The test species used in this study included representatives of several major protistan taxa, such as alveolates, stramenopiles, kinetoplastids, and cryptomonads (Table ). Cultures of all species were obtained from Carolina Biological Supply (Burlington, N.C.), except the ciliate Euplotes sp., which was isolated from the Sippewisset salt marsh, Cape Cod, Mass.
Classification of strains used for this study
We tested several standard and modified fixative mixtures (11
) for the ability to impart sufficient rigidity to the test cells. The list included paraformaldehyde (tested at 2, 4, and 10%; also tested at 2% supplemented with 0.2% glutaraldehyde), formalin (tested at the same concentrations and combinations as described for paraformaldehyde), Schaudinn's fixative (2 parts saturated aqueous HgCl2
, 1 part 100% ethanol, and 1% [final concentration] glacial acetic acid; 1 part of this fixative was diluted with 3 parts distilled water and added to the sample at a ratio of 1:1 [vol/vol]), Steve's fixative (76% saturated aqueous HgCl2
, 20% formaldehyde, and 4% glacial acetic acid mixed with 3 parts distilled water and added to the sample at a ratio of 1:1 [vol/vol]), and Bouin's fixative mixture (buffered formaldehyde saturated with picric acid and 2% glacial acetic acid added immediately before fixation) tested at 50% with or without the addition of 0.1 to 0.2% glutaraldehyde. The fixation time in all cases was 45 min at 4°C.
FISH probes and probe design.
FISH was used to visualize the marine ciliate Euplotes
sp. both in environmental samples and mixed with other test protists (Table ) as negative controls. We designed two oligonucleotide probes to target different regions of the 18S rRNA. A set of probes (nucleotides, 18 to 22; GC contents, 50 to 60%; nucleotide-nucleotide Tm
s, ≥57°C) was designed for each Euplotes
small-subunit (SSU) rRNA sequence retrieved from GenBank by use of the online tool Primer3 (19
). Generated probes were checked against the GenBank sequence collection by a standard nucleotide-nucleotide BLAST search (1
) and were compared to an accessibility map of the SSU rRNA of Escherichia coli
for hints of probe target sites with promising high signal intensities (8
). The potential for hairpin and dimer formation of selected oligonucleotides was assessed by use of the program mfold, v. 3.0 (21
). From the original 54 probe candidates, two oligonucleotides were chosen that fulfilled the general criteria of potentially successful probes (10
). The probe Eupl240 (5′-TCATCTCAGTAGACCTTGCG-3′) had no mismatches with the SSU rRNA of all but one Euplotes
species (E. raikovi
) but exhibited a 3-bp mismatch with the next closest sequence in GenBank. The probe Eupl1780 (5′-GACAGTCCAAAGAGGTTCAC-3′) matched all 33 Euplotes
sp. SSU rRNA sequences deposited in GenBank and had a mismatch of 3 bp with the next closest GenBank sequence. The probes used were purified by high-performance liquid chromatography and labeled with Cy-3 at the 5′ end by the manufacturer (Integrated DNA Technologies, Coralville, Iowa).
Other probes used included the universal Cy-3-labeled eukaryotic probe Euk1209R (5′-GGGCATCACAGACCTG-3′) (9
) and its Cy-3-labeled complement as a nonsense probe.
We used a standard protocol for in situ hybridization (16
), with several important modifications. Preserved samples were placed in wells with bottoms made of polycarbonate membranes (25-mm diameter; 0.4-μm pore size) (Costar Transwells; Corning Inc., Corning, N.Y.). The wells were placed on the base of standard 25-mm-diameter glass filtration units (Millipore, Bedford, Mass.) (Fig. ). To ensure an even cell distribution on the well's membrane, a 5.0-μm-pore-size 25-mm-diameter cellulose nitrate filter (Whatman, Newton, Mass.) was inserted between the well's bottom and the glass base. The excess of fixative was removed by gentle filtration (<200 mm Hg), always leaving a small amount of liquid covering the membrane. The cells were washed by gradually replacing the remains of the fixative with 1× phosphate-buffered saline (PBS) (0.14 M NaCl, 2.7 mM KCl, 4.3 mM Na2
, 1.4 mM KH2
PO4, pH 7.4). This was achieved in three to five cycles (in the case of Bouin's fixative, washing was continued until the yellow coloration disappeared), each consisting of adding 2 ml of fresh PBS and gently removing it by applying a weak vacuum. We found it essential for the preservation of cell shape to never expose the membrane surface (and thus cells) to drying. The washing buffer was then gradually replaced with a hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl [pH 8], 30% formamide [the optimal concentration was found in preliminary experiments], and 0.01% sodium dodecyl sulfate). This was done in two cycles, each consisting of adding and then removing 2 ml of the fresh buffer. The wells, with approximately 300 μl of hybridization buffer, were transferred to a six-well Transwell tissue culture tray, and 50 μl of probe solution (30 ng μl−1
in molecular-grade distilled water) was added to each well. The wells received either a universal eukaryotic, nonsense, or Euplotes
-specific probe (separately). The wells with probes added were transferred into glass inserts (stacking Stender dish; 27-mm inner diameter; volume, 8 ml), which were placed in a tightly sealed incubation chamber (plastic jar; 45-mm inner diameter; volume, 70 ml) containing a piece of hybridization buffer-saturated tissue paper at the bottom of the chamber. The incubation chambers with the wells were incubated for 2 h at 46°C. After incubation, 2 ml of a preheated (48°C) washing buffer (0.9 M NaCl, 20 mM Tris-HCl [pH 8], 5 mM EDTA, 0.01% sodium dodecyl sulfate) was added to the wells, which were further incubated for 10 min at 48°C. The wells were then placed back on the base of the filtration units, and their contents were washed by several cycles of addition and then removal of distilled water. As before, special care was taken not to expose the filter to the air. With approximately 300 μl of the last wash still in the well, 500 μl of a DAPI (4′,6′-diamidino-2-phenylindole) solution (1.5 μg ml−1
; Sigma-Aldrich, St. Louis, Mo.) was added and incubated in the dark for 5 min at room temperature. The DAPI solution was removed by washing the filter three times with 1 ml of distilled water using the vacuum filtration unit as described above.
FIG. 1. Several essential steps for localization of target cells in the combined FISH-SEM method. The images show the Transwell filtration system used to collect and process cells (A), marking of the circular area with the target cell(s) in the middle (B and (more ...) Preparing cells for SEM.
The probe-stained and DAPI-counterstained cells were dehydrated as follows. The distilled water remaining in the well after DAPI staining was gradually replaced by washing the filter two times with 30% ethanol, using the vacuum filtration system as described above. The wells were transferred to the tissue culture tray, a fresh portion (2 ml) of 30% ethanol was added, and the wells were left in the dark for 12 min. This process was sequentially repeated with 50, 70, 85, 95, and 100% ethanol (the last step was repeated three times for 15 min each). As before, it was essential that the filter not be exposed to air at any point during the procedure. After the last portion of 100% ethanol was removed by use of a vacuum (except for a small amount that was just enough to cover the membrane), the wells were quickly placed into a tissue culture tray, covered with 2 ml of a mixture of 100% ethanol-hexamethyldisilazane (Electron Microscopy Science, Ft. Washington, Pa.) (1:1 [vol/vol]), and allowed to be infiltrated for 20 min in the dark. This was followed by two 20-min infiltration steps with 100% hexamethyldisilazane. At this point, cells were allowed to air dry, as this caused no significant cell distortion. In this state, the filter can be stored in a dark and dry place at room temperature for at least 1 week with no losses in fluorescence intensity.
The air-dried filters were cut out of the wells with a dissecting knife and mounted on an adhesive silicone spacer (Schleicher & Schuell MicroScience, Riviera Beach, Fla.) affixed to a glass microscope slide. The filters were scanned under appropriate epifluorescent illumination on Zeiss Axioplan 2MOT and Axioskop 50 compound microscopes equipped with an HBO 100-W mercury lamp; ×10 Neofluar, ×40 (dry) Neofluar, ×40 (oil) F-Fluar, and ×100 Apo objectives; DAPI- and Cy-3-specific filter sets; and an Hitachi ORCA cooled charge-coupled device camera (Hamamatsu, Hamamatsu City, Japan) operated by the OpenLab software package (Improvision Inc., Lexington, Mass.). Once the positively Cy-3-stained target cell was located by use of the ×10 and ×40 (dry) objectives, a filter area of approximately 5 mm in diameter around the cell was marked with a needle; these and other steps are illustrated in Fig. . The marked area was cut out under a dissection microscope (Zeiss Stemi 2000-C) with a dissecting knife. The cut-out piece was checked once again under the fluorescence microscope (×10 and ×40 dry objectives) to confirm the presence and location of the target cell(s). At this point, a digital photograph of the target cell(s) and surrounding areas was taken, with special care taken to capture small landmarks (atypical cell aggregations, unusually shaped particles, etc.). This facilitated locating the cell under SEM. The cut-out piece was then attached to a carbon adhesive tab, mounted on an SEM specimen holder, and sputter coated with 10 to 15 nm of gold-palladium (60:40) by use of a Tousimis Samsputter 2A. SEM was performed on an Amray AMR-1000 scanning electron microscope. The target cells were located at a low SEM magnification, using photographs taken with the epifluorescence microscope.
Some filters were used to examine the Cy-3-labeled cells by epifluorescence rather than SEM. These were mounted on a glass slide, a mixture of 1 part VectaShield (Vector Laboratories, Burlingame, Calif.) and 4 parts Citifluor AF1 (Citifluor, London, United Kingdom) (vol:vol) was added, and a coverslip was gently applied at an angle. This allowed the use of high-numerical-aperture (NA) lenses (×40 oil and ×100 oil) to obtain high-quality epifluorescence images of the target cells. Throughout the project, the principal charge-coupled device camera parameters remained constant (the exposure time was 0.2 s, and all other parameters were at their default settings), except in control preparations, in which cases the exposure time was increased to 2.0 s to avoid featureless blank photos and achieve at least some visualization of the control specimens.
Target cell recovery.
We evaluated the usefulness of our FISH protocol to detect and quantify the target cells in complex species mixes and in environmental samples. The species mixes were prepared using the strains listed in Table . Known numbers of Euplotes sp. cells were added to vials with 3 ml of artificial protistan mixes to achieve concentrations of 2, 4, and 8 Euplotes sp. cells/ml. The contents of five vials for each Euplotes sp. concentration were fixed and hybridized to Euplotes-specific probes as well as to the universal and nonsense probes as described above. The negative control contained no probe. All filters were counterstained with DAPI (see above) to obtain the total protistan count. The resulting filters were scanned to examine the specific and nonspecific staining of target and nontarget cells and to count the Cy-3-labeled Euplotes spp.
In addition, 10 Euplotes sp. cells were added to four samples (10 ml each) obtained from Sippewisset salt marsh, which were subsequently stained with the two Euplotes-specific probes. Controls included environmental samples with naturally occurring Euplotes spp. only. For each probe and control treatment, duplicate samples were processed as described above, and duplicate filters were prepared and scanned to detect the Cy-3-labeled target and nontarget cells.