest that improperly cured silicone tubing can release catalytic byproducts which can be toxic to plant cells, and that the BY2 tobacco cells represent a suitable model system for studies of materials biocompatibility.     

The mechanics of pulling a glass micropipette.

The pulling of micropipette electrodes from glass tubing has been treated as a problem of viscous flow coupled with the Newtonian dynamics of the pulling apparatus. Analytical solutions are given from which the taper profile, tip diameter, and pulling time can be obtained. The physical principles of operation of micropipette pullers are discussed.     

Diethylpyrocarbonate—An effective agent for the sterilization of different types of nutrient media

A simple and convenient method has been tested for the steriltzation of nutrient media for long-term cultivation of plant cells. Diethylpyrocarbonate is suitable for this task in concentrations about 1000 mg l^-1 The cells cultivated for 15 subsequent passages on media treated by DPC had the same growth parameters, production pattern and ability to transform exogeneous organic compounds as did the controls. The method is suitable for the preparatian of both liquid and agar media, for stabilization of stock solutions and for sterilization of cultivation vessels and tubing.Abbreviations DPC diethylpyrocarbonate - medium MS nutrient medium according to Murashige and Skoog - NAA naphtaleneacetic acid     

The Effect of Equilibration Time and Tubing Material on Soil Gas Measurements

The collection of soil vapor samples representative of in-situ conditions presents challenges associated with the unavoidable disturbance of the subsurface and potential losses to the atmosphere. This article evaluates the effects of two variables that influence the concentration of volatile organic compounds in soil vapor samples: equilibration time and tubing material. The time for three types of soil vapor probes (i.e., macro-purge, mini-purge, and post-run tubing probes [PRT]) to equilibrate with subsurface conditions was assessed by installing probes and collecting multiple samples over a 72-hour period. The effect of tubing material was evaluated by collocating soil vapor probes constructed with different tubing material and collecting samples over several months. We recommend that soil vapor probes constructed with a sand filter-pack and bentonite seal (i.e., macro-purge probe) equilibrate for 24 to 48 hours prior to sample collection. Post-run tubing (PRT) probes equilibrated within one to two hours while a new probe design, (i.e., mini-purge probe) equilibrated and could be sampled after only 30 minutes for screening assessments. Nylaflow, Teflon®, polyetheretherketone (PEEK), and stainless-steel tubing had comparable trichloroethene (TCE) concentrations over all sampling time frames. We recommend that copper tubing be avoided and polyethylene only be used for screening assessments.     

Investigating movement in the laboratory: dispersal apparatus designs and the red flour beetle,Tribolium castaneum

The natural dispersal of Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) has been emulated in the laboratory for more than 50 years, using a simple dispersal apparatus. This has typically comprised of a starting container (initial resource or patch) connected by tubing, which contains thread for the animals to climb into a tube and hence to an end container. That is, beetles move to a new viable resource or patch from an inter‐patch zone or non‐viable habitat. We modified this basic apparatus design to test the effect of tubing length and tubing insertion angle on the dispersal rate and proportion of successful dispersers. We expected that the proportion of successful dispersers would be repeatable within each apparatus design, and that increasing tubing length and steepness of the insertion angle would reduce dispersal rate and success across apparatus designs. Dispersal increased linearly through time, similarly so for both males and females. The design with the most vertical tubing insertion angle had a lower proportion of successful dispersers. Tubing length also had a negative relationship with dispersal success (as judged by insects reaching the end container), but a significant reduction in dispersal success was only apparent between the shortest and longest tubing between containers. We suggest that locating and climbing the vertical section of string before they can enter the tubing between containers restricts dispersal and that at higher densities, insects exhibit greater inclination to climb. This type of apparatus has flexible design tolerances and further potential to study the dispersal of other small insect species that primarily use pedestrian locomotion.     

Preliminary investigations of preconcentration-capillary electrophoresis-mass spectrometry

Analyte preconcentration on-line with capillary electrophoresis-3-mass spectrometry (PPC-CE-MS) is described. Preconcentration cartridges were fabricated from PTFE tubing filled with ca. 1–2 mm bed of reversed-phase C₁₈ HPLC packing or polymeric reversed-phase beads. The particle size of the stationary phase was of larger dimension than the internal diameter of the CE capillary. Therefore, PC-CE capillaries were assembled without frit material and held together by friction. The wide applicability of on-line PC-CE-MS is demonstrated by the analysis of solutions containing peptides, proteins, and synthetic analogues of putative metabolites of the neuroleptic agent haloperidol.     

离心式机械单采血浆管路、贮存袋的研究

目的：研究单采血浆中两种耗材－管路和贮存袋，材料和方法：以压力监测器接头（DPM）、导管流量和贮存袋耐寒性的研究关键。并与国内外同类产品或材料作比较，结果：DPM必须具有适宜的透气性（感应时间≤2s）、阻血性（能耐受40Kpa,40s），滤除率（0.5um粒子，≥90％），导管流量的准确性（与机器设定值误差＜5％），稳定性（采集过程中流量误差＜5％）可通过控制导管内径（3.08-3.24mm)和选择弹性PVC材料来解决，贮存袋选用耐寒PVC料。扫描电镜证实韧性断裂。结论：研制成的管路和PCS^2机器适配能取代进口同类产品，研制的贮存袋能低温保存血浆，破损率小于2‰。     

Use of a silicone tubing sensor to control methanol concentration during fed batch fermentation of Pichia pastoris

A silicone tubing sensor controlled a constant methanol concentration in a fermenter up to 72 hours without the need for on-line gas chromatography or complex feeding schemes based on dissolved oxygen spikes. Methanol concentration was controlled up to 1.0% (v/v) with control around a given set point of ± 0.24%. The length of tubing, airflow through the tubing, pump speed and medium formulation had no effect on the control of methanol concentration.