APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1984, p. 771-7760099-2240/84/100771-06$02.00/0Copyright C 1984, American Society for Microbiology

Vol. 48, No. 4

High-Pressure-Temperature Gradient Instrument: Use forDetermining the Temperature and Pressure Limits of Bacterial

GrowthA. ARISTIDES YAYANOS,* R. VAN BOXTEL, AND ALLAN S. DIETZ

Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093

Received 17 January 1984/Accepted 12 July 1984

A pressurized temperature gradient instrument allowed a synoptic determination of the effects oftemperature and pressure on the reproduction of bacteria. The instrument consisted of eight pressure vesselshoused parallel to each other in an insulated aluminum block in which a linear temperature gradient was

supported. For a given experiment, eight pressures between 1 and 1,100 bars were chosen; the lineartemperature gradient was established over an interval within -20 to 100°C. Pure cultures and naturalpopulations were studied in liquid or solid medium either in short (ca. 2-cm) culture tubes or in long (76.2-cm)glass capillaries. In the case of a pure culture, experiments with the pressurized temperature gradientinstrument determined values of temperature and pressure that bounded its growth. Feasibility experimentswith mixed populations of bacteria from water samples from a shallow depth of the sea showed that theinstrument may be useful in identifying the extent to which a natural population is adapted to the temperaturesand pressures at the locale of origin of the sample. Additional conceived uses of the instrument includedsynoptic determinations of cell functions other than reproduction and of biochemical activities.

The bounds of the biosphere are in part determined byvalues of temperature and pressure that destroy or severelyinhibit life processes (1, 7, 8, 10). Yet no known individualspecies grows over the entire temperature and pressure (TP)domain within which life is found. Furthermore, pairs ofspecies exist within disjoint sets of TP values. For example,there is no point on the TP plane at which both Bacillusstearothermophilus (19a) and the deep-sea isolate MT-41(16, 17) reproduce. Thus, conditions of temperature andpressure create remarkable disparate habitats. Two prob-lems, particularly in the ecology of marine bacteria, can bebetter addressed with an improved knowledge of the effectsof temperature and pressure. First, to what extent cantemperature and pressure affect the biogeography of a spe-cies through lethal effects and by inhibiting reproduction?Second, can the bacteria in a sample from a given depth ofthe sea be at least partly separated into a class of trueinhabitants and a class of invaders on the basis of theirresponse to temperature and pressure? Answering thesequestions may also be relevant to the understanding of life inthe deep earth (10, 12) and possibly the upper atmosphere (7)and to the searching for life in other planetary environments(2, 6, 20).Temperature gradients provide a range of incubation tem-

peratures for the study of bacterial growth (3, 13, 14). Weherein describe a pressurized temperature gradient instru-ment (PITG) that allowed the study of microbial growth andreproduction and may possibly allow the study of othercellular and biochemical processes as a function of bothtemperature and pressure. The apparatus is compact, easy touse, sufficiently portable to encourage shipboard applica-tions, and may someday help to answer the above twoquestions.

* Corresponding author.

MATERIALS AND METHODS

PTG apparatus. The PTG is shown in Fig. 1. Eightpressure vessels were housed parallel to each other along thesame temperature gradient between the two separately ther-mostatted ends of the apparatus. The incubations werecarried out in these eight pressure vessels. Details formaking this apparatus were as follows.The apparatus consisted of the following components: (i)

an aluminum block with nine cylindrical holes (Fig. lc and2); (ii) pressure vessels in eight of the holes; (iii) a thermistorprobe in the center hole for checking the temperaturegradient; (iv) channels (Fig. 2), one in each end of thealuminum block, to allow the circulation of fluids thermo-statted at two different temperatures; (v) a manifold ofvalves to allow pressurization of the vessels; and (vi) thermalinsulation.The aluminum block (4 by 4 by 30 in. [ca. 10.16 by 10.16

by 76.2 cm]) was machined in two steps. First, nine parallelholes 0.794 cm in diameter were gun drilled through the 76.2-cm length of the block. The relative positioning of the nineholes in the aluminum block is shown in Fig. 2. The secondstep was to drill holes into each end of the aluminum block toform channels for the circulation of thermostatted fluid. Thedetails are shown in Fig. 2. The machined aluminum blockwas insulated with polystyrene foam 5.1 cm thick on all sidesand encased in a wooden box (Fig. 1).The pressure vessels were made from high-pressure tubing

(5/16-in. [ca. 0.794-cm] outer diameter.) of type 316 stainlesssteel, which has excellent resistance to corrosion. One endof each tube was threaded and capped with a fitting havingtwo female connections. One female connection was for thetube pressure vessel, and the other was for 1/16-in (ca. 0.159-cm) outer diameter high-pressure tubing. This fitting (EC inFig. lb) was made in our machine shop. A similar type ofconnection could have been made with a type 20F-9463

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772 YAYANOS, VAN BOXTEL, AND DIETZ

FIG. 1. Photographs of the PTG. Panel a shows the chilled end(CE) of the gradient and the heated end (HE). The thermostattedheater (TH) and the thermostatted cooler (TC) were mounted on theinsulated gradient. Also seen in panel are the valve manifold (VM)for pressurizing the vessels and the fitting (P) (19) for connecting thesystem to a high-pressure pump and gauge. Panel b shows the valvemanifold (VM), the polystyrene foam insulation (U), and the therm-istor probe (TM) for determining the temperature along the length ofthe aluminum block. The vessels were compressed through connec-tions in the end caps (EC). Panel c shows the end of the apparatusthrough which the samples were loaded. U is the polystyrene foaminsulation, AB is the aluminum block, TW the thermometer well,and PRPC the pin-retained piston closures (Fig. 3), which allowedeasy access to the pressure vessels. The small amount of spaceoccupied by these closures can be appreciated by a comparison withthe end caps (EC) on the opposite end of the pressure vessels(visible in panel b).

connector (Autoclave Engineers, Inc., Erie, Pa.) fitted witha type 15-21AF1HM4 adapter (High Pressure EquipmentCo., Erie, Pa.). Pressurization was done through the connec-tions on the capped ends via the manifold of valves (Fig. la).The other end of each high-pressure tube was machined toaccomodate a piston with an 0-ring seal and with a crosshole for a retaining pin (made of type 17-4 PH type stainlesssteel, which has a high shear strength). The pin-retainedpiston closures shown in Fig. lc and 3 allowed easy access tothe inside of the pressure vessels.The pressure in the vessels was determined with a Heise

bourdon tube gauge (Heise Bourdon Tube Co., Newtown,Conn.) accurate to +1.38 bars up to 1,380 bars. (1 atm =1.01325 bars = 1.01325 x 105 N/M2 = 0.101325 MPa). Thetemperature along the length of the temperature gradient wasdetermined with a model YS1709 thermistor probe andmodel 5650 digital thermometer (Markson Science, Phoenix,Ariz.) to an accuracy of ±0.10C. After the linearity of thegradient was established, the temperature of the gradient insubsequent experiments with the apparatus was determinedby reading only two temperatures, one with a YS1709 probe(at the cold end) and the other with a YS1703 probe (at thewarm end). The thermoregulated baths used were a Haake(Haake, Inc., Saddle Brook, N.J.) model FT (warm end) anda Neslab (Neslab Instruments, Inc., Portsmouth, N.H.)model RTE-8 refrigerated circulator (cold end).

Bacterial cultures and media. Cultures of the deep-seaisolate CNPT-3 (15) were maintained in type 2216 marinebroth (Difco Laboratories, Detroit, Mich.) at pH 7.0 and in anutrient medium solidified with silica gel (5) at 2 to 4°C and587 bars. Inoculations of culture media for maintenance ofthe deep-sea strain and for experiments with it were done atatmospheric pressure. Even though isolate CNPT-3 growsslowly at this pressure, manipulations were done close to0°C and were done as quickly as possible, usually within 15to 30 min. Being able to work with cultures under shortperiods of decompression is important if PTG instrumentsare to be useful with isolates from hadal regions (16).An artificial seawater was used to make gelatin gels

described below. The artificial seawater contained NaCl (24g), MgCl2 * 6H20 (5.3 g), MgSO4 * 7H20 (7 g), KNO3 (1 g),and KC1 (0.7 g) brought to a total volume of 1,000 ml withwater (Nanopure grade; Barnstead, Boston, Mass.).

Bacillus stearothermophilus was purchased from theAmerican Type Culture Collection (ATCC 7953). Cultureswere grown in Difco nutrient broth and in Difco nutrientagar. In some experiments glucose was added to the nutrient

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FIG. 2. Details of the two principal modifications made to thealuminum block block. Nine holes (0.794 cmn or 5/16 in.) were gundrilled at the locations shown through the entire 30-in. (76.2-cm)length of the block. Holes of 1/4 in. (0.64 cm) and 3/16 in. (0.48 cm)in diameter were drilled into each end of the aluminum block at thepositions shown. The drilled holes were capped with set screw plugsto form a channel. Thermostatted fluid was circulated through thechannel via the hose bibs.

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HIGH-PRESSURE-TEMPERATURE GRADIENT INSTRUMENT 773

media to a final concentration of 0.1%. Stock solutions ofglucose (50 g/liter) were prepared from reagent grade glucose(Mallinkrodt) and water (Nanopure grade) and then steril-ized by filtration through 0.22-,um pore size filters (MilliporeCorp., Bedford, Mass.).Growth of bacterial colonies in gels. Gels were inoculated

with bacteria and aspirated into glass tubes (7-mm outerdiameter, 76.2 cm long), the ends of which were pluggedwith rubber plungers from tuberculin syringes (Becton-Dickenson Co., Rutherford, N.J.). Care was taken to ex-clude air bubbles.Three types of gels were used: Difco marine agar and

Difco nutrient broth agar, nutrient silica gels (5), and nutrientgelatin gels (120 g of gelatin per liter of artificial seawatermixed with 2 parts of type 2216 marine broth (16). Only thesilica gels and gelatin gels have been found to be suitable forstudying the barophilic psychrophiles (15, 16).Determination of the number of bacterial colonies in gels.

The colonies in gels were visualized with a Wild M-8dissection microscope. (Wild, Heerbrugg, Switzerland). In-spection revealed the segment of a gel (and hence thetemperature range) over which colonies grew.Growth of bacteria in liquid culture along a temperature

gradient. Bacterial cultures were incubated until the latelogarithmic or early stationary phase of growth was reached.The cells were diluted with cold medium at 0°C and dis-pensed into small culture tubes (see below). None of thetubes used was entirely satisfactory because of the problemof hermetically sealing them. We used glass culture tubes(catalog no. 14958A; Fisher Scientific, Pittsburg, Pa.) andpolyethylene microcentrifuge tubes (catalog no. MC-2; ColeScientific, Calabasas, Calif.). The glass tubes were 6 by 50mm, and the MC-2 tubes (capped) were 31 mm long. Theglass tubes were capped with Parafilm. The MC-2 tubescame with plug caps, which formed a tight seal when firmlyinserted. Immediately after being filled, the tubes wereplaced into the pressure vessels and compressed. Thus, eachtube occupied a segment of the temperature gradient. Thepressure, drifting due to thermal equilibration, was adjusteduntil it stabilized. At the end of the incubation period, thecontents of each tube were mixed and assayed for theconcentration of bacterial cells with a Coulter particlecounter (Coulter Electronics, Inc., Hialeah, Fla.). The tem-

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perature of each tube was determined from its position alongthe gradient and taken to be that at the midpoint of the tube.

Determination of bacterial concentrations with a particlecounter. The concentration of cells in the liquid cultures wasdetermined with a model ZH Coulter counter with anaperture tube with a 30-,um-diameter orifice. Samples ofcultures were diluted with a counting solution (artificialseawater containing 0.05% sodium azide and 0.025% For-malin). Results obtained immediately after dilution into thecounting medium were identical to those obtained up to 20hours after dilution. Corrections for coincidence counting bythe Coulter counter were experimentally determined andapplied where necessary.

RESULTSCharacteristics of the temperature gradient. The linearity,

stability, and vessel-to-vessel comparability of the tempera-ture gradient were determined. The data in Fig. 4 show thatthe temperature gradient was linear. The stability of thegradient was a function of the quality of the thermostattedbaths. The following data were typical of the stabilityachieved in this study. Over a 10-day period one end of thegradient had a temperature of -4.17 (± 0.26)°C, and theother end had a temperature of 17.81 (± 0.13)°C where thevalues within parentheses are the standard deviations basedon 10 temperature determinations. Finally, the temperaturein the pressure vessels was compared with that measured inthe center hole. The mean difference for 46 comparisons was0.07°C, with a standard deviation of 0.07°C.Growth in liquid cultures. The data in Fig. 5 were obtained

after an incubation of liquid cultures of the deep-sea isolateCNPT-3 for 300 h. The initial titer at all temperatures was 5x 105 cells per ml in the experiments at 518 and 690 bars and7 x 106 cells per ml in the experiment at 587 bars. At 518bars, growth was absent at temperatures greater than 12.5 to13°C; at 587 bars, growth was absent at temperatures greaterthan 12 to 15°C; and at 690 bars, growth was absent attemperatures greater than 14 to 18°C. The upper temperaturelimit at which isolate CNPT-3 grew increased with increasingpressure if only the results from the experiments at 518 and690 bars are compared. This was corroborated by the resultsdescribed below.Growth of bacterial colonies in gels. The data in Fig. 6 show

the formation of colonies by the deep-sea isolate CNPT-3 ina gelatin-artificial seawater gel along a temperature gradientand at pressures between 1 and 1,050 bars. The data were

obtained with two separate incubations of the PTG. Theupper temperatures allowing formation of colonies at 518,

FIG. 3. Details of the pin-retained piston closure for the pressurevessels. The 0-ring and the groove dimensions allowed a piston-type seal inside of the high-pressure tube (0.794-cm outer diameter).

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774 YAYANOS, VAN BOXTEL, AND DIETZ

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FIG. 5. Results with liquid cultures in the PTG. A suspension ofthe deep-sea bacterial isolate CNPT-3 was prepared with type 2216marine broth and then dispensed into small culture tubes. Thesewere placed end to end in a pressure vessel of the PTG. After anincubation of 10 days at high pressures, the vessels were decom-pressed, the tubes were opened, and the concentrations of bacteriawere determined with a Coulter counter. Symbols: 0, 518 bars; 0,587 bars; 0, 690 bars.

587, and 690 bars (Fig. 6) were very close to the uppertemperatures allowing growth in liquid cultures at thosepressures (Fig. 5). Although the agreement between experi-ments done in liquid cultures and those done in gels ofgeletin was close, we believe that further work is needed tounderstand the problems of working in continuous gels alonga temperature gradient and to determine statistically if thereare any differences between data from gels and data fromliquid cultures.

Reproducibility of the TP boundary values determined ingels. The measurement of pressure presented no difficultyand was easily achievable within the accuracy of the gauges,as discussed above. The greatest source of inaccuracy waswith the measurement of temperature, and experiments weredone to determine the size of the inaccuracy.

Nutrient broth agar containing glucose and KNO3 wasinoculated with B. stearothermophilus from a culture grownovernight at 55°C in nutrient broth. The inoculated nutrientagar was aspirated into eight glass tubes (76.2 mm long, 7-mm outer diameter), which were placed in the eight pressurevessels of the PTG after the agar hardened. The tubes werekept at atmospheric pressure in the PTG along a temperaturegradient ranging from 33.8 to 76.3°C. Two observers deter-mined the lower and upper temperatures at which coloniesformed in each of the eight tubes. The mean low temperaturewas 40.56 (+ 0.23)°C and 39.85 (+ 0.66)°C according toobservers 1 and 2, respectively, where the values withinparenthesis are the standard deviations. The mean hightemperature was 69.05 (+ 0.23)°C and 69.01 (+ 0.22)°Caccording to observers 1 and 2, respectively.Growth of CFU from natural populations. Samples of

seawater at approximately 19.5°C from the end of the pier atScripps Institution were collected on three different days andused to inoculate gels made with gelatin, silica gel, and agar.Judging from the use of gelatin and silica gel with extremepsychrophiles (15, 16), these gels with nutrients shouldreveal the presence of any psychrophiles. The gelatin gelswere incubated for 120 h in the PTG. The distribution ofcolonies along the nutrient gels is shown in Fig. 7. The lowerbounds determined with agar gels were not consideredmeaningful because of possible thermal inactivation duringinoculation of the agar when it was 45°C. The upper tempera-

ture bounds in gelatin were indeterminate because the gela-tin liquified. The tubes with silica gel at the lower pressuresdid not allow any easy resolution of an upper boundarytemperature for reasons unknown.

DISCUSSION

We have described the PTG and shown that it can provideinformative synoptic views of bacterial growth in the TPplane. Data similar to those obtained with this PTG instru-ment could be obtained with conventionally used pressurevessels and temperature incubators only with much greatercapital expense and labor.The use of one instrument to view both temperature and

pressure effects requires additional studies to better defineany limitations such as arising from the use of long gels.Effects of concern include pH changes, compression, ther-mal expansion, and the Soret effect (4). After these effectsare better defined, the method can be further improved withcomputer-assisted methods. For example, colonies developat different rates as a function of temperature and pressureand, therefore, have different densities and sizes along a tubein the PTG during the early stages of growth. Computer-assisted data acquisition with an image analyzer or a densi-tometer may be useful in quantifying the distribution ofcolonies and colony sizes in long gels. Thus, relative growthrate information and the temperature and pressure of maxi-mal growth rate may be obtained from a single incubationwith the PTG. In the case of more involved designs of PTGs,real-time monitoring of bacterial activity along the length ofeach pressure vessel might prove useful.

Alternative designs of PTGs could be used to provide

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the pressure range between 1 and 950 bars. The other set of data (-)was obtained between 600 and 1,050 bars. The star is at thetemperature (ca. 2°C) and pressure (ca. 580 bars) of the likely naturalenvironment (15, 18) of isolate CNPT-3. The colder end of thegradient was not at a low enough temperature at pressures below 600bars to allow for a determination of the minimum temperature ofgrowth.

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HIGH-PRESSURE-TEMPERATURE GRADIENT INSTRUMENT 775

synoptic views of enzymatic activity, to characterize micro-bial functions other than growth, and to isolate in a singleapparatus bacteria capable of growth at a variety of tempera-tures and pressures.

Results from incubations of isolate CNPT-3 in the PTGshowed that the PTG can provide useful information forbiogeographical studies and for characterization. IsolateCNPT-3 could reproduce in cold (<8°C), shallow seas, coldbathyal and abyssal deep seas, and portions of the deep Suluand Mediterranean Seas having temperatures near 10°C.Many characteristics of growth of isolate CNPT-3 in the TPplane were easily determined with the PTG. Among thefeatures revealed by the data in Fig. 6 were the following. (i)The deep-sea bacterium is psychrophilic to a variable extentdepending on the pressure (15). (ii) The greatest interval oftemperature over which the isolate grew was 18.5°C, be-tween -1.5°C and 17°C at a constant pressure of ca. 600bars. (iii) The pressure at which this greatest temperaturerange of growth occurred was close to the pressure of 580bars (indicated by a star in Fig. 6) from which the organismoriginated (18). (iv) The largest interval of pressure overwhich the isolate grew was about 1,000 bars and was at atemperature of about 7°C. (v) The temperature (about 8°C) atwhich the organism became obligately barophilic (did notgrow at atmospheric pressure) was easily found. Character-istic (v) supports our previous assertion (16) that obligatebarophily must be defined at a reference temperature, andcharacteristic (i) leads us to suggest here that definitions ofpsychrophily must be made at a reference pressure. Furtherwork is needed to determine whether these definitions needto be further qualified by the effects of the chemical compo-sition of the medium or by the type of energy metabolismused by the organism.We investigated the feasibility of the method in answering,

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in the PTG. The points on the graph bound the TP domain in whichcolonies grew in nutrient gels inoculated with a natural population ofbacteria from a near-shore sea-surface sample of seawater. Nocolonies developed in the temperature-pressure regions (lower rightcorner of the graph) where deep-sea bacteria grow. Colonies weregrown (0) in nutrient agar containing 2216 marine broth, (A) in silicagels containing yeast extract, and (-) in gelatin. The star is at 1bar and 19°C, the environmental conditions from where the naturalpopulation of bacteria originated.

even to a limited degree, the question of what fraction of apopulation is allochthonous (with respect to temperature andpressure only). In the future we hope to investigate thisapplication of the method by comparing the TP domains ofnatural populations from a depth profile of the sea. In theabsence of data from such a profile we compared the TPdomain of a natural population (Fig. 7) of bacteria from theshallow coastal Pacific Ocean with the TP domain of theabyssal-depth isolate CNPT-3 (Fig. 6), with the thermalsensitivity of isolate CNPT-3 (15), and with some growthcharacteristics of the bathyal-depth isolate SC-2 (18). Thecomparisons revealed the following. (i) The environment(indicated by a star in Fig. 7) of the shallow temperate waternatural population is hostile (15) to the deep-sea isolateCNPT-3. (ii) There were in the natural population bacteriathat grew at pressures as much as 500 bars above the 1 atmorigin of the mixed population, but none that grew at highpressures and 2°C. A comparison with the isolate SC-2 (18)from a 1,957-m depth also shows that no member of thenatural population (Fig. 7) grew at 200 bars and 2°C-conditions under which isolate SC-2 grew. That is, within theresolution of the technique no bacteria of the cold deep seawere detected in samples of a shallow, temperate portion ofthe sea. (iii) Bacteria in the natural population occupying a19.5°C environment were capable of growth up to 40.5°C anddown to 3.5°C at atmospheric pressure. The range overwhich growth was observed became narrower as the pres-sure increased. The growth range for the deep-sea isolateCNPT-3 was narrow at atmospheric pressure and widest atthe pressure of its depth of origin.The interpretation of the results with natural populations

will require much further investigation and only may bepossible after a determination of the effects of nutrientconditions, a determination of the extent to which a naturalpopulation must be concentrated before study in the PTG,and a determination of whether the bacteria capable offorming colonies are, indeed, representative of the naturalcommunity of bacteria, most of which may not be capable offorming colonies. We imagine that the characterization ofnatural populations in this way will be useful, particularly soin the analysis of samples from microbial communities in thevicinity of hydrothermal vents (9), where there is intermixingof thermophilic and psychrophilic bacteria.

In conclusion PTGs appeared to be valuable tools for bothmicrobial physiology and ecology. PTGs can be inexpensive-ly fabricated and can be small. These features should en-hance their use both in the field and in the laboratory.

ACKNOWLEDGMENTSThis work was supported by grant OCE-8208419 from the Nation-

al Science Foundation and by a contract from Sandia NationalLaboratories, a prime contractor for the U.S. Department of Ener-gy-We thank Jonathan Trent for helpful comments.

LITERATURE CITED

1. Brock, T. D. 1967. Life at high temperatures. Science 158:1012-1019.

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3. Davey, C. B., and R. J. Miller. 1966. Correlation of temperature-dependent water properties and the growth of bacteria. Nature(London) 209:638.

4. de Groot, S. R. 1963. Thermodynamics of irreversible process-es. North-Holland Publishing Co., Amsterdam.

5. Dietz, A. S., and A. A. Yayanos. 1978. Silica gel medium forisolating and studying bacteria under hydrostatic pressure.

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Appl. Environ. Microbiol. 36:966-968.6. Henderson-Sellers, A. 1983. The origin and evolution of plane-

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10. Marquis, R. E. 1982. Microbial barobiology. Bioscience 32:267-271.

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12. Morita, R. Y. 1976. Survival of bacteria in cold and moderatehydrostatic pressure environments with special reference topsychrophilic and barophilic bacteria, p. 285-288. In T. R. G.Gray and J. R. Postgate (ed.), The survival of vegetative

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14. Oppenheimer, C. H., and W. Drost-Hansen. 1960. A relationshipbetween multiple temperature optima for biological systems andthe properties of water. J. Bacteriol. 80:21-24.

15. Yayanos, A. A., and A. S. Dietz. 1982. Thermal inactivation of adeep-sea barophilic bacterium, isolate CNPT-3. Appl. Environ.Microbiol. 43:1481-1489.

16. Yayanos, A. A., and A. S. Dietz. 1983. Death of a hadal deep-seabacterium after decompression. Science 220:497-498.

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18. Yayanos, A. A., A. S. Dietz, and R. Van Boxtel. 1982. Depen-dence of reproduction rate on pressure as a hallmark of deep-seabacteria. Appl. Environ. Microbiol. 44:1356-1361.

19. Yayanos, A. A., and R. Van Boxtel. 1982. Coupling device forquick high pressure connections to 100 MPa. Rev. Sci. Instrum.53:704-705.

19a.Yayanos, A. A., R. Van Boxtel, and Allan S. Dietz. 1983.Reproduction of Bacillus stearothermophilus as a function oftemperature and pressure. Appl. Environ. Microbiol. 46:1357-1363.

20. Young, R. S. 1976. Viking on Mars: a preliminary survey. Am.Scientist 64:620-627.

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