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Rapid enzymatic detection of faecal pollution
~
Pergamon
Wal. Sci. Tech. Vol. 34. No. 7-8. pp. 169-171, 1996. Copyright © 1996 fAWQ. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved.
PH: S0273-1223(96)00741-X
0273-1223/96 $15'00 + 0'00
RAPID ENZYMATIC DETECTION OF FAECAL POLLUTION Chery1M. Davies and Simon C. Apte Centre for Advanced Analytical Chemistry, CSIRO Division of Coal and Energy Technology, Private Mail Bag 7, Menai, NSW 2234, Australia
ABSTRACT Field trials were carried out using a I-hour f1uorimetric assay of ~-D-galactosidase activity alongside conventional membrane filtration to detect faecal coliforms in beachwater samples. The ultimate aim of the study was to test the reliability of the assay with a view to its use in the field to assess the compliance of coastal bathing waters with the guideline concentrations. The assay had a 99% success rate at detecting pass/fail at 300 faecal coliforms per 100 mI. using a threshold fluorescence of 60.3 nM. A good correlation (r=0.90) between faecal coliform concentration and fluorescence assay results was obtained. The assay provides a rapid. simple and inexpensive method for the detection of sewage polIution in marine waters. and with the aid of portable instrumentation. it may be performed in the field. alIowing real time monitoring of water quality. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd.
KEYWORDS Enzymatic activity; faecal coliforms; indicator organisms; monitoring; water quality. INTRODUCTION Sewage contamination of bathing waters is traditionally assessed by the enumeration of indicator bacteria such as faecal coliforms and enterococci. Traditional culturing techniques for the detection of these bacteria take in excess of 24 hours before results are obtained. The hydrolysis of labelled chromogenic or fluorogenic substrates by coliform-derived ~-D-galactosidase releases coloured or fluorescent products, respectively, which are visible by eye (Edberg et ai., 1988). These reactions form the basis of techniques such as Colilert and Coliquik for the detection and enumeration of total coliforms and Escherichia coli. Whilst these techniques are simpler to perform than conventional culturing techniques, they still require 18-24 hours to complete. This represents an unsatisfactory delay, presenting problems when deciding whether beaches are free of contamination. Instrumental detection of the hydrolysis products in enzyme assays significantly reduces detection times (Berg and Fiksdal, 1988; Apte and Batley, 1994). The following study describes the field trials of a rapid I-hour method for the assessment of sewage contamination in bathing waters. The method is based on the detection of faecal coliforms using a fluorogenic assay of ~-D-galactosidaseactivity (Apte and Batley, 1994). The ultimate aim of the study was to test the reliability of the assay with a view to its use in the field to assess the compliance of coastal bathing waters with the guideline concentrations, a mean of 300 faecal coliforms per 100 mI, for three samples collected with a maximum value in anyone sample of 2000 faecal coliforms per 100 mI (NSW Department of Health, 1982). 169
C. M. DAVIES and S. C. APTE
170
MATERIALS AND METHODS The rapid faecal colifonn assay Beta-D-galactosidase activity was measured using a 1 h assay carried out at 44.5°C, and employing the fluorescent substrate 4_methylumbelliferyl-~-D-galactoside (MUGal) (Apte and Batley, 1994). For each sample the assays were performed in triplicate, in 12 ml sterile test tubes, and consisted of 5 ml of sample, 1 ml of piperazine-N,N'bis(2-ethanesulphonic acid) (PIPES) buffer (pH 7.2), and 4 ml of 0.75 mM MUGal (Sigma Chemical Co., St Louis, Missouri), with appropriate reagent and .sam.ple bla?ks. The tUb~s were incubated in a circulating water bath at 44.5°C ± 0.5°C for I hour. Followmg mcubatlon, the reaction was stopped by cooling to room temperature and adjusting the pH to 10. The fluorescence intensities were measured using a Perkin Elmer LS-5 Luminescence Spectrometer at an excitation wavelength of 375 nm (slit width 10 nm), and an emission wavelength 465 nm (slit width 20 nm). The spectrometer readings were adjusted by subtracting the values of the reagent and background blanks, converted to concentrations of 4-methylumbelliferone (MU) using a calibration cQrve of the spectrometer readings versus standards of known MU concentration, and expressed as fluorescence (nanomolar (nM) MU) liberated by the enzyme. Field trials From September 1995 to January 1996, a trial of the rapid faecal colifonn assay was carried out on samples collected along a stretch of the New South Wales coastline which receives sewage discharges from treatment plants serving populations between 75,000 and 190,000. A total of 254 samples were collected and analysed in the laboratory by the rapid faecal colifonn assay as described above, and conventional membrane filtration by standard methods (APHA, 1992). RESULTS AND DISCUSSION The results of the field trials are given in Figure 1. Log fluorescence was plotted against log number of faecal colifonns per 100 ml. The vertical line at log 2.5 represents the compliance level of 300 faecal coliforms (cfu) per 100 ml (NSW Department of Health, 1982). This concentration corresponds to the fluorescence of 60.3 nM, which is represented by the horizontal line. This criterion for pass/fail is derived from previous trials. Points within quadrants a) and c) represent samples for which the two methods of enumeration were in agreement as to whether the sample passed or failed the guidelines. Points within quadrants b) and d) represent samples for which the two methods of enumeration disagreed, the false positives and false negatives, respectively, with respect to the assay. The results indicate a good correlation (r=0.90) between faecal coliform concentration and fluorescence assay results and a 99% success rate at detecting pass/fail at 300 faecal colifonns per 100 mI, using a threshold fluorescence of 60.3 nM. The outlier results comprised less than 1% false-positives and false• negatives. The rates of false-positives and false-negatives are acceptable for a routine method. However, at low concentrations of faecal colifonns, there is always an appreciable assay response which cannot be attributed solely to the presence of these bacteria. Recent work has focussed on elucidating potential interferences in the assay (Davies et al., 1994, 1995a, 1995b). Possible causes of interference include algae, marine bacteria, cell-free enzyme, and viable, non-culturable faecal coliforms. A number of agents and treatments have been identified ~y us, which reduce interference in the assay response yet have little effect on the response from faecal cohfonns. These have been evaluated and further field trials of the improved assay are currently underway. Not only will this reduce the possibility of false positive results, it will also improve the ability of the assay to detect low bacterial numbers.
Rapid enzymatic detection
171
A .portable test instrument has been developed, which is able to be operated in the back of a vehicle, and drIven to beaches where sampling and analysis may take place. Trials of the portable assay protocol in the field are underway.
5
b)
c)
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3 en Q)
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. ..,.
'-
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.31
0
a)
o
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• •• •
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• •
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.,
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• d)
234
567
Log cfu per 100 ml
Figure I. Field trials: correlation between fluorescence assay and conventional microbiological data for faecal coliforms in beachwater samples.
CONCLUSIONS The described faecal coliform assay provides a rapid, simple and inexpensive method for the detection of sewage pollution in marine waters. With the aid of newly developed portable instrumentation, analyses may be performed in the field, offering great potential as an 'early warning' for sewage contamination, and for real time monitoring of water quality. ACKNOWLEDGEMENT This study was funded in part by Sydney Water Corporation, Australia. The technical assistance of Angela O'Connell is gratefully acknowledged. REFERENCES APHA (1992). Standard Methods for the Examination of Water and Wastewater, 18th edn. American Public Health Association, Washington, D.C. Apte, S. C. and Batley, G. E. (1994). Rapid detection of sewage contamination in marine waters using a fluorimetric assay of P-D• galactosidase activity. Sci. Total Environ. 141, 175-180. Berg, J. D. and Fiksdal, L. (1988). Rapid detection of total and fecal coliforms in water by enzymatic hydrolysis of 4• methylumbelliferyl-p-D-galactoside. Appl. Environ. Microbiol. 4, 2118-2122. Davies, C. M., Apte, S. C., Peterson, S. M. and Stauber, 1. L. (1994). Plant and algal interference in bacterial p-D-galactosidase and P-D-glucuronidase assays. Appl. Environ. Microbiol. 60, 3959-3964. Davies, C. M., Apte, S. C. and Peterson, S. M. (l995a). Possible interference of lactose-fermenting marine vibrios in coliform P• D-galactosidase assays. J. Appl. Bacteriol. 78, 387-393. Davies, C. M., Apte, S. C. and Peterson, S. M. (l995b). p-D-galactosidase activity of viable, non-culturable coliform bacteria in marine waters. Lett. Appl. Microbiol. 21, 99-102. Edberg, S. C., Allen, M. J. and Smith, D. B. (1988). Rapid, specific, defined substrate technology for the simultaneous detection of total coliforms and Escherichia coli. Toxicity Assess. 3, 565-580. NSW Department of Health (1982). Criteria/or bathing water: tidal bathing standards.
Pergamon
Wal. Sci. Tech. Vol. 34. No. 7-8. pp. 169-171, 1996. Copyright © 1996 fAWQ. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved.
PH: S0273-1223(96)00741-X
0273-1223/96 $15'00 + 0'00
RAPID ENZYMATIC DETECTION OF FAECAL POLLUTION Chery1M. Davies and Simon C. Apte Centre for Advanced Analytical Chemistry, CSIRO Division of Coal and Energy Technology, Private Mail Bag 7, Menai, NSW 2234, Australia
ABSTRACT Field trials were carried out using a I-hour f1uorimetric assay of ~-D-galactosidase activity alongside conventional membrane filtration to detect faecal coliforms in beachwater samples. The ultimate aim of the study was to test the reliability of the assay with a view to its use in the field to assess the compliance of coastal bathing waters with the guideline concentrations. The assay had a 99% success rate at detecting pass/fail at 300 faecal coliforms per 100 mI. using a threshold fluorescence of 60.3 nM. A good correlation (r=0.90) between faecal coliform concentration and fluorescence assay results was obtained. The assay provides a rapid. simple and inexpensive method for the detection of sewage polIution in marine waters. and with the aid of portable instrumentation. it may be performed in the field. alIowing real time monitoring of water quality. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd.
KEYWORDS Enzymatic activity; faecal coliforms; indicator organisms; monitoring; water quality. INTRODUCTION Sewage contamination of bathing waters is traditionally assessed by the enumeration of indicator bacteria such as faecal coliforms and enterococci. Traditional culturing techniques for the detection of these bacteria take in excess of 24 hours before results are obtained. The hydrolysis of labelled chromogenic or fluorogenic substrates by coliform-derived ~-D-galactosidase releases coloured or fluorescent products, respectively, which are visible by eye (Edberg et ai., 1988). These reactions form the basis of techniques such as Colilert and Coliquik for the detection and enumeration of total coliforms and Escherichia coli. Whilst these techniques are simpler to perform than conventional culturing techniques, they still require 18-24 hours to complete. This represents an unsatisfactory delay, presenting problems when deciding whether beaches are free of contamination. Instrumental detection of the hydrolysis products in enzyme assays significantly reduces detection times (Berg and Fiksdal, 1988; Apte and Batley, 1994). The following study describes the field trials of a rapid I-hour method for the assessment of sewage contamination in bathing waters. The method is based on the detection of faecal coliforms using a fluorogenic assay of ~-D-galactosidaseactivity (Apte and Batley, 1994). The ultimate aim of the study was to test the reliability of the assay with a view to its use in the field to assess the compliance of coastal bathing waters with the guideline concentrations, a mean of 300 faecal coliforms per 100 mI, for three samples collected with a maximum value in anyone sample of 2000 faecal coliforms per 100 mI (NSW Department of Health, 1982). 169
C. M. DAVIES and S. C. APTE
170
MATERIALS AND METHODS The rapid faecal colifonn assay Beta-D-galactosidase activity was measured using a 1 h assay carried out at 44.5°C, and employing the fluorescent substrate 4_methylumbelliferyl-~-D-galactoside (MUGal) (Apte and Batley, 1994). For each sample the assays were performed in triplicate, in 12 ml sterile test tubes, and consisted of 5 ml of sample, 1 ml of piperazine-N,N'bis(2-ethanesulphonic acid) (PIPES) buffer (pH 7.2), and 4 ml of 0.75 mM MUGal (Sigma Chemical Co., St Louis, Missouri), with appropriate reagent and .sam.ple bla?ks. The tUb~s were incubated in a circulating water bath at 44.5°C ± 0.5°C for I hour. Followmg mcubatlon, the reaction was stopped by cooling to room temperature and adjusting the pH to 10. The fluorescence intensities were measured using a Perkin Elmer LS-5 Luminescence Spectrometer at an excitation wavelength of 375 nm (slit width 10 nm), and an emission wavelength 465 nm (slit width 20 nm). The spectrometer readings were adjusted by subtracting the values of the reagent and background blanks, converted to concentrations of 4-methylumbelliferone (MU) using a calibration cQrve of the spectrometer readings versus standards of known MU concentration, and expressed as fluorescence (nanomolar (nM) MU) liberated by the enzyme. Field trials From September 1995 to January 1996, a trial of the rapid faecal colifonn assay was carried out on samples collected along a stretch of the New South Wales coastline which receives sewage discharges from treatment plants serving populations between 75,000 and 190,000. A total of 254 samples were collected and analysed in the laboratory by the rapid faecal colifonn assay as described above, and conventional membrane filtration by standard methods (APHA, 1992). RESULTS AND DISCUSSION The results of the field trials are given in Figure 1. Log fluorescence was plotted against log number of faecal colifonns per 100 ml. The vertical line at log 2.5 represents the compliance level of 300 faecal coliforms (cfu) per 100 ml (NSW Department of Health, 1982). This concentration corresponds to the fluorescence of 60.3 nM, which is represented by the horizontal line. This criterion for pass/fail is derived from previous trials. Points within quadrants a) and c) represent samples for which the two methods of enumeration were in agreement as to whether the sample passed or failed the guidelines. Points within quadrants b) and d) represent samples for which the two methods of enumeration disagreed, the false positives and false negatives, respectively, with respect to the assay. The results indicate a good correlation (r=0.90) between faecal coliform concentration and fluorescence assay results and a 99% success rate at detecting pass/fail at 300 faecal colifonns per 100 mI, using a threshold fluorescence of 60.3 nM. The outlier results comprised less than 1% false-positives and false• negatives. The rates of false-positives and false-negatives are acceptable for a routine method. However, at low concentrations of faecal colifonns, there is always an appreciable assay response which cannot be attributed solely to the presence of these bacteria. Recent work has focussed on elucidating potential interferences in the assay (Davies et al., 1994, 1995a, 1995b). Possible causes of interference include algae, marine bacteria, cell-free enzyme, and viable, non-culturable faecal coliforms. A number of agents and treatments have been identified ~y us, which reduce interference in the assay response yet have little effect on the response from faecal cohfonns. These have been evaluated and further field trials of the improved assay are currently underway. Not only will this reduce the possibility of false positive results, it will also improve the ability of the assay to detect low bacterial numbers.
Rapid enzymatic detection
171
A .portable test instrument has been developed, which is able to be operated in the back of a vehicle, and drIven to beaches where sampling and analysis may take place. Trials of the portable assay protocol in the field are underway.
5
b)
c)
~4 e: Q) (.)
3 en Q)
#:j:. ••&
. ..,.
'-
0
:::J 2 c;:: C>
.31
0
a)
o
1
• •• •
.~.
• •
-.
.,
-A'-
c..
• d)
234
567
Log cfu per 100 ml
Figure I. Field trials: correlation between fluorescence assay and conventional microbiological data for faecal coliforms in beachwater samples.
CONCLUSIONS The described faecal coliform assay provides a rapid, simple and inexpensive method for the detection of sewage pollution in marine waters. With the aid of newly developed portable instrumentation, analyses may be performed in the field, offering great potential as an 'early warning' for sewage contamination, and for real time monitoring of water quality. ACKNOWLEDGEMENT This study was funded in part by Sydney Water Corporation, Australia. The technical assistance of Angela O'Connell is gratefully acknowledged. REFERENCES APHA (1992). Standard Methods for the Examination of Water and Wastewater, 18th edn. American Public Health Association, Washington, D.C. Apte, S. C. and Batley, G. E. (1994). Rapid detection of sewage contamination in marine waters using a fluorimetric assay of P-D• galactosidase activity. Sci. Total Environ. 141, 175-180. Berg, J. D. and Fiksdal, L. (1988). Rapid detection of total and fecal coliforms in water by enzymatic hydrolysis of 4• methylumbelliferyl-p-D-galactoside. Appl. Environ. Microbiol. 4, 2118-2122. Davies, C. M., Apte, S. C., Peterson, S. M. and Stauber, 1. L. (1994). Plant and algal interference in bacterial p-D-galactosidase and P-D-glucuronidase assays. Appl. Environ. Microbiol. 60, 3959-3964. Davies, C. M., Apte, S. C. and Peterson, S. M. (l995a). Possible interference of lactose-fermenting marine vibrios in coliform P• D-galactosidase assays. J. Appl. Bacteriol. 78, 387-393. Davies, C. M., Apte, S. C. and Peterson, S. M. (l995b). p-D-galactosidase activity of viable, non-culturable coliform bacteria in marine waters. Lett. Appl. Microbiol. 21, 99-102. Edberg, S. C., Allen, M. J. and Smith, D. B. (1988). Rapid, specific, defined substrate technology for the simultaneous detection of total coliforms and Escherichia coli. Toxicity Assess. 3, 565-580. NSW Department of Health (1982). Criteria/or bathing water: tidal bathing standards.