Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2008, p. 5068–5077 Vol. 74, No. 160099-2240/08/$08.00�0 doi:10.1128/AEM.00208-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Quantification of Target Molecules Needed To Detect Microorganismsby Fluorescence In Situ Hybridization (FISH) and Catalyzed

Reporter Deposition-FISH�

Tatsuhiko Hoshino,1 L. Safak Yilmaz,2 Daniel R. Noguera,2 Holger Daims,1* and Michael Wagner1

Department fur Mikrobielle Okologie, Universitat Wien, Althanstrasse 14, A-1090 Wien, Austria,1 and Department of Civil andEnvironmental Engineering, University of Wisconsin, 1415 Engineering Dr., Madison, Wisconsin 537062

Received 23 January 2008/Accepted 8 June 2008

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that iswidely used to detect and quantify microorganisms in environmental samples and medical specimens byfluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low,causing dim fluorescence signals that are not detectable against the background fluorescence. This limitationis ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimalnumbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH havenot been determined previously. In this study, a novel competitive FISH approach was developed and used todetermine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cellrequired to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures andin activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found tobe 370 � 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 � 17016S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 � 1.5 to 14 � 2and from 36 � 6 to 54 � 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISHwas 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols usingoligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situin microbial cells).

Natural ecosystems teem with microbial life, and most mi-crobes have not yet been isolated and studied by traditionalmicrobiology methods. Among the molecular approaches thatenable cultivation-independent characterization of microor-ganisms, fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes (4, 14) has been one of themost powerful and widely used techniques (1). Thanks to itsintimate link with rRNA-based phylogeny, FISH can specifi-cally detect, identify, and quantify microbes at a wide range ofphylogenetic levels, ranging from whole domains to specificgenera and species. Furthermore, FISH has been key toanalyzing the spatial organization of microbial communities(24, 26) and has been combined with other techniques forstudying the physiology of uncultured microbes in situ (18,22, 23, 27, 38).

The success of FISH experiments in determining microbialcommunity structures is often measured as the fraction ofdetectable microbial cells. Usually, this fraction is determinedby comparing the number of microorganisms detected by FISHwith a universal or Bacteria-specific probe labeled with a singlefluorescent dye to the total number of cells labeled by a non-specific nucleic acid stain (16). In many ecosystems this frac-tion is well below 50% (see reference 11 and referencestherein). FISH can fail to detect microbes for a number of

different reasons (for a review, see reference 43). One preva-lent problem occurs when targeted organisms have a low cel-lular rRNA content, which causes a probe-stained microbialcell to emit only weak fluorescence that may be invisible to thenaked eye when the sample is viewed under a microscope.Electronic signal amplification, as performed by digital cam-eras or confocal laser scanning microscope (CLSM) detectors,can overcome this obstacle if the signal-to-noise ratio (S/Nratio) is high enough to distinguish the amplified fluorescentsignals of the cells from the (also amplified) backgroundautofluorescence. Unfortunately, many kinds of environmentalsamples contain large amounts of autofluorescent matter,which hampers the detection of dim probe-stained cells.

Cells with low rRNA contents are common in samples fromoligotrophic environments (31), where many microorganismseither have low metabolic activity or persist in a dormant state.Several methods to improve the sensitivity of FISH in suchsituations have been developed (3, 17, 28, 40, 49). A veryelegant, albeit relatively time-consuming, technique is FISHcombined with tyramide signal amplification (TSA), which ex-ploits peroxidase-labeled probes. In combination with an ad-ditional cell permeabilization step this method has found wideapplication as catalyzed reporter deposition (CARD)-FISH(30, 37, 44) in microbial ecology.

Despite these technical improvements, the minimal numbersof rRNA target molecules required to obtain a visible fluores-cence signal after FISH or CARD-FISH with rRNA-targetedprobes have not been determined yet. These numbers areespecially relevant for further modifications and new applica-

* Corresponding author. Mailing address: Department fur Mikro-bielle Okologie, Universitat Wien, Althanstrasse 14, A-1090 Wien,Austria. Phone: 43 1 4277 54392. Fax: 43 1 4277 54389. E-mail: [email protected].

� Published ahead of print on 13 June 2008.

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tions of FISH. Different FISH protocols are usually evaluatedand compared based on fractions of detectable cells (seeabove) and on the fluorescence intensities, which can be ex-pressed as relative units (RU) when digital image analysis isused (11, 31). The main disadvantage of these relative mea-sures is that the same samples and imaging setup must be usedto compare the results obtained. In contrast, the required min-imal rRNA content would be a direct and absolute measure ofsensitivity. After thorough experimental verification, this pa-rameter could be used to compare different FISH and signalamplification protocols even between laboratories using differ-ent samples and imaging equipment. Furthermore, the re-quired rRNA content would indicate whether a particularFISH protocol has the potential to detect targets other thanrRNA, such as mRNA, whose copy numbers are much lowerthan those of rRNA in bacterial cells (only about 2% of thetotal RNA in Escherichia coli cells is mRNA [6]).

Determining the rRNA content of probe-stained cells is nota straightforward task. In this study we developed a novelapproach to measure, based on FISH experiments and thetheory of hybridization thermodynamics, the numbers of 16SrRNA copies needed to detect the cells of a bacterial probe-targeted population. Our approach does not distinguish be-tween 16S rRNA in mature ribosomes and 16S rRNA in pre-cursor transcripts, because both types of molecules are targetsfor FISH (36). This method was used with E. coli cells todetermine and compare the sensitivities of conventional FISHand CARD-FISH.

MATERIALS AND METHODS

Bacterial strains, growth conditions, and cell fixation. E. coli strain JM109 wasgrown in 250-ml flasks in Luria-Bertani medium for up to 26 h at 37°C. Thecultures were shaken at 200 rpm. Growth was monitored by measuring theoptical density at 600 nm, and cells were harvested at different stages duringgrowth. Verrucomicrobium spinosum was grown as described by Daims et al. (11).Cells of each species were collected by centrifugation (10 min, 7,000 � g) andfixed for 8 h in a 3% formaldehyde solution as described elsewhere (13). Fixedcells were suspended in a 1:1 mixture of 1� phosphate-buffered saline (PBS) and96% (vol/vol) ethanol and stored at �20°C.

Oligonucleotide probes, FISH, and CARD-FISH. Probe EUB338-I (2) targetsmost known Bacteria and has been used in many studies where FISH was used todetect environmental bacteria. Therefore, we chose this probe to determine therequired rRNA copy numbers in most experiments; the exceptions were theexperiments performed with activated sludge, where probe Eco681 (15) was usedto specifically detect E. coli. Probe EUB338-I was 5� labeled with the sulfoindo-cyanine dye Cy3 or Cy5 or with horseradish peroxidase (HRP). Probe Eco681was labeled with Cy3 or Cy5. Labeled probes were obtained from ThermoHybaid (Interactiva Division, Ulm, Germany). Conventional FISH of biomassimmobilized on slides was performed as described by Daims et al. (13) inhybridization buffers containing 40% (vol/vol) formamide (FA) (probeEUB338-I) or 10% (vol/vol) FA (probe Eco681). The optimal FA concentrationsfor the probes were determined by recording the probe dissociation profiles withincreasing FA concentrations (see Fig. 3; data not shown for Eco681). Thehybridization time was 24 h (EUB338-I) or 3 h (Eco681).

For conventional FISH of suspended cells (45), fixed E. coli cell suspensionswere dehydrated in 50, 80, and 96% (vol/vol) ethanol. Between the ethanol stepsthe cells were centrifuged, and the supernatant was removed. The dehydratedcells were resuspended in 200-�l PCR tubes in hybridization buffer with 40%(vol/vol) FA. The probe concentration in the hybridization buffer was adjusted tovalues between 0.01 and 0.5 �M. After 24 h of hybridization at 46°C, the cellswere centrifuged (5 min, 7,000 � g), resuspended in prewarmed (48°C) washingbuffer, and incubated for 15 min at 48°C. The composition of the washing bufferwas the same as the composition of the buffer for FISH on slides. Subsequently,the cells were centrifuged again, the supernatant was discarded, and the cellswere washed once in ice-cold 1� PBS. Following another centrifugation step, thecells were finally resuspended in ice-cold 1� PBS. To measure the fluorescence

intensity of suspended cells after FISH, the cells were dispersed by sonication onice. Almost complete dispersion (i.e., disruption of cell aggregates) was con-firmed by microscopy. The fluorescence intensity of the sonicated suspension wasmeasured with an ND-3300 fluorospectrometer (NanoDrop Technologies, Wil-mington, DE). The fluorescence intensities of hybridized and sonicated cellsuspensions were also measured in an independent experiment that was per-formed without any washing step after the hybridization.

CARD-FISH of E. coli cells was performed on slides. To improve the com-parability to conventional FISH, the hybridization temperature for CARD-FISHwas adjusted to 46°C instead of the 35°C used in the original CARD-FISHprotocol with HRP-labeled oligonucleotide probes (30). This higher tempera-ture, which also required a shorter hybridization time to avoid a loss of HRPactivity, was used with success in a previous study (41). The CARD-FISH pro-tocol used was the protocol described by Sekar et al. (39), with the followingmodifications. All CARD-FISH experiments were performed on glass slidesinstead of membrane filters. The cells were permeabilized in a 3-mg/ml lysozymesolution at 4°C for 15 min, and achromopeptidase was not used. Subsequently,the cells were incubated in 0.01 M HCl at room temperature for 30 min. Thehybridization time was 3 h, and the hybridization buffer contained 20% (vol/vol)FA. The slides were washed at 48°C for 15 min in the same washing buffer thatwas used for conventional FISH. The cells were incubated in 1� PBS on ice for10 min and then in “substrate mixture” (1 part of Cy3-labeled tyramide and 200parts of amplification buffer) at 46°C for 45 min.

To determine dissociation profiles of Cy3- and Cy5-labeled probe EUB338-I,FISH was performed in hybridization buffers containing 30 to 90% (vol/vol) FAusing a hybridization time of 3 h. To determine the dissociation profile ofHRP-labeled EUB338-I, CARD-FISH was performed in hybridization bufferscontaining 20 to 50% FA. To determine the dissociation profile of Cy3- andCy5-labeled probe Eco681, FISH was performed with 0 to 60% FA.

Microscopy, cell counting, and digital image analysis. The cell concentrationsof fixed E. coli cell suspensions were determined by direct visual cell counting.For this purpose, the suspended cells were stained at room temperature for 5 minwith a 0.1% (wt/vol) solution of 4,6-diamidino-2-phenylindole (DAPI). Subse-quently, the suspension was diluted 1:500, and 5 ml of the diluted suspension wasfiltered onto a polycarbonate filter (pore size, 0.2 �m; diameter, 47 mm; typeGTTP; Millipore Corp., Bedford, MA). The immobilized cells on the filter wereobserved by confocal microscopy (see below). In each of four replicate experi-ments, 20 images were recorded, and the fluorescent cells in each image werecounted (every image contained approximately 200 cells). Finally, the cell con-centration in the original suspension was calculated from the average number ofcells per image, the known area of each image (in �m2), the known area of thefilter, the filtered volume of the diluted suspension, and the dilution factor.

Images of probe-stained cells on glass microscope slides and on polycarbonatefilters were recorded with a CLSM (LSM 510 Meta; Zeiss, Oberkochen, Ger-many) by using two HeNe lasers (543 and 633 nm) for detection of Cy3 and Cy5,respectively. The fluorescence intensity of the probe-stained cells (mean pixelbrightness per cell) in confocal images was measured, and probe dissociationcurves were obtained by using the digital image analysis program daime (12).

Competitive hybridization with Cy3- and Cy5-labeled probes for measuringthe sensitivity of conventional FISH. The sensitivity of conventional FISH (interms of the required 16S rRNA copy number) was determined by using acompetitive hybridization approach. The theoretical basis of this approach isdescribed in the Results and Discussion. First, the ratio of the equilibriumconstants for Cy3- and Cy5-labeled probes (KCy3/KCy5) was determined by car-rying out competitive FISH of the E. coli pure culture diluted 1:100 on glassmicroscope slides. The hybridization buffers contained Cy3- and Cy5-labeledEUB338-I at Cy3/(Cy3 � Cy5) ratios of 0.2 to 1. The total probe concentrationin the buffers was 0.5 �M. Following FISH, images of Cy3 probe-labeled cellswere recorded, and the fluorescence intensity conferred by the Cy3-labeledprobe was measured by image analysis. At least 200 cells were measured for eachratio of the Cy3- and Cy5-labeled probes.

In a subsequent experiment, aliquots (10 �l) of a mixture of fixed E. coli(diluted 1:100) and V. spinosum (diluted 1:50) cells were immobilized on glassslides. Competitive FISH was then carried out with Cy3- and Cy5-labeledEUB338-I at Cy3/(Cy3 � Cy5) ratios of 0.001 to 0.1. The total probe concen-tration in the hybridization buffers was 0.5 �M. After FISH the slides wereobserved by using the CLSM in epifluorescence mode with the optical filter setfor Cy3. The lowest probe ratio (i.e., the smallest amount of Cy3-labeled probe)that yielded a clearly visible fluorescence signal for E. coli was noted.

Activated sludge samples were obtained from an animal waste-renderingwastewater treatment plant (Plattling, Germany) and fixed immediately in a 3%formaldehyde solution. A suspension of fixed E. coli cells was diluted 1:50 withfixed activated sludge, and 10-�l aliquots of this mixture were immobilized on

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glass microscope slides for competitive FISH with Cy3- and Cy5-labeled Eco681at Cy3/(Cy3 � Cy5) ratios of 0.001 to 0.1 and with a total probe concentration of0.5 �M in the hybridization buffers. Following FISH, confocal images of the Cy3signal were recorded by using the same detector settings that were used in theexperiments with E. coli pure cultures and probe EUB338-I. The intensity ofthe fluorescence signal was measured by image analysis in the images where theprobe-stained E. coli cells were clearly visible against the autofluorescent back-ground of activated sludge.

Competitive hybridization with Cy- and HRP-labeled probes for measuringthe sensitivity of CARD-FISH. To determine the ratio of equilibrium constantsfor Cy3- and HRP-labeled probes (KCy3/KHRP) (see Results and Discussion), weperformed competitive hybridizations with E. coli pure-culture cells diluted 1:100and Cy3- and HRP-labeled EUB338-I. The Cy3/(Cy3 � HRP) probe ratio wasadjusted to values ranging from 0.01 to 1. These competitive hybridizations werecarried out using the CARD-FISH protocol, except that the detection step ofCARD-FISH (i.e., addition of tyramide) was omitted, because only the fluores-cence intensity conferred by the Cy3-labeled probe was measured after thehybridizations. The measured fluorescence intensities were plotted, and curvefitting based on equation 13 (see below) was performed with Sigma Plot 8.0(Systat Software, San Jose, CA).

To determine the detection limit of CARD-FISH, probe EUB338-I labeledwith Cy5 instead of Cy3 was used as a competitor for the HRP-labeled probe.Cy5 was required because Cy3-labeled tyramide had to be used for CARD-FISHto maintain comparability to the results of the conventional FISH experiments,which had been performed by using Cy3-labeled probes. The Cy5/(Cy5 � HRP)probe ratio was adjusted to values ranging from 0.003 to 0.06. CARD-FISH andthe detection step were carried out as described above.

RESULTS AND DISCUSSION

Dependence of fluorescence intensity (i.e., 16S rRNA con-tent) on the growth phase of E. coli. FISH with Cy3-labeledEUB338-I and E. coli cells which had been harvested at dif-ferent stages in the growth curve showed that the intensity ofprobe-conferred fluorescence decreased during the log phaseand reached a constant, low value in the stationary phase (Fig.1). Theoretically, the lower intensities might be explained byreduced permeability of the cells due to structural changes inthe cell wall in the stationary phase. However, this seemsunlikely as E. coli is a gram-negative bacterium and problemswith fluorochrome-labeled oligonucleotide probes and cellpermeabilization are restricted mostly to gram-positive organ-

isms and Archaea (9, 44). Thus, the decrease in fluorescenceintensity was most likely caused by a decrease in the cellular16S rRNA content. This is consistent with previous studies thatdemonstrated the use of FISH with oligonucleotide probes formonitoring changes in the 16S rRNA content of bacteria (7,33). All subsequent experiments were performed with station-ary-phase E. coli cells that were collected at 26 h. After fixationand resuspension of the cells, the concentration of E. coli was1.26 � 109 � 0.15 � 109 cells/ml.

Average cellular 16S rRNA content of E. coli. Aliquots ofstationary-phase E. coli cells were hybridized in suspensionwith different concentrations (0.01 to 0.5 �M) of Cy3-labeledEUB338-I. After the hybridization and washing steps, the cellswere sonicated, and the fluorescence intensities of the suspen-sions were measured. Parallel experiments were performedwithout the washing steps after the hybridizations. The fluo-rescence measured in the experiments with the washing stepswas conferred only by probes bound to 16S rRNA, whereas thefluorescence measured in the experiments without the washingsteps was emitted by probes that had bound to 16S rRNA andalso by remaining probe molecules in the supernatant. Thefluorescence intensities measured with and without the wash-ing steps were plotted against the known initial probe concen-trations in the hybridization buffers. The curve obtained in theexperiment without washing was linear over the whole range ofprobe concentrations (Fig. 2). The linearity of this curve dem-onstrates that there was no significant difference between theintensities of fluorescence emitted by hybridized and nonhy-bridized probe molecules and that possible quenching or othernegative effects of cell material were negligible in this experi-ment. This curve was used as a standard curve for determiningthe 16S rRNA content, as follows.

In the experiment with washing, the fluorescence intensity

FIG. 1. Optical density at 600 nm (�) and intensity of probe-conferred fluorescence (per cell) after FISH with Cy3-labeled probeEUB338-I (�) during the growth phases of an E. coli batch culture.The arrows indicate the growth phases (arrow 1, lag phase; arrow 2,logarithmic phase; arrow 3, stationary phase). One experiment wasperformed; for each data point the fluorescence of at least 200 cellswas measured. OD600, optical density at 600 nm.

FIG. 2. Intensity of probe-conferred fluorescence of a sonicated E.coli cell suspension with (E) and without (�) the washing steps afterFISH with Cy3-labeled probe EUB338-I. The fluorescence intensitieswere measured by fluorimetry. (Inset) Data for probe concentrationsbetween 0 and 0.1 �M, showing that the fluorescence without washingalso increased linearly at these low probe concentrations. The errorbars indicate the standard deviations (n � 3), and error bars smallerthan the symbols are not shown.

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reached a constant value (239 � 15 RU) at the higher probeconcentrations, indicating that all available binding sites on16S rRNA molecules were occupied by probes (Fig. 2). Thisintensity corresponds to a total probe concentration of 0.076 �0.005 �M in the standard curve (i.e., in the experiment withoutwashing). Based on Avogadro’s number (6.022 � 1023 mole-cules per mol), this probe concentration could be converted tothe absolute number of probe molecules that had bound to 16SrRNA (4.56 � 1013 � 0.28 � 1013 molecules ml�1). Then,dividing this probe concentration by the cell concentration ofthe suspension, we found that the average 16S rRNA contentper cell was 37,000 � 4,500 copies. Bremer and Dennis (8)inferred the ribosome content of E. coli from other measuredparameters and found that cells from a fast-growing culture(doubling time, 24 min) contained about 72,000 ribosomes/cell,whereas cells from a slow-growing culture (doubling time, 100min) contained only about 6,800 ribosomes/cell. Based onthese numbers, one would expect stationary-phase cells to con-tain a lower number of 16S rRNA copies than the numberdetermined by our method. However, the rate of ribosomedegradation in starved E. coli cells depends strongly on thecomposition of the nutrient medium (19). Ramagopal (34)isolated ribosomes from E. coli grown in Luria-Bertani me-dium and showed (by directly quantifying the ribosomes) thatthe ribosome content at 24 h after inoculation was as high as82% of the ribosome content in the logarithmic growth phase.In our study, E. coli was also grown in Luria-Bertani medium,and the cells used to determine the 16S rRNA content wereharvested after 26 h. Furthermore, ribosome degradation leadsto a transient accumulation of high-molecular-weight RNA-containing particles in starved E. coli cells (25). We cannotexclude the possibility that rRNA-targeted probes also hybrid-ized to such degradation intermediates. The spatial structureof partially degraded rRNA may differ from the structure inintact ribosomes, and structural modifications, such as dimer-ization (42), occur in mature ribosomes at different growthstages of E. coli. Therefore, it is difficult to predict whichfraction of the 16S rRNA molecules was accessible to theoligonucleotide probe, and the 16S rRNA copy number re-ported here should be considered a minimum estimate of theaverage copy number in the cells analyzed.

Theoretical framework for competitive hybridizations. Ourapproach for quantifying the sensitivity of FISH relied on thecompetition between differently labeled probes targeting thesame site on the 16S rRNA. Below we describe the thermody-namic model for these competition experiments, which isbased on models used previously for predicting the affinity ofFISH probes (47, 48) and for simulating probe dissociationprofiles (46).

The competitive hybridization of two probes can be de-scribed by equations 1 and 2, where P, T, and H are the probe,target, and probe/target hybrid, respectively (subscripts 1 and 2indicate the competing probes). In FISH, the probe and targetcan have folded and unfolded conformations (47), both ofwhich are included in the definitions of P and T in equations 1and 2 but are not shown for the sake of simplicity. If probes areadded in excess of the target {i.e., [P1]0 � [P2]0 [T]0}, then[P1]0 � [P1] � [H1] � [P1] and [P2]0 � [P2] � [H2] � [P2],where [P1]0 and [P2]0 are the initial probe concentrations in thehybridization buffer. Thus, the equilibrium constants, K1 and

K2, of the hybridizations can be described by equations 3 and4. These constants correspond to the overall equilibrium con-stants of the main processes that occur during in situ hybrid-ization (47). Dividing equation 3 by equation 4 and rearrangingresult in equation 5, which describes the ratio of the hybrids.

P1 � T7 H1 (1)

P2 � T7 H2 (2)

K1 �H1�

P1�0T�(3)

K2 �H2�

P2�0T�(4)

H1�

H2��

K1P1�0

K2P2�0(5)

In competitive hybridizations, the experimental response isobtained as the fluorescence of one of the probes. Let thisprobe be probe P1. To link the response of P1 to ribosomequantity, we started with the mass balance of probe and targetas described by equations 6 and 7, where [PT] and [TT] are thetotal probe and target concentrations, respectively. The parti-tioning of total target into free (T) and hybridized (H1 and H2)forms (equation 7) does not always allow a unique solution forthe intended derivation of ribosome quantity from the exper-imental response. However, in the ideal case, when the rRNAtargets are nearly saturated {i.e., [T] � 0; hence, [T] �� [H1] �[H2]}, equation 7 simplifies to equation 8. Then it is possible todetermine the fraction of targets hybridized to the fluorescentprobe ([H1]/[TT]) as a function of equilibrium constants andprobe concentrations only, as shown in equation 9. The right-most term of this equation is obtained by substituting [H1] and[H2] from the corresponding expressions in equations 3 and 4.

PT� � P1�0 � P2�0 (6)

TT� � T� � H1� � H2� (7)

TT� � H1� � H2� (8)

H1�

TT��

H1�

H1� � H2��

K1P1�0

K1P1�0 � K2P2�0(9)

Equation 9 can be rearranged in two steps. First, dividing boththe numerator and the denominator on the rightmost side by [PT]results in equation 10, where R is the probe ratio (i.e., Ri �[Pi]0/[PT]). Second, equation 11 is obtained by substituting R2 �1 � R1 in equation 10, dividing the nominator and denomina-tor by K2, and rearranging. This equation allows quantificationof rRNA targets from competitive hybridization experiments ifthe K1/K2 ratio is known.

H1�

TT��

K1R1

K1R1 � K2R2(10)

H1�

TT��

K1

K2R1

1 � �K1

K2� 1�R1

(11)

Finally, assuming that fluorescence intensity (F) is propor-



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tional to the fraction of targets hybridized to the fluorescentprobe (i.e., [H1]/[TT]), it can be described by equation 12,where is a proportionality factor and ε is the backgroundfluorescence. Combining equations 11 and 12 results in equa-tion 13, which allows quantification of the K1/K2 ratio fromcompetitive hybridization experiments. However, the criticalassumption that nearly all rRNAs are hybridized with probes(equation 8) must be fulfilled when equations 11 and 13 areused. This assumption is true if two conditions are met: (i) theamount of at least one probe is greater than the amount of thetarget, and (ii) this probe is at the high-fluorescence plateau ofits dissociation profile.

F � H1�

TT�� ε (12)

F �

K1

K2R1

1 � �K1

K2� 1�R1

� ε (13)

FA dissociation profiles. The molecular weight of HRP-labeled oligonucleotide probes is approximately 10 timeshigher than that of Cy-labeled probes. Therefore, the K valuesof the probes were expected to differ significantly, which wouldlead to different probe dissociation profiles. Since the hybrid-ization stringency had to be adjusted to ensure that the probeswere at their high-fluorescence plateaus, FA dissociation pro-files of Cy- and HRP-labeled EUB338-I probes were deter-mined and compared after conventional FISH (Cy3 and Cy5)and CARD-FISH (HRP). As shown in Fig. 3, a high-fluores-cence plateau for both Cy-labeled probes was observed withFA concentrations up to 50%. In contrast, the signal of theHRP-labeled probe decreased rapidly at FA concentrations

above 30% and disappeared at an FA concentration of 50%.To verify that the rapid decrease in signal intensity was notcaused by inhibition of HRP by higher FA concentrations, wealso determined the dissociation profile of the HRP-labeledprobe at room temperature. In this experiment the signal in-tensities were higher than those at 46°C, confirming that HRPwas not inhibited by FA and that the observed decrease in thesignal intensity at 46°C was caused by probe-target dissociation(data not shown). Consequently, to ensure that the mathemat-ical modeling was valid, competition between Cy3 and Cy5 wasexamined using 40% FA, while competition between Cy-la-beled probes and HRP-labeled probes was examined using20% FA. Note that the required minimal numbers of 16SrRNA copies found in this study for conventional FISH andCARD-FISH could be accurately determined despite the dif-ferent FA concentrations used (40% versus 20%), because inall experiments all probes were at the plateaus of their disso-ciation profiles.

Estimation of equilibrium constant ratios. Since the molec-ular weights and the chemical structures of Cy3- and Cy5-labeled probes are comparable, it was anticipated that the KCy3

and KCy5 equilibrium constants would be similar. According toequation 13, if this condition is met, then the intensity offluorescence conferred by a Cy3-labeled probe should linearlydepend on the fraction of the Cy3-labeled probe (R1 � Cy3/[Cy3 � Cy5]) in the probe mixture. Figure 4A shows the resultsof competitive hybridizations with E. coli cells and mixtures ofCy3- and Cy5-labeled EUB338-I probes, confirming that thereis a linear correlation between R1 and fluorescence intensity.Therefore, we consider the ratio of KCy3 to KCy5 to be 1. Fromequation 5, it also follows that in all competitive hybridizationexperiments with Cy3- and Cy5-labeled probes, the ratio ofhybridization products was equal to the ratio of the initialconcentrations of the two probes.

The KCy3/KHRP ratio was also determined experimentally bycompetitive FISH with Cy3- and HPR-labeled EUB338-Iprobes (Fig. 4B). A coefficient of determination of 0.995 indi-cates that the curve fit based on equation 13 matched theexperimental data very well. Accordingly, the KCy3/KHRP ratiowas found to be 16.2. This is consistent with the probe disso-ciation profiles shown in Fig. 3 since the lower melting point ofthe HRP-labeled probe indicates lower stability and hence asmaller equilibrium constant.

Sensitivity of conventional FISH. The minimal rRNA con-tent required for FISH depends on the type of environmentalsample being analyzed. Planktonic cells immobilized on glassslides or by filtration are observed, after FISH, against a back-ground with a relatively low level of autofluorescence (if noinorganic particles or phototrophic microbes exhibiting strongautofluorescence are present). In contrast, samples such asactivated sludge or sediment samples can contain relativelylarge amounts of fluorescing matter, which makes the obser-vation of probe-stained cells more difficult. Hence, cells with alow rRNA content may be detectable on glass slides or filtersbut may be overlooked in more complex samples. To deter-mine the required minimal 16S rRNA content of planktoniccells immobilized on glass slides, we performed a competitiveFISH experiment with Cy3- and Cy5-labeled EUB338-I probesand a mixture of E. coli and V. spinosum cells. V. spinosum,which is not detected by EUB338-I (11), was used to obtain a

FIG. 3. Dissociation profiles of Cy3-labeled (� and solid line),Cy5-labeled (E and dashed and dotted line), and HRP-labeled (� anddashed line) probe EUB338-I after conventional FISH (Cy3 and Cy5)or CARD-FISH (HRP) with a hybridization temperature of 46°C anda washing temperature of 48°C. The profiles were determined with E.coli cells in hybridization experiments in which increasingly stringenthybridization and washing conditions were used. For each data pointthe mean fluorescence intensity of at least 1,000 cells was determined.Error bars (standard deviations; n � 3) are not shown as they aresmaller than the symbols. Fluorescence intensities were normalized bydefining the intensity measured with the lowest FA concentration usedin each experiment as 1.



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weakly autofluorescent background like that often seen in non-targeted organisms after FISH on slides or filters. The Cy3probe-stained E. coli cells could clearly be distinguished byepifluorescence microscopy from the unlabeled V. spinosumcells if the probe ratio (R1 � Cy3/[Cy3 � Cy5]) was �0.01.According to equation 11 and with KCy3 � KCy5, the R1 ratioalso corresponds to the ratio of 16S rRNA molecules hybrid-ized to the Cy3-labeled probe. Thus, based on the previouslydetermined average 16S rRNA content of E. coli (i.e., 37,000 �4,500 copies per cell), we found that 370 � 45 16S rRNAmolecules per cell had hybridized to the Cy3-labeled probewhen R1 was 0.01. This value can be considered the minimal16S rRNA content of planktonic cells required for conven-tional FISH if the cells are observed with a common epifluo-rescence microscope. The detection limit may be slightly lowerunder optimal conditions if the background fluorescence isvery low and highest-quality microscope equipment is used.

To determine the minimal 16S rRNA content required forFISH with a complex environmental sample, activated sludgewas spiked with E. coli cells. Subsequently, these cells weredetected by FISH and observed against the relatively strongautofluorescence of the activated sludge biomass. However, forthis purpose, a probe specific for E. coli (Eco681) had to beused instead of EUB338-I. The Cy3 probe-stained E. coli cellsbecame visible against the fluorescent background with an R1

ratio of 0.07, but a ratio of 0.1 was required to easily spot E. colicells that were embedded in thick sludge flocs. With an R1

value of 0.1, the intensity of fluorescence conferred by Cy3-labeled Eco681 was 59.4 � 0.8 RU. In general, the fluores-cence signal of Eco681 was dimmer than that of EUB338-I, asshown in Fig. 5 as a function of R1. Thus, from Fig. 5 it followsthat the fluorescence observed with Eco681 at an R1 value of0.1 would be achieved with EUB338-I at an R1 value of 0.038.Based on this ratio (0.038), we calculated that 1,400 � 170 16S

rRNA molecules per cell would be needed to unambiguouslydetect E. coli cells in activated sludge with the EUB338-Iprobe.

As mentioned above, a probe ratio of at least 0.01 was

FIG. 4. (A) Fluorescence intensities measured in a competitive hybridization experiment (conventional FISH) with E. coli cells and mixturesof Cy3- and Cy5-labeled EUB338-I. The mean intensity of fluorescence (per cell) conferred by the Cy3-labeled probe was quantified. The equationand the coefficient of determination of the regression line are indicated. The experiment was performed to verify that KCy3/KCy5 was 1 (see the textfor details). For each data point the fluorescence intensity of at least 200 cells was determined. Error bars (standard deviations) are not shown asthey were smaller than the symbols. (B) Fluorescence intensities measured in a competitive hybridization experiment (conventional FISH) withE. coli cells and mixtures of Cy3- and HRP-labeled EUB338-I. The mean intensity of fluorescence (per cell) conferred by the Cy3-labeled probewas quantified. For each data point the fluorescence intensity of at least 200 cells was determined. The equation and the coefficient ofdetermination for the curve fitted based on equation 13 are indicated. Data points obtained for probe ratios (Cy3/[Cy3 � HRP]) of �0.02 (‚) wereexcluded from the regression, because at these ratios the Cy3-labeled probe was not in high excess of the target molecules. Note that no backgroundfluorescence was recorded, because the excitation laser of the CLSM had been set to a very low intensity to prevent bleaching of the sample.Therefore, the regression curve was forced to pass through the origin. The experiment was performed to determine KCy3/KHRP. The error barsindicate the standard deviations (n � 3). The gray lines indicate the 95% confidence limits.

FIG. 5. Fluorescence intensities measured in competitive hybridiza-tion experiments (conventional FISH). One experiment (E) was donewith mixtures of Cy3- and Cy5-labeled EUB338-I hybridized to mixed E.coli and V. spinosum cultures. Data points obtained for probe ratios(Cy3/[Cy3 � Cy5]) of �0.02 (‚) were excluded from the regression,because at these ratios the Cy3-labeled probe was not in high excess of thetarget molecules. In the other experiment (�), mixtures of Cy3- andCy5-labeled Eco681 were hybridized to activated sludge spiked with E.coli cells. The mean intensity of fluorescence (per cell) conferred by theCy3-labeled probe was quantified. The dashed lines indicate the lowestsignal intensities needed to visually detect E. coli cells in the presence ofthe autofluorescence of V. spinosum (S/N � 1.5) and to detect E. coli cellsagainst the fluorescent background of activated sludge (S/N � 3). Theequation and the coefficient of determination for the regression line areindicated. For each data point the fluorescence intensity of at least 200cells was determined. The error bars (standard deviations) were smallerthan the symbols and are not shown.



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needed to clearly distinguish E. coli from V. spinosum by FISHwith probe EUB338-I on glass slides. This probe ratio corre-sponded to a fluorescence intensity of approximately 30 RU.Based on this value and on a background fluorescence intensityof 20.1 (Fig. 5), an S/N ratio of at least 1.5 was required todetect E. coli. This value is consistent with the results of Per-nthaler et al. (32), who found that an S/N ratio of at least 1.3was needed to detect and automatically count probe-stainedplanktonic cells on filters. The intensity of the E. coli cellswhich were detected by probe Eco681 in the activated sludgewas approximately 60 RU, which would have corresponded toan S/N ratio of 3 if FISH had been done with a pure culture onglass slides. These S/N ratios were used to estimate the 16SrRNA copy numbers needed for CARD-FISH as describedbelow.

Sensitivity of CARD-FISH. As HRP-labeled probe mole-cules are larger than probes labeled with Cy fluorochromes,microbial cells must be permeabilized by incubating the samplewith enzymes such as lysozyme prior to CARD-FISH. Thispermeabilization step might cause leakage of ribosomes andrRNA precursor molecules from the cells. To check this, FISHwith Cy3-labeled EUB338-I was performed using the conven-tional FISH and CARD-FISH protocols with the same amountof FA (40%) in the hybridization buffer. Only the TSA step ofthe CARD-FISH protocol was omitted, because a Cy3-labeledprobe was used in this experiment. Subsequently, the fluores-cence intensities of the probe-stained E. coli cells were mea-sured and compared. Indeed, after the CARD-FISH protocolthe average intensity of probe-conferred fluorescence was 67% �9% of the value obtained with conventional FISH. Theoreti-cally, the decrease could have been caused by the shorterhybridization time (24 h for conventional FISH versus 3 h forCARD-FISH) and/or by the permeabilization with lysozyme.Therefore, to assess the impact of hybridization time, we per-formed conventional FISH for 3 and 24 h and found that theshorter hybridization time resulted in only a 10% decrease inthe fluorescence intensity. Thus, the larger decrease observedwith the CARD-FISH protocol was most likely caused by areduced 16S rRNA content due to the permeabilization step.The loss of target molecules must be considered in the CARD-FISH sensitivity calculations. Therefore, we determined twodetection limits for this method. The “practical” detection limitindicates how many target molecules that a cell must containprior to the permeabilization step to be detectable by CARD-FISH. This value was calculated based on the original 16SrRNA content (i.e., 37,000 � 4,500 copies per E. coli cell). The“ideal” detection limit is the number of remaining target mol-ecules per cell (after the permeabilization step) required toobtain a visible fluorescence signal. This value is the maximalsensitivity of CARD-FISH that could theoretically be achievedif no permeabilization was needed. It was determined based ona 16S rRNA content that was 67% of the original content (i.e.,24,800 � 4,100 16S rRNA molecules per E. coli cell).

As with conventional FISH, the minimal 16S rRNA contentrequired for CARD-FISH was determined by performing acompetitive FISH experiment. In this case, competitive hy-bridizations were performed with Cy5- and HRP-labeledEUB338-I and E. coli cells on glass slides. As KCy3 and KCy5 areidentical (see above), the experimentally determined KCy3/KHRP ratio of 16.2 was assumed to be applicable to the com-

petition experiment with Cy5- and HRP-labeled probes (i.e.,KCy5/KHRP � 16.2). Then, using equation 11, the percentagesof 16S rRNA molecules hybridized to the HRP-labeled probein these competitive hybridizations were calculated (i.e., 100 �[HHRP]/[TT]). These calculated percentages were then plottedagainst the measured intensities of HRP probe-conferred flu-orescence (Fig. 6). As with conventional FISH (Fig. 5), a clearcorrelation was obtained. This demonstrates that the quantifi-cation of target molecules by CARD-FISH is not hampered byeffects such as fluorescence quenching or exhaustion or satu-ration of HRP in the signal amplification step. The conven-tional FISH experiments had revealed that an S/N ratio of 1.5was needed to detect E. coli cells on glass slides and to distin-guish E. coli from a probe nontarget population. According toFig. 6, an S/N ratio of 1.5 corresponded to a fluorescenceintensity of 39 RU after CARD-FISH. At this signal intensity,0.036% of the 16S rRNA molecules had hybridized to theHRP-labeled probe (Fig. 6). From this it follows that the idealdetection limit of CARD-FISH of planktonic cells is as low as8.9 � 1.5 target molecules per cell. The practical detectionlimit, which considers the necessity of cell permeabilizationcausing a loss of target molecules, is slightly higher, 14 � 2target molecules per cell. The same calculations repeated foran S/N ratio of 3 showed that 36 � 6 target molecules per cell(ideal detection limit) to 54 � 7 target molecules per cell(practical detection limit) would be required for successfulCARD-FISH in activated sludge. Consequently, CARD-FISHwas 26- to 41-fold more sensitive than conventional FISH inour experiments. These values are similar to the results ob-tained by Lebaron et al. (21) (12-fold) and by Schonhuber et al.(37) (20-fold), who determined the increase in sensitivity dueto CARD-FISH by measuring fluorescence intensities withoutquantifying 16S rRNA copy numbers.

FIG. 6. Fluorescence intensities measured in a competitive hybrid-ization experiment (CARD-FISH) with E. coli cells and mixtures ofCy5- and HRP-labeled probe EUB338-I. The mean intensity of fluo-rescence (per cell) conferred by the HRP-labeled probe after TSA wasquantified. The dashed lines indicate the signal intensities that corre-spond to S/N ratios of 1.5 and 3. The equation and the coefficient ofdetermination for the regression line are indicated. Note that the x axisdoes not show the initial probe concentration ratio (as in Fig. 4 and 5)but instead shows the percentage of HRP-labeled probe that bound to16S rRNA inside E. coli cells. This percentage was calculated based onequation 11 and the experimentally determined KCy3/KHRP (Fig. 4B).For each data point the fluorescence intensity of at least 200 cells wasdetermined. Error bars (standard deviations) are not shown as theywere smaller than the symbols.



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Concluding remarks and outlook. In this study, we deter-mined how many 16S rRNA molecules are required for twoapplications of FISH and CARD-FISH with oligonucleotideprobes: the detection of planktonic cells on glass slides and thedetection of single cells in activated sludge flocs. The valueobtained here for conventional FISH on glass slides (approx-imately 370 16S rRNA copies per cell) is low compared to thedetection limit of flow cytometers (at least those which wereused before 1995), which were used to sort probe-stained bac-terial pure cultures (several thousand ribosomes per cell) (4).This is consistent with the findings of Zheng and Harata (50),who reported that a CLSM could detect as few as 82 rhoda-mine molecules in an area of 1.4 �m2, which is similar to thearea of a single bacterial cell in a two-dimensional image.Moreover, the detection of the E. coli cells in our FISH ex-periments on glass slides was facilitated by the almost completeabsence of fluorescent background. The great influence ofbackground autofluorescence on the sensitivity of FISH isshown by the much higher 16S rRNA content needed for FISHin activated sludge (approximately 1,400 copies per cell), whichis closer to the range indicated by Amann et al. (4). Activatedsludge represents a structurally relatively complex type of en-vironmental sample and resembles other complex samples,such as aquatic biofilms. Thus, the minimal 16S rRNA copynumbers determined for activated sludge can serve as refer-ence values for many standard applications of FISH andCARD-FISH. However, the sensitivity of these methods mightbe significantly lower with extreme samples, such as biofilmscontaining large numbers of phototrophic microbes or algae,whose pigments show much stronger autofluorescence than theactivated sludge biomass and matrix.

In addition to the type of sample, the sensitivity of FISH andCARD-FISH depends on technical factors, such as the opticalquality of the microscope and the fluorochrome used for probelabeling or the detection step. For example, Cy3 is much brighterthan the green dyes fluorescein isothiocyanate and FLUOS [5(6)-carboxyfluorescein-N-hydroxysuccinimide ester] (35). Further-more, the rRNA content determines the brightness of FISH sig-nals in combination with other parameters, like the cell wallstructure, the cell fixation method, quenching effects, probe affin-ity, and the accessibility of the probe binding sites on the rRNA(43, 44, 47, 48). Experiments should be performed to identify therelative importance of these factors with the goal of further op-timizing FISH and tailoring diagnostic FISH protocols to specifictarget organisms. For quantification of rRNA copy numbers, ourwork provides the theoretical and technical backbone for suchstudies.

Another important factor, which affects the sensitivity ofFISH, is the hybridization efficiency of the oligonucleotideprobe used. Some probes yield dim fluorescence signals be-cause of low probe affinity, which is a function of not only theprobe sequence but also the accessibility of the probe targetsite due to structural constraints in the ribosome (47). Hence,to yield visible signals after FISH, such dim probes may require16S rRNA copy numbers for their target organisms higher thanthose determined here for probe EUB338-I, which yields mod-erate to high fluorescence intensities (15). On the other hand,Yilmaz et al. (48) showed that the brightness of so-called dimprobes can be enhanced significantly by longer hybridizationtimes, which accounts for the slower hybridization kinetics of

such probes. Therefore, it should be noted that if probes otherthan EUB338-I are used in the competitive hybridization ap-proach, the hybridization time and stringency must be adjustedto ensure that the hybridization efficiency is optimal, becauseonly then would the results be independent of the probe used.

Interestingly, the dissociation profiles of Cy- and HRP-la-beled probes were significantly different, although the nucleo-tide sequences of the probes were identical (Fig. 3). Theseresults imply that, despite the signal amplification, CARD-FISH may yield only dim fluorescence signals if the hybridiza-tion stringency (e.g., FA concentration) is the same as thatused for conventional FISH with Cy-labeled probes. One couldargue that this effect may be less pronounced if CARD-FISHis performed at temperatures lower than those used in thisstudy. However, direct comparison with the same hybridizationand washing temperatures clearly showed that the stability ofthe rRNA-probe hybrid was lower for the HRP-labeled probe.One reason for this could be that the large molecule HRPnegatively affects the base pairing between the nucleotidesclose to the 5� end of the probe and the complementary nu-cleotides at the probe target site. This would decrease theequilibrium constant (i.e., K value) of probe target hybridiza-tion and, therefore, contribute to the altered probe dissocia-tion profile. Moreover, it might reduce the specificity of anHRP-labeled probe if the base mismatches with nontargetorganisms are located close enough to the 5� end of the probe.Our observations strongly suggest that the specificity of HRP-labeled probes should be carefully verified and that the optimalhybridization conditions should be determined separately forHRP- and Cy-labeled probes.

Applications of our competitive FISH technique are notnecessarily restricted to determining the sensitivity of FISH.Previous research has shown that cellular metabolic activityand rRNA content correlate in some bacteria, whereas in otherbacteria this is not the case (7, 8, 36). This was found by usingthe signal intensity after FISH as a simple indicator of ribo-some content. Determining absolute rRNA copy numbers in-stead of only relative fluorescence units could improve thecomparability of such studies.

Several studies have shown that CARD-FISH enables in situdetection of mRNA in microbial cells. Most of these studiesrelied on means to further enhance the sensitivity, such aspolynucleotide probes labeled with many HRP molecules (29,44) or two rounds of TSA-mediated signal amplification (20).These approaches suffer from disadvantages such as the re-duced specificity of polynucleotide probes (compared to oligo-nucleotides) and the need for additional time-consuming steps.However, based on the high sensitivity of “normal” CARD-FISH with oligonucleotide probes, this method alone couldtheoretically be sufficient for detecting mRNA in bacterialcells. Nevertheless, detection of bacterial mRNA by CARD-FISH with oligonucleotide probes and only one signal ampli-fication step has been reported only once (5). Recently, evenconventional FISH using probes labeled with a single near-infrared dye was used to detect mRNA in pure bacterial cul-tures (10). Future studies may show whether this approach,which does not include any signal amplification step, is alsosuitable for analyzing uncultured microbes in environmentalsamples. General problems with mRNA detection by FISHcould be (i) the low stability of mRNA and its degradation



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during the relatively long CARD-FISH or conventional FISHprocedures, which take place at temperatures far above roomtemperature, and (ii) leakage of mRNA out of the cells. Ex-periments carried out in this study revealed that 16S rRNAmolecules may leak out of cells permeabilized for CARD-FISH. As mRNA is often smaller than 16S rRNA or the sizesof 16S rRNA and mRNA are similar, it appears likely that thepermeabilization can also result in a significant loss of mRNAmolecules. Furthermore, the lower stability of the probe-targetduplex with the HRP label (Fig. 3) also suggests that hybrid-ization conditions should be specifically optimized for HRP-labeled probes. Therefore, developing new protocols that min-imize mRNA loss and maximize probe-target duplex stabilitymight be the key to successful, specific, and efficient FISHdetection of mRNA in microorganisms.

ACKNOWLEDGMENTS

T.H. was supported by a JSPS postdoctoral fellowship for researchabroad. Support from National Science Foundation grant CBET-0636533 to D.R.N. and L.S.Y. is also acknowledged.

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Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ...

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