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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


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

0167-7012 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0167-7012( 00 )00152-4
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.
¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112 87

access to their target, but they might carry fewer Sensitivity of FISH can be further increased using
labels. There are different ways of labeling. Direct enzymatic signal amplification. This method was
fluorescent labeling is most commonly used and is developed by Kagiyama et al. (1993) for cytogenetic
also the fastest, cheapest and easiest way because it investigations and later adapted for the detection of
does not require any further detection steps after bacteria by Yamaguchi et al. (1996). A digoxygenin-
hybridization. One or more fluorescent dye mole- labeled oligonucleotide is detected by an anti-digox-
cules are directly bound to the oligonucleotide either ygenin antibody that is coupled to alkaline phospha-
chemically during synthesis through an aminolinker tase. This enzyme converts HNPP (2-hydroxy-3-
at the 59-end of the probe [Fig. 2(a)], or enzymatical- naphtoic acid-29-phenylanilide phosphate) to its de-
ly using terminal transferase to attach fluorescently phosphorylated form that generates bright-red fluo-
labeled nucleotides at the 39-end [Fig. 2(b)] (Moter rescence in combination with Fast Red TR. Fluores-
et al., 1998a). Coupling of Fluorescein–Isothio- cence was reported to be eight times more intensive
cyanate (FITC) to the oligonucleotide via an 18- than that of oligonucleotides carrying a single label.
carbon spacer may increase signal intensity as com- This approach of enzymatic signal amplification was
pared to a directly conjugated probe (Deere et al., further improved by a technique called ‘Tyramide
1998). An increase in fluorescence signal has also Signal Amplification (TSA)’ system. Oligonucleo-
been reported by labeling probes at both ends, one tides were labeled with horseradish peroxidase
fluorescent molecule at the 39-end and four mole- (HRP) that used fluorescein–tyramide as substrate
cules at the 59-end using appropriate spacers to ¨
[Fig. 2(d)] (Schonhuber et al., 1997). The TSA
prevent quenching of fluorescence (Spear et al., system resulted in a 10–20-fold increase in signal
1999). Fluorescence labeled oligonucleotides are intensity. However, the number of positive cells was
now commercially available. They can be stored at clearly reduced compared to the conventional use of
2 208C in the dark for several months. In some monolabeled probes. This might be due to insuffi-
cases, indirect detection is helpful; it has been shown cient penetration of the high molecular weight
that the sensitivity of the FISH assays can be reagents into the bacterial cells. Although per-
increased by linking the probe to a reporter molecule meabilization of cells by lysozyme improved signal
like digoxygenin (DIG) that is then detected by a intensity, this approach does not seem applicable to
fluorescent antibody [Fig. 2(c)] (Zarda et al., 1991). 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
Fig. 2. Direct, (a) and (b), and indirect, (c)–(e), labeling of probes observation of multicolour FISH, multibandpass
using digoxygenin (DIG), horseradish peroxidase (HRP) or filters may be used. The combined fluorochromes
Tyramide Signal Amplification (TSA). 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- ¨


(for review see Gobel, 1991 and Amann et al.,
ground problems and bleed-through. The brightest, 1995). With steadily growing sequence data entries
most photostable dye should be used for low-abun- in public or commercially available databases
dance targets. Dyes commonly used for FISH in (Maidak et al., 2000; Van de Peer et al., 2000), the
microbiology are fluorescein-derivates (Fluorescein– 16S rRNA gene offers the greatest ease and accuracy
Isothiocyanate (FITC), 5-(-6-)carboxyfluorescein–N- in identification for most microorganisms. This is
hydroxysuccimide-ester (FluoX)) and rhodamine-de- especially important when dealing with mixed bac-
rivates (Tetramethyl–Rhodamine–Isothiocyanate terial populations or unculturable organisms. The
(TRITC), Texas Red), and more recently cyanine high copy number of 16S rRNA in each replicating
dyes like Cy3 and Cy5 (Table 1). The latter ones and metabolically active cell usually offers sufficient
have been shown to be superior to the classical dyes target to visualize single bacterial cells even with
because they provide significant brighter staining and monolabeled oligonucleotides in FISH. The choice
are very stable to photobleaching (Wessendorf and of target site and design of probes should be done
Brelje, 1992). Blue fluorescent counterstaining can with greatest care. Appropriate software for rational
be performed with aromatic diamidines like 49,6- development of probes like the ARB program pack-
diamidino–2-phenylidole dihydrochloride (DAPI) age (see Strunk, O., Gross, O., Reichel, B., May, M.,
that non-intercalatively binds to DNA with great Hermann, S., Stuckmann, N., Nohhoff, B., Ginhart,
¨
affinity (Zimmer and Wahnert, 1986). For further T., Vilbig, A., Lenke, M., Ludwig, T., Bode, A.,
advice regarding fluorescent probes see Cullander Schleifer, K.-H., Ludwig, W. ARB: a software en-
(1999). vironment for sequence data; http: / / www.mik-
ro.biologie.tu-muenchen.de. Department of Micro-
3.3. Ribosomal RNA as a target for FISH biology, Technical University Munich, Munich, Ger-
many.) are available. Practicality and limitations of
In microbiology the most commonly used target 16S rRNA comparative sequence analysis, and the
molecule for FISH is 16S rRNA because of its design of rRNA probes has been reviewed before
genetic stability, its domain structure with conserved (Weisburg et al., 1991; Amann et al., 1995; Fredricks
and variable regions, and its high copy number and Relman, 1996; von Wintzingerode et al., 1997).
(Woese, 1987). Oligonucleotide probes for each Other targets like 23S rRNA (Manz et al., 1993;
taxonomic level, between bacterial and archaeal, Amann et al., 1995; Nordentoft et al., 1997; Lemke
down to genus-specific and species-specific can be et al., 1997; Trebesius et al., 1998), 18S rRNA
designed according to the region of rRNA targeted (Lischewski et al., 1996; Lischewski et al., 1997; Li

Table 1
Fluorochromes used for the detection of microorganisms by FISH
Fluorochrome Wavelength a Color
Excitation (nm) Emission (nm)
AMCA 351 450 Blue
Fluorescein–isothiocyanate (FITC) 492 528 Green
5-(-6-)carboxyfluorescein–N-hydroxysuccimide- 488 520 Green
ester (FluoXE)
Tetramethyl–rhodamine–isothiocyanate (TRITC) 557 576 Red
Texas Red  578 600 Red
Cyanine Dyes
Cy3E 550 570 Orange / red
Cy5E 651 674 Infrared
a
Wavelengths may vary slightly depending on the respective manufacturer. Emission spectra might change in different solvents or sample
conditions (Cullander, 1999).
¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112 89

et al., 1997a; Spear et al., 1999) and recently mRNA like mild acid hydrolysis with 1 M HCl or treatment
(Wagner et al., 1998) have also been successfully with mutanolysin or 1,4 dithio-L-threitol (Macnaug-
detected by FISH. 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
3.4. Fixation species are strain dependent, analysis of complex
environmental samples containing these bacteria
Prior to hybridization bacteria have to be fixed and remains difficult (Macnaughton et al., 1994; De Los
permeabilized for penetration of the fluorescent Reyes et al., 1997).
probes into the cell and to protect the RNA from For FISH on tissue sections, pretreatment pro-
degradation by endogenous RNAses. Fixation can cedures have been applied to increase accessibility of
employ precipitating agents like ethanol or methanol, the probe to the target and to reduce non-specific
cross-linking agents like aldehydes, or a mixture binding. For paraffin sections, de-waxing with xylene
thereof. Fixation conditions may vary dependent prior to the hybridization step is mandatory (Boye et
upon the target organism and the type of sample or al., 1998). An additional step for paraffin as well as
tissue. Effective fixation is crucial for satisfactory for cryo-sections is pretreatment with proteinase K
FISH results, but unfortunately it is difficult to (Loy et al., 1996). Recently, FISH has been applied
optimize. Optimal fixation should result in good to tissue embedded in cold polymerizing resin
probe penetration, retention of the maximal level of (Moter et al., 1998b). In these plastic sections,
target RNA, and maintenance of cell integrity and visualization of bacteria and excellent histological
morphologic detail. In general a 3–4% (v / v) form- conservation of the tissue was achieved without any
aldehyde or paraformaldehyde solution is sufficient digestive pretreatment (Figs. 5–9).
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., 3.6. Hybridization
1992; Brown-Howland et al., 1992; Roller et al.,
1994) and additional pretreatments might be neces- Hybridization must be carried out under stringent
sary for permeabilization (see below). conditions for proper annealing of the probe to the
target sequence. For this crucial step of the FISH
procedure, preheated hybridization buffer is applied
3.5. Specimen preparation and pretreatment to the sample containing fluorescently labeled probes
complementary to the target RNA. Stringency can be
For better attachment of specimens to glass slides, adjusted by varying either the formamide concen-
it is recommended to treat the surfaces first with a tration or the hybridization temperature. Formamide
coating agent. Chemicals that have been successfully decreases the melting temperature by weakening the
used include gelatin (Amann et al., 1990b), poly-L- hydrogen bonds, thus enabling lower temperatures to
lysine (Lee et al., 1999) or silanating agents (Moter be used with high stringency.
et al., 1998b). If cell suspensions are investigated, The hybridization takes place in a dark humid
bacteria are fixed in suspension, spotted onto micro- chamber, usually at temperatures between 378C and
scopic slides, air dried, and dehydrated in an ethanol 508C. Hybridization time varies between 30 min and
series. In some cases, e.g. for Gram-positive cells, an several hours. Afterwards slides are briefly rinsed
additional enzymatic treatment with lysozyme with distilled water to remove unbound probe. Post-
¨
(Schonhuber et al., 1997; Wagner et al., 1998), hybridization stringency washes are performed as
lysostaphin, or an enzyme mixture (Krimmer et al., required. To reduce the amount of toxic waste,
1999) may be necessary to open the peptidoglycan stringency of the washing buffer can be regulated by
layer. For mycolic acid-containing Actinomycetales varying the salt concentration (Lathe, 1985) instead
like the Mycobacterium complex, Norcardia or for of by using formamide. Finally, slides are rinsed
Microthrix parvicella, permeabilization techniques 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 epifluorescence microscopy (Manz et al., 2000).
available. Another microscope used for FISH is the confocal
laser scanning microscope (CLSM). By restricting
3.7. Documentation the collected signal to a thin section of the object
investigated, out-of-focus fluorescence is removed,
For microscopy, a conventional epifluorescence leading to sharp images. This is useful for thick
microscope equipped with narrow-band-pass filter samples or those with a high background, like sludge
sets for multicolour FISH can be used. Use of a flocs, biofilms or tissue sections (Wagner et al.,
charged coupled device (CCD) camera and appro- 1993; Wagner et al., 1994; Manz et al., 1995;
priate image analysis software allows digitalization Ghiorse et al., 1996; Lawrence et al., 1998; Moter et
and manipulation of images. This is currently the al., 1998b). Software packages are available that
most sensitive system and can be helpful for critical allow three-dimensional reconstruction of images
samples where low signal intensity is expected. It (Fig. 8). However, due to fast bleaching the use of
has been used for microscopic enumeration of micro- CLSM often requires higher fluorescence signal
organisms (Li et al., 1997a) and for measuring the yield. For examples of the different microscope
activity of single cells in biofilms by quantification techniques mentioned above see Fig. 7. FISH signals
of rRNA content (Poulsen et al., 1993). Recently, can also be recorded using flow cytometry. Although
digital analysis of the spatial arrangement of bacteria the flow cytometer does not give any information
was further improved using widefield deconvolution 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 97

4. Problems and pitfalls: what can go wrong? (Fig. 3) (Graf et al., 1998). Therefore the interpreta-
tion of fluorescent signals regarding fungi has to be
4.1. False positive results done with caution. Nevertheless, specific detection of
moulds and yeasts in cell cultures, in experimentally
4.1.1. Autofluorescence infected tissues, and in spiked human blood has been
Regarding autofluorescent material, FISH clearly reported (Lischewski et al., 1996; Lischewski et al.,
has limitations. The most striking problem is auto- 1997). However, when dealing with unknown mixed
fluorescence of microorganisms themselves. This has bacterial populations or tissue specimens, there is a
been reported for a variety of moulds and yeasts clear need to check for autofluorescence prior to the
(Fig. 3) (Margo and Bombardier, 1985; Graham, FISH procedure.
1983) for certain bacteria like members of the genus
Pseudomonas (for example, see Brown and Low- 4.1.2. Lack of specificity
bury, 1996), Legionella (Edelstein, 1985; Wilkinson Accuracy and reliability of FISH is highly depen-
et al., 1990), for Rhodospirillum centenum (Albrecht- dent on the specificity of the oligonucleotide probe.
¨
Buehler, 1996), for cyanobacteria (Schonhuber et al., Careful design and critical evaluation of new probes
1999) and for archaeal species like methanogenes are essential. Positive controls as well as closely
(Sorensen et al., 1997) the latter ones complicating related strains harbouring target sequences with few
FISH analysis of environmental microbiota. Auto- mismatches to the respective probe as negative
fluorescence can also be found in material surround- controls should be included in every FISH experi-
ing the bacteria in environmental samples, like ment. It is important to note that an oligonucleotide
activated sludge, plants or even drinking water and is is only as precise as the database from which it is
caused by natural fluorescent biological or inorganic derived. Due to the inaccuracy of some reported
debris (Vesey et al., 1997). Similarly various tissues sequences and the rapid expansion of existing data-
that contain elastin and collagen or blood cells like bases (Bhatia et al., 1997), probe sequences should
erythrocytes and eosinophile granulocytes usually be checked on a regular basis using the newest
exhibit bright autofluorescence (Van de Lest et al., version of sequencing data. Dealing with fastidious
1995; Monici et al., 1995). Although some back- or uncultured organisms, it is useful to check spe-
ground autofluorescence may be helpful working as a cificity first using other hybridization assays like
counterstain allowing orientation within tissue sec- dot-blot hybridizations (Lischewski et al., 1996;
tions (Figs. 6 and 9), it generally decreases the Moter et al., 1998a; Wullings et al., 1998). If there
signal-to-noise ratio and masks specific fluorescent are still difficulties with adjusting the stringency of
signal. In cytogenetic and immunologic studies, the probes, resequencing of control strains and
tissue autofluorescence has been successfully elimi- isolates may also be required. Therefore design and
nated by subtracting it pixel by pixel during digital evaluation of new probes should be done in lab-
¨ ¨ et al., 1995; Van de Lest
image processing (Szollosi oratories with much experience in microbiological
et al., 1995). and molecular biological methods.
Thorough analyses of the phenomenon of auto- Despite meticulous probe design and testing,
fluorescence and how to avoid interference with binding of probes to as-yet undescribed organisms
FISH are rare, however, growth media, fixation cannot be ruled out, when analyzing mixed bacterial
methods and mounting media seem to have dramatic populations. To ensure that the correct bacterium is
influence on the signal intensities (Sorensen et al., detected, two specific probes that each target a
1997; Graf et al., 1998). Use of narrow-band filter different position of the 16S rRNA and are labeled
sets and signal amplification systems are recom- with different fluorochromes can be used. Cells that
mended to overcome the problem of autofluoresc- are detected by both probes and therefore exhibit
¨
ence (Sorensen et al., 1997; Schonhuber et al., double fluorescence can be regarded as target organ-
1999). For Candida albicans it has been shown that, isms (Neef et al., 1996). This approach is, however,
depending on the fixation and excitation wavelength, restricted by the limited availability of specific target
autofluorescence can also vary within a single strain 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 it is mandatory to evaluate and optimize every newly
designed probe with the respective reference organ-
4.2.1. Insufficient penetration ism and with proper negative controls before it is
Low signal intensity may be the consequence of applied to mixed or unknown samples.
insufficient probe penetration into the bacterial cell, Since self-annealing and hairpin formation of the
which depends on the structure of the bacterial cell probe itself can also lead to low signal intensities,
wall. Gram-negative organisms usually do not pose newly designed oligonucleotides should be checked
any problem as they are permeable even for poly- using appropriate software (Fuchs et al., 1998).
ribonucleotide probes. For Gram-positive microorga-
nisms, particularly those exhibiting long or medium 4.2.3. Low rRNA content
chain mycolic acids, special fixation and pretreat- Although normally highly abundant, the rRNA
ment may be necessary (see above). FISH has been content of bacterial cells may vary considerably not
performed on Gram-positive cocci (Beimfohr et al., only between species, but also within cells of one
1993; Krimmer et al., 1999), Corynebacterium (Rol- strain according to their physiologic state which is
ler et al., 1994), Actinomyces (De Los Reyes et al., directly correlated with growth rate (DeLong et al.,
1997; Schuppler et al., 1998), Bacillus (Felske et al., 1989; Kemp et al., 1993; Wallner et al., 1993;
1998) and Listeria (Wagner et al., 1998). Poulsen et al., 1993). Low physiological activity can
However, cell wall characteristics can sometimes thereby result in low signal intensity or false nega-
be used advantageously; FISH has been used to tive results. For detection of slow-growing species,
estimate the state of bacterial cell walls (Bidnenko et bright fluorescent dyes like Cy3 are recommended.
al., 1998). Using HRP-labeled probes Lactococcus To further increase signal intensity and thus sen-
lactis was not detectable by FISH unless it was sitivity, one can use multiple labeling. Two or more
infected by a virulent bacteriophage leading to specific probes labeled with the same fluorescent dye
expression of autolytic enzymes and permeabiliza- are applied to increase the number of fluorescent
tion of cell walls. The percentage of hybridized cells molecules per cell (Lee et al., 1993). This technique
indicated the level of autolysis in culture. is of course limited by the availability of target sites
for specific probes.
4.2.2. Higher order structure of target or probe As mentioned above, sensitivity can also be
Due to the three-dimensional structure of the enhanced using signal amplification systems or poly-
rRNA, not all sequences are equally accessible for ribonucleotide probes (see Section 3.1).
probes. Loop and hairpin formation as well as
rRNA-protein interactions hinders hybridization 4.2.4. Photobleaching
leading to differential sensitivity of oligonucleotide Many fluorochromes will fade rapidly upon excita-
probes. This explains why probes that performed tion leading to irreversible destruction over time.
well in hybridization formats using denatured RNA With exposure times of several seconds or even
or DNA did not give satisfactory results in FISH minutes, this may have a critical effect, particularly
(Frischer et al., 1996). A systematic study addressing on photomicrographs. To circumvent this problem,
this problem was published by Fuchs et al. (1998). the use of narrow band filter sets, photostable
The authors created more than 200 adjacent oligo- cyanine dyes (as mentioned above), and antifading
nucleotide probes, specific for different locations on mounting media are strongly recommended.
E. coli 16S rRNA, and measured their signal inten-
sities in FISH using flow cytometry. Relative to the 4.2.5. Use of bacterial probes
brightest probe, the oligonucleotides were classified The best control for false negative results due to
into six brightness classes and an accessibility map methodological problems is the use of a bacterial
of E. coli 16S rRNA was established. Since the 16S probe. If hybridization with an universal probe gives
rRNA is highly conserved, this map can facilitate a satisfactory results in FISH, fixation, probe penetra-
rational probe design not only for E. coli, but also for tion and rRNA content of bacterial cells are not the
other microorganisms. However, as the authors state, limiting factors. EUB338 (Amann et al., 1990a) is
¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112 99

the probe most commonly used for this purpose. al., 1998) and lake snow of a high mountain lake
However, recent analyses indicated that some bac- (Weiss et al., 1996), in soils (Felske et al., 1998) and
terial phyla including Planctomycetales and Ver- on root surfaces (Macnaughton et al., 1996). How-
rucomicrobia are missed by this probe (Daims et al., ever, when analyzing complex environmental mi-
1999). Therefore it might be necessary to use a set of crohabitats autofluorescence can be problematic.
bacterial probes to get a more comprehensive view FISH not only provides snapshots of a certain
when analysing complex microbial communities. To moment but has also been used to monitor bacterial
control non-specific binding of the eubacterial probe consortia in continuous-flow cultures of seawater
to bacterial 16S rRNA or to cell components other sediment (Bruns and Berthe-Corti, 1998), during
than nucleic acids, the complementary probe, called seasonal changes in a high mountain lake (Pernthaler
NON338 (Wallner et al., 1993) can be used and it et al., 1998), and planktonic composition in response
should not give any signal in FISH. ¨
to enhanced protozoan grazing (Jurgens et al., 1999).
Furthermore, uncultivated species or new, recently
defined bacterial phyla have been detected in en-
5. Applications vironmental samples using FISH, like members of
the Holophaga /Acidobacterium phylum (Ludwig et
It is beyond the scope of this article to review all al., 1997), an uncultured Bacillus in Dutch grassland
possible applications of FISH. Therefore the selec- soils (Felske et al., 1998), and recently Aquabac-
tion of works cited is rather subjective and should terium in a Berlin (Germany) drinking water system
highlight the versatility of the method. (Kalmbach et al., 1999).
All these studies have great implications for
5.1. Microbial diversity in environmental samples understanding the composition and ecology of natu-
ral bacterial consortia and their population dynamics
It is well accepted that the number of bacterial in response to influences of nature or mankind.
species in the environment by far exceeds the
number of described species. For a long time, the 5.2. Microbial diversity in wastewater treatment
magnitude of microbial diversity was underesti-
mated. In various environmental samples, micro- Ardern and Lockett (1914) developed the first
scopic total cell counts exceeded the number of activated sludge system for purification of waste-
culturable bacteria by several orders of magnitude water in Manchester. However, the role of microbial
(Kogure et al., 1979; Ferguson et al., 1984; Staley consortia in this process is still not completely
and Konopka, 1985). Comparative rRNA analysis understood. Because culture-based techniques were
has revolutionized the description of mixed bacterial found to be too selective to give a comprehensive
consortia. However, most techniques involve crucial and authentic picture of the entire microbial com-
steps like nucleic acid extraction or PCR amplifica- ¨
munity (Wagner et al., 1993; Kampfer et al., 1996),
tion that may be biased as well (for a review see von rRNA-based population analysis has found increas-
Wintzingerode et al., 1997). Because FISH gives a ingly wide application in analysis of such environ-
detailed picture of the microenvironments without ments like bioreactors and wastewater treatment
any selective purification or amplification steps, it plants. Sequences that were retrieved from molecular
has been extensively used in the field of environmen- analysis using PCR-based techniques were used to
tal diversity research and only a few examples can be create new oligonucleotide probes for FISH inves-
mentioned in this review. It has been used worldwide tigation of bacterial communities, e.g. in activated
to investigate microbial communities in natural sludge (Amann et al., 1996; Snaidr et al., 1997;
environments such as aquatic habitats, e.g. popula- Schuppler et al., 1998). The number and spatial
tions in Wadden Sea sediments (Llobet-Brossa et al., distribution of nitrite-oxidizing and ammonia-oxi-
1998), in fjords (Ramsing et al., 1996), bacterioplan- dizing bacteria, responsible for key steps in micro-
¨
kton in river- or seawater (Glockner et al., 1996; bial nitrogen cycling, were analyzed in a nitrifying
Alfreider et al., 1996; Lemke et al., 1997; Kenzaka et fluidized bed reactor and in activated sludge
100 ¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112

(Schramm et al., 1998; Juretschko et al., 1998). within amoebae that were recovered from a sputum
Recently, Bond et al. (1999) identified groups of sample of a patient with pneumonia (Springer et al.,
bacteria in sludge with enhanced biological phos- 1992). This demonstrates that amoebae should not
phorus removal. Other groups focused on mycolic- only be recognized as human pathogens themselves
acid containing actinomycetes that are thought to be but also as vectors for a variety of bacterial
involved in filamentous foaming in sewage treatment symbionts. This might be the case for facultative
plants (De Los Reyes et al., 1997; De Los Reyes et endosymbionts like Legionella pneumophila which
al., 1998; Schuppler et al., 1998). Also members of has been vizualized within Acanthamoeba castellani
the domain Archaea, particularly methanogens, have (Grimm et al., 1998) and within ciliate Tetrahymena
been found by FISH in anaerobic digesters and pyriformis (Manz et al., 1995). In nutritionally poor
within anaerobic sludge granules, despite their strong aquatic habitats, legionellae are able to enter a viable
autofluorescence (Sorensen et al., 1997; Rocheleau et but unculturable state, in which they are not detect-
al., 1999). Recently, the combined use of FISH and able by conventional culturing techniques. Resuscita-
microsensors allowed analyses of bacterial com- tion of dormant cells back to the culturable state was
munities and metabolic activities simultaneously, successfully monitored by FISH within the natural
thereby revealing anaerobes in anoxic microniches host Acanthamoeba castellanii (Steinert et al., 1997).
within aerobic environments (Schramm et al., These observations may have great implications for
1999b). the epidemiology of this potential pathogen.
Using different ISH strategies, bacterial endo-
5.3. Symbiotic bacteria symbionts were detected in the gills of the protob-
ranch bivalve Solemya reidii. This organism served
Most obligate microbial symbionts are as-yet as a model to evaluate a metacrylate embedding and
uncultured. Using the 16S rRNA approach, they can acetone de-embedding (MEADE) technique to in-
be identified and phylogenetically classified. How- crease signal intensities and improve the sensitivity
ever, because bacteria other than symbionts, e.g. of ISH on tissue sections (Warren et al., 1998).
ingested bacteria or intestinal microflora, are found Recently, Methanobrevibacter-related Archaea were
in protozoa or complex eukaryotes, it is difficult to detected by FISH in the hindgut contents of Re-
differentiate them from true symbionts. The localiza- ticulitermes speratus, a termite collected in the Japan
tion of microorganisms within different compart- archipelago (Shinzato et al., 1998).
ments of the host by FISH can prove their symbiotic
nature, as shown by Amann et al. (1991) who 5.4. FISH in medicine
identified uncultured bacterial endosymbionts of the
genus Holospora within the ciliate Paramecium 5.4.1. Analysis of complex microbial communities
caudatum (their natural habitat). Since Holospora
species were detected within the nucleus of the 5.4.1.1. Oral cavity
ciliate, it could be clearly distinguished from ing- Population analysis by FISH has proven particu-
ested bacteria present in food vacuoles. Similarly, larly useful for description of the normal flora or that
FISH was used to demonstrate that Paramecium of mixed microbial infections. This has been shown
caudatum also harbours Caedibacter caryophila, a for the oral cavity harbouring more than 300 differ-
killer endosymbiont that is phylogenetically related ent bacterial species, many of which can be compli-
to the genus Rickettsia (Springer et al., 1993). cated to culture or are not yet cultured. Oral in-
Recently, novel bacterial endosymbionts also related fections such as periodontitis and gingivitis are
to Rickettsia have been demonstrated in Acan- associated with specific microbial consortia. Use of
thamoeba spp. (Fritsche et al., 1999). Interestingly, culturing techniques exclusively has yielded an
the respective amoeba strains were isolated from underestimate of the microbial diversity. The utility
scrapings of infected human corneal tissue. Sar- of FISH has been shown for Gram-negative an-
cobium lyticum, a bacterium that is closely related to aerobes, like Porphyromonas gingivalis, Bacteroides
the genus Legionella, has also been demonstrated forsythus and Prevotella intermedia in subgingival
¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112 101

plaque material of periodontitis patients (Gersdorf et development of intestinal flora between breast-fed
al., 1993a,b). Further molecular-based analyses re- and formula-fed infants were studied (Harmsen et
vealed an unexpected diversity of yet-to-be cultured al., 2000). However, the physiologically relevant
oral treponemes in patients with rapidly progressive spatial distribution of the human gut flora and
periodontitis (Choi et al., 1994). The high numbers intestinal biofilm still remains largely unexplored.
and different morphologies of these spirochetes were Several approaches have been successfully applied
demonstrated by FISH on smears of subgingival in animals to study the spatial distribution of bacteria
plaque material (Fig. 4) (Moter et al., 1998a). in the intestine and to detect enteropathogens within
Furthermore, organization and spatial distribution of this complex habitat. Escherichia coli has been
oral treponemes could be studied within subgingival visualized in cryosections of the mouse large intes-
biofilms (Fig. 5). However, because epidemiological tine using probes targeting specific regions of the
data on the association of treponemes with oral 23S rRNA. In this study, E. coli cells were observed
disease alone do not prove their pathogenic potential, in mucus overlying the epithelial cells without direct
the same oligonucleotide probes were applied to attachment to the epithelium (Poulsen et al., 1994).
tissue sections of biopsies from heel bulbs of cattle Spirochetes of the genus Brachyspira /Serpulina
suffering from digital dermatitis (Figs. 6–8). This were specifically visualized within intestines of pigs
chronic, tissue-destructive, and difficult-to-treat dis- with swine dysentery and colonic spirochaetosis
ease is associated with a predominantly anaerobic (Boye et al., 1998). Brachyspira ( Serpulina)
mixed flora very similar to that found in periodon- pilosicoli was found to colonize and invade the
titis. A layering of the different bacterial groups surface epithelium and crypts of inoculated pigs
could be demonstrated within the tissue, indicating whereas Serpulina intermedia inoculated pigs re-
that certain phylogenetic groups of treponemes are vealed no abnormalities (Jensen et al., 2000).
more likely to invade tissue and maintain the disease Another study analyzed the colonization of the colon
than are other treponemes, rods or cocci (Moter et in mice experimentally infected with Salmonella
al., 1998b). typhimurium. An avirulent, streptomycin-resistant
Salmonella strain was compared to its lipopolysac-
5.4.1.2. Gastro-intestinal flora charide (LPS)-deficient mutant (Licht et al., 1996).
Because the intestine is the most heavily colonized Both strains were equally able to proliferate in the
part in the human body, the healthy intestinal flora intestinal mucus and signal intensity in FISH indi-
and biofilm formation in human gut are of great cated a similar ribosome content. Whereas the wild-
interest. Again, culturing techniques underestimate type colonized the epithelial cells on the first day, the
the actual number of bacteria (Harmsen et al., 1999). LPS-deficient strain stayed in the layer close to the
To date most FISH investigations on human gut flora colonic lumen and did not adhere to the epithelium,
have been restricted to feces. The amount of suggesting that LPS might enable Salmonella to
Bifidobacterium spp. and of the flavonoid degrading better penetrate the intestinal mucosal layer and bind
bacterium Eubacterium ramulus were estimated in to epithelial cells. A similar approach was used to
fecal samples using genus- or species-specific investigate the influence of Klebsiella pneumonia
probes, respectively (Langendijk et al., 1995; Sim- capsule expression during the colonization of mouse
mering et al., 1999). Variations of bacterial popula- large intestine (Favre-Bonte´ et al., 1999). In another
tions, including Bacteroides and Clostridium spp., study using experimentally infected mice, Nordentoft
Streptococcus, Lactococcus and the Clostridium et al. (1997) tried to establish a diagnostic FISH for
coccoides-Eubacterium rectale group in human feces detection of Salmonella. Due to high sequence
were monitored over an 8-month period (Franks et homology, 16S rRNA does not discriminate most
al., 1998). This technique was further improved by species of Enterobacteriaceae. Therefore, the authors
development of automated image acquisition and used a sequence from the 23S rRNA as a target for a
analysis software allowing fast and accurate micro- specific probe detecting 49 of 55 Salmonella
scopic enumeration of groups of intestinal bacteria serovars but none of the 43 other investigated species
(Jansen et al., 1999). Recently, the differences in 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 FISH was 100%, whereas the sensitivity was moder-
colon and lung tissue from experimentally infected ately lower.
mice as well as from animals with a history of
salmonellosis. 5.4.2. Detection of pathogens within tissues or
Other pathogenic organisms, Yersinia pestis, Y. sterile compartments
pseudotuberculosis and Y. enterocolitica, were suc-
cessfully identified down to the species level by 5.4.2.1. Implanted medical devices
combination of two probes targeting 16S or 23S Infections of implanted biomaterial like intraven-
rRNA in spiked stool and throat swab specimens, ous catheters, arthroplasties or orthopedic implants,
and in cryosections of experimentally infected mice are a common cause of septicemia and malfunction
(Trebesius et al., 1998). However, the authors stated of medical devices. Due to their ability to adhere and
that PCR-based techniques might be more suitable form biofilms on biomaterial, staphylococci are the
for detection of pathogenic yersiniae at concentra- most frequently isolated bacteria in polymer-associ-
tions below 10 5 cells ml 21 . This is particularly true ated infections. However, diagnosis of staphylococci
for detection of pathogens within a mixed bacterial can be difficult when culture fails, possibly because
flora that might contain organisms phylogenetically these organisms may form small-colony variants
closely related to the target species. with reduced growth rate. In these cases FISH can be
more sensitive than culturing techniques. Moreover,
it can differentiate between infection of an implanted
5.4.1.3. Respiratory tract infections device or contamination of the sample by showing
Haemophilus influenzae is a common cause of adherent staphylococci on the device. Using 16S
upper and lower respiratory tract infections. Using rRNA directed probes, Staphylococcus aureus and
FISH, non-capsulated, non-typeable H. influenzae Staphylococcus epidermidis were specifically iden-
strains were visualized in cryosections of adenoid tified within experimental biofilms (Krimmer et al.,
tissue of 10 children during symptom-free intervals 1999). It was further shown by FISH that small-
(Forsgren et al., 1994). Bacteria were detected in colony variants of S. aureus persist intracellularily
macrophage-like cells in subepithelial cell layers and within a human endothelial cell line. In the same
found to infiltrate the reticular crypt epithelium. The study S. epidermidis was visualized in paraffin
same authors investigated the identity of these sections of connective tissue samples of a patient
infected cells using FISH for detection of with hip arthroplasty loosening.
Haemophilus and immunofluorescence for the de-
tection of CD 14 expressing cells (Forsgren et al., 5.4.2.2. Blood cultures
1996). The results indicated that Haemophilus may Recently, the applicability of FISH for the identifi-
persist within cells of the monocyte / macrophage cation of pathogenic microorganisms in positive
lineage. blood culture bottles was examined by two indepen-
Recently, a set of oligonucleotide probes was dent groups (Kempf et al., 2000; Jansen et al., 2000).
designed for the specific detection of pathogens The panel of genus- and species-specific oligonu-
commonly isolated from cystic fibrosis (CF) patients, cleotide probes used covered most of the pathogens
including Pseudomonas aeruginosa, Burkholderia typically found in septic patients, including staphylo-
cepacia, Stenotrophomonas maltophilia, cocci, streptococci, enterococci, Enterobacteriaceae
Haemophilus influenzae, Streptococcus pyogenes, and fungi. The authors report an excellent sensitivity
Staphylococcus aureus, and Candida albicans. In a and specificity of the FISH analysis that yielded an
clinical trial with sputum samples and throat swab identification within 25 min or 2.5 h, respectively,
specimens, these probes proved to be useful for the whereas species identification by conventional cul-
rapid and specific detection of bacteria causing acute turing usually takes 24–72 h to be completed. This
exacerbations in CF patients (Hogardt et al., 2000). rapid identification of causative pathogens in blood
Compared to culturing techniques the specificity of 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- and immunolabeling. While FISH allows identifica-
terocolitica was detected within infected cell lines, tion of individual bacteria, immunofluorescence (IF)
rat spleens and patient biopsies (Odinot et al., 1998). could provide additional information regarding an-
In this case plasmid DNA was biotin-labeled by nick tigen expression. The combination of both tech-
translation, generating fragments of 100–500 bp in niques was optimized and evaluated for the identifi-
length. Animal pathogens detected by ISH include cation of bacteria in artificial mixtures by flow
intracellular organisms in enterocytes of pigs suffer- cytometry (Wallner et al., 1996). Since antibodies
ing from proliferative enteropathy (Gebhart et al., are frequently specific at the species level and below,
1994), Anaplasma marginale in bovine erythrocytes this combination allows fast and automated identifi-
(Ge et al., 1995) and Chlamydia in avian tissues cation at a wide range of phylogenetic levels. FISH
(Theil et al., 1996). and IF were also simultaneously applied for the
microscopic detection of Bacteroides fragilis mixed
with E. coli (Ramage et al., 1998). Eubacterial or
6. Perspectives species-specific oligonucleotide probes were used for
FISH, followed by treatment with either monoclonal
6.1. FISH and function antibodies or a polyclonal antiserum specific for a
common antigen (cAg) of B. fragilis. Because signal
A further improvement of the technique is the intensity is dependant on culture conditions, on
combination of in situ identification of bacteria with pretreatment, and on washing steps during the pro-
additional information on their functional state, like cedure, the authors state that the combination of the
gene expression, metabolism or antigen expression. two techniques is a research rather than a diagnostic
The use of FISH in combination with microsensor tool. However, in situ identification combined with
measurements has already been mentioned (see analysis of antigenic variation, such as virulence
Section 5.2). These needle-shaped devices detect determinant expression, without cultivation could
gradients of specific compounds like oxygen, hydro- help in understanding mechanisms of bacterial patho-
gen sulfide, nitrate, nitrite or pH directly within genicity.
activated sludge flocs and biofilms. Combined with A technique for analysis of virulence factors was
FISH these analyses permitted study and monitoring developed by Wagner et al. (1998). Conventional
of metabolic activities, changes in population struc- FISH with a 16S rRNA-directed probe for the
ture and growth of biofilm over time (Santegoeds et identification of Listeria monocytogenes was com-
al., 1998; Schramm et al., 1999a; Schramm et al., bined with in situ detection of mRNA of the iap
1999b). (invasion associated protein) gene, coding for a
Microautoradiography can be used to visualize virulence factor in the same species. Due to the
metabolic activity. Defined cell mixtures or activated instability and low copy number of mRNA as
sludge are incubated with radioactively labeled or- compared to rRNA, a highly sensitive FISH tech-
ganic and inorganic substrates such as glucose, nique was developed. Antisense polyribonucleotide
acetate or bicarbonate. After fixation of samples and probes, internally labeled with digoxygenin during
preparation of slides, radioactivity can be visualized reverse transcription, were hybridized to whole bac-
by application and development of an autoradiog- terial cells and detected by HRP-labeled anti-DIG
raphic emulsion. Using this technique simultaneously antibodies and the fluorogenic TSA system as de-
with FISH, radiolabeled substrate uptake was shown scribed above [see Section 3.1, Fig. 2(e)]. This
to be confined to certain bacterial species and could technique is rather delicate and has to be optimized
be monitored within activated sludge under aerobic for each new target species. However, it is a
versus anaerobic conditions (Lee et al., 1999) and in promising tool for studying in situ gene expression in
natural picoplankton communities (Ouverney and complex environments. Recently, another fluorescent
Fuhrman, 1999). marker molecule, green fluorescent protein (GFP)
Another approach applicable to analyses of medi- has found increasingly wide application. By intro-
cally relevant samples is the combined use of FISH ducing the respective gene into plasmids or chromo-
¨
A. Moter, U.B. Gobel / Journal of Microbiological Methods 41 (2000) 85 – 112 105

somal DNA of an organism of interest, the expressed bone, PNAs diffuse through hydrophobic cell walls
GFP can be used to visualize and monitor promoter and allow detection of mycobacteria by FISH with-
activity or gene expression at the single cell level. out any pretreatment. Using specific, fluorescently
This reporter gene assay was combined with FISH labeled PNAs, differentiation between tuberculous
for the analysis of activated sludge (Eberl et al., and non-tuberculous mycobacteria was possible in
1997). In defined model biofilms, the spatial dis- smears of mycobacterial cultures, and directly in
tribution of organisms, time course of promoter smear-positive sputum samples within a few hours as
induction, promoter expression and bacterial interac- well (Stender et al., 1999a,b; Drobniewski et al.,
tions could be analyzed (Moller et al., 1998). A 2000). This technique constitutes an improvement in
similar approach was chosen to identify Pseudo- routine diagnosis of tuberculosis and may help to
monas putida cells by FISH and to visualize simul- establish FISH as a valuable, rapid and cost-effective
taneously the horizontal transfer of a gfp-tagged method in clinical microbiology.
plasmid within an experimental biofilm (Christensen
et al., 1998). This approach will have an important 6.3. Future
impact on structure-function analyses of complex
microbial communities. As compared to microbiological culture or in vitro
Recently, FISH was used to analyze the subcellu- amplification of nucleic acids, FISH is fast, cheap
lar cell-cycle dependant localization of DNA seg- and easy to carry out. It should be especially
ments of the E. coli chromosome (Niki et al., 2000). valuable for the detection of slow-growing, fastidi-
ous or unculturable bacteria. If applied to blood or
6.2. FISH with peptide nucleic acid ( PNA) probes cerebrospinal fluids, it could provide rapid and
accurate diagnosis of infections in critically ill
Recently, peptide nucleic acid probes have been patients. A big step towards implementation into
introduced into hybridization techniques. PNAs are clinical microbiology will be made if FISH could be
DNA analogs with an uncharged polyamide back- automated. Systems for automated image analysis
bone instead of sugar phosphates. They are stable to have already been tested. However, automation of
degradation, hybridize to complementary sequences sample processing as well as plausible control of
with higher affinity and have different hybridization results using expert systems are required. A first
characteristics. So far they have been used for a wide generation of hybridization instruments is available,
range of applications including PCR clamping, but they have not been used for microbiological
Southern blotting, detection of mutations and DNA samples yet (Capodieci et al., 1998). Besides reduc-
purification (for review see Corey, 1997). Usually tion of manual work, automated FISH would allow a
PNA probes shorter than conventional oligonucleo- high turnover and improved reproducibility of pro-
tides are required for specific binding. They hence cessing clinical samples in the laboratory. However,
seem to be an interesting alternative to conventional despite its many advantages FISH will not replace
oligonucleotide probes. This was demonstrated by culture-based methods in the near future.
Prescott and Fricker (Prescott and Fricker, 1999)
using biotin-labeled PNA probes combined with the
TSA system for specific and sensitive detection of E. 7. Summary
coli in water samples. This highly sensitive approach
detected rRNA in bacterial cells long after cell death. FISH provides information about the presence,
One of the major drawbacks of FISH using number, morphology and spatial distribution of
oligonucleotide probes is the variable and sometimes microorganisms. Although its application is fast and
insufficient penetration of probes into bacteria de- cheap, thorough controls are crucial to ensure quality
pending on their cell wall characteristics. This is a of the results. Therefore, development and evaluation
problem particularly seen in Gram-positive species of probes for diagnostic purposes should be restricted
and acid-fast bacilli. PNAs may be instrumental in to expert reference laboratories with facilities for
overcoming this problem. Due to their neutral back 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- K.-H., 1991. Identification in situ and phylogeny of uncultured
bacterial endosymbionts. Nature 351, 161–164.
ture of complex microbiological communities in
Amann, R.I., Ludwig, W., Schleifer, K.-H., 1995. Phylogenetic
humans, animals and in the environment. It has identification and in situ detection of individual microbial cells
successfully been used to detect pathogens in smears without cultivation. Microbiol. Rev. 59, 143–169.
or tissue preparations from humans and animals. It Amann, R., Snaidr, J., Wagner, M., Ludwig, W., Schleifer, K.-H.,
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by a grant (01KI9318) from the Bundesministerium Identification of some of the major groups of bacteria in
¨ Bildung und Forschung (UBG), Humboldt Uni-
fur efficient and nonefficient biological phosphorus removal acti-
¨ zu Berlin and the Korber
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