- Email: [email protected]
Use of fluorochrome-labeled rRNA targeted oligonucleotide probe and tyramide signal amplification to improve sensitivity of fluorescence in situ hybridization
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 98, No. 4, 282–286. 2004
Use of Fluorochrome-Labeled rRNA Targeted Oligonucleotide Probe and Tyramide Signal Amplification to Improve Sensitivity of Fluorescence in situ Hybridization WIWAT JUPRAPUTTASRI,1 SUPAPON CHEEVADHANARAK,1 PAWINEE CHAIPRASERT,1 MORAKOT TANTICHAROEN,2 AND SOMKIET TECHKARNJANARUK2* School of Bioresources, King Mongkut University of Technology Thonburi, Bangkhuntien, Bangkok 10150, Thailand 1 and Biochemical Engineering and Pilot Plant Research and Development Unit, National Center for Genetics Engineering and Biotechnology, King Mongkut University of Technology Thonburi, Bangkhuntien, Bangkok 10150, Thailand 2 Received 27 May 2004/Accepted 20 July 2004
A tyramide signal amplification (TSA) system was used in combination with a conventional fluorochrome-labeled 16S rRNA oligonucleotide probe to increase the sensitivity of fluorescence in situ hybridization. TSA was performed after hybridization resulted in a low fluorescence signal intensity. In contrast to the horseradish peroxidase-tyramide signal amplification (HRP-TSA) system and biotin-tyramide signal amplification (biotin-TSA) system, no additional expensive probe labeling was required. A whole cell hybridization technique was used to compare the fluorescence signal obtained using a monolabeled probe with that obtained using the TSA system. The fluorescence signal of the probe obtained using the TSA system was much higher than that obtained using the monolabeled probe. The technique was successfully applied to the in situ detection of microbial communities in anaerobic sludge. It was demonstrated that TSA resulted in an increased in sensitivity, as the fluorescence signal intensity was much higher than that obtained using a conventional probe. [Key words: 16S rRNA probe, fluorescence in situ hybridization, tyramide signal amplification, sensitivity improvement]
Fluorescence in situ hybridization (FISH) using rRNAtargeted oligonucleotide probes has been increasing used in several fields of microbiology, including public health, biotechnology, and environment for the detection, enumeration, and localization of specific target sequences in whole fixed cells without cultivation (1, 2). In situ hybridization can most reliably be used for physiologically active bacteria. The high cellular ribosome content of most microorganisms facilitates a culture-independent identification of individual cells based on rRNA-targeted oligonucleotide monolabeled with a fluorescent dye. Good detection yields have also been obtained in nutrient-rich environments such as activated sludge (3) or lake snow (4). In bulk soil, however, only a small fraction of the total bacteria community (1%) has been detected by rRNA-targeted probes (5). Several bacteria growing in natural environments tend to grow slowly and may adapt by the formation of resting or dormant cells such as dwarf cells or cysts (6), and have small amounts of ribosomal RNA (7). Since the signal intensity obtained by hybridization with
rRNA-targeted probes depends on the content of ribosomes, a low cellular rRNA content can be one reason for the low detection yield. Attempts have been made to increase the fluorescence signal intensity using (i) multiple probes targeting to different target sites on the rRNA molecule and (ii) indirect labeling with a reporter molecule. Among those attempts, tyramide signal amplification (TSA) was used successfully to enhance fluorescence signal with either a biotin-labeled (8) or a horseradish peroxidase-labeled oligonucleotide probe (9). However, individual probes of interest must be labeled with either horseradish peroxidase (HRP) or biotin, and this is more expensive than conventional fluorochrome-labeled oligonucleotides probes. In this study, we evaluated a method of signal amplification based on the TSA system in which the conventional fluorochrome-labeled oligonucleotide probe was used for in situ hybridization. No additional HRP- or biotin-labeled oligonucleotide probe was required as previously reported, and TSA was performed when the FISH signal intensity was low. In additions, its application to natural samples from anaerobic sludge was also evaluated.
* Corresponding author. e-mail: [email protected] phone: +66-2-452-3452 fax: +66-2-452-3455 The costs of publication of this article were supported in part by Grants-in-Aid for Publication of Scientific Research Results from the Japan Society for the Promotion of Science (JSPS) (no. 163041).
MATERIALS AND METHODS Strains and cultivation 282
Pure cultures of Escherichia coli,
VOL. 98, 2004
IMPROVED SENSITIVITY OF FLUORESCENCE IN SITU HYBRIDIZATION
Desulfovibrio desulfuricans, Bacillus subtilis, Bacillus spp. and Methanosaeta concilii DSM3671 were used to evaluate the applicability of the TSA system. E. coli, Bacillus spp. and B. subtilis were grown aerobically in the Luria–Bertani medium at 37°C. D. desulfuricans was grown anaerobically in medium C at 30°C. M. concilii DSM3671 was grown anaerobically in sterile digestion fluid with 0.05% acetic acid at 37°C. Cells were harvested at logarithmic phase to obtain cells with a high ribosome content. Anaerobic sludge was sampled from a laboratory-scale hybrid anaerobic reactor, which has been fed with cassava starch wastewater. Oligonucleotide probes The Bacterial-specific probe EUB338 (10), the Archaea-specific probe ARC915 (11) and the sulfate reducing bacteria-specific probe SRB385 (12), were used in this study. Fluorescein-labeled probes were purchased from Interactiva (Ulm, Germany). For TSA, horseradish peroxidase-labeled anti-FITC (anti-FITC-HRP) antibody, biotinyl-tyramide and streptavidin-fluorescein (SA-FITC) were purchased from NEN Life Science Products (Boston, MA, USA). Fluorescence in situ hybridization Samples were fixed with freshly prepared 4% paraformaldehyde in PBS as previously described (13). The samples were immobilized on glass slides coated with poly-L-lysine, and treated with ethanol series. FISH was performed at 46°C in a hybridization buffer containing 0.9 M NaCl, 20 mM Tris–HCl (pH 7.2) and 0.01% sodium dodecyl sulfate (SDS) for 1–2 h. Probe concentration was 50 ng for 10 ml of hybridization solution. After hybridization, the slides were washed at 48°C for 15 min in washing buffer containing 0.9 M NaCl, 20 mM Tris–HCl (pH 7.2), and 0.01% SDS. Washing buffer was removed with dH2O. All cells were stained with 4¢,6-diamidino2-phenylindole (DAPI) as previously described (13). Finally, the slides were air-dried and mounted with an anti-fading solution (Slow Fade; Molecular Probes, Eugene, OR, USA). Samples were viewed under an Olympus BX60 microscope with appropriate filters. Images were captured with an Olympus DP50 digital camera system using Viewfinder Lite 1.0 software (Olympus, Tokyo) and processed using Studio Lite 1.0 software (Olympus). For a direct comparison, double images were acquired from the same microscopic fields at probe and DAPI excitation wavelengths. Identical exposure times were used for the analysis of parallel samples FISH and FISH-TSA. Mean intensities of fluorescent cells were analyzed from the captured images using Line Profile analysis of Olympus MicroImage software (Olympus). About 50 cells were measured for each slide. The final images were prepared with Adobe PhotoShop 4.0 software for presentation (Adobe, Mountain View, CA, USA). TSA amplification protocol When the FISH signal intensity was low, TSA reaction was performed further on the hybridized slides. Prior to TSA reaction, cell permeabilization was performed to allow large molecules of anti-FITC-HRP antibody to enter the cells. The cells were treated with lysozyme prior to TSA, covered with 10 ml of lysozyme–TE solution (0.1 mg of lysozyme [Sigma; 50,000 U/mg] per ml in Tris–EDTA buffer [TE], which contained 100 mM Tris–HCl [pH 8.2] and 50 mM EDTA), incubated at 0°C for 10 s. The enzyme reaction was stopped by rinsing the slides with TE solution. Then, the slides were incubated with TNB (0.1 M Tris–HCl [pH 7.5], 0.15 M NaCl, 0.5% blocking reagent) for 15 min and rinsed briefly with TNT buffer (0.1 M Tris– HCl [pH 7.5], 0.15 M NaCl, 0.05% Tween 20). Fixed cells on slides were incubated with anti-FITC-HRP antibody (1 : 250 [vol/vol] in TNB buffer) at room temperature for 30 min. The slides were washed by immersion in TNT buffer for 5 min at room temperature, and the cells were incubated with biotinyl-tyramide (1:25 in amplification buffer supplied by NEN Life Science Products) for 10 min at room temperature. The slides were then rinsed with TNT buffer for 5 min, and SA-FITC (1 : 250 [vol/vol] in TNB buffer) was added. After 30 min incubation at room temperature, the slides
283
were rinsed with TNT buffer and immersed in the same buffer for 5 min.
RESULTS AND DISCUSSION Whole-cell hybridization in combination with TSA system The combination of FITC-labeled, rRNA-targeted oligonucleotide probes and the TSA system was first evaluated by whole-cell hybridization. Artificial mixed cultures of gram-negative, gram-positive bacteria and Euryarchaeota were used. The TSA system was applied after cells were hybridized with fluorescence-labeled oligonucleotides. The fluorescence signal obtained using one monolabeled probe was compared with that obtained using the TSA system. The fluorescence signal intensity obtained using one monolabeled probe was low, whereas the use of the TSA system resulted in a marked increase in fluorescence signal intensity of target cells that was 1.6- to 2.7-fold higher than that obtained using monolabeled probes (Table 1 and Fig. 1). In the absence of a lysozyme treatment prior to TSA, the fluorescence signal of paraformaldehyde-fixed cells with the FITC-oligo-TSA system was significantly lower and the staining was quite uneven (data not shown). Similar observations were reported previously for the HRP-TSA system (9) and the biotin-TSA system (8). The lysozyme pretreatment protocol was required for hybridization to increase the permeability of fixed cells and allow the anti-FITC-HRP antibody to penetrate into the cells. Control experiments were performed without using a probe to check for nonspecific binding and peroxidase activity. Nonspecific binding was observed for streptavidinFITC and was eliminated by treating fixed samples with avidin to block endogenous biotin prior to hybridization. To reduce nonspecific staining due to biotin, the streptavidinFITC could be omitted, and with the anti-FITC-HRP antibody, a more direct detection can be achieved using tyramides labeled with fluorescent dyes instead of biotinyl-tyramide. Application of TSA system to environmental samples To further evaluate the applicability of the TSA system, fixed samples of anaerobic sludge were hybridized and compared with those hybridized with FITC-labeled oligonucleotide probes. When applied to the detection of bacteria, hybridization with a monolabeled EUB338-FITC probe resulted in a very low fraction of hybridized cells, and the fluorescence signal was very low (Fig. 2). This was probably due to the low RNA content of these cells since TSA resulted in a significant increase in the hybridized cell fraction TABLE 1. Comparison of average intensities between monolabeled probe and TSA system Mean intensitya Strain Monolabeled TSA Ratiob probe Escherichia coli 1374 ± 175 3673 ± 147 2.7 Desulfovibrio desulfuricans 1521 ± 205 2499 ± 355 1.6 a Mean intensities of fluorescence signals from 50 cells for each slide were analyzed using Line Profile Analysis of MicroImage software. b Ratio of intensity of monolabeled probe to that of TSA system.
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JUPRAPUTTASRI ET AL.
J. BIOSCI. BIOENG.,
FIG. 1. Epifluorescence micrographs of D. desulfuricans and E. coli. Bar: 10 mm. (A) D. desulfuricans hybridize with fluorescein-monolabeled probe SRB385; (B) D. desulfuricans hybridize with fluorescein-monolabeled probe SRB385 and TSA system; (C) E. coli hybridize with fluorescein-monolabeled probe EUB338; (D) E. coli hybridize with fluorescein-monolabeled probe EUB338 and TSA system. DAPI staining (left) and epifluorescence micrographs (right) are shown for identical microscopic fields. Exposure times were 20 ms for DAPI and 100 ms for fluorescein-labeled probe.
(Fig. 2). The use of the TSA system resulted in a marked increase in fluorescence signal intensity and yielded heterogeneous fluorescence signals. Similar observations of heterogeneous fluorescence signals were previously reported (8, 9). This heterogeneity was probably due to the accessibility of
the anti-FITC-HRP antibody rather than probe accessibility. Conclusion In this study, the application of TSA system in the combination with a conventional fluorochromelabeled probe successfully increased fluorescence signal intensity for hybridization. The system consisted of a conven-
VOL. 98, 2004
IMPROVED SENSITIVITY OF FLUORESCENCE IN SITU HYBRIDIZATION
285
FIG. 2. Epifluorescence micrographs of anaerobic sludge. Bar: 10 mm. (A) Sludge hybridize with fluorescein-labeled probe EUB338; (B) sludge hybridize with fluorescein-labeled probe EUB338 and TSA system. DAPI staining (left) and epifluorescence micrographs (right) are shown for identical microscopic fields. Exposure times were 20 ms for DAPI and 200 ms for fluorescein-labeled probe.
tional FITC-labeled oligonucleotide probe, anti–FITC-HRP antibody, biotinyl-tyramide, and streptavidin-fluorescein. No additional expensive probe labeling such as HRP or biotin, for the probe of interest was required. The commonly available, anti-FITC-HRP antibody can be generally used. The amplification step can be performed on hybridized samples following the visualization of monolabeled probe with in low signal intensity. In addition, instead of using of biotinyltyramide, a more direct detection can be achieved using tyramide labeled with fluorescent dyes. The fluorescence signal of the probe obtained using the TSA system was much higher than that obtained with using a monolabeled probe. The technique was successfully applied to the in situ detection of microbial communities in environmental samples. It was demonstrated that TSA increased sensitivity, as the fluorescence signal intensity was much higher than that obtained using a conventional probe, and the signal can overcome the autofluorescence background. The method, however, still has some limitations, as the optimal cell permeabilization protocol needs to be established to achieve a more homogenous staining. The approach provides an alternative method of improving the sensitivity of FISH.
ACKNOWLEDGMENTS This study was supported by a research grant from the National Center for Genetics Engineering and Biotechnology, Thailand. W. Jupraputtasri was supported by a postgraduate scholarship from the National Center for Genetics Engineering and Biotechnology. We thank Professor Preeda Malasit (Medical Biotechnology Unit, Mahidol University, Thailand) for providing chemical reagents and for fruitful discussions on hybridization techniques.
REFERENCES 1. Amann, R. I., Ludwig, W., and Schleifer, K.-H.: Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev., 59, 143–169 (1995). 2. Lipski, A., Friedrich, U., and Aldendrof, K.: Application of rRNA-targeted oligonucleotide probes in biotechnology. Appl. Microbiol. Biotechnol., 56, 40–57 (2001). 3. Wagner, M., Erhart, R., Manz, W., Amann, R., Lemmer, H., Wedi, D., and Schleifer, K.-H.: Development of an rRNAtargeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge. Appl. Environ. Microbiol., 60, 792–800 (1994). 4. Weiss, P., Schweitzer, B., Amann, R., and Simon, M.: Identification in situ and dynamics of bacteria on limnetic organic aggregates (lake snow). Appl. Environ. Microbiol., 62, 1998– 2005 (1996). 5. Hahn, D., Amann, R. I., and Zeyer, J.: Whole-cell hybrid-
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6. 7.
8.
9.
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ization of Frankia strains with fluorescence- or digoxigeninlabeled, 16S rRNA-targeted oligonucleotide probes. Appl. Environ. Microbiol., 59, 1709–1716 (1993). Roszak, D. B. and Colwell, R. R.: Survival strategies of bacteria in the natural environment. Microbiol. Rev., 51, 365– 379 (1987). Lee, S. and Kemp, P. F.: Single-cell RNA content of natural marine planktonic bacteria measured by hybridization with multiple 16S rRNA targeted fluorescent probes. Limnol. Oceanogr., 39, 869–879 (1994). Lebaron, P., Catala, P., Fajon, C., Joux, F., Baudart, J., and Bernard, L.: A new sensitive, whole-cell hybridization technique for detection of bacteria involving a biotinylated oligonucleotide probe targeting rRNA and tyramide signal amplification. Appl. Environ. Microbiol., 63, 3274–3278 (1997). Schonhuber, W., Fuchs, B., Juretschko, S., and Amann, R.: Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification. Appl. Environ. Mi-
J. BIOSCI. BIOENG.,
crobiol., 63, 3268–3273 (1997). 10. Amann, R. I., Krumholz, L., and Stahl, D. A.: Fluorescentoligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol., 172, 762–770 (1990). 11. Amann, R., Snaidr, J., Wagner, M., Ludwig, W., and Schleifer, K.-H.: In situ visualization of high genetic diversity in a natural microbial community. J. Bacteriol., 178, 3496– 3500 (1996). 12. Amann, R. I., Stromley, J., Devereux, R., Key, R., and Stahl, D. A.: Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms. Appl. Environ. Microbiol., 58, 614–623 (1992). 13. Wagner, M., Amann, R., Lemmer, H., and Schleifer, K.-H.: Probing activated sludge with proteobacteria-specific oligonucleotides: inadequacy of culture-dependent methods for describing microbial community structure. Appl. Environ. Microbiol., 59, 1520–1525 (1993).
Use of Fluorochrome-Labeled rRNA Targeted Oligonucleotide Probe and Tyramide Signal Amplification to Improve Sensitivity of Fluorescence in situ Hybridization WIWAT JUPRAPUTTASRI,1 SUPAPON CHEEVADHANARAK,1 PAWINEE CHAIPRASERT,1 MORAKOT TANTICHAROEN,2 AND SOMKIET TECHKARNJANARUK2* School of Bioresources, King Mongkut University of Technology Thonburi, Bangkhuntien, Bangkok 10150, Thailand 1 and Biochemical Engineering and Pilot Plant Research and Development Unit, National Center for Genetics Engineering and Biotechnology, King Mongkut University of Technology Thonburi, Bangkhuntien, Bangkok 10150, Thailand 2 Received 27 May 2004/Accepted 20 July 2004
A tyramide signal amplification (TSA) system was used in combination with a conventional fluorochrome-labeled 16S rRNA oligonucleotide probe to increase the sensitivity of fluorescence in situ hybridization. TSA was performed after hybridization resulted in a low fluorescence signal intensity. In contrast to the horseradish peroxidase-tyramide signal amplification (HRP-TSA) system and biotin-tyramide signal amplification (biotin-TSA) system, no additional expensive probe labeling was required. A whole cell hybridization technique was used to compare the fluorescence signal obtained using a monolabeled probe with that obtained using the TSA system. The fluorescence signal of the probe obtained using the TSA system was much higher than that obtained using the monolabeled probe. The technique was successfully applied to the in situ detection of microbial communities in anaerobic sludge. It was demonstrated that TSA resulted in an increased in sensitivity, as the fluorescence signal intensity was much higher than that obtained using a conventional probe. [Key words: 16S rRNA probe, fluorescence in situ hybridization, tyramide signal amplification, sensitivity improvement]
Fluorescence in situ hybridization (FISH) using rRNAtargeted oligonucleotide probes has been increasing used in several fields of microbiology, including public health, biotechnology, and environment for the detection, enumeration, and localization of specific target sequences in whole fixed cells without cultivation (1, 2). In situ hybridization can most reliably be used for physiologically active bacteria. The high cellular ribosome content of most microorganisms facilitates a culture-independent identification of individual cells based on rRNA-targeted oligonucleotide monolabeled with a fluorescent dye. Good detection yields have also been obtained in nutrient-rich environments such as activated sludge (3) or lake snow (4). In bulk soil, however, only a small fraction of the total bacteria community (1%) has been detected by rRNA-targeted probes (5). Several bacteria growing in natural environments tend to grow slowly and may adapt by the formation of resting or dormant cells such as dwarf cells or cysts (6), and have small amounts of ribosomal RNA (7). Since the signal intensity obtained by hybridization with
rRNA-targeted probes depends on the content of ribosomes, a low cellular rRNA content can be one reason for the low detection yield. Attempts have been made to increase the fluorescence signal intensity using (i) multiple probes targeting to different target sites on the rRNA molecule and (ii) indirect labeling with a reporter molecule. Among those attempts, tyramide signal amplification (TSA) was used successfully to enhance fluorescence signal with either a biotin-labeled (8) or a horseradish peroxidase-labeled oligonucleotide probe (9). However, individual probes of interest must be labeled with either horseradish peroxidase (HRP) or biotin, and this is more expensive than conventional fluorochrome-labeled oligonucleotides probes. In this study, we evaluated a method of signal amplification based on the TSA system in which the conventional fluorochrome-labeled oligonucleotide probe was used for in situ hybridization. No additional HRP- or biotin-labeled oligonucleotide probe was required as previously reported, and TSA was performed when the FISH signal intensity was low. In additions, its application to natural samples from anaerobic sludge was also evaluated.
* Corresponding author. e-mail: [email protected] phone: +66-2-452-3452 fax: +66-2-452-3455 The costs of publication of this article were supported in part by Grants-in-Aid for Publication of Scientific Research Results from the Japan Society for the Promotion of Science (JSPS) (no. 163041).
MATERIALS AND METHODS Strains and cultivation 282
Pure cultures of Escherichia coli,
VOL. 98, 2004
IMPROVED SENSITIVITY OF FLUORESCENCE IN SITU HYBRIDIZATION
Desulfovibrio desulfuricans, Bacillus subtilis, Bacillus spp. and Methanosaeta concilii DSM3671 were used to evaluate the applicability of the TSA system. E. coli, Bacillus spp. and B. subtilis were grown aerobically in the Luria–Bertani medium at 37°C. D. desulfuricans was grown anaerobically in medium C at 30°C. M. concilii DSM3671 was grown anaerobically in sterile digestion fluid with 0.05% acetic acid at 37°C. Cells were harvested at logarithmic phase to obtain cells with a high ribosome content. Anaerobic sludge was sampled from a laboratory-scale hybrid anaerobic reactor, which has been fed with cassava starch wastewater. Oligonucleotide probes The Bacterial-specific probe EUB338 (10), the Archaea-specific probe ARC915 (11) and the sulfate reducing bacteria-specific probe SRB385 (12), were used in this study. Fluorescein-labeled probes were purchased from Interactiva (Ulm, Germany). For TSA, horseradish peroxidase-labeled anti-FITC (anti-FITC-HRP) antibody, biotinyl-tyramide and streptavidin-fluorescein (SA-FITC) were purchased from NEN Life Science Products (Boston, MA, USA). Fluorescence in situ hybridization Samples were fixed with freshly prepared 4% paraformaldehyde in PBS as previously described (13). The samples were immobilized on glass slides coated with poly-L-lysine, and treated with ethanol series. FISH was performed at 46°C in a hybridization buffer containing 0.9 M NaCl, 20 mM Tris–HCl (pH 7.2) and 0.01% sodium dodecyl sulfate (SDS) for 1–2 h. Probe concentration was 50 ng for 10 ml of hybridization solution. After hybridization, the slides were washed at 48°C for 15 min in washing buffer containing 0.9 M NaCl, 20 mM Tris–HCl (pH 7.2), and 0.01% SDS. Washing buffer was removed with dH2O. All cells were stained with 4¢,6-diamidino2-phenylindole (DAPI) as previously described (13). Finally, the slides were air-dried and mounted with an anti-fading solution (Slow Fade; Molecular Probes, Eugene, OR, USA). Samples were viewed under an Olympus BX60 microscope with appropriate filters. Images were captured with an Olympus DP50 digital camera system using Viewfinder Lite 1.0 software (Olympus, Tokyo) and processed using Studio Lite 1.0 software (Olympus). For a direct comparison, double images were acquired from the same microscopic fields at probe and DAPI excitation wavelengths. Identical exposure times were used for the analysis of parallel samples FISH and FISH-TSA. Mean intensities of fluorescent cells were analyzed from the captured images using Line Profile analysis of Olympus MicroImage software (Olympus). About 50 cells were measured for each slide. The final images were prepared with Adobe PhotoShop 4.0 software for presentation (Adobe, Mountain View, CA, USA). TSA amplification protocol When the FISH signal intensity was low, TSA reaction was performed further on the hybridized slides. Prior to TSA reaction, cell permeabilization was performed to allow large molecules of anti-FITC-HRP antibody to enter the cells. The cells were treated with lysozyme prior to TSA, covered with 10 ml of lysozyme–TE solution (0.1 mg of lysozyme [Sigma; 50,000 U/mg] per ml in Tris–EDTA buffer [TE], which contained 100 mM Tris–HCl [pH 8.2] and 50 mM EDTA), incubated at 0°C for 10 s. The enzyme reaction was stopped by rinsing the slides with TE solution. Then, the slides were incubated with TNB (0.1 M Tris–HCl [pH 7.5], 0.15 M NaCl, 0.5% blocking reagent) for 15 min and rinsed briefly with TNT buffer (0.1 M Tris– HCl [pH 7.5], 0.15 M NaCl, 0.05% Tween 20). Fixed cells on slides were incubated with anti-FITC-HRP antibody (1 : 250 [vol/vol] in TNB buffer) at room temperature for 30 min. The slides were washed by immersion in TNT buffer for 5 min at room temperature, and the cells were incubated with biotinyl-tyramide (1:25 in amplification buffer supplied by NEN Life Science Products) for 10 min at room temperature. The slides were then rinsed with TNT buffer for 5 min, and SA-FITC (1 : 250 [vol/vol] in TNB buffer) was added. After 30 min incubation at room temperature, the slides
283
were rinsed with TNT buffer and immersed in the same buffer for 5 min.
RESULTS AND DISCUSSION Whole-cell hybridization in combination with TSA system The combination of FITC-labeled, rRNA-targeted oligonucleotide probes and the TSA system was first evaluated by whole-cell hybridization. Artificial mixed cultures of gram-negative, gram-positive bacteria and Euryarchaeota were used. The TSA system was applied after cells were hybridized with fluorescence-labeled oligonucleotides. The fluorescence signal obtained using one monolabeled probe was compared with that obtained using the TSA system. The fluorescence signal intensity obtained using one monolabeled probe was low, whereas the use of the TSA system resulted in a marked increase in fluorescence signal intensity of target cells that was 1.6- to 2.7-fold higher than that obtained using monolabeled probes (Table 1 and Fig. 1). In the absence of a lysozyme treatment prior to TSA, the fluorescence signal of paraformaldehyde-fixed cells with the FITC-oligo-TSA system was significantly lower and the staining was quite uneven (data not shown). Similar observations were reported previously for the HRP-TSA system (9) and the biotin-TSA system (8). The lysozyme pretreatment protocol was required for hybridization to increase the permeability of fixed cells and allow the anti-FITC-HRP antibody to penetrate into the cells. Control experiments were performed without using a probe to check for nonspecific binding and peroxidase activity. Nonspecific binding was observed for streptavidinFITC and was eliminated by treating fixed samples with avidin to block endogenous biotin prior to hybridization. To reduce nonspecific staining due to biotin, the streptavidinFITC could be omitted, and with the anti-FITC-HRP antibody, a more direct detection can be achieved using tyramides labeled with fluorescent dyes instead of biotinyl-tyramide. Application of TSA system to environmental samples To further evaluate the applicability of the TSA system, fixed samples of anaerobic sludge were hybridized and compared with those hybridized with FITC-labeled oligonucleotide probes. When applied to the detection of bacteria, hybridization with a monolabeled EUB338-FITC probe resulted in a very low fraction of hybridized cells, and the fluorescence signal was very low (Fig. 2). This was probably due to the low RNA content of these cells since TSA resulted in a significant increase in the hybridized cell fraction TABLE 1. Comparison of average intensities between monolabeled probe and TSA system Mean intensitya Strain Monolabeled TSA Ratiob probe Escherichia coli 1374 ± 175 3673 ± 147 2.7 Desulfovibrio desulfuricans 1521 ± 205 2499 ± 355 1.6 a Mean intensities of fluorescence signals from 50 cells for each slide were analyzed using Line Profile Analysis of MicroImage software. b Ratio of intensity of monolabeled probe to that of TSA system.
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JUPRAPUTTASRI ET AL.
J. BIOSCI. BIOENG.,
FIG. 1. Epifluorescence micrographs of D. desulfuricans and E. coli. Bar: 10 mm. (A) D. desulfuricans hybridize with fluorescein-monolabeled probe SRB385; (B) D. desulfuricans hybridize with fluorescein-monolabeled probe SRB385 and TSA system; (C) E. coli hybridize with fluorescein-monolabeled probe EUB338; (D) E. coli hybridize with fluorescein-monolabeled probe EUB338 and TSA system. DAPI staining (left) and epifluorescence micrographs (right) are shown for identical microscopic fields. Exposure times were 20 ms for DAPI and 100 ms for fluorescein-labeled probe.
(Fig. 2). The use of the TSA system resulted in a marked increase in fluorescence signal intensity and yielded heterogeneous fluorescence signals. Similar observations of heterogeneous fluorescence signals were previously reported (8, 9). This heterogeneity was probably due to the accessibility of
the anti-FITC-HRP antibody rather than probe accessibility. Conclusion In this study, the application of TSA system in the combination with a conventional fluorochromelabeled probe successfully increased fluorescence signal intensity for hybridization. The system consisted of a conven-
VOL. 98, 2004
IMPROVED SENSITIVITY OF FLUORESCENCE IN SITU HYBRIDIZATION
285
FIG. 2. Epifluorescence micrographs of anaerobic sludge. Bar: 10 mm. (A) Sludge hybridize with fluorescein-labeled probe EUB338; (B) sludge hybridize with fluorescein-labeled probe EUB338 and TSA system. DAPI staining (left) and epifluorescence micrographs (right) are shown for identical microscopic fields. Exposure times were 20 ms for DAPI and 200 ms for fluorescein-labeled probe.
tional FITC-labeled oligonucleotide probe, anti–FITC-HRP antibody, biotinyl-tyramide, and streptavidin-fluorescein. No additional expensive probe labeling such as HRP or biotin, for the probe of interest was required. The commonly available, anti-FITC-HRP antibody can be generally used. The amplification step can be performed on hybridized samples following the visualization of monolabeled probe with in low signal intensity. In addition, instead of using of biotinyltyramide, a more direct detection can be achieved using tyramide labeled with fluorescent dyes. The fluorescence signal of the probe obtained using the TSA system was much higher than that obtained with using a monolabeled probe. The technique was successfully applied to the in situ detection of microbial communities in environmental samples. It was demonstrated that TSA increased sensitivity, as the fluorescence signal intensity was much higher than that obtained using a conventional probe, and the signal can overcome the autofluorescence background. The method, however, still has some limitations, as the optimal cell permeabilization protocol needs to be established to achieve a more homogenous staining. The approach provides an alternative method of improving the sensitivity of FISH.
ACKNOWLEDGMENTS This study was supported by a research grant from the National Center for Genetics Engineering and Biotechnology, Thailand. W. Jupraputtasri was supported by a postgraduate scholarship from the National Center for Genetics Engineering and Biotechnology. We thank Professor Preeda Malasit (Medical Biotechnology Unit, Mahidol University, Thailand) for providing chemical reagents and for fruitful discussions on hybridization techniques.
REFERENCES 1. Amann, R. I., Ludwig, W., and Schleifer, K.-H.: Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev., 59, 143–169 (1995). 2. Lipski, A., Friedrich, U., and Aldendrof, K.: Application of rRNA-targeted oligonucleotide probes in biotechnology. Appl. Microbiol. Biotechnol., 56, 40–57 (2001). 3. Wagner, M., Erhart, R., Manz, W., Amann, R., Lemmer, H., Wedi, D., and Schleifer, K.-H.: Development of an rRNAtargeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge. Appl. Environ. Microbiol., 60, 792–800 (1994). 4. Weiss, P., Schweitzer, B., Amann, R., and Simon, M.: Identification in situ and dynamics of bacteria on limnetic organic aggregates (lake snow). Appl. Environ. Microbiol., 62, 1998– 2005 (1996). 5. Hahn, D., Amann, R. I., and Zeyer, J.: Whole-cell hybrid-
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6. 7.
8.
9.
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