2010 Progress Report: Assessment of Microbial Pathogens in Drinking Water using Molecular Methods Coupled with Solid Phase Cytometry
EPA Grant Number:
Assessment of Microbial Pathogens in Drinking Water using Molecular Methods Coupled with Solid Phase Cytometry
Pyle, Barry H
, Ford, Timothy E.
Pyle, Barry H
Montana State University - Bozeman
Little Big Horn College
EPA Project Officer:
Klieforth, Barbara I
March 1, 2008 through
February 28, 2011
(Extended to February 28, 2013)
Project Period Covered by this Report:
June 10, 2010 through June 9,2011
Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens and Cyanobacteria and Their Toxins in Drinking Water (2007)
To develop and evaluate innovative approaches for quantitative assessment of pathogens in drinking water sources, using fluorescent in situ hybridization (FISH) coupled with the Solid Phase Laser Cytometer (ScanRDI) and other molecular methods.
Target organisms: Escherichia coli O157:H7, Helicobacter pylori, Legionella pneumophila, Mycobacterium avium, Aeromonas hydrophila, Giardia lamblia, Cryptosporidium parvum.
Background: The ScanRDI is a Solid Phase Laser Cytometer that detects appropriately labeled cells on a 25 mm diameter black polycarbonate membrane filter. The instrument scans an entire filter in 3-4 minutes, detecting and mapping individual fluorescent particles. The software system discriminates between cells and debris, and then locates particles to be viewed with an epifluorescent microscope, allowing the operator to directly validate the results.
Because the ultimate objective of this project is to screen for multiple pathogens on a single membrane, optimization of methods focuses on protocols that can be used for a range of organisms. These protocols include permeabilization techniques, hybridization buffers and hybridization incubation temperatures and times.
Direct epifluorescent microscopy is used during optimization. In addition to enumeration, images are taken with the Zeiss Axioskop to be analyzed with the Metamorph Software (Molecular Devices) for fluorescent intensity data. Because previous work has demonstrated that SYBR Green fluoresces at sufficient intensity to be detected by the ScanRDI (3), fluorescent intensity of cells labeled by an experimental method is sometimes compared to fluorescent intensity of SYBR Green.
Detection of E. coli with the ScanRDI
As has been reported previously by this lab, the ScanRDI detected less than 10% of the E. coli cells labeled with FISH probes coupled directly to fluorescein or Alexa 488. Use of polyamide nucleic acid (PNA) probes was unacceptable due to high numbers of artifacts. Therefore, from the beginning of this reporting period, tyramide signal amplification (TSA) was used to enhance the signal output and increase the numbers of detectable cells. The TSA technique uses oligonucleotide probes directly coupled with horse radish peroxidase (HRP) or a probe that can be coupled later with HRP, e.g., biotin, which binds to avidin-HRP, or fluorescein, which is intended to interact with a-fluorescein-HRP antibody. HRP converts fluorescein labeled tyramide into an activated intermediate that binds to adjacent proteins. Multiple deposition of fluorescein occurs in a short time producing a large signal amplification.
Optimization of TSA on filter membranes
Many reported FISH methods are carried out in suspension or after fixation to microscope slides (1, 4-6, 8) and the ScanRDI requires an intact 25 mm filter membrane for analysis. Using E. coli and a Eubacterial probe coupled with HRP (EUB 388-HRP) obtained from Thermo Scientific, TSA amplification was optimized following modification of techniques reported by Baudart, et. al. (2) by adjusting the concentration of both the EUB probe and tyramide-fluorescein. The Baudart technique directly hybridizes cells on polyester membrane filters (AES-Chemunex). These particular filters are important in the procedure because, in contrast to commonly available polycarbonate filters, they resist degradation during the permeabilization and hybridization procedures. In brief, the Baudart protocol combined a direct viable count procedure to increase ribosomal concentration preceding fixation. Fixation and permeabilization was done by placing the membranes directly on 4% paraformaldehyde, followed by a series of ethanol solutions (50, 80 and 94%) and lysozyme 5000 units/ml). With the Baudart method, the hybridization buffer was placed on the membrane containing fixed permeabilized cells and covered with a coverslip to prevent evaporation followed by a wash at 48°C. Hybridization of E. coli with a Eubacterial probe coupled to HRP (EUB 388-HRP) showed optimal fluorescence at 20 ng/µl after 2 hour incubation at 46°C (Fig. 1).
Antibiotic preincubation Antibiotic treatment to increase ribosomal content in the cells is one method of increasing FISH intensity. Because some organisms react differently with different antibiotic treatments work was done comparing use of nalidixic acid and chloramphenicol (7). As Table 1 shows, preincubation with both chloramphenicol and nalidixic acid (NA) did increase the minimum fluorescent intensity more than cells with no preincubation, and the intensity was 60 to 70% of the intensity of SYBR Green stained cells.
Chloramphenicol 30 min
NA 30 min
NA 60 min
NA 120 min
Fluorescent antibody labeling and FISH Hybridization of Cryptosporidium parvum
Cryptosporidium oocysts were obtained from Sterling Laboratories, fluorescent antibody (FAb) from LifeSpan Biosciences and CRY-HRP conjugate probe from Thermo Scientific.Cryptosporidium, detected in water as a protozoan oocyst, has unique barriers to FISH probes. According to previously published protocols for Cryptosporidium (4, 9), permeabilization with hot 50% ethanol produced the most intense fluorescence of the methods tested. Although fixation in suspension with hot ethanol lyses E. coli, attempts to fix E. coli on membranes with this procedure showed some promise. It was determined, however, that the standard procedure for fixation of E. coli described above also was superior to the hot ethanol method in Cryptosporidium. Further permeabilization was done with E. coli and Cryptosporidium following the Baudart method.
Optimization procedures were analyzed based on Metamorph data (see above). The FAb concentration for labeling Cryptosporidium was optimized at a dilution of 1:100 (see Fig. 1), to minimize background fluorescence and maximize fluorescent intensity. The optimization of FISH probe concentration, shown in Fig. 2, was done using the hybridization buffer suggested by Deere, et.al. (4) at a final concentration of 1 pmol/µl although sufficient fluorescent intensity for use with the ScanRDI was produced at 0.5 and 0.2 pmol/µl when compared to fluorescent intensity of SYBR Green. To conserve the probe, hybridizations can be done at those concentrations.
Optimization of FISH-TSA with L. pneumophila The FISH-TSA method optimized for E. coli on filter membranes did not properly label Legionella pneumophila cells. Further work was done with L. pneumophila fixed and permeabilized in suspensions, followed by FISH-TSA using glass slides for hybridization and microscopy. It was found that addition of H2O2 incubation before the lysozyme incubation could increase fluorescence intensity of L. pneumophila cells. Addition of 0.01M HCl incubation to stop all enzymatic activities after the lysozyme incubation also increased fluorescent intensity. The optimum concentration of LEGPNE1-HRP was determined to be 35 ng/µl.
Further work is being done to optimize FISH for use with Cryptosporidium, including TSA-CY3 labeling and labeling oocysts with both FAb and FISH at once. Simultaneous detection of E. coli and Cryptosporidium as well as other target organisms using FISH techniques and the ScanRDI will be investigated. The optimization for L. pneumophila cells will be adapted for the membrane filter method for use with the ScanRDI and for other target bacterial pathogens. Procedures for Giardia will be added in the near future. Sampling and analysis of water from the Crow Indian Reservation, applying the procedures that have been developed, will start this summer (2011).
1. Amann, R. I., B. Zarda, D. A. Stahl, and K.-H. Schleifer. 1992. Identification of Individual Prokaryotic Cells by Using Enzyme-Labeled, rRNA-Targeted Oligonucleotide Probes. Applied and Environmental Microbiology 58:3007-3011.
2. Baudart, J., J. Coallier, P. Laurent, and M. Prévost. 2002. Rapid and Sensitive Enumeration of Viable Diluted Cells of Members of the Family Enterobacteriaceae in Freshwater and Drinking Water. Applied & Environmental Microbiology 68:5057.
3. Broadaway, S. C., S. A. Barton, and B. H. Pyle. 2003. Rapid staining and enumeration of small numbers of total bacteria in water by solid-phase laser cytometry. Applied and Environmental Microbiology 69:4272-4273.
4. Deere, D., G. Vesey, M. Milner, K. Williams, N. Ashbolt, and D. Veal. 1998. Rapid method for fluorescent in situ ribosomal RNA labelling of Cryptosporidium parvum. Journal of Applied Microbiology 85:807-818.
5. Moreno, Y., M. A. Ferrus, J. L. Alonso, A. Jimenez, and J. Hernandez. 2003. Use of fluorescent in situ hybridization to evidence the presence of Helicobacter pylori in water. Water Research 37:2251-2256.
6. Moter, A., and U. B. Gobel. 2000. Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. Journal of Microbiological Methods 41:85-112.
7. Ouverney, C. C., and J. A. Fuhrman. 1997. Increase in fluorescence intensity of 16S rRNA in situ hybridization in natural samples treated with chloramphenicol. Applied and Environmental Microbiology 63:2735-2740.
8. Pernthaler, J., F. O. Glockner, W. Schonhuber, and R. Amann. 2001. Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes. Methods in Microbiology, Vol 30 30:207-226.
9. Vesey, G., N. Ashbolt, E. J. Fricker, D. Deere, K. L. Williams, D. A. Veal, and M. Dorsch. 1998. The use of a ribosomal RNA targeted oligonucleotide probe for fluorescent labelling of viable Cryptosporidium parvum oocysts. Journal of Applied Microbiology 85:429-440.
No journal articles submitted with this report: View all 6 publications for this project
water, drinking water, watersheds,toxics, toxic substances: pathogens, bacteria,biology,monitoring, analytical, measurement methods,northwest, MT, EPA Region 8
Progress and Final Reports:
2009 Progress Report