Membrane integrity

Flow cytometry

Membrane integrity

Flow cytometry

10° 101 102 103 104 FL2

10° 101 102 103 104 FL2

Fluorescent activated cell sorting

Molecular Analysis -e.g., DGGE

Figure 2 FISH involves whole cell hybridization with fluorescent oligonucleotide probes targeted against specific bacterial groups and species (left-hand scheme). The fluorescent probe hybridized cells may be visualized and/or counted using fluorescent microscopy and image analysis. The right-hand scheme illustrates how the viability of the cells may be assessed using functional probes that can also be visualized by fluorescent microscopy (A). FISH-labeled or functional probe-labeled cells may also be detected and enumerated using the flow cytometer (FCM). (B) shows a dot blot of fecal cells that were hybridized with a Bifidobacterium-specific probe. Following FCM the cells can be sorted according to the functional properties based on the probe stains, and subjected to further analysis. Abbreviation: DGGE, denaturing gradient gel electrophoresis.

after a stringent washing step the amount of target rRNA is quantified. The membrane can be rehybridized with a general bacterial probe, and the amount of population-specific rRNA detected with the specific probe is expressed as a fraction of the total bacterial RNA. Quantification of the absolute and relative (as compared to total rRNA) amounts of a specific rRNA reflects the abundance of the target population. Consequently this technique does not represent a direct measure of cell number since cellular rRNA content varies with the current environmental conditions and the physiological activity of the cells at the time of sampling (67). Dot-blot hybridization has been successfully used to quantify rRNA from human fecal and cecal samples (68,69). It was found that strict anaerobic bacterial populations represented by the Bacteroides, Clostridium leptum and Clostridium coccoides groups were significantly lower in the cecum (right colon) than in the feces, while the Lactobacillus group was significantly higher in the feces than in the cecum (68).

In contrast to dot-blot hybridization, FISH is applied to morphologically intact cells and thus provides a quantitative measure of the target organism without the limitation of culture-dependent methods (Fig. 2) (15,70). Following fixation, bacteria from any given sample can be hybridized with an appropriate probe or set of probes. The fixation allows permeabilization of the cell membrane and thus facilitates the accessibility of the fluorescent probes to the target sequence. For some Gram-positive bacteria, especially lactobacilli, additional pre-treatments including the use of cell wall lytic enzymes e.g., lysozyme, mutanolysin, protease K or a mixture is needed (71-73). Prior to hybridization, the cells can be either immobilized on gelatine-treated glass slides or simply kept in suspension when analyzed by FCM. The oligonucleotide probe is labeled covalently at the 50 end with a fluorescent dye, such as fluorescein iso(thio)cyanate, while any necessary competitor probes are unlabeled. The stringency, i.e., conditions of hybridization that increase the specificity of binding between the probe and its target sequence, can be adjusted by varying either the hybridization temperature or formamide concentration. Under highly stringent conditions oligonucleotide probes can discriminate closely related target sites. Post-hybridization stringency can be achieved by lowering the salt concentration in the washing buffer in order to remove unbound probe and avoid unspecific binding.

Quantification of FISH Signals

Over the past years, significant methodological improvements of the probe fluorescent-conferred signal have been reported. These include the use of brighter fluorochromes including Cy3 and Cy5 (74,75), and unlabeled helper oligonucleotide probes (76) that bind adjacent to and increase the accessibility of the selected target site. Horseradish peroxidase labeled probes and tyramide signal amplification (also termed CARD-FISH) can be used to significantly enhance the signal intensity of hybridized cells (77). However, the latter requires effective permeabilization for the large enzyme-probe complex to enter the cell with the risk of damaging and lysing fixed cells. A further possibility is the use of peptide nucleic acid (PNA) probes which can confer very bright signals to the cell (78,79). However, currently PNA probes are rather expensive and previously published oligonucleotide probes cannot be simply translated into PNA probes.

Epifluorescence microscopy is the standard method by which fluorescent-stained cells are enumerated; however, the method is time consuming and subjective (56,57). This technique has been improved by development of automated image acquisition and analysis software allowing accurate microscopic enumeration of fecal bacterial cells (73). Alternatively, FCM offers a potential platform for high-resolution, high throughput identification and enumeration of microorganisms using fluorescent rRNA-targeted oligonucleotides with the possibility of cell sorting (40,80-84).

An FCM method for direct detection of the anaerobic bacteria in human feces was first described over a decade ago (85). A membrane-impermeable nucleic acid dye propidium iodide (PI) was used in combination with the intrinsic scatter parameters of the cells to discriminate fecal cells from large particles. Coupling FCM results and image analysis, the authors showed that most of the particles detected with a large forward scatter value corresponded to aggregates most likely representing mucus fragments and undigested dietary compounds. They confirmed by means of cell sorting that the Pi-stained cells (fecal cells) corresponded to a 2-D surface area of <1.5 mm2 while the unstained particles (aggregates) were around 5.0 mm2 (85). The work highlighted the potential of FCM to study anaerobic fecal bacteria without culturing. Despite this valuable work and to quote from Shapiro "the subject matter may stink, but the method is superb" (86), the application of FCM to study the intestinal microbiota is still in development.

FISH-FCM was applied to detect and accurately quantify both fecal and mucosa-associated bacteria, and statistical analysis showed a high correlation between the FCM counts and microscopic counts (Fig. 2) (37,44,84). Using FCM, several thousands of cells can be counted accurately in a few seconds. Following the hybridization step, fecal cells are stained with a nucleic acid dye, for example PI, SYTO BC, and TOTO-1, to detect the total cells and subsequently spiked with standard beads of known size and concentration. The beads are thus used as an internal standard to calibrate the measured volume and to determine the absolute count of the probe-detected cells (40,87). In addition to the determination of the absolute cell counts, the fluorescence intensity signal can also be quantified using fluorescent beads with known fluorescent intensities (86). This is of major importance for determining optimal hybridization conditions for newly designed probes (37,82,88). FCM is becoming a popular method for high-resolution, high throughput identification of microorganisms using fluorescent rRNA-targeted oligonucleotides.

Application of FISH to Study the GI-Tract Ecosystem

During the last five years, hybridization studies with rRNA-targeted probes have provided significant knowledge about the composition and structure of the gut microbiota. A large panel of oligonucleotide probes specific for various genera predominant in the GI tract have been designed and validated (Table 2), and have been used intensively in these studies.

The uniqueness and complexity of the human gut microbiota revealed by fingerprinting techniques were supported by results of analysis using nucleic-acid probe-based methods. These studies revealed that the majority of fecal bacteria belong to the Clostridium coccoides-Eubacterium rectale group and the Clostridium leptum group (~ 20-30% each), Bacteroides (~ 10%), Atopobium and bifidobacteria groups in that order of abundance (81,89,91,96,97). The Clostridium coccoides-Eubacterium rectale probe (Erec482) (Table 2) covers Eubacterium hallii, Lachnospira and Ruminococcus members, while the Clostridium leptum group comprises members of Ruminococcus species and Faecalibacterium prausnitzii (89,98). In particular members of C. coccoides-E. rectale, C. leptum, and the Bacteroides groups constituted more than half of the fecal microbiota. Atopobium and bifidobacteria groups comprised typically 4-5% each. The Lactobacillus-Enterococcus group, Enterobacteriaceae, Phascolarctobacterium and relatives, and Veillonella were less dominant (0.1 to a few percent) (90,91). However, differences in the occurrence of these bacterial groups have been reported by different research groups. These deviations may be due to the different methods or probes used, but it is also likely that the observed variance is due to the differences in the genetic background, lifestyle,

Table 2 FISH Probes Used to Study the Gastrointestinal Microbiota


Probe sequence (5"-3")

Target organism

% Formamide



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