Bubble formation in crabs induced by limb motions after decompression

In vivo bubble formation was studied in the megalopal stage of the crab Pachygrapsus crassipes. The animals were equilibrated with elevated argon, nitrogen, or helium pressures then rapidly decompressed to atmospheric pressure. Voluntary motions induced bubble nucleation in leg joints after exposures to as low as 2 atm nitrogen (gauge pressure). Delays of several minutes sometimes passed between decompression and bubble formation. Mechanically stimulating the animals to move their legs increased this bubble formation, whereas immobilizing the legs before gas equilibration prevented it, even in animals decompressed from 150 atm nitrogen. We conclude that preformed nuclei are not responsible for bubbles developing in the legs of this animal. Instead, tribonucleation of bubbles apparently occurs as a result of limb motions at relatively low gas supersaturations.     

Effects of helium and other inert gases on Echinosphaerium nucleofilium (protozoa, heliozoa).

The effects of helium, nitrogen, argon and krypton on Echinosphaerium nucleofilum (Heliozoa) have been studied at partial pressures of 10-130 atm. Additional experiments have been carried out with hydrostatic pressure alone. Helium causes shortening of the axopods over the whole range of pressures, and damage to the cell body at pressures of 60-90 atm, both with a maximum at 80 atm. These effects cannot be explained in terms of hydrostatic pressure alone; a 'pressure reversal' effect may be operating, causing the peak at 80 atm. Nitrogen also causes both cell damage and axopod shortening, the severity increasing with increasing pressure. Argon and krypton cause cell damage but no shortening. The order of potency for cell damage is krypton greater than argon greater than nitrogen greater than helium. It is suggested that there may be tuo sites of action, possibly the microtubules (for axopod shortening) and the cell membrane (for cell damage). In appropriate mixtures of helium and argon, both the cell damage usually caused by argon, and the axopod shortening usually caused by helium, are prevented. Possible mechanisms include the effects of hydrostatic pressure on gas solubility coefficients, reversal of the effects of the gases by the increase in total pressure, and competition for sites of action.     

Intracellular Bubble Formation: Differences in Gas Supersaturation Tolerances Between Tetrahymena and Euglena1

Cells of Tetrahymena pyriformis, T. thermophila, and Euglena gracilis were saturated with nitrogen gas at pressures up to 300 atm and rapidly decompressed. Damage was assessed by measuring post-decompression cell fragmentation or viability. Occurrence of intracellular bubbles was determined by cinephotomicrography performed during the decompression or by direct observations afterwards. The extreme gas supersaturations induced led to intracellular bubble formation and rupture in cells of Tetrahymena that contained food vacuoles, but only with supersaturations of 175 atm or higher; 225 atm left few cells intact. Bubbles were never observed in cells of Euglena or in Tetrahymena cells freed of food vacuoles, even when they were decompressed from substantially higher nitrogen supersaturations. Cells of Euglena were most resistant and were unaffected by supersaturations up to 250 atm.     

Intracellular gas supersaturation tolerances of erythrocytes and resealed ghosts.

Intact mammalian, avian, and amphibian erythrocytes were saturated with up to 300 atm nitrogen or argon gas and rapidly decompressed. Despite the profuse nucleation of gas bubbles in the suspending fluid, no evidence of intracellular gas bubble nucleation was found; all or most of the cells remained intact and little or no hemoglobin escaped. Internal bubbles were similarly absent from resealed ghosts of human erythrocytes as shown by lack of disintegration and by retention of an entrapped fluorescent compound. The absence of bubbles may indicate that much of the internal water does not have the same nucleation properties as external water.     

Promotion of gas bubble formation by ingested nuclei in the ciliate, Tetrahymena pyriformis

Cells of the ciliate Tetrahymena pyriformis were suspended with carmine or graphite particles or with Halobacterium gas vesicles, all of which promote bubble formation in aqueous suspensions when tested with 10 atm and above (0.1-0.5 X 10(7) Pa) (carmine and graphite) or 25 atm and above (gas vesicles) of nitrogen supersaturations. All three particles were ingested, but only the gas vesicles promoted intracellular gas bubble formation if the cells containing them were nitrogen or methane saturated in a slow stepwise fashion prior to rapid decompression. Cell rupture did not occur until gas saturation pressures greater than 25 atm were used; this suggests that the ciliate pellicle and cytoplasm cannot resist the mechanical forces of an expanding gas phase induced by decompression from between 25 and 50 atm and thus provides an estimate of the physical strength of these cellular components. The inability of the ingested carmine, graphite, and collapsed gas vesicles to induce intracellular gas bubble formation suggests that the phagocytic process somehow altered them. This procedure may thus provide a tool for the study of early events in the digestive processes of ciliates.     

Rupture of the cell envelope by induced intracellular gas phase expansion in gas vacuolate bacteria.

Using a new approach, we estimated the physical strength of the cell envelopes of three species of gram-negative, gas vacuolate bacteria (Microcyclus aquaticus, Prosthecomicrobium pneumaticum, and Meniscus glaucopis). Populations of cells were slowly (0.5 to 2.9 h) saturated with argon, nitrogen, or helium to final pressures up to 100 atm (10, 132 kPa). The gas phases of the vesicles remained intact and, upon rapid (1 to 2 s) decompression to atmospheric pressure, expanded and ruptured the cells; loss of colony-forming units was used as an index of rupture. Because the cell envelope is the cellular component most likely to resist the expanding intracellular gas phase, its strength can be estimated from the minimum gas pressures that produce rupture. The viable counts indicated that these minimum pressures were between 25 and 50 atm; the majority of the cell envelopes were ruptured at pressures between 50 and 100 atm. Cells in which the gas vesicles were collapsed and the gas phases were effectively dissolved by rapid compression tolerated decompression from much higher gas saturations. Cells that do not normally possess gas vesicles (Escherichia coli) or that had been prevented from forming them by addition of L-lysine to the medium (M. aquaticus) were not harmed by decompression from gas saturation pressures up to 300 atm.     

Pressure and Temperature Effects on Growth and Methane Production of the Extreme Thermophile Methanococcus jannaschii

The marine archaebacterium Methanococcus jannaschii was studied at high temperatures and hyperbaric pressures of helium to investigate the effect of pressure on the behavior of a deep-sea thermophile. Methanogenesis and growth (as measured by protein production) at both 86 and 90°C were accelerated by pressure up to 750 atm (1 atm = 101.29kPa), but growth was not observed above 90°C at either 7.8 or 250 atm. However, growth and methanogenesis were uncoupled above 90°C, and the high-temperature limit for methanogenesis was increased by pressure. Substantial methane formation was evident at 98°C and 250 atm, whereas no methane formation was observed at 94°C and 7.8 atm. In contrast, when argon was substituted for helium as the pressurizing gas at 250 atm, no methane was produced at 86°C. Methanogenesis was also suppressed at 86°C and 250 atm when the culture was pressurized with a 4:1 mix of H₂ and CO₂, although limited methanogenesis did occur when the culture was pressurized with H₂.     

Promotion of gas bubble formation by ingested nuclei in the ciliate,Tetrahymena pyriformis

Cells of the ciliateTetrahymena pyriformis were suspended with carmine or graphite particles or with Halobacterium gas vesicles, all of which promote bubble formation in aqueous suspensions when tested with 10 atm and above (0.1−0.5×10⁷ Pa) (carmine and graphite) or 25 atm and above (gas vesicles) of nitrogen supersaturations. All three particles were ingested, but only the gas vesicles promoted intracellular gas bubble formation if the cells containing them were nitrogen or methane saturated in a slow stepwise fashion prior to rapid decompression. Cell rupture did not occur until gas saturation pressures greater than 25 atm were used; this suggests that the ciliate pellicle and cytoplasm cannot resist the mechanical forces of an expanding gas phase induced by decompression from between 25 and 50 atm and thus provides an estimate of the physical strength of these cellular components. The inability of the ingested carmine, graphite, and collapsed gas vesicles to induce intracellular gas bubble formation suggests that the phagocytic process somehow altered them. This procedure may thus provide a tool for the study of early events in the digestive processes of ciliates.     

Growth responses of Neurospora crassa to increased partial pressures of the noble gases and nitrogen

Buchheit, R. G. (Union Carbide Corp., Tonawanda, N.Y.), H. R. Schreiner, and G. F. Doebbler. Growth responses of Neurospora crassa to increased partial pressures of the noble gases and nitrogen. J. Bacteriol. 91:622-627. 1966.-Growth rate of the fungus Neurospora crassa depends in part on the nature of metabolically "inert gas" present in its environment. At high partial pressures, the noble gas elements (helium, neon, argon, krypton, and xenon) inhibit growth in the order: Xe > Kr> Ar > Ne > He. Nitrogen (N(2)) closely resembles He in inhibitory effectiveness. Partial pressures required for 50% inhibition of growth were: Xe (0.8 atm), Kr (1.6 atm), Ar (3.8 atm), Ne (35 atm), and He ( approximately 300 atm). With respect to inhibition of growth, the noble gases and N(2) differ qualitatively and quantitatively from the order of effectiveness found with other biological effects, i.e., narcosis, inhibition of insect development, depression of O(2)-dependent radiation sensitivity, and effects on tissue-slice glycolysis and respiration. Partial pressures giving 50% inhibition of N. crassa growth parallel various physical properties (i.e., solubilities, solubility ratios, etc.) of the noble gases. Linear correlation of 50% inhibition pressures to the polarizability and of the logarithm of pressure to the first and second ionization potentials suggests the involvement of weak intermolecular interactions or charge-transfer in the biological activity of the noble gases.     

Gas supersaturation tolerances in amoeboid cells before and after ingestion of bubble-promoting particles.

Macrophages and other cells are capable of ingesting a variety of solids from their external environment. When such phagocytic processes occur in animals, they can lead to phagocytosis from the respiratory or the digestive tract of particles containing minute air emobli that may serve as bubble nuclei upon exposure of the animal to conditions of gas supersaturation. To test whether this is possible, gas supersaturation tolerances were determined for murine macrophages and macrophage-like tumor cells, and for cells of the slime mold Dictyostelium discoideum, before and after phagocytosis of particles that were effective in inducing bubble formation in nitrogen-supersaturated aqueous suspensions. After phagocytosis, the ability of the particles to induce bubble formation was completely abolished. All three cell types essentially retained their normal high resistance to bubble formation; even nitrogen supersaturations in excess of 150 atm (1.55 x 10(7) Pa) did not lead to internal bubbles. Alterations of the particle surfaces and unique properties of the intracellular fluid appear to be the underlying cause of the extremely high gas supersaturation tolerances observed.     

Gas supersaturation tolerances in amoeboid cells before and after ingestion of bubble-promoting particles

Macrophages and other cells are capable of ingesting a variety of solids from their external environment. When such phagocytic processes occur in animals, they can lead to phagocytosis from the respiratory or the digestive tract of particles containing minute air emobli that may serve as bubble nuclei upon exposure of the animal to conditions of gas supersaturation. To test whether this is possible, gas supersaturation tolerances were determined for murine macro-phages and macrophage-like tumor cells, and for cells of the slime moldDictyostelium discoideum, before and after phagocytosis of particles that were effective in inducing bubble formation in nitrogensupersaturated aqueous suspensions. After phagocytosis, the ability of the particles to induce bubble formation was completely abolished. All three cell types essentially retained their normal high resistance to bubble formation; even nitrogen supersaturations in excess of 150 atm (1.55 × 10⁷ Pa) did not lead to internal bubbles. Alterations of the particle surfaces and unique properties of the intracellular fluid appear to be the underlying cause of the extremely high gas supersaturation tolerances observed.     

Rapid compressions in a captive bubble apparatus are isothermal.

Captive bubbles are commonly used to determine how interfacial films of pulmonary surfactant respond to changes in surface area, achieved by varying hydrostatic pressure. Although assumed to be isothermal, the gas phase temperature (Tg) would increase by >100 degrees C during compression from 1 to 3 atm if the process were adiabatic. To determine the actual change in temperature, we monitored pressure (P) and volume (V) during compressions lasting <1 s for bubbles with and without interfacial films and used P x V to evaluate Tg. P x V fell during and after the rapid compressions, consistent with reductions in n, the moles of gas phase molecules, because of increasing solubility in the subphase at higher P. As expected for a process with first-order kinetics, during 1 h after the rapid compression P x V decreased along a simple exponential curve. The temporal variation of n moles of gas was determined from P x V >10 min after the compression when the two phases should be isothermal. Back extrapolation of n then allowed calculation of Tg from P x V immediately after the compression. Our results indicate that for bubbles with or without interfacial films compressed to >3 atm within 1 s, the change in Tg is <2 degrees C.     

The secretion of inert gas into the swim-bladder of fish

The composition of the gas mixture secreted into the swim-bladders of several species of fish has been determined in the mass spectrometer. The secreted gas differed greatly from the gas mixture breathed by the fish in the relative proportions of the chemically inert gases, argon, neon, helium, and nitrogen. Relative to nitrogen the proportion of the very soluble argon was increased and the proportions of the much less soluble neon and helium decreased. The composition of the secreted gas approaches the composition of the gas mixture dissolved in the tissue fluid. A theory of inert gas secretion is proposed. It is suggested that oxygen gas is actively secreted and evolved in the form of minute bubbles, that inert gases diffuse into these bubbles, and that the bubbles are passed into the swim-bladder carrying with them inert gases. Coupled to a preferential reabsorption of oxygen from the swim-bladder this mechanism can achieve high tensions of inert gas in the swim-bladder. The accumulation of nearly pure nitrogen in the swim-bladder of goldfish (Carassius auratus) is accomplished by the secretion of an oxygen-rich gas mixture followed by the reabsorption of oxygen.     

THE RELATION OF EXERCISE TO BUBBLE FORMATION IN ANIMALS DECOMPRESSED TO SEA LEVEL FROM HIGH BAROMETRIC PRESSURES

1. Bullfrogs (Rana catesbiana) and rats have been subjected to high barometric pressures and studied for bubble formation on subsequent decompression to sea level. Pressures varying from 3 to 60 pounds per square inch, in excess of atmospheric pressure, were used. 2. Muscular activity after decompression is necessary for bubble formation in bullfrogs after pressure treatment throughout the above range. Anesthetized frogs remained bubble-free following decompression. Rats compressed at 15 to 45 pounds per square inch likewise did not contain bubbles unless exercised on return to sea level. 3. Bubbles form without voluntary muscular activity in anesthetized rats previously subjected to pressure of 60 pounds per square inch. Small movements involved in breathing and other vital activities are believed sufficient to initiate bubbles in the presence of very high supersaturations of N₂. 4. Bubbles appear (with exercise) in rats previously compressed at 15 pounds per square inch, and in bullfrogs subjected to pressure at levels as low as 3 pounds per square inch above atmospheric pressure. The percentage drop in pressure necessary for bubble formation is less in compressed animals than in those decompressed from sea level to simulated altitudes. 5. The action of exercise on bubble formation in compressed frogs and rats is attributed to mechanical factors associated with muscular activity, combined with the high supersaturation of N₂. CO₂ probably is not greatly involved, since its concentration does not reach supersatuation, as it does at high altitude. 6. Anoxia following decompression from high barometric pressures has no observable facilitating effect on bubble formation.     

Development of the brine shrimp Artemia is accelerated during spaceflight

Developmentally arrested brine shrimp cysts have been reactivated during orbital spaceflight on two different Space Shuttle missions (STS-50 and STS-54), and their subsequent development has been compared with that of simultaneously reactivated ground controls. Flight and control brine shrimp do not significantly differ with respect to hatching rates or larval morphology at the scanning and transmission EM levels. A small percentage of the flight larvae had defective nauplier eye development, but the observation was not statistically significant. However, in three different experiments on two different flights, involving a total of 232 larvae that developed in space, a highly significant difference in degree of flight to control development was found. By as early as 2.25 days after reactivation of development, spaceflight brine shrimp were accelerated, by a full instar, over ground control brine shrimp. Although developing more rapidly, flight shrimp grew as long as control shrimp at each developmental instar or stage.     

The effect of helium and of hydrogen at high pressure on the cell division of Tetrahymena pyriformis W.

The rate of cell division of Tetrahymena growing in an observational high pressure vessel was measured at selected pressures of helium, hydrogen and at high hydrostatic pressure. Pressures greater than 100 atm reduced the rate of division, but the gases inhibited division to a lesser degree than pure hydrostatic pressure. Hydrogen's effect was distinguishable from that of hydrostatic pressure at 130 atm or more, while helium's effect appeared at 175 atm. These inert gases probably counteract the action of pressure by stabilising apolar pressure-labile targets.     

Effect of ventilation with soluble and diffusible gases on the size of air emboli

Pulmonary hypertension resulting from venous air embolism is known to increase after ventilation with highly soluble and diffusible gases. Exacerbation of the hypertension could be due to further blockage of the circulation if the bubbles enlarge as a result of ingress of gas by diffusion. This mechanism has been frequently cited but lacks direct proof. To determine directly whether intravascular air bubbles actually enlarge when highly soluble and diffusible gases are inspired, we used microscopy to measure the size of gas emboli in vivo. When air bubbles were injected into the right atrium, the bubbles that appeared in pulmonary arterioles were larger during ventilation with helium or nitrous oxide than with air. Air bubbles injected into the pulmonary artery enlarged when the inspired gas was changed to helium or nitrous oxide. The direction, magnitude, and timing of changes in bubble size were consistent with a net diffusion of gas into the bubbles. These data support the idea that venous air emboli enlarge during ventilation with soluble and diffusible gases and thereby cause further vascular obstruction.     

Effects of Hyperbaric Pressure on a Deep-Sea Archaebacterium in Stainless Steel and Glass-Lined Vessels

The effects of hyperbaric helium pressures on the growth and metabolism of the deep-sea isolate ES4 were investigated. In a stainless steel reactor, cell growth was completely inhibited but metabolic gas production was observed. From 85 to 100°C, CO₂ production proceeded two to three times faster at 500 atm (1 atm = 101.29 kPa) than at 8 atm. At 105°C, no CO₂ was produced until the pressure was increased to 500 atm. Hydrogen and H₂S were also produced biotically but were not quantifiable at pressures above 8 atm because of the high concentration of helium. In a glass-lined vessel, growth occurred but the growth rate was not accelerated by pressure. In most cases at temperatures below 100°C, the growth rate was lower at elevated pressures; at 100°C, the growth rates at 8, 250, and 500 atm were nearly identical. Unlike in the stainless steel vessel, CO₂ production was exponential during growth and continued for only a short time after growth. In addition, relatively little H₂ was produced in the glass-lined vessel, and there was no growth or gas production at 105°C at any pressure. The behavior of ES4 as a function of temperature and pressure was thus very sensitive to the experimental conditions.     

Growth and development of Atlantic mud crab larvae fed natural Zooplankton prey

Atlantic mud crab larvae (Panopeus herbstii H. Milne Edwards) were fed either brine-shrimp nauplii or natural Zooplankton in the laboratory and in large, field-deployed enclosures. Larvae developed fastest in 1440-1, field-deployed enclosures. By 9 days post hatching, more than 90% of the enclosure larvae had reached zoea Stage IV compared to only 50% of the larvae that were fed brine shrimp in small bowls. Larvae fed natural Zooplankton in either 40-1 tanks developed more slowly and were still in zoea Stage II after 9 days. Larvae that were fed natural Zooplankton in small bowls lost weight during the course of the experiment. Even though larvae developed most rapidly in field-deployed enclosures, there was no significant difference between the rate of dry-weight growth of larvae fed brine shrimp in 50-ml bowls and larvae fed natural zooplankton in enclosures. The greater stage-specific dry weight of larvae fed brine shrimp may have been related to the relative energy content of the two diets, while differences in the rates of development may have been related to the respective spatial scales of the different culture techniques. Diel cycles in temperature within the enclosures may have also had an effect on the rate of development.     

A new gas lesion syndrome in man, induced by "isobaric gas counterdiffusion".

Normal men have been found to develop pruritis and gas bubble lesions in the skin, and disruption of vestibular function, when breathing nitrogen or neon with oxygen while surrounded by helium at increased ambient pressure. This phenomenon, which occurs at stable ambient pressures, at 1 or many ATA, has been designated the "isobaric gas counterdiffusion syndrome." In a series of analyses and experiments in vivo and in vitro the cause of the syndrome has been established as due to gas accumulation and development of gas bubbles in tissues as a result of differences in selective diffusivities, for various respired and ambient gases, in the tissue substances between capillary blood and the surrounding atmosphere. The phenomenon here described in man is an initial stage of a process shown later in animals to progress to continuous, massive, lethal, intravascular gas embolization.