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Anal. Chem. 1991, 63,526-529
Detection of Bacteria by Ion Mobility Spectrometry A. Peter Snyder* a n d Donald B. Shoff
U S . Army Chemical Research, Development and Engineering Center, Aberdeen Proving Ground, Maryland 21010-5423 Gary A. Eiceman Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003-0003 David A. Blyth a n d J o h n A. P a r s o n s GeoCenters, Inc., Ft. Washington, Maryland 20744 INTRODUCTION A fundamental tool in microorganism analyses from a microbiological perspective is the assay of their inherent intracellular and extracellular enzymes. Enzyme assay interrogations have served microbiologists for many decades as a major cornerstone for microorganism taxonomy, detection, and identification purposes. In vitro and in vivo enzymatic assays can be performed on solid or liquid media with varying degrees of specificity, sensitivity, and analysis time. Methods that are currently used in the characterization of viable microorganisms range from straightforward, simple test tube/ agar plate procedures to sophisticated instrumental techniques (1-20). Table I provides a capsule summary of some of the important methods and figures of merit in the characterization of viable microorganisms. Characterization methods are listed (first seven entries in Table I) that do not rely on inherent bacterial enzymes while the enzymatic methods listed in Table I, in comparison, are competitive in terms of bacterial detection limits and times needed for a positive response at the detection limit. Table I reflects the concept that instrumental or microbiological methods are used generally to exploit a well-established property of the analyte. For example, for an enzymatic reaction on a substrate that produces a colored product, the common methods of detection would be either a visual or a spectrophotometric analysis. A fluorescent product would usually require a hand-held ultraviolet (UV) lamp, fluorimetric or laser system, and an electrochemical analysis of a biochemical reaction would produce a pH change. The analytical technique of spectrophotometry has an important role in the detection of fecal coliform bacteria by enzyme/substrate reactions. Fecal coliform bacteria belong to the Enterobacteriaceae and are comprised of E. coli and Klebsiella, Enterobacter, and Citrobacter species. A wellestablished method for their detection in suspect contaminated waters is the reaction of the @-galactosidase extracellular enzyme with that of (o-nitropheny1)galactopyranoside(ONPG) (15, 19, 20). The enzyme cleaves the substrate to yield a colorless galactopyranoside sugar and the yellow o-nitrophenol (ONP) products, and the ONP is spectrophotometrically detected. Ion mobility spectrometry (IMS) is a straightforward, analytical vapor detection technique. Neutral analyte vapors enter the device and are ionized, usually by a 63Niring. The ions are electrically gated and "drift" through an antiparallel flow of buffer gas (air or nitrogen). The ions are focused by an electrical field about the heated, cylindrical drift region and are registered by a Faraday cup detector. The entire process, from vapor sampling to the detection event, takes place a t ambient or near-ambient pressure, and therefore atmospheric pressure ionization chemistry characterizes the ion formation process. Ions are partitioned primarily according to their mass and shape and are characterized by their corrected drift times (typically in milliseconds) or ion mobilities. St. Louis and Hill (21) provide an excellent overview and comprehensive treatment of IMS in terms of theory,
ionization chemistry, sample introduction devices, and applications. A relatively new concept is explored where the potential for IMS is applied to the detection of living microorganisms. A recent report by Lawrence (22) details an investigation by using a laboratory ion mobility spectrometer in the differentiation of normal and bacteria-infected red oak wood. By applying thermal desorption to the wood samples, the latter type of wood displayed two additional intense lower mobility (longer drift time) peaks than in the ion mobility spectrum of the former wood type. No conclusions concerning the chemical and/or biochemical origins of this phenomenon were presented. In the present report, however, IMS is used to interrogate the classical bacterial enzyme/substrate reaction by probing the product analyte in a manner unconventional with that of modem microbiological, clinical, and analytical microorganism detection techniques. In the present investigation, advantage is taken of the fact that ONP exhibits a relatively high vapor pressure (0.54 Torr at 40 " C ) and it was found that this property of ONP allows for its sampling by IMS technology. A portable, hand-held IMS was used as the detector, and the biochemical reaction was simple in design. Microliter amounts of an E. coli bacteria suspension and an ONPG substrate solution were deposited on a piece of filter paper in a vial, and after a short incubation time, the vial was introduced to the inlet of the IMS detector. The relatively small, simple-indesign, and sensitive IMS bacterial detection scheme provides ramifications for the monitoring of water and wastewater sources and for the medical and clinical communities in the rapid detection of microorganisms. EXPERIMENTAL SECTION Instrumentation. A hand-held IMS from Graseby Ionics, Ltd. (Watford,UK), and an IMS/mass spectrometer (MS) from PCP, Inc. (W. Palm Beach, FL), were described previously (23)and were used under the same operating conditions. Materials a n d Reagents. o-Nitrophenol and (o-nitrophenyl)-@-D-galactopyranoside were obtained from Aldrich Chemical Co., Inc. (Milwaukee,WI), and Sigma Chemical Co. (St. Louis, MO), respectively. The filter paper support was Whatman No. 5 from Whatman International, Ltd. (Maidstone, UK). A sterile phosphate buffered saline solution (0.7% NaCl) at pH 7.4 (phosphate-bufferedsaline (PBS))was used for all solutions and bacterial suspensions. Procedures. Pure E. coli suspensions (ATCC 11303) were prepared by growth in a nutrient broth solution for 48 h, which was supplemented with 0.5% lactose sugar for induction of the @-galactosidaseenzyme. The bacterial growth was centrifuged and the pellet was washed three times with PBS. The bacteria was suspended in PBS for experimental analyses. Protective gloves were used in the handling of the organisms during growth, in the preparation of stock suspensions,and in the experimental protocol. Sterile conditions and apparatus were used from the bacterial growth to the experimental protocol procedures. The filter paper was baked at 150 "C overnight in a glass vial. The ONPG solution was prepared at a concentration of 2 mg/mL in sterile PBS containing 0.1% of the surfactant Tween 20. Two microliters of the ONPG solution was added to a 13-
0003-2700/9110363-0526$02.50/0 0 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 5, MARCH 1, 1991
Table I. Viable Microorganism Characterization Techniques total no. of bacteria 107 107 10" 1 103-1 04 103 1-5 80 105 104 108 2.7 x 104 104 104 105 1
5 x 107 105 10 7
time, h 0.5 0.25 1
0.5
0.03 0.5 >5 8.5 1 1 0.4 3 9 4 4.25 24 0.5 0.25 0.25
technique
response
organism growth polarized light scattering excitation-emission matrix 3-laser flow-through cytometry fiber-optic immunoassay filter fluorescent antibodies polymerase chain reaction gas chromatography radiometry electrochemical light-addressablepotentiometric sensor enzyme-linked lectinosorbent assay H2/C02 evolution glucuronidase enzyme extracellular enzyme @-galactosidase aminopeptidase enzymes extracellular enzymes extracellular enzymes with nutrients
electrical impedance Mueller matrix fluorescence fluorescence fluorescence fluorescence radiolabel film exposure ethanol metabolite 14C02metabolite dye-based pH change redox potential lectin-conjugate visual, gas bubbles fluorescence colorimetric colorimetric fluorescence fluorescence fluorescence
mm-diameter sterile filter paper disk placed on the bottom of a 5-mL glass vial. A 2-pL aliquot from a suspension of E. coli in sterile PBS or 2 r L of PBS as a control was added to the filter disk, and the bottle was sealed with a screw cap. After an incubation period at 40-42 "C, the headspace of the bottle was sampled with the hand-held IMS by removing the cap and immediately placing the vial opening at the inlet of the IMS unit. Suspensions of E. coli ranged from 200 to 2 X lo4 cells/mL, and the incubation times ranged from 10 to 30 min. Nutrient broth agar plates were used to enumerate bacteria. Aliquots of a 2-mL PBS suspension with 2 pL of E. coli from each stock bacterial suspension were plated and counted after a 48-h incubation. This resulted in a total number of E. coli cells that provided an approximation of that in the 2-wL aliquot applied to the filter paper in the experimental vial. Mobility spectra of the ONP product were collected continuously (24) in the negative ion mode after a predetermined incubation time until the product ion intensity returned to base line (ca. 30 s at a rate of 2 mobility spectrum scans/s). The areas of all the ONP peaks for an experiment were summed in order t o obtain a measure of total ONP released from the galactosidaseONPG reaction. An equivalent number of blank mobility spectra areas were summed, where the same time frame for the area calculation was used as that of the ONP signal, and subtracted from the total ONP area.
RESULTS AND DISCUSSION Mobility Spectra from E . coli and ONPG. The negative ion mobility spectra obtained with an IMS/MS for the reaction between ONPG and the in vivo E. coli 0-galactosidase enzyme are shown in Figure 1. Headspace vapors from buffered ONPG on a filter paper disk without bacteria consisted only of the two reactant ion peaks (Figure 1A) a t drift times of 18.12 and 19.99 ms or reduced mobilities (KO)of 2.44 and 2.21 cm2/(V s), respectively. These peaks comprised 10 constituents resembling those seen previously in IMS/MS investigations with negative ions in air (25). The dominant ions in a mass spectrum of this sample were the base peak, (HzO)z.O,- ( m / z 68, 33.7% total ion intensity) and H20. C02-O; ( m / z 94, 26.9%). The next most intense ion ( m / z 70, not identified) represented 11.5% of the total ion intensity and was considered inconsequential as were all other reactant ions of low abundance. The relevance of the ONPG background spectrum was that, in the absence of the @-galactosidase enzyme, the hydrolysis of ONPG to ONP did not occur, and thus, no detectable levels of volatile decomposition products were observed. The addition of an E. coli suspension to the ONPG substrate caused a change in the composition of headspace vapors. Samples containing 2 x 105bacterial cells remained at ambient
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Figure 1. Analysis of headspace vapors by IMSlMS from ONPG with and without E . coli. The mobility spectra are shown for ONP product vapors from ONPG before (frame A) and after (frame 6 ) addition of bacteria. In frame C, the mass-resolved mobility spectrum is shown for the ion mobility spectrum in frame B.
temperature for 5 min, and IMS/MS analysis of the headspace vapors exhibited a peak in addition to the reactant ion peaks as shown in Figure 1B. This peak appeared a t a drift time of 25.99 ms (KO of 1.70 cm2/(V s)). The mass-resolved mobility spectrum for this sample Figure IC) showed that the peak a t a KOof 1.70 cm2/(V s) was due to an m / z 171 ion and is most likely an M.OZ- species where M is a neutral gaseous ONP molecule. At high vapor levels of ONP,a dimer ion (Mf) was discernible (not shown). The KOvalue and mass-resolved mobility spectrum for authentic ONP (not shown) were identical with those found in Figure 1B and 1C. The only previous IMS/MS studies with nitrophenols was that of Karasek e t al. (26), who examined p-nitrophenol (PNP) in nitrogen a t 208 OC. Since the conditions of temperature, gas composition, and vapor concentration were significantly different from the present work, comparison of findings is largely irrelevant. For example, P N P yielded product ions in the positive ion mode while comparable behavior was not observed with ONP in air a t ambient temperature. Consequently, these findings gave preliminary support t o the concept that vapor products that arise from reactions between ONPG and the in vivo 0-galactosidase enzyme can be detected by IMS and serve as a marker for the presence of E. coli. The use of negative ion IMS for detection of ONP vapors from microbiological samples is advantageous since the number of naturally occurring potential interferences (i.e., strong electron-capturing species with reduced mobilities comparable to ONP) in clinical or environmental circumstances may be expected to be fewer and less widespread than
528
ANALYTICAL CHEMISTRY, VOL. 63, NO. 5, MARCH 1, 1991
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a W
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Figure 2. Mobility spectra for ONP vapor from before (control, bottom trace) and after (top trace) the addition of an E . coli suspension to an ONPG solution on filter paper at the following number of bacterial cells and incubation times: (A) 200 cells, 30 min; (B) 1000 cells, 10 min; and (C) 26 000 cells, 10 min. hcubation
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Flgure 3. Plot of IMS response versus E. coli bacteria concentration. The response was measured as the sum of the peak areas/incubation time from sampling of the reaction vessel by the hand-held IMS with an inlet sample flow of ca. 0.5 Llmin. The data points indicated with letters A-C refer to mobility spectra in Figure 2. Lines connect the median of each group of data points for the 10- and 15-min incubation times.
with those that produce a response in the positive ion mode. The possible utility of IMS in actual microbiological applications is also enhanced by the generation of ONP for which few, if any, naturally occurring sources exist. Response Curves for E . coli and ONPG. The relationship between IMS response and bacteria concentration, in cells/milliliter, is shown in Figures 2 and 3. Peak heights for the ONP product ion were proportional to bacteria levels (Figure 2 ) , and scatter (Figure 3) was comparable to previous fluorescence-based methods (17). This scatter also could be attributed to the unrefined means of sampling headspace vapors from incubation vessels and might be improved through modifications in this portion of the sampling procedure. Nonetheless, a proportional trend is clear (Figure 3) and suggests that the IMS may be suitable for semiquantitative determinations of E.coli with only 10-30-min incubation times for 200+ cells/mL. Clearly, longer incubation times favored better detection limits although lengthy incubation times detract from the central attraction of this method, speed. The mobility spectra corresponding to data points identified with letters in Figure 3 are shown in Figure 2 and include the background control spectrum (lower trace) for each measurement. Growth in the product ion for lo00 (frame B) and 26 000 (frame C) E.coli cells was evident in the upper traces in Figure 2. The mobility spectrum for 200 cells (frame A)
showed a visible product ion (top trace) that could have been but was not enhanced further through the scale expansions or improved parameters in digital signal averaging. Thus, under the present experimental conditions, the detection limit should be considered to be ca. 200 cells for a 15-30-min incubation. In comparison to the techniques in Table I, the concept of microorganism detection with IMS appears to be attractive with respect to sensitivity and speed. At the levels of bacteria in Figures 2 and 3, the absence of cluster ions (27), fragmentation reactions (28),or multiple product ions such as dimer ions suggests that further improvements in detection limits could occur without the complications of complex ion source chemistry (25), which have been found to be analyte concentration dependent for certain chemical species. The possibility of enhanced detection limits in negative ion IMS through the use of chloride ions (29) is worthy of consideration in the IMS detection of ONP. The growth of E. coli in a medium that includes lactose represents an ideal condition for the organism to mFnufacture and express the @-galactosidaseenzyme in a considerable amount. The enzyme is said to be induced and the presence of lactose in coliform growth media inherently provides an optimal condition for detection protocols. "Real-world" conditions, however, seldom provide for this luxury, and therefore, minimal to negligible amounts of @-galactosidase enzyme are found in E.coli collected from outdoor community and environmental waters. For detection methods based on enzyme reactions, what is normally required is that the E. coli organisms in suspect water samples are grown in the presence of lactose prior to or during the detection protocol ( I , 9,13, 15,19,20,30,31). However, by probing the @-galactosidase enzyme with 4-(methylumbelliferyl)-@-~-galact~side, Berg and Fiksdal (18) showed that the addition of lactose, nutrient broth, and the detergent sodium lauryl sulfate provided a sufficient means for the detection of the 4-methylumbelliferone fluorescent product from noninduced E. coli in environmental water samples in a 15-20-min assay. To this end, E. coli organisms were grown in nutrient broth without lactose and were tested with the ONPG biochemical reaction with the IMS technique. Enzyme inducers/enhancers, detergents or growth media were not added to the experimental protocols. Generally, an order of magnitude decrease of the product ion area/time parameter was observed (data not shown) with the same bacterial concentration with respect to E. coli grown in the presence of lactose (Figure 3). The next stage of development in this concept involves the exploration of the effects of growth media and the effects of growth media, detergents, and enzymatic inducers in the assay itself. In addition to lactose, enzymatic inducers such as isopropyl-P-D-thiogalactopyranoside and (thiomethy1)-@-Dgalactopyranoside also serve to facilitate the production of the @-galactosidaseenzyme (32-34) in the presence of ONPG and appear to be candidate compounds in the refinement of the general IMS technique. Optimization of experimental parameters, such as the support matrix, reaction vessel, and volume, and sample transfer to the IMS could lower detection limits, increase the speed of reaction, and streamline the entire experimental process. Another dimension in this concept is the use of other enzyme/substrate combinations, e.g., glucosidase/ONP-glucopyranoside, to add further specificity to detection schemes.
ACKNOWLEDGMENT The efforts of Maryalice Miller in the preparation and enumeration of E. coli samples is gratefully acknowledged. Assistance from Dennis M. Davis in data reduction is also appreciated. Registry No. @-Galactosidase,9031-11-2; (o-nitrophenyl)-PD-galactopyranoside,369-07-3; o-nitrophenol, 88-75-5.
Anal. Chem. 1991, 63,529-532
LITERATURE CITED
(1) Cady, P.; Dufour, s. W.; Shaw, J.; Kraeger, S.J. J . Clin. Microbiol. 1978, 7 , 265-272. (2) Fraatr, R. J.; Prakash, G.; Ailen, F. S . A m . Biotechnol. Lab. 1988, 6 , 24-28. (3) Shelly, D. C.; Quaries, J. M.; Warner, I. M. Anal. Left. 1981, 14(B13), 1111-1 124. (4) Steinkamp. J. A.; Fulwyler, M. J.; Couker. J. R.; Hiebert, R . D.; Horney, J. L.; Mullaney. P. F. Rev. Sci. Instrum. 1973, 44, 1301-1310. (5) Regina, F. J.; Lin, S. H.: Bolts, J. M. Abstracts of Papers, 199th National Meeting of the American Chemical Society, Boston, MA, April 22-27, 1990; American Chemical Society: Washington, DC, 1990; Abstract No. 51 (6) Lim. L. C. L.; Pennell, D. R.; Schell. R. F. J . Clin. Microbia/. 1990, 28, 670-675. Bej, A. K.; Steffan, R. J.; DiCesare, J.; Haff, L.; Atlas, R . M. Appi. Environ. Microbiol. 1990, 56, 307-314. Newman, J. S.:O'Brien, R . T. Appl. Microbiol. 1975, 30, 584-588. Bachrach. U.; Bachrach, Z. Appl. Microbiol. 1974, 2 8 , 169-171. Maoyu. Y . ; Zhang. Y. Appl. Environ. Microbiol. 1989. 55, 2082-2085. Libby, J. M.; Wada, H. G. J . Clin. Microbiol. 1989, 2 7 , 1456-1459. Graham, K.; Keller, K.; Ezzel, J.; Doyle, R . Eur. J . Clin. Microbiol. 1984, 3 , 210-212. Feng. P. C. S.: Hartman, P. A. Appl. Environ. Microbiol, 1982, 43, 1320- 1329. Warren, L. S.;Benoit, R . E.; Jessee, J. A. Appl. Environ. Microbiol. 1978, 3 5 , 136-141. Edberg, S. E.: Allen. M. J.; Smith, D. B. Toxic. Assess. 1988, 3 , 565-580. Godsey. J. H.; Matteo, M. R.; Shen, D.;Tolman, G.; Gohlke, J. R. J . Clin. Microbiol. 1981, 13, 483-490. Snyder, A. P.: Wang. T. T.: Greenberg, D. B. Appl. Environ. Microbiol. 1988. 5 1 . 969-977. Berg, J. D.; Fiksdal, L. Appl. Environ. Microbiol. 1968, 5 4 , 21 18-2122.
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(19) Edberg, S.C.; Edberg, M. M. Yale J . Biol. Med. 1988, 67, 389-399. (20) Covert, T. C.: Shadix, L. C.; Rice, E. W.; Haines, J. R.; Freyberg, R. W. Appl. Environ. Microbiol. 1989, 55, 2443-2447. (21) St. Louis, R . H.; HI4 H. H., Jr. Crit. Rev. Anal. Chem. 1990, 21, 321-355. (22) Lawrence, A. H. J . Pulp Paper Sci. 1989. 15, J196-J199. (23) Eiceman, G. A.; Blyth, D. A.; Shoff, D. B.; Snyder, A. P. Anal. Chem. 1990, 62, 1374-1379. (24) Eiceman, G. A.; Snyder, A. P.; Biyth, D. B. Int. J . Environ. Anal. Chem. 1990, 38, 415-425. (25) Eiceman, G. A.; Shoff, D. 6.; Harden, C. S.;Snyder, A. P.;Martinez, P. M.; Fleischer, M. E.; Watkins, M. L. Anal. Chem. 1989, 61. 1093- 1099. (26) Karasek. F. W.; Kim, S. H.; Hill, H. H..Jr. Anal. Chem. 1976, 48, 1133-1 137. (27) Preston, J. M.; Rajadhyax, L. Anal. Chem. 1988, 6 0 , 31-34. (28) Eiceman, G. A.; Shoff, D. 6.; Harden, C. S.;Snyder, A. P. Int. J . Mass Spectrom. Ion Proc. 1968, 8 5 , 265-275. (29) Lawrence, A. H.; Neudorfl, P. Anal. Chem. 1988, 6 0 , 104-109. (30) Standard Methods for the Examination of Water and Wastewater, 16th ed.; Greenberg, A. E., Trussell, R. R., Clesceri, L. S.,Eds.; American Public Health Assoc.: Washington, DC, 1985; pp 876-886. (31) Rice, E. W.; Allen, M. J.; Edberg, S.C. Appl. Environ. Microbiol. 1990, 56. 1203-1205. (32) Novick, A.; Weiner. M. Proc. Natl. Acad. Sci. U . S . A . 1957, 4 3 , 553-566. (33) Bello, J. Science 1960, 165, 240-241. (34) Pardee, A. B.; Prestidge, L. S. Biochim. Biophys. Acta 1961, 49, 77-88.
RECEIVED for review August 2, 1990. Accepted November 19, 1990.
Extraction of Methane from Seawater Using Ultrasonic Vacuum Degassing Manfred Schmitt* Geochemische Analysen, Wilhelmstrasse 36, 0-3160 Lehrte, FRG
Eckhard Faber Bundesanstalt fur Geowissenschaften und Rohstoffe, Stilleweg 2, 0-3000 Hannover 51, FRG
Reiner Botz and Peter Stoffers Geologisch-PalaontologischesInstitut, Universitat Kiel, Ohlshausenstrasse 40-60, 0-2300 Kiel, FRG
INTRODUCTION Methane (and higher hydrocarbons) is a well-known constituent of marine and freshwater sediments and the overlying water column (1-5). Degassing of sediments has been shown to deliver reliable information on both the gas quantities and the stable isotopic composition of methane in sediments (6-8). Hydrocarbon gases in sediments generally consist of more than 80% methane, with 6 13CH4between -100% and -20% (8), depending on the thermal or bacterial origin and the postgenetic history of gases. Although the saturation concentration of methane in water is near 23 mg/L, such high values are virtually never found. Methane concentrations of ocean waters usually range from several nanograms/liter to several micrograms/liter (4,9,10). This may be explained by low methane input into the water, gas release into the atmosphere before equilibration, and/or oxidation of methane in the water column. It has been shown that high methane concentrations in seawater are characteristic of active hydrothermal areas. Mapping of methane concentration anomalies in seawater has proved to be a reliable indicator for active hydrothermal areas (9,10). In general, the methane concentration in seawater is determined directly on board research vessels, as sample storage is not necessary then. To extract the dissolved gases, a helium
stripping method is commonly used (11).It requires relatively large amounts of purified helium, which is lost after stripping to the atmosphere. To recover methane from the carrier gas (helium), an adsorption-desorption technique is required. This method delivers reliable results on the methane concentration in water samples. However, isotopic fractionation of the extracted methane is possible due to incomplete adsorption-desorption processes. To overcome this problem, a new water-degassing system has been developed: methane (and other dissolved gases) is extracted in a vacuum system while the water sample is exposed to ultrasonic energy. A capillary gas chromatograph is used to determine the methane concentration. The main advantage of the new system is that within a short period of time (approximately 20 min/sample) reproducible data are available for the methane concentration in water. This may contribute to further cruise planning in the evaluation of hydrothermal areas and furthermore provides isotopically unfractionated methane for later stable carbon isotope investigations.
EXPERIMENTAL SECTION Water Sampling. Water samples are taken by Niskin bottles in water depths ranging from the surface down to 6000 m. After
that, the water is transferred from the Niskin bottles through short silicone tubes into evacuated glass bottles. As the analysis time
0003-2700/91/0363-0529$02.50100 1991 American Chemical Society
