plant surface microbiology.pdf

24 Microbial Community Analysis in the Rhizosphere 453
These glass slides are immersed in 50, 80 and 96 % ethanol for 3 min each and
stored at room temperature. Oligonucleotide probes (Table 1) labeled with
Cy3, Cy5 or 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS) at
the 5¢¢-end are used. The oligonucleotides are stored in distilled water at a
concentration of 50 ng/ml (Amann et al. 1990).
FISH was performed as described in detail, e.g., by Wagner et al. (1993) at
46 °C for 90 min in hybridization buffer (20 mM Tris-HCl, pH 7.2, 0.01 % SDS
and 5 mM EDTA) containing 0.9 M NaCl and formamide at the percentages
shown in Table 1. Hybridization was followed by a stringent washing step at
48 °C for 15 min. The washing buffer was removed by rinsing the slides with
distilled water. Counterstaining with DAPI and mounting in Citifluor AF1
(Citifluor Ltd., London, UK) was performed as described previously (Aßmus
et al. 1995).
The microscopic in situ analysis can be performed using an LSM 410 or
LSM 510 inverted confocal laser scanning microscope (Zeiss, Jena, Germany),
equipped with two lasers (Ar-ion UV; Ar-ion visible; HeNe) supplying excitation
wavelengths at 365, 488, 543 and 633 nm, respectively. Sequentially
recorded images are assigned to the respective fluorescence color and then
merged into a true color display. All image combining and processing is performed
with the standard software provided by Zeiss.
Using the general cell/DNA staining with DAPI and FISH with probes specific
for the domain bacteria and group-specific probes (Table 1),bacteria can simultaneously
be localized and identified at the rhizoplane.In addition to the groupspecific
probes, in situ binding genus- and species-specific oligonucleotide
probes are available for a number of root-associated and symbiotic bacteria
(Ludwig et al. 1998; Hartmann et al. 2000). Figure 2A shows the localization of
Azospirillum brasilense in the wheat rhizosphere by FISH (combination of two
differently labeled oligonucleotide probes Eub338-Cy3 and Abras1420-Cy5)
and CSLM. The application software “orthogonal view” of the LSM 510 (Zeiss,
Germany) allows the display of optical cuts through the sample in xz and yz sections
(Fig.2B).The localization within the tissue is clearly visible.
2.2 Immunofluorescence Labeling Combined with Fluorescence in Situ
Hybridization
The combination of FISH, which allows a phylogenetic identification of bacteria
from the phylum down to the species level, with immunological
approaches extends the in situ identification to the individual strain level, if
strain-specific antisera or special monoclonal antibodies are applied. Antibodies
directed against bacterial surface antigens can be created by using,
e.g., UV-inactivated bacteria as antigens (Schloter et al. 1995; Hartmann et al.
1997). In addition, antibodies can also be created to identify specific enzymes,
e.g., denitrifying enzymes (Bothe et al. 2000) and thus add a phenotypic or