Journal

of
Microbiological
Journal of Microbiological Methods 41 (2000) 85–112
Methods
www.elsevier.com / locate / jmicmeth

Invited review

Fluorescence in situ hybridization (FISH) for direct visualization of

microorganisms
Annette Moter*, Ulf B. Gobel
¨
¨ Mikrobiologie und Hygiene, Universitatsklinikum
Institut f ur ¨ Charite´ , Humboldt-Universitat
¨ zu Berlin, Dorotheen Str. 96,
D-10117 Berlin, Germany
Received 28 January 2000; received in revised form 10 April 2000; accepted 17 April 2000

Abstract

As a technique allowing simultaneous visualization, identification, enumeration and localization of individual microbial
cells, fluorescence in situ hybridization (FISH) is useful for many applications in all fields of microbiology. FISH not only
allows the detection of culturable microorganisms, but also of yet-to-be cultured (so-called unculturable) organisms, and can
therefore help in understanding complex microbial communities. In this review, methodological aspects, as well as problems
and pitfalls of FISH are discussed in an examination of past, present and future applications.  2000 Elsevier Science B.V.
All rights reserved.

Keywords: FISH; Fluorescence; In situ hybridization; 16S rRNA; Probes

1. Introduction already used successfully in clinical microbiology to

The ultimate goal of diagnostic microbiology is
the rapid and accurate identification of bacteria in
their natural environments. Culture-based methods
are time consuming and are often too selective,
particularly for fastidious or yet-to-be cultured bac-
teria, and therefore this approach does not reflect the
exact composition of mixed bacterial communities or
microbial diversity in infections (Wagner et al.,
1993; Choi et al., 1994). During recent years,
molecular techniques like PCR and subsequent hy-
bridization or sequencing have revolutionized all
fields of microbiology, and sensitive detection and
exact identification of bacteria are possible. They are
*Corresponding author. Tel.: 149-30-2093-4708; fax: 149-30-
2093-4703.
E-mail address: annette.moter@charite.de (A. Moter).
detect slow growing organisms (e.g. Mycobacteria),
or organisms that are difficult to culture, e.g.
Tropheryma whippelii. However, these methods do
not provide information about morphology, number,
spatial distribution or the cellular environment of the
organisms. Microscopic analyses using bacterial
stains like Gram or Ziehl-Neelsen stains are old but
quite useful techniques. They are fast and cheap,
combining direct visualization and crude characteri-
zation of the bacteria by providing information on
the structure of their cell wall. Due to the paucity of
morphological distinctions among bacteria, such
simple microscopic methods do not allow the reliable
identification. This obstacle can be overcome by
immunofluorescence using species-specific monoclo-
nal antibodies. This technique in turn is often
hampered by unspecific binding, and it depends
heavily on phenotypic antigen variation and expres-

0167-7012 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
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86 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112

sion. In addition the target microorganism must be phylogenetic, ecologic, diagnostic, and environmen-
cultured first to raise a specific antibody. Further- tal studies in microbiology (Amann et al., 1990b).
more, the size of antibodies can restrict easy access
to their target antigens in tissues or biofilms
(Szwerinski et al., 1985). Moreover, it has been 3. Technical aspects: How does it work?
shown that cross-reactivity was a severe problem
when applying monoclonal antibodies to study com- Fluorescence in situ hybridization detects nucleic
plex microbiota. Therefore, this method can not be acid sequences by a fluorescently labeled probe that
regarded as completely culture independent. In con- hybridizes specifically to its complementary target
trast FISH combines the precision of molecular sequence within the intact cell. The procedure in-
genetics with the visual information from micro- cludes the following steps (Fig. 1): (i) fixation of the
scopy, to permit visualization and identification of specimen; (ii) preparation of the sample, possibly
individual microbial cells within their natural mi- including specific pretreatment steps; (iii) hybridiza-
crohabitat or diseased tissue. tion with the respective probes for detecting the
respective target sequences; (iv) washing steps to
remove unbound probes; (v) mounting, visualization
and documentation of results. In the following
2. History sections we concentrate on the most commonly used
techniques only.
In situ hybridization (ISH) was independently
developed by two research groups (Pardue and Gall, 3.1. Probes and labeling
1969; John et al., 1969). Radiolabeled DNA or 28S
RNA was hybridized to cytological preparations The choice of probes for FISH must consider
from Xenopus oocytes and detected by mi- specificity, sensitivity and ease of tissue penetration.
croautoradiography. This technique allowed nucleic A typical oligonucleotide probe is between 15 and 30
acid sequences to be examined inside cells without base pair (bp) in length and is generated on an
altering the cell’s morphology or the integrity of its automated synthesizer. Short probes have an easier
various compartments. Since then, ISH has been
modified for studies of chromosomal evolution,
chromosome analysis of tumors and leukaemias, and
cytogenetic studies of a wide range of species. It was
finally introduced into bacteriology by Giovannoni et
al. (1988), who was the first to use radioactively
labeled rRNA-directed oligonucleotide probes for the
microscopic detection of bacteria. With the develop-
ment of fluorescent labels (Landegent et al., 1984;
Pinkel et al., 1986; Pinkel et al., 1988), radioactive
labels were steadily supplanted by non-isotopic dyes.
In 1989, DeLong first used fluorescently labeled
oligonucleotides for the detection of single microbial
cells. Compared to the radioactive probes, fluores-
cent probes are safer, they offer better resolution and
do not need additional detection steps. Moreover,
fluorescent probes can be labeled with dyes of
different emission wavelength thus enabling detec-
tion of several target sequences within a single
hybridization step. Over the last decade, sensitivity Fig. 1. Flow chart of a typical FISH procedure. See text for
and speed have made FISH a powerful tool for details.
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A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
access to their target, but they might carry fewer
labels. There are different ways of labeling. Direct
fluorescent labeling is most commonly used and is
also the fastest, cheapest and easiest way because it
does not require any further detection steps after
hybridization. One or more fluorescent dye mole-
cules are directly bound to the oligonucleotide either
chemically during synthesis through an aminolinker
at the 59-end of the probe [Fig. 2(a)], or enzymatical-
ly using terminal transferase to attach fluorescently
labeled nucleotides at the 39-end [Fig. 2(b)] (Moter
et al., 1998a). Coupling of Fluorescein–Isothio-
cyanate (FITC) to the oligonucleotide via an 18-
carbon spacer may increase signal intensity as com-
pared to a directly conjugated probe (Deere et al.,
1998). An increase in fluorescence signal has also
been reported by labeling probes at both ends, one
fluorescent molecule at the 39-end and four mole-
cules at the 59-end using appropriate spacers to
prevent quenching of fluorescence (Spear et al.,
1999). Fluorescence labeled oligonucleotides are
now commercially available. They can be stored at
2 208C in the dark for several months. In some
cases, indirect detection is helpful; it has been shown
that the sensitivity of the FISH assays can be
increased by linking the probe to a reporter molecule
like digoxygenin (DIG) that is then detected by a
fluorescent antibody [Fig. 2(c)] (Zarda et al., 1991).
Fig. 2. Direct, (a) and (b), and indirect, (c)–(e), labeling of probes
using digoxygenin (DIG), horseradish peroxidase (HRP) or
Tyramide Signal Amplification (TSA).
87
Sensitivity of FISH can be further increased using
enzymatic signal amplification. This method was
developed by Kagiyama et al. (1993) for cytogenetic
investigations and later adapted for the detection of
bacteria by Yamaguchi et al. (1996). A digoxygenin-
labeled oligonucleotide is detected by an anti-digox-
ygenin antibody that is coupled to alkaline phospha-
tase. This enzyme converts HNPP (2-hydroxy-3-
naphtoic acid-29-phenylanilide phosphate) to its de-
phosphorylated form that generates bright-red fluo-
rescence in combination with Fast Red TR. Fluores-
cence was reported to be eight times more intensive
than that of oligonucleotides carrying a single label.
This approach of enzymatic signal amplification was
further improved by a technique called ‘Tyramide
Signal Amplification (TSA)’ system. Oligonucleo-
tides were labeled with horseradish peroxidase
(HRP) that used fluorescein–tyramide as substrate
¨
[Fig. 2(d)] (Schonhuber et al., 1997). The TSA
system resulted in a 10–20-fold increase in signal
intensity. However, the number of positive cells was
clearly reduced compared to the conventional use of
monolabeled probes. This might be due to insuffi-
cient penetration of the high molecular weight
reagents into the bacterial cells. Although per-
meabilization of cells by lysozyme improved signal
intensity, this approach does not seem applicable to
certain Gram-positive species and hence for analysis
of many mixed bacterial populations. The sensitivity
of FISH in natural samples was significantly in-
creased using polyribonucleotide probes labeled with
several fluorochrome molecules (DeLong et al.,
1999). Probably the most sensitive approach is the
combined use of polyribonucleotide probes, internal-
ly labeled with digoxygenin, with the tyramide signal
amplification system. This technique was successful-
ly used for the detection and visualization of a
virulence factor in Listeria [Fig. 2(e)] (Wagner et al.,
1998).
3.2. Fluorescent dyes
Fluorochromes with different excitation and emis-
sion maxima allow simultaneous detection of two or
more microorganisms. For simultaneous microscopic
observation of multicolour FISH, multibandpass
filters may be used. The combined fluorochromes
should have sharp emission peaks to prevent spectral
88 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
overlap between probes, thereby eliminating back-
ground problems and bleed-through. The brightest,
most photostable dye should be used for low-abun-
dance targets. Dyes commonly used for FISH in
microbiology are fluorescein-derivates (Fluorescein–
Isothiocyanate (FITC), 5-(-6-)carboxyfluorescein–N-
hydroxysuccimide-ester (FluoX)) and rhodamine-de-
rivates (Tetramethyl–Rhodamine–Isothiocyanate
(TRITC), Texas Red), and more recently cyanine
dyes like Cy3 and Cy5 (Table 1). The latter ones
have been shown to be superior to the classical dyes
because they provide significant brighter staining and
are very stable to photobleaching (Wessendorf and
Brelje, 1992). Blue fluorescent counterstaining can
be performed with aromatic diamidines like 49,6-
diamidino–2-phenylidole dihydrochloride (DAPI)
that non-intercalatively binds to DNA with great
¨
affinity (Zimmer and Wahnert, 1986). For further
advice regarding fluorescent probes see Cullander
(1999).
3.3. Ribosomal RNA as a target for FISH
In microbiology the most commonly used target
molecule for FISH is 16S rRNA because of its
genetic stability, its domain structure with conserved
and variable regions, and its high copy number
(Woese, 1987). Oligonucleotide probes for each
taxonomic level, between bacterial and archaeal,
down to genus-specific and species-specific can be
designed according to the region of rRNA targeted
Table 1
Fluorochromes used for the detection of microorganisms by FISH
Fluorochrome
AMCA
Fluorescein–isothiocyanate (FITC)
5-(-6-)carboxyfluorescein–N-hydroxysuccimide-
ester (FluoXE)
Tetramethyl–rhodamine–isothiocyanate (TRITC)
Texas Red 
Cyanine Dyes
Cy3E
Cy5E
a
Wavelengths may vary slightly depending on the respective manufacturer. Emission spectra might change in different solvents or sample
conditions (Cullander, 1999).
¨
(for review see Gobel, 1991 and Amann et al.,
1995). With steadily growing sequence data entries
in public or commercially available databases
(Maidak et al., 2000; Van de Peer et al., 2000), the
16S rRNA gene offers the greatest ease and accuracy
in identification for most microorganisms. This is
especially important when dealing with mixed bac-
terial populations or unculturable organisms. The
high copy number of 16S rRNA in each replicating
and metabolically active cell usually offers sufficient
target to visualize single bacterial cells even with
monolabeled oligonucleotides in FISH. The choice
of target site and design of probes should be done
with greatest care. Appropriate software for rational
development of probes like the ARB program pack-
age (see Strunk, O., Gross, O., Reichel, B., May, M.,
Hermann, S., Stuckmann, N., Nohhoff, B., Ginhart,
T., Vilbig, A., Lenke, M., Ludwig, T., Bode, A.,
Schleifer, K.-H., Ludwig, W. ARB: a software en-
vironment for sequence data; http: / / www.mik-
ro.biologie.tu-muenchen.de. Department of Micro-
biology, Technical University Munich, Munich, Ger-
many.) are available. Practicality and limitations of
16S rRNA comparative sequence analysis, and the
design of rRNA probes has been reviewed before
(Weisburg et al., 1991; Amann et al., 1995; Fredricks
and Relman, 1996; von Wintzingerode et al., 1997).
Other targets like 23S rRNA (Manz et al., 1993;
Amann et al., 1995; Nordentoft et al., 1997; Lemke
et al., 1997; Trebesius et al., 1998), 18S rRNA
(Lischewski et al., 1996; Lischewski et al., 1997; Li
Wavelength a Color
Excitation (nm) Emission (nm)
351 450 Blue
492 528 Green
488 520 Green
557 576 Red
578 600 Red
550 570 Orange / red
651 674 Infrared
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A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
et al., 1997a; Spear et al., 1999) and recently mRNA
(Wagner et al., 1998) have also been successfully
detected by FISH.
3.4. Fixation
Prior to hybridization bacteria have to be fixed and
permeabilized for penetration of the fluorescent
probes into the cell and to protect the RNA from
degradation by endogenous RNAses. Fixation can
employ precipitating agents like ethanol or methanol,
cross-linking agents like aldehydes, or a mixture
thereof. Fixation conditions may vary dependent
upon the target organism and the type of sample or
tissue. Effective fixation is crucial for satisfactory
FISH results, but unfortunately it is difficult to
optimize. Optimal fixation should result in good
probe penetration, retention of the maximal level of
target RNA, and maintenance of cell integrity and
morphologic detail. In general a 3–4% (v / v) form-
aldehyde or paraformaldehyde solution is sufficient
for most Gram-negative bacteria. For Gram-positive
organisms, ethanol (50%), ethanol / formalin (9:1 v /
v) or heat treatment is recommended (Jurtshuk et al.,
1992; Brown-Howland et al., 1992; Roller et al.,
1994) and additional pretreatments might be neces-
sary for permeabilization (see below).
3.5. Specimen preparation and pretreatment
For better attachment of specimens to glass slides,
it is recommended to treat the surfaces first with a
coating agent. Chemicals that have been successfully
used include gelatin (Amann et al., 1990b), poly-L-
lysine (Lee et al., 1999) or silanating agents (Moter
et al., 1998b). If cell suspensions are investigated,
bacteria are fixed in suspension, spotted onto micro-
scopic slides, air dried, and dehydrated in an ethanol
series. In some cases, e.g. for Gram-positive cells, an
additional enzymatic treatment with lysozyme
¨
(Schonhuber et al., 1997; Wagner et al., 1998),
lysostaphin, or an enzyme mixture (Krimmer et al.,
1999) may be necessary to open the peptidoglycan
layer. For mycolic acid-containing Actinomycetales
like the Mycobacterium complex, Norcardia or for
Microthrix parvicella, permeabilization techniques
89
like mild acid hydrolysis with 1 M HCl or treatment
with mutanolysin or 1,4 dithio-L-threitol (Macnaug-
hton et al., 1994; Schuppler et al., 1998; Erhart et al.,
1997) have been evaluated. Since the optimal fixa-
tion and pretreatment procedures for the G 1 C rich
species are strain dependent, analysis of complex
environmental samples containing these bacteria
remains difficult (Macnaughton et al., 1994; De Los
Reyes et al., 1997).
For FISH on tissue sections, pretreatment pro-
cedures have been applied to increase accessibility of
the probe to the target and to reduce non-specific
binding. For paraffin sections, de-waxing with xylene
prior to the hybridization step is mandatory (Boye et
al., 1998). An additional step for paraffin as well as
for cryo-sections is pretreatment with proteinase K
(Loy et al., 1996). Recently, FISH has been applied
to tissue embedded in cold polymerizing resin
(Moter et al., 1998b). In these plastic sections,
visualization of bacteria and excellent histological
conservation of the tissue was achieved without any
digestive pretreatment (Figs. 5–9).
3.6. Hybridization
Hybridization must be carried out under stringent
conditions for proper annealing of the probe to the
target sequence. For this crucial step of the FISH
procedure, preheated hybridization buffer is applied
to the sample containing fluorescently labeled probes
complementary to the target RNA. Stringency can be
adjusted by varying either the formamide concen-
tration or the hybridization temperature. Formamide
decreases the melting temperature by weakening the
hydrogen bonds, thus enabling lower temperatures to
be used with high stringency.
The hybridization takes place in a dark humid
chamber, usually at temperatures between 378C and
508C. Hybridization time varies between 30 min and
several hours. Afterwards slides are briefly rinsed
with distilled water to remove unbound probe. Post-
hybridization stringency washes are performed as
required. To reduce the amount of toxic waste,
stringency of the washing buffer can be regulated by
varying the salt concentration (Lathe, 1985) instead
of by using formamide. Finally, slides are rinsed
with water again, dried and mounted. Mounting
90 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
Fig. 3. Autofluorescence of paraformaldehyde-fixed Candida albicans cells. Note the different intensity of autofluorescence (kindly
provided by Dr. B. Graf).
media containing antifading agents are commercially
available.
3.7. Documentation
For microscopy, a conventional epifluorescence
microscope equipped with narrow-band-pass filter
sets for multicolour FISH can be used. Use of a
charged coupled device (CCD) camera and appro-
priate image analysis software allows digitalization
and manipulation of images. This is currently the
most sensitive system and can be helpful for critical
samples where low signal intensity is expected. It
has been used for microscopic enumeration of micro-
organisms (Li et al., 1997a) and for measuring the
activity of single cells in biofilms by quantification
of rRNA content (Poulsen et al., 1993). Recently,
digital analysis of the spatial arrangement of bacteria
was further improved using widefield deconvolution
epifluorescence microscopy (Manz et al., 2000).
Another microscope used for FISH is the confocal
laser scanning microscope (CLSM). By restricting
the collected signal to a thin section of the object
investigated, out-of-focus fluorescence is removed,
leading to sharp images. This is useful for thick
samples or those with a high background, like sludge
flocs, biofilms or tissue sections (Wagner et al.,
1993; Wagner et al., 1994; Manz et al., 1995;
Ghiorse et al., 1996; Lawrence et al., 1998; Moter et
al., 1998b). Software packages are available that
allow three-dimensional reconstruction of images
(Fig. 8). However, due to fast bleaching the use of
CLSM often requires higher fluorescence signal
yield. For examples of the different microscope
techniques mentioned above see Fig. 7. FISH signals
can also be recorded using flow cytometry. Although
the flow cytometer does not give any information
about morphology or spatial distribution of micro-
 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112 91

Fig. 4. Fluorescence in situ hybridization of subgingival plaque from a periodontitis patient. (a) Simultaneous hybridization with eubacterial
probe EUB338 FITC (green) for visualization of different bacterial morphologies at single-cell resolution and TRE I Cy3 (yellow) for the
detection of phylogenetic group I treponemes, most of which are as-yet uncultured. (b) The same plaque material, hybridized with TRE I FITC
(green) and TRE II Cy3 (yellow), the latter detecting oral treponemes of phylogenetic group II (Moter et al., 1998a). It should be noted that
the classification of treponemes into phylogenetic groups based on the 16S rRNA data (according to Choi et al., 1994), correlate well with
morphologic differences as shown in this figure (with permission, Moter et al., 1998a).
92 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112

Fig. 5. FISH on a subgingival biofilm using probes EUB338 FluoX and TRE I Cy3 . (a) Cross-section at low magnification, with the carrier
material seen on the bottom and the border towards the gingiva at the top, showing a high number of group I treponemes. (b) Higher
magnification of this highly organized area at the border towards the gingival tissue, showing group I treponemes (yellow), surrounded by
other bacteria stained by the eubacterial probe (green). Blood cells (dark) are grouped around a dense core of bacteria in the center.
 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112 93

Fig. 6. FISH on semithin sections of biopsies from digital dermatitis (DD) lesions. The tissue was fixed and embedded in cold polymerizing
resin (with permission, Moter et al., 1998b). Simultaneous hybridization with EUB338 FITC and DDK4 Cy3 , a probe specific for treponemes
associated with digital dermatitis. (a) Note the stratification with EUB338-positive bacteria (green) predominantly at the outside and
DDK4-positive treponemes (yellow) within stratum corneum and the stratum spinosum. The arrow indicates a blood vessel. (b) At higher
magnification, large numbers of microorganisms are visible (upper part of the microphotograph). Single spirochetes seem to invade the tissue
through the intercellular spaces. Epidermal cells exhibiting week autofluorescence with black nuclei allow orientation within the tissue. (c)
Microcolony of DDK4-positive treponemes adjacent to the healthy tissue.
94 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112

Fig. 7. FISH on semithin sections of biopsies from digital dermatitis (DD) lesions. A slide analyzed using three different microscopic
techniques (Fig. 4). (a) Microphotograph taken by conventional epifluorescence microscopy showing large numbers of treponemes between
necrotic epidermal cells. (b) Same region shown in a higher resolution as achieved by a CCD camera. (c) Identification of single bacterial
cells by confocal laser microscopy (bar, 5 mm).

organisms, it can be useful for automated, quantita- for sorting at high frequency (Amann et al., 1990a;
tive analysis of bacteria in suspensions or mixed Wallner et al., 1993; Wallner et al., 1997; Snaidr et
planktonic populations and it has the unique potential al., 1999).
 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112 95

Fig. 8. FISH on semithin sections of biopsies from digital dermatitis (DD) lesions. TRE I-positive (green) and DDK4-positive (yellow)
treponemes surrounding an epithelial cell. (a) Epifluorescence microscopy. (b) Adjacent section of the identical biopsy analyzed by CLSM
(DDK4-treponemes only). (c) Three-dimensional reconstruction of the same image. Stereoscopic view allows a spatial impression of the
tissue (bars, 5 mm).
96 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112

Fig. 9. Visualization by FISH of Treponema pallidum in a section of rabbit testis. (a) Green autofluorescence of cells allows orientation
within the tissue. (b) FISH with a Cy3-labeled probe specific for Treponema pallidum showing the bacteria between the tubuli seminiferi.
 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
4. Problems and pitfalls: what can go wrong?
4.1. False positive results
4.1.1. Autofluorescence
Regarding autofluorescent material, FISH clearly
has limitations. The most striking problem is auto-
fluorescence of microorganisms themselves. This has
been reported for a variety of moulds and yeasts
(Fig. 3) (Margo and Bombardier, 1985; Graham,
1983) for certain bacteria like members of the genus
Pseudomonas (for example, see Brown and Low-
bury, 1996), Legionella (Edelstein, 1985; Wilkinson
et al., 1990), for Rhodospirillum centenum (Albrecht-
¨
Buehler, 1996), for cyanobacteria (Schonhuber et al.,
1999) and for archaeal species like methanogenes
(Sorensen et al., 1997) the latter ones complicating
FISH analysis of environmental microbiota. Auto-
fluorescence can also be found in material surround-
ing the bacteria in environmental samples, like
activated sludge, plants or even drinking water and is
caused by natural fluorescent biological or inorganic
debris (Vesey et al., 1997). Similarly various tissues
that contain elastin and collagen or blood cells like
erythrocytes and eosinophile granulocytes usually
exhibit bright autofluorescence (Van de Lest et al.,
1995; Monici et al., 1995). Although some back-
ground autofluorescence may be helpful working as a
counterstain allowing orientation within tissue sec-
tions (Figs. 6 and 9), it generally decreases the
signal-to-noise ratio and masks specific fluorescent
signal. In cytogenetic and immunologic studies,
tissue autofluorescence has been successfully elimi-
nated by subtracting it pixel by pixel during digital
¨ ¨ et al., 1995; Van de Lest
image processing (Szollosi
et al., 1995).
Thorough analyses of the phenomenon of auto-
fluorescence and how to avoid interference with
FISH are rare, however, growth media, fixation
methods and mounting media seem to have dramatic
influence on the signal intensities (Sorensen et al.,
1997; Graf et al., 1998). Use of narrow-band filter
sets and signal amplification systems are recom-
mended to overcome the problem of autofluoresc-
¨
ence (Sorensen et al., 1997; Schonhuber et al.,
1999). For Candida albicans it has been shown that,
depending on the fixation and excitation wavelength,
autofluorescence can also vary within a single strain
97
(Fig. 3) (Graf et al., 1998). Therefore the interpreta-
tion of fluorescent signals regarding fungi has to be
done with caution. Nevertheless, specific detection of
moulds and yeasts in cell cultures, in experimentally
infected tissues, and in spiked human blood has been
reported (Lischewski et al., 1996; Lischewski et al.,
1997). However, when dealing with unknown mixed
bacterial populations or tissue specimens, there is a
clear need to check for autofluorescence prior to the
FISH procedure.
4.1.2. Lack of specificity
Accuracy and reliability of FISH is highly depen-
dent on the specificity of the oligonucleotide probe.
Careful design and critical evaluation of new probes
are essential. Positive controls as well as closely
related strains harbouring target sequences with few
mismatches to the respective probe as negative
controls should be included in every FISH experi-
ment. It is important to note that an oligonucleotide
is only as precise as the database from which it is
derived. Due to the inaccuracy of some reported
sequences and the rapid expansion of existing data-
bases (Bhatia et al., 1997), probe sequences should
be checked on a regular basis using the newest
version of sequencing data. Dealing with fastidious
or uncultured organisms, it is useful to check spe-
cificity first using other hybridization assays like
dot-blot hybridizations (Lischewski et al., 1996;
Moter et al., 1998a; Wullings et al., 1998). If there
are still difficulties with adjusting the stringency of
the probes, resequencing of control strains and
isolates may also be required. Therefore design and
evaluation of new probes should be done in lab-
oratories with much experience in microbiological
and molecular biological methods.
Despite meticulous probe design and testing,
binding of probes to as-yet undescribed organisms
cannot be ruled out, when analyzing mixed bacterial
populations. To ensure that the correct bacterium is
detected, two specific probes that each target a
different position of the 16S rRNA and are labeled
with different fluorochromes can be used. Cells that
are detected by both probes and therefore exhibit
double fluorescence can be regarded as target organ-
isms (Neef et al., 1996). This approach is, however,
restricted by the limited availability of specific target
sequences for the respective microorganism.
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A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
4.2. False negative results
4.2.1. Insufficient penetration
Low signal intensity may be the consequence of
insufficient probe penetration into the bacterial cell,
which depends on the structure of the bacterial cell
wall. Gram-negative organisms usually do not pose
any problem as they are permeable even for poly-
ribonucleotide probes. For Gram-positive microorga-
nisms, particularly those exhibiting long or medium
chain mycolic acids, special fixation and pretreat-
ment may be necessary (see above). FISH has been
performed on Gram-positive cocci (Beimfohr et al.,
1993; Krimmer et al., 1999), Corynebacterium (Rol-
ler et al., 1994), Actinomyces (De Los Reyes et al.,
1997; Schuppler et al., 1998), Bacillus (Felske et al.,
1998) and Listeria (Wagner et al., 1998).
However, cell wall characteristics can sometimes
be used advantageously; FISH has been used to
estimate the state of bacterial cell walls (Bidnenko et
al., 1998). Using HRP-labeled probes Lactococcus
lactis was not detectable by FISH unless it was
infected by a virulent bacteriophage leading to
expression of autolytic enzymes and permeabiliza-
tion of cell walls. The percentage of hybridized cells
indicated the level of autolysis in culture.
4.2.2. Higher order structure of target or probe
Due to the three-dimensional structure of the
rRNA, not all sequences are equally accessible for
probes. Loop and hairpin formation as well as
rRNA-protein interactions hinders hybridization
leading to differential sensitivity of oligonucleotide
probes. This explains why probes that performed
well in hybridization formats using denatured RNA
or DNA did not give satisfactory results in FISH
(Frischer et al., 1996). A systematic study addressing
this problem was published by Fuchs et al. (1998).
The authors created more than 200 adjacent oligo-
nucleotide probes, specific for different locations on
E. coli 16S rRNA, and measured their signal inten-
sities in FISH using flow cytometry. Relative to the
brightest probe, the oligonucleotides were classified
into six brightness classes and an accessibility map
of E. coli 16S rRNA was established. Since the 16S
rRNA is highly conserved, this map can facilitate a
rational probe design not only for E. coli, but also for
other microorganisms. However, as the authors state,
it is mandatory to evaluate and optimize every newly
designed probe with the respective reference organ-
ism and with proper negative controls before it is
applied to mixed or unknown samples.
Since self-annealing and hairpin formation of the
probe itself can also lead to low signal intensities,
newly designed oligonucleotides should be checked
using appropriate software (Fuchs et al., 1998).
4.2.3. Low rRNA content
Although normally highly abundant, the rRNA
content of bacterial cells may vary considerably not
only between species, but also within cells of one
strain according to their physiologic state which is
directly correlated with growth rate (DeLong et al.,
1989; Kemp et al., 1993; Wallner et al., 1993;
Poulsen et al., 1993). Low physiological activity can
thereby result in low signal intensity or false nega-
tive results. For detection of slow-growing species,
bright fluorescent dyes like Cy3 are recommended.
To further increase signal intensity and thus sen-
sitivity, one can use multiple labeling. Two or more
specific probes labeled with the same fluorescent dye
are applied to increase the number of fluorescent
molecules per cell (Lee et al., 1993). This technique
is of course limited by the availability of target sites
for specific probes.
As mentioned above, sensitivity can also be
enhanced using signal amplification systems or poly-
ribonucleotide probes (see Section 3.1).
4.2.4. Photobleaching
Many fluorochromes will fade rapidly upon excita-
tion leading to irreversible destruction over time.
With exposure times of several seconds or even
minutes, this may have a critical effect, particularly
on photomicrographs. To circumvent this problem,
the use of narrow band filter sets, photostable
cyanine dyes (as mentioned above), and antifading
mounting media are strongly recommended.
4.2.5. Use of bacterial probes
The best control for false negative results due to
methodological problems is the use of a bacterial
probe. If hybridization with an universal probe gives
satisfactory results in FISH, fixation, probe penetra-
tion and rRNA content of bacterial cells are not the
limiting factors. EUB338 (Amann et al., 1990a) is
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A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
the probe most commonly used for this purpose.
However, recent analyses indicated that some bac-
terial phyla including Planctomycetales and Ver-
rucomicrobia are missed by this probe (Daims et al.,
1999). Therefore it might be necessary to use a set of
bacterial probes to get a more comprehensive view
when analysing complex microbial communities. To
control non-specific binding of the eubacterial probe
to bacterial 16S rRNA or to cell components other
than nucleic acids, the complementary probe, called
NON338 (Wallner et al., 1993) can be used and it
should not give any signal in FISH.
5. Applications
It is beyond the scope of this article to review all
possible applications of FISH. Therefore the selec-
tion of works cited is rather subjective and should
highlight the versatility of the method.
5.1. Microbial diversity in environmental samples
It is well accepted that the number of bacterial
species in the environment by far exceeds the
number of described species. For a long time, the
magnitude of microbial diversity was underesti-
mated. In various environmental samples, micro-
scopic total cell counts exceeded the number of
culturable bacteria by several orders of magnitude
(Kogure et al., 1979; Ferguson et al., 1984; Staley
and Konopka, 1985). Comparative rRNA analysis
has revolutionized the description of mixed bacterial
consortia. However, most techniques involve crucial
steps like nucleic acid extraction or PCR amplifica-
tion that may be biased as well (for a review see von
Wintzingerode et al., 1997). Because FISH gives a
detailed picture of the microenvironments without
any selective purification or amplification steps, it
has been extensively used in the field of environmen-
tal diversity research and only a few examples can be
mentioned in this review. It has been used worldwide
to investigate microbial communities in natural
environments such as aquatic habitats, e.g. popula-
tions in Wadden Sea sediments (Llobet-Brossa et al.,
1998), in fjords (Ramsing et al., 1996), bacterioplan-
¨
kton in river- or seawater (Glockner et al., 1996;
Alfreider et al., 1996; Lemke et al., 1997; Kenzaka et
99
al., 1998) and lake snow of a high mountain lake
(Weiss et al., 1996), in soils (Felske et al., 1998) and
on root surfaces (Macnaughton et al., 1996). How-
ever, when analyzing complex environmental mi-
crohabitats autofluorescence can be problematic.
FISH not only provides snapshots of a certain
moment but has also been used to monitor bacterial
consortia in continuous-flow cultures of seawater
sediment (Bruns and Berthe-Corti, 1998), during
seasonal changes in a high mountain lake (Pernthaler
et al., 1998), and planktonic composition in response
¨
to enhanced protozoan grazing (Jurgens et al., 1999).
Furthermore, uncultivated species or new, recently
defined bacterial phyla have been detected in en-
vironmental samples using FISH, like members of
the Holophaga /Acidobacterium phylum (Ludwig et
al., 1997), an uncultured Bacillus in Dutch grassland
soils (Felske et al., 1998), and recently Aquabac-
terium in a Berlin (Germany) drinking water system
(Kalmbach et al., 1999).
All these studies have great implications for
understanding the composition and ecology of natu-
ral bacterial consortia and their population dynamics
in response to influences of nature or mankind.
5.2. Microbial diversity in wastewater treatment
Ardern and Lockett (1914) developed the first
activated sludge system for purification of waste-
water in Manchester. However, the role of microbial
consortia in this process is still not completely
understood. Because culture-based techniques were
found to be too selective to give a comprehensive
and authentic picture of the entire microbial com-
¨
munity (Wagner et al., 1993; Kampfer et al., 1996),
rRNA-based population analysis has found increas-
ingly wide application in analysis of such environ-
ments like bioreactors and wastewater treatment
plants. Sequences that were retrieved from molecular
analysis using PCR-based techniques were used to
create new oligonucleotide probes for FISH inves-
tigation of bacterial communities, e.g. in activated
sludge (Amann et al., 1996; Snaidr et al., 1997;
Schuppler et al., 1998). The number and spatial
distribution of nitrite-oxidizing and ammonia-oxi-
dizing bacteria, responsible for key steps in micro-
bial nitrogen cycling, were analyzed in a nitrifying
fluidized bed reactor and in activated sludge
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A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
(Schramm et al., 1998; Juretschko et al., 1998).
Recently, Bond et al. (1999) identified groups of
bacteria in sludge with enhanced biological phos-
phorus removal. Other groups focused on mycolic-
acid containing actinomycetes that are thought to be
involved in filamentous foaming in sewage treatment
plants (De Los Reyes et al., 1997; De Los Reyes et
al., 1998; Schuppler et al., 1998). Also members of
the domain Archaea, particularly methanogens, have
been found by FISH in anaerobic digesters and
within anaerobic sludge granules, despite their strong
autofluorescence (Sorensen et al., 1997; Rocheleau et
al., 1999). Recently, the combined use of FISH and
microsensors allowed analyses of bacterial com-
munities and metabolic activities simultaneously,
thereby revealing anaerobes in anoxic microniches
within aerobic environments (Schramm et al.,
1999b).
5.3. Symbiotic bacteria
Most obligate microbial symbionts are as-yet
uncultured. Using the 16S rRNA approach, they can
be identified and phylogenetically classified. How-
ever, because bacteria other than symbionts, e.g.
ingested bacteria or intestinal microflora, are found
in protozoa or complex eukaryotes, it is difficult to
differentiate them from true symbionts. The localiza-
tion of microorganisms within different compart-
ments of the host by FISH can prove their symbiotic
nature, as shown by Amann et al. (1991) who
identified uncultured bacterial endosymbionts of the
genus Holospora within the ciliate Paramecium
caudatum (their natural habitat). Since Holospora
species were detected within the nucleus of the
ciliate, it could be clearly distinguished from ing-
ested bacteria present in food vacuoles. Similarly,
FISH was used to demonstrate that Paramecium
caudatum also harbours Caedibacter caryophila, a
killer endosymbiont that is phylogenetically related
to the genus Rickettsia (Springer et al., 1993).
Recently, novel bacterial endosymbionts also related
to Rickettsia have been demonstrated in Acan-
thamoeba spp. (Fritsche et al., 1999). Interestingly,
the respective amoeba strains were isolated from
scrapings of infected human corneal tissue. Sar-
cobium lyticum, a bacterium that is closely related to
the genus Legionella, has also been demonstrated
within amoebae that were recovered from a sputum
sample of a patient with pneumonia (Springer et al.,
1992). This demonstrates that amoebae should not
only be recognized as human pathogens themselves
but also as vectors for a variety of bacterial
symbionts. This might be the case for facultative
endosymbionts like Legionella pneumophila which
has been vizualized within Acanthamoeba castellani
(Grimm et al., 1998) and within ciliate Tetrahymena
pyriformis (Manz et al., 1995). In nutritionally poor
aquatic habitats, legionellae are able to enter a viable
but unculturable state, in which they are not detect-
able by conventional culturing techniques. Resuscita-
tion of dormant cells back to the culturable state was
successfully monitored by FISH within the natural
host Acanthamoeba castellanii (Steinert et al., 1997).
These observations may have great implications for
the epidemiology of this potential pathogen.
Using different ISH strategies, bacterial endo-
symbionts were detected in the gills of the protob-
ranch bivalve Solemya reidii. This organism served
as a model to evaluate a metacrylate embedding and
acetone de-embedding (MEADE) technique to in-
crease signal intensities and improve the sensitivity
of ISH on tissue sections (Warren et al., 1998).
Recently, Methanobrevibacter-related Archaea were
detected by FISH in the hindgut contents of Re-
ticulitermes speratus, a termite collected in the Japan
archipelago (Shinzato et al., 1998).
5.4. FISH in medicine
5.4.1. Analysis of complex microbial communities
5.4.1.1. Oral cavity
Population analysis by FISH has proven particu-
larly useful for description of the normal flora or that
of mixed microbial infections. This has been shown
for the oral cavity harbouring more than 300 differ-
ent bacterial species, many of which can be compli-
cated to culture or are not yet cultured. Oral in-
fections such as periodontitis and gingivitis are
associated with specific microbial consortia. Use of
culturing techniques exclusively has yielded an
underestimate of the microbial diversity. The utility
of FISH has been shown for Gram-negative an-
aerobes, like Porphyromonas gingivalis, Bacteroides
forsythus and Prevotella intermedia in subgingival
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A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
plaque material of periodontitis patients (Gersdorf et
al., 1993a,b). Further molecular-based analyses re-
vealed an unexpected diversity of yet-to-be cultured
oral treponemes in patients with rapidly progressive
periodontitis (Choi et al., 1994). The high numbers
and different morphologies of these spirochetes were
demonstrated by FISH on smears of subgingival
plaque material (Fig. 4) (Moter et al., 1998a).
Furthermore, organization and spatial distribution of
oral treponemes could be studied within subgingival
biofilms (Fig. 5). However, because epidemiological
data on the association of treponemes with oral
disease alone do not prove their pathogenic potential,
the same oligonucleotide probes were applied to
tissue sections of biopsies from heel bulbs of cattle
suffering from digital dermatitis (Figs. 6–8). This
chronic, tissue-destructive, and difficult-to-treat dis-
ease is associated with a predominantly anaerobic
mixed flora very similar to that found in periodon-
titis. A layering of the different bacterial groups
could be demonstrated within the tissue, indicating
that certain phylogenetic groups of treponemes are
more likely to invade tissue and maintain the disease
than are other treponemes, rods or cocci (Moter et
al., 1998b).
5.4.1.2. Gastro-intestinal flora
Because the intestine is the most heavily colonized
part in the human body, the healthy intestinal flora
and biofilm formation in human gut are of great
interest. Again, culturing techniques underestimate
the actual number of bacteria (Harmsen et al., 1999).
To date most FISH investigations on human gut flora
have been restricted to feces. The amount of
Bifidobacterium spp. and of the flavonoid degrading
bacterium Eubacterium ramulus were estimated in
fecal samples using genus- or species-specific
probes, respectively (Langendijk et al., 1995; Sim-
mering et al., 1999). Variations of bacterial popula-
tions, including Bacteroides and Clostridium spp.,
Streptococcus, Lactococcus and the Clostridium
coccoides-Eubacterium rectale group in human feces
were monitored over an 8-month period (Franks et
al., 1998). This technique was further improved by
development of automated image acquisition and
analysis software allowing fast and accurate micro-
scopic enumeration of groups of intestinal bacteria
(Jansen et al., 1999). Recently, the differences in
101
development of intestinal flora between breast-fed
and formula-fed infants were studied (Harmsen et
al., 2000). However, the physiologically relevant
spatial distribution of the human gut flora and
intestinal biofilm still remains largely unexplored.
Several approaches have been successfully applied
in animals to study the spatial distribution of bacteria
in the intestine and to detect enteropathogens within
this complex habitat. Escherichia coli has been
visualized in cryosections of the mouse large intes-
tine using probes targeting specific regions of the
23S rRNA. In this study, E. coli cells were observed
in mucus overlying the epithelial cells without direct
attachment to the epithelium (Poulsen et al., 1994).
Spirochetes of the genus Brachyspira /Serpulina
were specifically visualized within intestines of pigs
with swine dysentery and colonic spirochaetosis
(Boye et al., 1998). Brachyspira ( Serpulina)
pilosicoli was found to colonize and invade the
surface epithelium and crypts of inoculated pigs
whereas Serpulina intermedia inoculated pigs re-
vealed no abnormalities (Jensen et al., 2000).
Another study analyzed the colonization of the colon
in mice experimentally infected with Salmonella
typhimurium. An avirulent, streptomycin-resistant
Salmonella strain was compared to its lipopolysac-
charide (LPS)-deficient mutant (Licht et al., 1996).
Both strains were equally able to proliferate in the
intestinal mucus and signal intensity in FISH indi-
cated a similar ribosome content. Whereas the wild-
type colonized the epithelial cells on the first day, the
LPS-deficient strain stayed in the layer close to the
colonic lumen and did not adhere to the epithelium,
suggesting that LPS might enable Salmonella to
better penetrate the intestinal mucosal layer and bind
to epithelial cells. A similar approach was used to
investigate the influence of Klebsiella pneumonia
capsule expression during the colonization of mouse
large intestine (Favre-Bonte´ et al., 1999). In another
study using experimentally infected mice, Nordentoft
et al. (1997) tried to establish a diagnostic FISH for
detection of Salmonella. Due to high sequence
homology, 16S rRNA does not discriminate most
species of Enterobacteriaceae. Therefore, the authors
used a sequence from the 23S rRNA as a target for a
specific probe detecting 49 of 55 Salmonella
serovars but none of the 43 other investigated species
including members of Enterobacteriaceae. Sal-
102 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
monellae were visualized within paraffin sections of
colon and lung tissue from experimentally infected
mice as well as from animals with a history of
salmonellosis.
Other pathogenic organisms, Yersinia pestis, Y.
pseudotuberculosis and Y. enterocolitica, were suc-
cessfully identified down to the species level by
combination of two probes targeting 16S or 23S
rRNA in spiked stool and throat swab specimens,
and in cryosections of experimentally infected mice
(Trebesius et al., 1998). However, the authors stated
that PCR-based techniques might be more suitable
for detection of pathogenic yersiniae at concentra-
tions below 10 5 cells ml 21 . This is particularly true
for detection of pathogens within a mixed bacterial
flora that might contain organisms phylogenetically
closely related to the target species.
5.4.1.3. Respiratory tract infections
Haemophilus influenzae is a common cause of
upper and lower respiratory tract infections. Using
FISH, non-capsulated, non-typeable H. influenzae
strains were visualized in cryosections of adenoid
tissue of 10 children during symptom-free intervals
(Forsgren et al., 1994). Bacteria were detected in
macrophage-like cells in subepithelial cell layers and
found to infiltrate the reticular crypt epithelium. The
same authors investigated the identity of these
infected cells using FISH for detection of
Haemophilus and immunofluorescence for the de-
tection of CD 14 expressing cells (Forsgren et al.,
1996). The results indicated that Haemophilus may
persist within cells of the monocyte / macrophage
lineage.
Recently, a set of oligonucleotide probes was
designed for the specific detection of pathogens
commonly isolated from cystic fibrosis (CF) patients,
including Pseudomonas aeruginosa, Burkholderia
cepacia, Stenotrophomonas maltophilia,
Haemophilus influenzae, Streptococcus pyogenes,
Staphylococcus aureus, and Candida albicans. In a
clinical trial with sputum samples and throat swab
specimens, these probes proved to be useful for the
rapid and specific detection of bacteria causing acute
exacerbations in CF patients (Hogardt et al., 2000).
Compared to culturing techniques the specificity of
FISH was 100%, whereas the sensitivity was moder-
ately lower.
5.4.2. Detection of pathogens within tissues or
sterile compartments
5.4.2.1. Implanted medical devices
Infections of implanted biomaterial like intraven-
ous catheters, arthroplasties or orthopedic implants,
are a common cause of septicemia and malfunction
of medical devices. Due to their ability to adhere and
form biofilms on biomaterial, staphylococci are the
most frequently isolated bacteria in polymer-associ-
ated infections. However, diagnosis of staphylococci
can be difficult when culture fails, possibly because
these organisms may form small-colony variants
with reduced growth rate. In these cases FISH can be
more sensitive than culturing techniques. Moreover,
it can differentiate between infection of an implanted
device or contamination of the sample by showing
adherent staphylococci on the device. Using 16S
rRNA directed probes, Staphylococcus aureus and
Staphylococcus epidermidis were specifically iden-
tified within experimental biofilms (Krimmer et al.,
1999). It was further shown by FISH that small-
colony variants of S. aureus persist intracellularily
within a human endothelial cell line. In the same
study S. epidermidis was visualized in paraffin
sections of connective tissue samples of a patient
with hip arthroplasty loosening.
5.4.2.2. Blood cultures
Recently, the applicability of FISH for the identifi-
cation of pathogenic microorganisms in positive
blood culture bottles was examined by two indepen-
dent groups (Kempf et al., 2000; Jansen et al., 2000).
The panel of genus- and species-specific oligonu-
cleotide probes used covered most of the pathogens
typically found in septic patients, including staphylo-
cocci, streptococci, enterococci, Enterobacteriaceae
and fungi. The authors report an excellent sensitivity
and specificity of the FISH analysis that yielded an
identification within 25 min or 2.5 h, respectively,
whereas species identification by conventional cul-
turing usually takes 24–72 h to be completed. This
rapid identification of causative pathogens in blood
cultures allows earlier appropriate antimicrobial
 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112 103

therapy thus possibly improving the prognosis of hepatopancreatic tubular epithelic cells (Loy et al.,
septicemic patients. 1996).
The role of uncultured treponemes in digital
dermatitis in cows and of Brachyspira /Serpulina in
5.4.3. Fungi
pigs has been discussed above (see Sections 5.4.1.1
Opportunistic fungal pathogens like Candida spp.
and 5.4.1.2, respectively).
are a constant threat for immunocompromised pa-
tients. Diagnosis of disseminated disease is still
5.4.5. Plant pathogens
difficult since blood cultures often remain negative.
Bacterial ring rot disease in potatoes is caused by
Histological detection of Candida does not differen-
Clavibacter michiganensis subsp. sepedonicus. The
tiate the fungal elements to the species level. Since
fast and accurate identification of this organism is
susceptibility to antifungal agents differs in Candida
important for disease control and eradication. Diag-
spp., a fast and accurate identification is desirable.
nosis was improved by application of FISH using
Despite the phenomenon of autofluorescence (dis-
16S-rRNA directed probes and subsequent immuno-
cussed above), C. albicans /tropicalis and C. parap-
fluorescence using a specific monoclonal antibody on
silosis were specifically detected in cryosections of
the same slide, because two independent criteria
deep organs in experimentally infected mice and in
ensured exact identification (Li et al., 1997b).
spiked human blood after lysis of blood cells and
Ralstonia solanacearum, a bacterium that is
subsequent filtration using polycarbonate filters (Lis-
phylogenetically closely related to the genus Pseudo-
chewski et al., 1996; Lischewski et al., 1997). In
monas, is the causative agent of bacterial wilt or
these cases the oligonucleotide probes were directed
brown rot disease in potatoes. For FISH detection of
to 18S rRNA.
this pathogen, 23S-rRNA directed probes were de-
Aureobasidium pullulans, another yeast-like fun-
signed. In combination with indirect immunofluores-
gus that grows on living leaves, has been studied
cence using a polyclonal antibody targeting Ral-
using FISH. Though this fungus does not have any
stonia solanacearum, identification of the organisms
pathogenic potential, it might well be of importance
within soil, in water samples and in root tissue of the
in phytopathology because this ubiquitous organism
weed host Solanum dulcamara (bittersweet) was
could act as a biological control agent of plant
possible (Wullings et al., 1998).
pathogens. Quantitative imaging including statistical
analysis allowed comprehensive analysis of growth
5.4.6. Detection of pathogens using non-fluorescent
of A. pullulans on leaf surfaces (Li et al., 1997a;
hybridization
Spear et al., 1999).
A few medically relevant studies using probes
Furthermore, Sterflinger and Hain (1999) de-
labeled with digoxygenin or enzymes should also be
veloped oligonucleotide probes for the FISH de-
mentioned briefly. In 1988, Mycoplasma pneumo-
tection of black yeasts and meristematic fungi that
niae-DNA was localized within diseased gingiva of
were grown on glass and plastic surfaces.
periodontitis patients by in situ hybridization using a
biotin-labeled probe (Saglie et al., 1988). A single
5.4.4. Veterinary medicine report of ISH detection of Mycobacterium leprae in
Necrotizing pancreohepatitis (NHP) is a severe sections of skin biopsies (Arnoldi et al., 1992) was
disease of farm-raised Pacific white shrimp, Penaeus reported using an immunoenzymatic detection.
vannamei. This economically important condition Chlamydia trachomatis was localized with digox-
has lead to severe reduction of shrimp production ygenin-labeled probes in subsynovial cell layers of
and has been found in Texas and in Central and tissue from patients with Reiter’s syndrome (Beutler
South America. It has been associated with an as-yet et al., 1995). Karttunen et al. (1996) used a 520 bp
uncultured Rickettsia-like microorganism, the NHP- PCR product that was labeled with digoxygenin to
bacterium. FISH of infected P. vannamei shrimp visualize Helicobacter pylori in gastric biopsy speci-
visualized the NHP-bacteria in the cytoplasm of mens. Using a catalyzed reporter deposition signal
104 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
amplification system (CARD-ISH), Yersinia en-
terocolitica was detected within infected cell lines,
rat spleens and patient biopsies (Odinot et al., 1998).
In this case plasmid DNA was biotin-labeled by nick
translation, generating fragments of 100–500 bp in
length. Animal pathogens detected by ISH include
intracellular organisms in enterocytes of pigs suffer-
ing from proliferative enteropathy (Gebhart et al.,
1994), Anaplasma marginale in bovine erythrocytes
(Ge et al., 1995) and Chlamydia in avian tissues
(Theil et al., 1996).
6. Perspectives
6.1. FISH and function
A further improvement of the technique is the
combination of in situ identification of bacteria with
additional information on their functional state, like
gene expression, metabolism or antigen expression.
The use of FISH in combination with microsensor
measurements has already been mentioned (see
Section 5.2). These needle-shaped devices detect
gradients of specific compounds like oxygen, hydro-
gen sulfide, nitrate, nitrite or pH directly within
activated sludge flocs and biofilms. Combined with
FISH these analyses permitted study and monitoring
of metabolic activities, changes in population struc-
ture and growth of biofilm over time (Santegoeds et
al., 1998; Schramm et al., 1999a; Schramm et al.,
1999b).
Microautoradiography can be used to visualize
metabolic activity. Defined cell mixtures or activated
sludge are incubated with radioactively labeled or-
ganic and inorganic substrates such as glucose,
acetate or bicarbonate. After fixation of samples and
preparation of slides, radioactivity can be visualized
by application and development of an autoradiog-
raphic emulsion. Using this technique simultaneously
with FISH, radiolabeled substrate uptake was shown
to be confined to certain bacterial species and could
be monitored within activated sludge under aerobic
versus anaerobic conditions (Lee et al., 1999) and in
natural picoplankton communities (Ouverney and
Fuhrman, 1999).
Another approach applicable to analyses of medi-
cally relevant samples is the combined use of FISH
and immunolabeling. While FISH allows identifica-
tion of individual bacteria, immunofluorescence (IF)
could provide additional information regarding an-
tigen expression. The combination of both tech-
niques was optimized and evaluated for the identifi-
cation of bacteria in artificial mixtures by flow
cytometry (Wallner et al., 1996). Since antibodies
are frequently specific at the species level and below,
this combination allows fast and automated identifi-
cation at a wide range of phylogenetic levels. FISH
and IF were also simultaneously applied for the
microscopic detection of Bacteroides fragilis mixed
with E. coli (Ramage et al., 1998). Eubacterial or
species-specific oligonucleotide probes were used for
FISH, followed by treatment with either monoclonal
antibodies or a polyclonal antiserum specific for a
common antigen (cAg) of B. fragilis. Because signal
intensity is dependant on culture conditions, on
pretreatment, and on washing steps during the pro-
cedure, the authors state that the combination of the
two techniques is a research rather than a diagnostic
tool. However, in situ identification combined with
analysis of antigenic variation, such as virulence
determinant expression, without cultivation could
help in understanding mechanisms of bacterial patho-
genicity.
A technique for analysis of virulence factors was
developed by Wagner et al. (1998). Conventional
FISH with a 16S rRNA-directed probe for the
identification of Listeria monocytogenes was com-
bined with in situ detection of mRNA of the iap
(invasion associated protein) gene, coding for a
virulence factor in the same species. Due to the
instability and low copy number of mRNA as
compared to rRNA, a highly sensitive FISH tech-
nique was developed. Antisense polyribonucleotide
probes, internally labeled with digoxygenin during
reverse transcription, were hybridized to whole bac-
terial cells and detected by HRP-labeled anti-DIG
antibodies and the fluorogenic TSA system as de-
scribed above [see Section 3.1, Fig. 2(e)]. This
technique is rather delicate and has to be optimized
for each new target species. However, it is a
promising tool for studying in situ gene expression in
complex environments. Recently, another fluorescent
marker molecule, green fluorescent protein (GFP)
has found increasingly wide application. By intro-
ducing the respective gene into plasmids or chromo-
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A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
somal DNA of an organism of interest, the expressed
GFP can be used to visualize and monitor promoter
activity or gene expression at the single cell level.
This reporter gene assay was combined with FISH
for the analysis of activated sludge (Eberl et al.,
1997). In defined model biofilms, the spatial dis-
tribution of organisms, time course of promoter
induction, promoter expression and bacterial interac-
tions could be analyzed (Moller et al., 1998). A
similar approach was chosen to identify Pseudo-
monas putida cells by FISH and to visualize simul-
taneously the horizontal transfer of a gfp-tagged
plasmid within an experimental biofilm (Christensen
et al., 1998). This approach will have an important
impact on structure-function analyses of complex
microbial communities.
Recently, FISH was used to analyze the subcellu-
lar cell-cycle dependant localization of DNA seg-
ments of the E. coli chromosome (Niki et al., 2000).
6.2. FISH with peptide nucleic acid ( PNA) probes
Recently, peptide nucleic acid probes have been
introduced into hybridization techniques. PNAs are
DNA analogs with an uncharged polyamide back-
bone instead of sugar phosphates. They are stable to
degradation, hybridize to complementary sequences
with higher affinity and have different hybridization
characteristics. So far they have been used for a wide
range of applications including PCR clamping,
Southern blotting, detection of mutations and DNA
purification (for review see Corey, 1997). Usually
PNA probes shorter than conventional oligonucleo-
tides are required for specific binding. They hence
seem to be an interesting alternative to conventional
oligonucleotide probes. This was demonstrated by
Prescott and Fricker (Prescott and Fricker, 1999)
using biotin-labeled PNA probes combined with the
TSA system for specific and sensitive detection of E.
coli in water samples. This highly sensitive approach
detected rRNA in bacterial cells long after cell death.
One of the major drawbacks of FISH using
oligonucleotide probes is the variable and sometimes
insufficient penetration of probes into bacteria de-
pending on their cell wall characteristics. This is a
problem particularly seen in Gram-positive species
and acid-fast bacilli. PNAs may be instrumental in
overcoming this problem. Due to their neutral back
105
bone, PNAs diffuse through hydrophobic cell walls
and allow detection of mycobacteria by FISH with-
out any pretreatment. Using specific, fluorescently
labeled PNAs, differentiation between tuberculous
and non-tuberculous mycobacteria was possible in
smears of mycobacterial cultures, and directly in
smear-positive sputum samples within a few hours as
well (Stender et al., 1999a,b; Drobniewski et al.,
2000). This technique constitutes an improvement in
routine diagnosis of tuberculosis and may help to
establish FISH as a valuable, rapid and cost-effective
method in clinical microbiology.
6.3. Future
As compared to microbiological culture or in vitro
amplification of nucleic acids, FISH is fast, cheap
and easy to carry out. It should be especially
valuable for the detection of slow-growing, fastidi-
ous or unculturable bacteria. If applied to blood or
cerebrospinal fluids, it could provide rapid and
accurate diagnosis of infections in critically ill
patients. A big step towards implementation into
clinical microbiology will be made if FISH could be
automated. Systems for automated image analysis
have already been tested. However, automation of
sample processing as well as plausible control of
results using expert systems are required. A first
generation of hybridization instruments is available,
but they have not been used for microbiological
samples yet (Capodieci et al., 1998). Besides reduc-
tion of manual work, automated FISH would allow a
high turnover and improved reproducibility of pro-
cessing clinical samples in the laboratory. However,
despite its many advantages FISH will not replace
culture-based methods in the near future.
7. Summary
FISH provides information about the presence,
number, morphology and spatial distribution of
microorganisms. Although its application is fast and
cheap, thorough controls are crucial to ensure quality
of the results. Therefore, development and evaluation
of probes for diagnostic purposes should be restricted
to expert reference laboratories with facilities for
culturing as well as molecular techniques. Thus far
106 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112
FISH has yielded important insights into the struc-
ture of complex microbiological communities in
humans, animals and in the environment. It has
successfully been used to detect pathogens in smears
or tissue preparations from humans and animals. It
should be pointed out that the number of target
organisms has to be above the detection level as with
any other microscopic technique. However, we feel
optimistic that FISH will soon become a powerful
diagnostic tool for microbiologic diagnosis of human
or animal infections, and for the assessment of the
population structure of complex microbial com-
munities.
Acknowledgements
The authors thank M. Wagner for access to and
help with the CLSM and A. Friedmann, G. Leist, B.
Riep, V. Sambri, K. Schrank and J. Wecke for
providing clinical samples. The critical reading and
helpful suggestions of R.J. Palmer, K. Heuner and C.
¨ S., 1998. Estimation of the state of the bacterial cell wall by
Schlotelburg are appreciated. We thank C. Hefen-
brock, G. Fiedler and A. Pohlisch for excellent
technical assistance. This work was supported in part
by a grant (01KI9318) from the Bundesministerium
¨ Bildung und Forschung (UBG), Humboldt Uni-
fur
¨ zu Berlin and the Korber
¨ vated sludge systems. Appl. Environ. Microbiol. 65, 4077–
versitat European Research
Award (UBG).
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