192
L. Giuliano et aI.
ty in the original sample, the data presented give
a further contribution for checking the previously proposed method.
Materials and Methods
Sampling Site and Procedure
The DYFAMED station (430 25' Nand 070 52' E),
is an oligotrophic pelagic station located in the
Ligurian Sea (northwestern Mediterranean Sea),
28 miles from the coast of Nice. Despite the general oligotrophic conditions, some eutrophication events usually occur in the euphotic layers
of the sampled area in spring time (Minas and
Minas 1990; Morel and Andre 1991; Prieur 1981).
During the DYFABAC 07 cruise (IS to 21 May
1996), aboard the Tethys II research vessel, water
samples were collected with 61 Niskin bottles
attached to a CTD (conductivity-temperaturedepth) rosette from 30 m depth layer. Processing
of samples on board the research vessel was previously described (Bianchi and Giuliano 1996).
Direct Bacterial Counts
Formalin-fixed subsamples of 5 to 20 ml were filtered with black membrane filters (pore-size.
0.22 Jll11; diameter, 25 mm; Nudepore) and
stained with 4'.6' -diamidino-2-phenylindole
(DAPI), and bacterial abundances were determined by epifluorescence microscopy {Porter
and Feig 1980}.
Design and Characterization
of Oligonucleotide Probes
Original samples were diluted in unamended,
previously filtered, sterilized seawater to the
extent of extinction in order to obtain enrichments of numerically representative oligotrophic
marine bacteria. Detailed information about the
utilized dilution culture technique, the DNA
extraction, amplification, cloning and the analyses of the amplified products were previously
reported (Giuliano et al. 1998). Briefly, water
samples for DNA extraction were obtained from
the highest positive seawater dilution of the natural samples. After filtration of the diluted sample, the total genomic DNA was extracted by following the protocol of Fuhrman et aI. (1988).
Almost full-length bacterial16S rDNA fragments
were amplified by PCR from the extracted DNA
by using two general bacterial 168 rDNA
primers, 16F27 and 16R1492 (Lane 1991). After a
previous analysis of restriction fragments
(RFLP), eight clones were selected and their 16S
rDNA inserts were fully sequenced. The clone
identifications, EMBL accession numbers, and
nearest relatives in the database are presented in
the Table. Six either clone- or group-specific
oligonucleotide probes were constructed and
evaluated with the RDP programme
CHECKYROBE (Larsen et al 1994). The
sequences and target positions of the designed
probes are given in the Table. After optimization
and specificity study. the digoxigenin-l1-ddUTP
(DIG)-labelled probes, purchased from Gibco
BRL (Germany) were utilized for the screening of
the clone libraries. For in situ hybridization. the
same probes were labelled with the indo carbocyanine dye Cy3.
FISH
The FISH of filter sections from the 30 m depth
sample with oligonucleotide probes, counterstaining with DAPI. mounting for microscopic
evaluation and epifluorescence microscopy were
performed following the protocol of Glockner et
aL (1996). Cells were observed using an Axioplan
epifluorescence microscope (Zeiss, lena,
Germany) equipped with a SOW mercury high
pressure bulb and specific mter sets (DAPI: Zeiss
01, CY3: Zeiss 15). Colour photomicrographs
were done on Kodak Ektachrome 1600
(Rochester, NY, USA). Exposure times were 15 s
for DAP! and 20 s for CY3.
Results
Cell density determined by DAPI staining was
9.6x10 s cells ml-I.An increase of mean cell number ml- 1 at a second survey, to 1.98x10 6 cells ml- 1 ,
in agreement with an increase of the chlorophyll
a values (data not shown) indicated a bloom
event evolution in the sampled area, at the sampIing time.
Between 15 and 23% of all DAPI-stained
objects (mean, 19%) were detected by the groupspecific G Rb probe, and between 20 and 28%
L. Giuliano et aI.
ty in the original sample, the data presented give
a further contribution for checking the previously proposed method.
Materials and Methods
Sampling Site and Procedure
The DYFAMED station (430 25' Nand 070 52' E),
is an oligotrophic pelagic station located in the
Ligurian Sea (northwestern Mediterranean Sea),
28 miles from the coast of Nice. Despite the general oligotrophic conditions, some eutrophication events usually occur in the euphotic layers
of the sampled area in spring time (Minas and
Minas 1990; Morel and Andre 1991; Prieur 1981).
During the DYFABAC 07 cruise (IS to 21 May
1996), aboard the Tethys II research vessel, water
samples were collected with 61 Niskin bottles
attached to a CTD (conductivity-temperaturedepth) rosette from 30 m depth layer. Processing
of samples on board the research vessel was previously described (Bianchi and Giuliano 1996).
Direct Bacterial Counts
Formalin-fixed subsamples of 5 to 20 ml were filtered with black membrane filters (pore-size.
0.22 Jll11; diameter, 25 mm; Nudepore) and
stained with 4'.6' -diamidino-2-phenylindole
(DAPI), and bacterial abundances were determined by epifluorescence microscopy {Porter
and Feig 1980}.
Design and Characterization
of Oligonucleotide Probes
Original samples were diluted in unamended,
previously filtered, sterilized seawater to the
extent of extinction in order to obtain enrichments of numerically representative oligotrophic
marine bacteria. Detailed information about the
utilized dilution culture technique, the DNA
extraction, amplification, cloning and the analyses of the amplified products were previously
reported (Giuliano et al. 1998). Briefly, water
samples for DNA extraction were obtained from
the highest positive seawater dilution of the natural samples. After filtration of the diluted sample, the total genomic DNA was extracted by following the protocol of Fuhrman et aI. (1988).
Almost full-length bacterial16S rDNA fragments
were amplified by PCR from the extracted DNA
by using two general bacterial 168 rDNA
primers, 16F27 and 16R1492 (Lane 1991). After a
previous analysis of restriction fragments
(RFLP), eight clones were selected and their 16S
rDNA inserts were fully sequenced. The clone
identifications, EMBL accession numbers, and
nearest relatives in the database are presented in
the Table. Six either clone- or group-specific
oligonucleotide probes were constructed and
evaluated with the RDP programme
CHECKYROBE (Larsen et al 1994). The
sequences and target positions of the designed
probes are given in the Table. After optimization
and specificity study. the digoxigenin-l1-ddUTP
(DIG)-labelled probes, purchased from Gibco
BRL (Germany) were utilized for the screening of
the clone libraries. For in situ hybridization. the
same probes were labelled with the indo carbocyanine dye Cy3.
FISH
The FISH of filter sections from the 30 m depth
sample with oligonucleotide probes, counterstaining with DAPI. mounting for microscopic
evaluation and epifluorescence microscopy were
performed following the protocol of Glockner et
aL (1996). Cells were observed using an Axioplan
epifluorescence microscope (Zeiss, lena,
Germany) equipped with a SOW mercury high
pressure bulb and specific mter sets (DAPI: Zeiss
01, CY3: Zeiss 15). Colour photomicrographs
were done on Kodak Ektachrome 1600
(Rochester, NY, USA). Exposure times were 15 s
for DAP! and 20 s for CY3.
Results
Cell density determined by DAPI staining was
9.6x10 s cells ml-I.An increase of mean cell number ml- 1 at a second survey, to 1.98x10 6 cells ml- 1 ,
in agreement with an increase of the chlorophyll
a values (data not shown) indicated a bloom
event evolution in the sampled area, at the sampIing time.
Between 15 and 23% of all DAPI-stained
objects (mean, 19%) were detected by the groupspecific G Rb probe, and between 20 and 28%