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Applied and Environmental Microbiology, May 2000, p. 2211-2215, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Simultaneous Direct Counting of Total and Specific Microbial Cells in Seawater, Using a Deep-Sea Microbe as Target

Akihiko Maruyama1,* and Michinari Sunamura1,2

Bioconsortium Analysis Program, National Institute of Bioscience and Human Technology, Higashi, Tsukuba, Ibaraki 305-8566,1 and Mitsubishi Kasei Institute of Life Science, Machida, Tokyo 194-8511,2 Japan

Received 1 November 1999/Accepted 2 March 2000


    ABSTRACT
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To rapidly and accurately enumerate total and specific microbes in aquatic samples, fluorescent in situ hybridization was combined with direct counting via direct immobilization of cells on a polymer-coated Nuclepore filter. The technique, named FISH-DC, achieved almost complete recovery of total cells and reproducibility of Psychrobacter pacificensis cells of deep-sea origin (error, <= 3%) in a mixed culture and in natural seawater. Target cells immobilized on the filter were also successfully enumerated after stringent 3-cycle hybridization and even after a 16-month preservation at -30°C.


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Whole-cell fluorescent in situ hybridization (FISH) is one of the most widely used methods for detecting and monitoring of specific microbial cells in applied, environmental, and ecological microbiology (e.g., see references 1, 2, 5, 6, 20, and 21). In situ detection of individual microbial cells using 16S rRNA sequence information was reviewed in detail by Amann et al. (3). FISH, as used for detection of microbes in natural aquatic samples, generally consists of such key techniques as cell fixation, concentration, drop or transfer onto a polymer-coated slide, immobilization, refixation or relaxation of the cell membrane, hybridization with fluorescent oligonucleotide probes, washing and removing of unhybridized probes, and detection and counting of hybridized fluorescent cells. For aquatic samples, such as oligotrophic river, lake, marine, and drinking water samples, it is indispensable that cells be concentrated through filtration prior to quantitative FISH. In general, cells in such samples were transferred manually from a filter to a polymer-coated slide, as in the technique used for fingerprinting. It remains unclear, however, whether cell transfer from a filter or immobilization on a polymer-coated slide is a reliable and reproducible method for the enumeration of total and target microorganisms and whether the cells are adequately retained on the slide during the subsequent hybridization and washing processes.

Direct counting (DC) using DNA-specific fluorochromes, such as 3,6-bis(dimethylamino)acridinium chloride (acridine orange) (9) and 4',6-diamidino-2-phenylindole (DAPI) (22), is now widely used to count total microbial cells in aquatic samples (10) and has proved effective in clarifying the localization and variation of total microbial populations in the ocean, particularly in such unexplored regions as tropical marine lakes (25) and deep-sea hydrothermal vents (13, 16). For counting total viable but nonculturable microorganisms in nature, the direct viable counting method was developed based on DC (11). Both the DC and the direct viable counting methods use direct cell trapping from an aquatic sample onto a Nuclepore filter in order to achieve highly reliable enumeration and direct counting of stained cells under an epifluorescent microscope. However, this trapping technique has not yet been utilized appropriately in FISH experiments.

Here, we report a simple, rapid, and highly reliable counting method, FISH-DC, for enumerating both total and specific microbial cells in aquatic samples, based on direct cell trapping and immobilization using polymer-coated Nuclepore filters. Through the development of species-specific oligonucleotide probes for a newly isolated deep-sea microorganism, Psychrobacter pacificensis (17), we demonstrate that the introduced target microbial cells are precisely counted by FISH-DC under both artificially mixed and natural microbial conditions.

Microbial strains. The following microbial strains were used: P. pacificensis NIBH P2K6T (=IFO 16270T), P2J2, P2J3, P2J13, P2K18, Psychrobacter glacincola ACAM 483T, Psychrobacter immobilis ATCC 43116T, Psychrobacter frigidicola ACAM 304T, Psychrobacter urativorans ATCC 15174T, and Psychrobacter phenylpyruvicus ATCC 23333T in the family Moraxellaceae (17), Pseudomonas aeruginosa IFO 12689T, Vibrio parahaemolyticus IFO 12711T, Bacillus marinus ATCC 29841T, and Saccharomyces cerevisiae IFO 10217.

Microbial cell preparation. A novel gamma -Proteobacteria species, P. pacificensis, abundant in psychrotrophic bacterial communities isolated from 5,000- to 6,000-m-deep seawater in the Japan Trench (15, 17), was used as a target microorganism. All Psychrobacter species were incubated at 10 to 20°C in 1/2 TZ medium (13), as were P. aeruginosa and V. parahaemolyticus. B. marinus was cultured at 20°C in marine broth (Difco, Detroit, Mich.) and S. cerevisiae at 30°C in yeast-malt extract broth (Difco). Cells in the early to middle growth phase were fixed with paraformaldehyde solution consisting of 15% paraformaldehyde (TAAB, Aldermaston, England) in phosphate-buffered saline (3× PBS [per liter]: 24 g of NaCl, 0.6 g of KCl, 4.32 g of Na2HPO4, 0.72 g of KH2PO4 [pH 7.4]). After addition of 2.5 ml of the paraformaldehyde solution to 10 ml of a culture sample (final concentration, 3%), cells were fixed at 4°C overnight. Cells were concentrated by centrifugation at 12,000 × g for 5 min and stored in ethanol at -30°C. Natural seawater microbes were collected with a 5-liter Niskin bottle from the innermost region of Tokyo Bay on 19 August 1998. Seawater sample was filtered through a plankton net (inner mesh size, ca. 100 µm), and microbial cells in 40 ml of filtrate were fixed immediately (i.e., while still on the boat) with 10 ml of the paraformaldehyde solution (final concentration, 3%) and stored at 4°C. Immediately before use, fixed microbial samples were filtered through a 10-µm-pore-size Nuclepore filter to eliminate phytoplankton and other debris. The cell density of free-living microbes in the final filtrate, i.e., a 0.2- to 10-µm fraction, was 1.6 × 106 cells ml-1. By using 0.2-µm-pore-size-filtered artificial seawater, densities of P. aeruginosa cells and seawater microbes were adjusted by dilution in the range of 80 to 100 cells in a microscopic field (ca. 100 by 100 µm), i.e., 8.1 × 105 cells ml-1 for P. aeruginosa and 6.3 × 105 cells ml-1 for seawater microbes (final cell densities), and samples were serially diluted for FISH-DC analysis. An equal number of P. pacificensis cells was added to each dilution sample, i.e., 2.9 (2.93±0.23) × 105 cells ml-1 for P. aeruginosa samples or 1.5 (1.50±0.09) × 105 cells ml-1 for seawater microbe samples.

                              
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  		TABLE 1.   Total and specific cell numbers estimated by FISH-DC using PLL-coated Nuclepore filters

Filter preparation and cell immobilization. A polymer-coated filter was prepared as follows: (i) a 47-mm-diameter Nuclepore black filter (pore size, 0.2 µm) was aseptically cut in four equal sections, (ii) each section was labeled with an identification number, (iii) a fan-shaped Nuclepore filter was then immersed in a 0.01% solution of poly-L-lysine (PLL; Sigma, St. Louis, Mo.) for 5 min, and (iv) the filter was dried and stored at room temperature in a 51-mm-diameter aseptic petri dish until use. The polymer-coated filter was placed in a glass filtration apparatus with an inner diameter of 10 mm, and microbial cells in each 2-ml sample were directly trapped onto the filter at a vacuum below 30 cm Hg (<40 kPa), as in DC. After air drying, microbial cells attached to the filter were immediately used for whole-cell hybridization or stored at -30°C in an aseptic petri dish.

Oligonucleotide probes. Probes for P. pacificensis strains were designed from 16S rDNA sequences, accession no. AB 016054 to 016059 (17), using probe check software available on the Ribosomal Database Project homepage (http://www.cme.msu.edu/RDP/). Three candidates were synthesized with an Oligo 1000M system (Beckman Coulter, Tokyo, Japan) and 5'-aminolinked with the fluorochromes fluorescein-5-isothiocyanate (FITC), tetramethyl rhodamine-5 (and -6)-isothiocyanate (TRITC), and/or indodicarbocyanine (Cy 5) by using trifluoroacetic acid aminolink cyanoethyl phosphoramidite (PE Biosystems, Chiba, Japan). After cross-checking through whole-cell hybridization with the target species and others, probe Psypac 469 (the number indicates a position in Escherichia coli numbering [12]), which consisted of the sequence 5'-TAA TGT CAT CGT CCC CGG G-3' (19-mer), was finally selected. The group-specific oligonucleotide probes Euba 338 (domain Bacteria) (3), Univ 519 (universal) (3), and Univ 1390 (universal) (28) were also prepared, as was the Cont probe (control), with the sequence 5'-GTG CCA GCA GCC GCG G-3' (16-mer).

In situ hybridization and detection. Hybridization solution (per microliter: 0.9 M NaCl, 5 mM EDTA, 0.5% sodium dodecyl sulfate, 50 mM sodium phosphate buffer [pH 7], 10× Denhardt solution [Sigma], 1 µg of poly(A) and 1 ng of FITC-, TRITC-, or Cy 5-labeled oligonucleotide) was prepared and supplemented with formamide (Sigma), depending on the probe sequence composition and hybridization stringency (23). Fifty microliters of the solution was dropped onto cells attached to the PLL-coated filter in a slide chamber, and cells were kept at 42 to 46°C, depending on the probes and experiments, under moist conditions for 4.5 h. The filter was removed from the slide and washed twice in aseptic plastic tubes containing 50 ml of wash solution (0.9 M NaCl, 0.1% sodium dodecyl sulfate, 50 mM sodium phosphate buffer [pH 7]) at 44°C for 30 min, using a rotary hybridization incubator (type HB; Taitec, Koshigaya, Japan). After the filter was desalted by dipping in pure water, microorganisms on the filter were stained with DAPI (final concentration, 5 µg ml-1) for 10 min. The filter was then washed again in 50 ml of water at room temperature for 15 min and set onto a slide, mounted with antiquenching reagents such as 1,4-diazabicyclo[2.2.2]octane (DABCO; Wako, Osaka, Japan) (1 g 100 ml-1 [10 ml of phosphate-buffered saline and 90 ml of glycerol]) and ProLong Antifade (Molecular Probes, Eugene, Oreg.). Microbial cells on the PLL-coated filter were then counted by the DC method (22), in which more than 1,500 (total count) or 500 (P. pacificensis count) cells in at least 20 microscopic fields were tallied, using a Zeiss Axioplan epifluorescence microscope, with excitation/emission filters of 360/460 (DAPI), 480/535 (FITC), 546/565 (TRITC), and 620/700 (Cy 5) nm. Microscopic cell images were also captured with a 6.7- by 6.7-µm-device-used shutterless high-resolution cooled charge-coupled device camera (MicroMAX-1300Y; Nippon Princeton Instruments, Nippon Roper, Chiba, Japan) and analyzed using IPLab version 3.1.2 software (Scanalytics, Inc., Fairfax, Va.) on a Power Macintosh G3.

Species specificity of the oligonucleotide probe Psypac 469 was examined by using five P. pacificensis strains and eight others, and appropriate hybridization conditions were determined for evaluating differences in the efficiency of cell retention on a PLL-coated filter with different treatment cycles, temperatures, and solutions. For probes Psypac 469, Univ 519, and Euba 338, we first selected a simple 1-cycle condition of hybridization at 45°C (without formamide) and washing at 45°C. This condition had the distinct advantage of allowing rapid simultaneous hybridization and characterization of target P. pacificensis cells, although a loss of stringency somehow appeared in the probe specificity check with the neighbor species P. glacincola, of Antarctic origin (4). Under both artificially mixed (Fig. 1a through d) and natural (Fig. 1e through h) microbial conditions, microbial cells that were concentrated and immobilized on PLL-coated filters were clearly visualized, not only with a DNA fluorochrome DAPI (Fig. 1a and e) but also with the probes Univ 519 (Fig. 1b and f) and Euba 338 (Fig. 1c and g) under the above-described simultaneous hybridization condition. Target P. pacificensis cells in the same filter sample were also identified with the Psypac 469 (Fig. 1d and h) under epifluorescence microscopy.


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  		FIG. 1.   Epifluorescent micrographs of total and specific microbial cells directly trapped on a polymer-coated Nuclepore filter. A target deep-sea microbe, P. pacificensis, was mixed with P. aeruginosa (a through d) or natural seawater microbes (e through h). Cells were simultaneously hybridized at 45°C with DNA probes of Cy 5-labeled Univ 519 (b and f), FITC-labeled Euba 338 (c and g) and P. pacificensis-specific TRITC-labeled Psypac 469 (d and h), washed at 45°C, and then stained with a DNA-specific fluorochrome DAPI (a and e). Psypac 469-hybridized cells appear larger than their actual size due to their greater fluorescent intensities.

For quantitative evaluation, P. pacificensis cells were added to a serially diluted solution of P. aeruginosa cells, trapped onto a PLL-coated filter, and subjected to simultaneous whole-cell hybridization. Using the Psypac 469 probe, the P. pacificensis cells introduced were finally detected in each dilution bottle, showing an excellent recovery rate of 99%, and the results were reproducible with an error of <= 3% (Fig. 2A). Slightly higher total cell counts (106% ± 7%) were seen with FISH-DC than with the general DC (Fig. 2A). This slight difference may have been due principally to the fact that the cell immobilization on filters during cell preparation for microscopic counting was more efficient in FISH-DC than in DC. Similar recovery efficiencies were achieved in experiments using gram-positive B. marinus as a background microbe (data not shown). It appeared that few cells were detached from the PLL-coated filter during hybridization, washing, and detection for these standard microbes.


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  		FIG. 2.   Recovery of both total and specific microbes through FISH-DC. Equal numbers of target P. pacificensis cells were added at 2.93 × 105 cells ml-1 to a serially diluted solution of P. aeruginosa (A) and at 1.50 × 105 cells ml-1 to natural seawater microbes obtained from Tokyo Bay (B). Cells were simultaneously hybridized at 45°C with the Univ 519, Euba 338, and Psypac 469 probes, washed at 45°C, and stained with DAPI (Fig. 1). Bars show the standard deviation (SD) in three different filter samples from a single dilution. In the two-dilution series, the average numbers of recovered target P. pacificensis cells were 2.90 × 105 cell ml-1 (99% recovery; SD = 3%) (A) and 1.46 × 105 cell ml-1 (97% recovery; SD = 2%) (B). In making a regression line for total cell counts in FISH-DC, each average number of P. pacificensis cells recovered was used as the origin at 0 on the x axis. Numbers of P. aeruginosa (A) and natural seawater microbes (B) in FISH-DC were obtained by subtracting Psypac 469-stainable cell numbers from DAPI-stainable total cell numbers. After preservation of the Tokyo Bay filter samples for 16 months at -30°C, P. pacificensis cell numbers almost corresponded to those estimated within a month, i.e., 101% (SD = 3%) for 3/4 dilution samples and 98% (SD < 1%) for 1/4 dilution samples, while apparent decreases were found in total cell numbers, i.e., 96% (SD = 5%) for 3/4 dilution samples and 91% (SD < 1%) for 1/4 dilution samples. Numbers of cells hybridized with the Cont probe were estimated with each sample before dilution and were assumed to be below the detection limit, i.e., <0.5% of the total DAPI counts.

Instead of culture strains, target P. pacificensis cells were introduced into natural seawater collected from Tokyo Bay. Recovery of target P. pacificensis cells was demonstrated to be high, >97%, and reproducible among samples, with an error of <= 3% (Fig. 2B). A slightly higher cell count (108% ± 4%) was also obtained with FISH-DC (Fig. 2B) than with DC. These findings indicated that cell detachment from the PLL-coated filter during the hybridization and washing was negligible. In addition to the cell retention efficiency, the presence of enormous numbers of virus-sized DNA particles is a known source of error in natural seawater samples (8, 14). Some DAPI-stainable particles may be tightly retained on a polymer-coated filter and detectable only by greater image intensity enhancement, rather than by manned direct microscopic observation. In the present experiment, however, the contribution of such virus-like particles was very low, probably <3%. Most of the DAPI-stainable microbial cells in these seawater samples were clearly visualized with both Euba and Univ probes (Fig. 1e through g). Although seawater samples from Tokyo Bay had been prefiltered with a 10-µm-pore-size filter before analysis, domain Bacteria cells appeared to be abundant in the small particle fraction of seawater.

To achieve greater stringency in hybridization for specifying target cells at the single-species level, in general, some specific probes may be needed for strict hybridization conditions differing from those for domain-specific or other types of probes. In such cases, more than 2 cycles of hybridization and washing were required for FISH. We therefore chose the Psypac 469 probe for determining whether serious cell detachment from a PLL-coated filter occurs during repeated hybridization processes. First, in order to discriminate between P. pacificensis and P. glacincola cells, we defined a more stringent hybridization condition consisting of hybridization at 46°C with a hybridization solution supplemented with 35% formamide. After repeated hybridization and washing under three different conditions for use with each of the Psypac 469, Enba 338, and Univ 1390 probes, numbers of target microbial cells immobilized on the filter were determined (Table 1). No significant difference in cell number was found between the simultaneous hybridization and 3-cycle hybridization methods, demonstrating that the present method was effective for accurate detection and counting of P. pacificensis cells at the single-species level as well as for characterizing cells using domain-specific probes. From these results, we conclude that FISH-DC has a great advantage over previous combinations of FISH and DC for detecting and counting of specific microbial cells, in addition to total microbial population, in aquatic samples.

The direct cell trapping and immobilization procedure demonstrated here was highly effective for counting both total and specific microbial cells. In indirect trapping, about 40% of the cells are estimated to be lost during transfer and hybridization (20). Glöckner et al., who first applied the direct trapping technique to FISH, reported that about 10% of the total microbes were detached from Nuclepore filters during transfer, hybridization, and washing (7). We have found that cell-retaining efficiencies vary with samples and handling skill in the case of nonpolymer-coated Nuclepore filters (e.g., see Table 1). We have also found significant detachment of S. cerevisiae cells compared to results with other smaller microbial cells with general FISH, suggesting that cell transfer and adhesion efficiency may vary with cell size and membrane features.

In low-cell-density samples, such as oligotrophic or mesotrophic lake, river, or seawater samples, we must enumerate the total cell population by using DC in order to indirectly estimate target cell numbers by FISH. Thus, for the enumeration of the total or specific microbial cells, FISH-DC is faster, simpler, and more reliable than previous combinations of FISH and DC. Furthermore, the FISH-DC preparation enables long-term preservation of fixed microbial cells by freezing. Using the Tokyo Bay samples that had been stored at -30°C, no apparent changes were found in hybridization and counting of P. pacificensis cells even after 16 months (Fig. 2). This is an outstanding merit for marine microbiologists, who depend on on-board research. With hydrothermal fluid samples obtained from the southern East Pacific Rise using a submersible "Alvin," we have succeeded in detecting both total and specific microbial cells using DAPI and domain- and group-specific probes in 6 months at -30°C after the direct immobilization treatment on board (data not shown). Although a portion of seawater microbes became invisible during preservation lasting as long as 16 months (Fig. 2), probably due to lower DNA and RNA content than that of the introduced cells, the preservation as a frozen form for FISH-DC is much better with natural microbes than that as a liquid form for general DC. For example, DAPI-stainable components in microbial particles from deep-sea water used to disappear within a half-year in liquid preservation at 4°C, even with cell fixatives.

Coupled with other recent molecular and cellular detection techniques, such as target gene or signal enhancement (e.g., see references 18, 20, 24, and 27) and cell staining with green fluorescent high-affinity nucleic acid dyes (19, 26), FISH-DC is expected to advance population analyses of both total and specific microorganisms in natural aquatic environments, even if they have only a small number of target sequences. Cellular or molecular detection using the Psypac 469 probe may also be useful for monitoring an indicator microbe such as P. pacificensis in deep-ocean circulation and upwelling in the Pacific Ocean (17).


    ACKNOWLEDGMENTS

This work was supported by a grant from the Industrial Science and Technology Frontier Program from AIST and NEDO, Japan. Field research was partly funded by a grant from STA, Japan.

We thank project leader Ryuichiro Kurane, as well as Takanori Higashihara and Masumi Kubo, of NIBH and Takashi Tsuji of MKILS for their support of the marine microbial study. Thanks are also due to J. P. Bowman and T. A. McMeekin, University of Tasmania, for providing ACAM strains.


    FOOTNOTES

* Corresponding author. Mailing address: Marine Microbiology and Ecology Group, National Institute of Bioscience and Human Technology, Higashi, Tsukuba, Ibaraki 305-8566, Japan. Phone: 81-298-61-6062. Fax: 81-298-61-6412. E-mail: maruyama{at}nibh.go.jp.


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Applied and Environmental Microbiology, May 2000, p. 2211-2215, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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