Illuminated microelectrode for tissue culture

Conventional glass microelectrodes acting as light guides have been used to facilitate the accurate approach and penetration of selected cells maintained in vitro. The device is a simple, inexpensive modification of conventional microelectrode holders and facilitates electrophysiological and microinjection studies of cells in vitro.     

On microelectrode ionophoresis

An equation for ionophoresis in large tip microelectrodes is derived from Nernst-Planck equations for the general case of a completely dissociated electrolyte. The relation between the release of ions and the applied electric current is mainly determined by two parameters: the transference number of the ions under consideration and the diffusional leak of the microelectrode. Also it is shown how the release of ions is affected by the concentration of the electrolyte within the electrode and that of the external solution. The equation describes the ionophoretic release of polyvalent spermine. In addition, new equations for tip potential and for tip resistance are derived.     

Microelectrode measurement of oxygen tensions in collagen-hepatocyte gels

Summary Microelectrodes have been used for the measurement of oxygen tension in various biological systems.(Silver, 1987) Although not previously reported, microelectrodes allow the direct measurement of oxygen tension profiles within collagen gels containing entrapped hepatocytes (collagcn-hepatocyte gels). These oxygen tension profiles, along with hepatocyte oxygen consumption data, allowed the estimation of a diffusion coefficient for oxygen in collagen-hepatocyte gels, D _g = 2.99 × 10^–5 cm²/s.     

The effects of varying oxygen tensions on organ cultures

Summary 1. Human embryonic organ slices survived better in organ culture when the oxygen tension was relatively low. Necrosis, presumably due to oxygen poisoning was seen at an oxygen pressure of 2.0 atmospheres.2. Slices of organs from adult laboratory animals varied in their reponse to high oxygen pressuresin vitro.3. In some organs, e. g. liver and kidney, considerably thicker slices survived in culture at pressures of 2.0 and 3.0 atmospheres than at 1.0 atmosphere.4. Some tissues, zona fasciculata of the adrenal cortex and brown fat, which do not survive at 1.0 atmosphere were found to do well at raised pressures of oxygen.5. Other tissues, notably the adrenal medulla, survived well at 1.0 atmosphere, but were destroyed by high oxygen pressure.6. Rat liver slices, cultivated under high oxygen pressure maintained a reasonably normal histological appearance for 10–14 days, but rapidly lost their glycogen content. Their oxygen consumption remained essentially the same for at least 6 days. Attempts to prolong the survival of liver slices by the use of feeder cultures and by extracts of liver were unsuccessful.7. Glucuronide synthesis rapidly fell off in slices of mouse liver maintained at 3.0 atmospheres. A similar result was obtained with guinea-pig kidney, but in this case, the addition of extra glucose to the medium had a marked effect in delaying the fall.

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Die Wirkungen verschiedener Sauerstoffspannungen auf Organkulturen

Kurzfassung Die Sauerstoffbedürfnisse embryonaler und adulter Organe sind recht unterschiedlich. Erstere werden durch hohe Sauerstoffspannungen getötet, letztere überleben vielfach nicht bei niedrigen Spannungen. Im günstigsten Falle können nur dünne Scheiben adulter Organe lebend erhalten werden, selbst wenn die Luft im Kulturgefäß durch Sauerstoff ersetzt wird. Organkulturen wurden daher in einer Druckkammer durchgeführt unter Sauerstoffpartialdrucken zwischen 0,2 und 4,0 Atmosphären (absolut). Die höheren Drücke begünstigten das Überleben einiger Organe von adulter Ratte und Maus, z. B. Nebennierenrinde, Leber und braunes Fett, welche bis zu Drücken von 1,0 Atmosphären nicht gut gedeihen. Andererseits überlebten embryonale Organe und bestimmte adulte Organe — wie etwa Nebennierenmark — besser bei relativ niedrigen Sauerstoffdrücken und wurden durch höhere Drücke stark geschädigt. Im Falle der adulten Leber wurden QO₂-Bestimmungen und Studien der bei der Glucuronid-Synthese beteiligten Enzyme benutzt, um die herkömmlichen histologischen Kriterien für das Überlebensvermögen nach mehreren Kulturtagen zu ergänzen. Es wird die Schlußfolgerung gezogen, daß die Anwendung hoher Sauerstoffdrücke von beträchtlichem Wert sein könnte für Arbeiten mit Organkulturen.

     

Effects of elevated oxygen tensions on acetylene reduction in Alnus incana-Frankia symbioses

An open flow-through gas system was used to determine the effect of C₂H₂ and elevated O₂ on acetylene reduction activity (ARA) and respiration of the intact, potted root system of Alnus incana (L.) Moench in symbiosis with Frankia Avcll or with a local source of Frankia . Both symbiotic systems responded to C₂H₂ by an immediate plateau range in ARA. The Plateau in ARA was in some cases followed by a decline of less extent than reported for many legumes. A concurrent decline in net respiration of the root system was on average 8% of the CO₂ efflux prior to C₂H₂ introduction.
Respiration of the root systems in both symbioses responded to elevated oxygen levels in the 10 kPa C₂H₂ atmosphere by an increase of up to 17% of the net respiration prior to C₂H₂ introduction in 21 kPa O₂. In contrast, the elevated oxygen levels resulted in an immediate drop in ARA followed by a minor increase to a stable level lower than that at the preceding, lower oxygen tension. The symbiosis with the local Frankia had lost all ARA when the partial pressure of O₂ exceeded 50 kPa, whereas the symbiosis with Avcll still had some activity at 80 kPa O₂. This difference in tolerance of elevated O₂ clearly shows that the oxygen exclusion mechanisms may be controlled by the microsymbiont in Alnus-Frankia symbioses. The symbiotic systems recovered ARA to a similar extent when returned from elevated O₂ levels to 21 kPa O₂.     

Metabolic consequences of agonistic behaviour: crab fights in declining oxygen tensions

The energetic consequences of fighting, which may depend on environmental conditions, can be an important factor shaping contest strategy and duration. Energy expenditure may be costly to fitness because it depletes reserves that could otherwise have been allocated to reproduction, and metabolites are produced that may constrain subsequent activities. We examined the variation in the metabolic consequences of fighting in relation to hypoxia. Contests were staged between pairs of size-matched male shore crabs Carcinus maenas L., under a range of water oxygen tensions (between 10 and 100% oxygen saturation) which crabs experience in their natural habitat. Fighting under normoxic and hypoxic conditions resulted in significantly elevated concentrations of haemolymph metabolites (L-lactate and glucose) compared with crabs at rest. However, these concentrations were much lower than in crabs that had been walking on a treadmill. Glycogen concentrations differed only under hypoxic conditions: glycogen stores were reduced in crabs after fighting and this reduction was similar to that after exercise on a treadmill. Contests were shorter when they were staged below a water P o2of 6.7 kPa ( approximately 30% normoxia). As water oxygen tensions were reduced, fighting crabs had greater concentrations of L-lactate and glucose in their blood and tissues whilst glycogen stores were reduced. Fights became shorter when crabs were exposed to severe hypoxia (P o2=2 kPa) for increasing lengths of time, and blood L-lactate concentrations increased. The results suggest that as fights progressed, crabs experienced an increasing metabolic debt, in the form of accumulation of L-lactate and a reduction in energy stores, which was amplified by hypoxic conditions. Copyright 1999 The Association for the Study of Animal Behaviour.     

Manipulation of oxygen tensions for in vitro cell culture using a hypoxic workstation

It is increasingly clear that oxygen tension exerts potent effects on many biologic processes in a range well above that at which aerobic metabolism is compromised. Cell culture ex vivo is traditionally performed in unstirred liquid media at ambient oxygen concentrations in the laboratory, with no attention to the level of oxygen experienced by the cells. This is certainly not reflecting physiology, and oxygenation may be further altered during cell handling and extraction procedures. The hypoxia-inducible factor pathway illustrates the potential for oxygen tension to have dramatic effects in terms of post-translational modification of proteins, and to influence a broad range of cellular pathways including those involved in substrate transport, metabolic pathways, growth factor signaling and differentiation. While the standard laboratory approach may remain suitable for many biologic applications, there are other situations in which more attention to oxygenation will be appropriate. This review discusses a workstation that allows investigators to manipulate oxygenation.     

Manipulation of oxygen tensions for in vitro cell culture using a hypoxic workstation

It is increasingly clear that oxygen tension exerts potent effects on many biologic processes in a range well above that at which aerobic metabolism is compromised. Cell culture ex vivo is traditionally performed in unstirred liquid media at ambient oxygen concentrations in the laboratory, with no attention to the level of oxygen experienced by the cells. This is certainly not reflecting physiology, and oxygenation may be further altered during cell handling and extraction procedures. The hypoxia-inducible factor pathway illustrates the potential for oxygen tension to have dramatic effects in terms of post-translational modification of proteins, and to influence a broad range of cellular pathways including those involved in substrate transport, metabolic pathways, growth factor signaling and differentiation. While the standard laboratory approach may remain suitable for many biologic applications, there are other situations in which more attention to oxygenation will be appropriate. This review discusses a workstation that allows investigators to manipulate oxygenation.     

Degradation of 2,5-dichlorobenzoic acid byPseudomonas aeruginosa JB2 at low oxygen tensions

From long-term chemostat experiments, variants ofPseudomonas aeruginosa JB2 were obtained which exhibited altered properties with respect to the metabolism of 2,5-dichlorobenzoic acid (2,5-DBA). Thus, unlike the original strain JB2-WT, strain JB2-var1 is able to grow in continuous culture on 2,5-DBA as the sole limiting carbon and energy source. Yet, at a dilution rate of 0.07 h^–1 and a dissolved oxygen concentration of 12 µM, even with this strain no steady states with 2,5-DBA alone could be established in continuous cultures. Yet another strain was obtained after prolonged continuous growth of JB2-var1 in the chemostat. It has improved 2,5-DBA degrading capabilities which become apparent only during growth in continuous culture: a lower apparent K _m for 2,5-DBA and lowered steady-state residual concentrations of 2,5 DBA. Although with this strain steady states were obtained at oxygen concentrations as low as 11 µM, at further lowered concentrations this was no longer possible. In C-limited continuous cultures of JB2-var1 or JB2-var2, addition of benzoic acid (BA) to the feed reduced the amounts of 2,5-DBA degraded, which was most apparent at low oxygen concentrations (< 30 µM). At higher dissolved oxygen concentrations the addition of BA resulted in increasing cell-densities but did not affect the residual steady state concentration of 2,5-DBA. Indeed, whole cell suspensions from chemostat cultures grown on BA plus 2,5-DBA did show a lower apparent affinity for 2,5-DBA than those from cultures grown on 2,5-DBA alone. These results indicate that in environments with low oxygen concentrations and alternative, more easily degradable, substrates the degradation rates of chloroaromatic compounds by aerobic organisms may be negatively affected.Abbreviations BA benzoic acid - 2,5-DBA 2,5-dichlorobenzoic acid - QO ₂ ^max maximum specific respiration rate     

In situ oxygen microelectrode measurements of bottom-ice algal production in McMurdo Sound, Antarctica

In-situ estimates of fast-ice algal productivity at Cape Evans, McMurdo Sound, in 1999 were lower than at the same site in previous years. Under-ice irradiance was between 0 and 8 µmol photons m^-2 s^-1; the ice was between 1.9 and 2.0 m thick and the algal biomass averaged 150 mg chl a m^-2, although values as high as 378 mg chl a m^-2 were recorded. Production on 11 and 12 November was between 0.053 and 1.474 mg C m^-2 h^-1. When the data from 11 November were fitted to a hyperbolic tangent function, a multilinear regression gave estimates for P_max of 0.571 nmol O₂ cm^-2 s^-1, an ! of 0.167 nmol O₂ cm^-2 s^-1 µmol^-1 photons m^-2 s^-1 and an E_k of 3.419 µmol photons m^-2 s^-1. A P_max of 2.674 nmol O₂ cm^-2 s^-1, an ! of 0.275 nmol O₂ cm^-2 s^-1 µmol^-1 photons m^-2 s^-1, r of 0.305 nmol O₂ cm^-2 s^-1 and an E_k of 9.724 µmol^-1 photons m^-2 s^-1 were estimated from the 12 November data. The sea-ice algal community was principally comprised of Nitzschia stellata, Entomoneis kjellmanii and Berkeleya adeliensis. Other taxa present included N. lecointei, Fragilariopsis spp., Navicula glaciei, Pleurosigma spp. and Amphora spp. Variations in the method for estimating the thickness of the diffusive boundary layer were not found to significantly affect the measurements of oxygen flux. However, the inability to accurately measure fine-scale variations in biomass is thought to contribute to the scatter of the P versus E data.     

Nitrogen-limited continuous culture ofRhodopseudomonas capsulata growing photosynthetically or heterotrophically under low oxygen tensions

A continuous culture apparatus is described, which allows cultivation of photosynthetic bacteria anaerobically in the light and semiaerobically in the dark at constant oxygen tensions. The growth-parameters 1. substrate concentration at half maximum growth rate (K _s) and 2. yield (Y) forRhodopseudomonas capsulata are calculated.An automatic control system of the oxygen partial pressure in the culture medium is elaborated. It is shown, that the discontinuous regulation with a control unit in connection with two magnetic valves, which give short pulses of either oxygen-free gas or gas mixed with oxygen, is an economic, practicable and reliable method.The yield coefficientY during growth limited by ammonium sulfate is variable due to the synthesis of nitrogen independent metabolites, such as poly--hydroxybutyrate. The storage of poly--hydroxybutyrate in continuous culture is a function of both the actual concentration of ammonia and of theC/N ratio. At very low growth rates (1/6 µ _m ) the poly--hydroxybutyrate content increases amounting to 33% of the dry weight.In semiaerobically dark grown cells (pO₂: 5 mm Hg) the protein and bacteriochlorophyll content increased definitely on dry weight basis with increasing growth rates. In contrast, in anaerobic light cultures only a small increase of the bacteriochlorophyll level but no change of the protein content per dry weight of cells was observed at increasing growth rates.