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An inexpensive method for ultra-rapid detection of microbial contamination in industrial fluids
hTternational Biodeterioration 25 (1989) 137-145
An Inexpensive Method for Ultra-Rapid Detection of Microbial Contamination in Industrial Fluids
A. P. F. Turner, M. Allen, B. H. Schneider, A. Swain & F. Taylor Bioelectronics Division, Biotechnology Centre, Cranfield Institute of Technology, Cranfield, Bedfordshire, MK43 0AL, UK
ABSTRACT Rapid methods for the detection of microbial contamination have received increasing attention in recent yearsfor applications in clinical microbiology, the food industry and for monitoring a diverse range of biodeterioration problems. The term 'rapid method' is generally accepted to mean a technique which offers substantial savings in labour and~or time over traditional microbiological practice," tests taking O.5-24 h fall into this category. A new generation of ultra-rapid methods, however, is emerging which permit realtime monitoring of microbiological contamination. This paper briefly reviews recent developments in ultra-rapid methodology, and describes in detail a new electrochemical method for monitoring microbial activity in a variety of water-based fluids, such as process waters, cutting fluids and sewage. A prototype instrument, the Biocheck, has been developedfor more in-depth testing of this electrochemical approach. Results obtained with a range of bacterial genera often associated with contamination are discussed, and a more detailed assessment of sewage taken from a local sewage treatment plant is presented.
INTRODUCTION
Traditional m e t h o d s for evaluating the microbiological status o f a given s a m p l e have relied heavily on culturing techniques, which entail 137 International Biodeterioration 0265-3036/89/$03.50© 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain.
138
A. P F. Turner, M. Allen, B. H. Schneider, A. Swain & F. Taylor
extended periods of incubation of two, three or more days. Such times are undesirable to manufacturers who have to pay storage costs on products awaiting despatch, to clinicians who need to prescribe antibiotics quickly and in industrial biodeterioration where action is required in real time. The trend has been towards more rapid methods of detecting microbial contamination, which enables savings in labour costs and in time. The methods available have been summarised by Harris & Kell (1985), Dziezak (1987) and Ramsay et al. (1987), and in general include any method taking less than 24 h. However, it is necessary to introduce the concept of ~ultra-rapid" technology, which, by providing an assessment of microbiological contamination in 5 min or less, approximates to real-time analysis. It is therefore necessary to measure a direct function of the bacterium, e.g. a direct count, assay of a particular chemical or to tap in directly to central metabolism. This excludes techniques such as impedance and conductance which require sample monitoring over extended periods of time. Examples of fully automated ultra-rapid systems are the Orbec urine screening system (Orbec Ltd, Sanderstead, Surrey), the Direct Epifluorescence Filter Technique (DEFT) which was developed by the National Institute for Research in Dairying, and the Lumac microbiological testing technique (Lumac, B. V., 6370 AC Schaesberg, the Netherlands). They all require sophisticated instruments costing in excess of £20 000. The Orbec system is a Coulter-counter type method. About 100 samples per hour can be analysed. One drawback is its inability to distinguish viable from non-viable micro-organisms. The D E F T technique involves filtration through a polycarbonate filter, where the bacteria are stained with acridine orange prior to counting using a fluorescence microscope. Generally, viable cells stain orange, and non-viable cells green. The introduction of semi-automatic counting by image analysis has overcome microscope operator fatigue as well as speeding up the analysis time. D E F T is not without its disadvantages, m a n y of which are dependent on the operator. A particular difficulty is experienced with heat-treated products containing Streptococci and Micrococci as these organisms tend to stain orange even after heat treatment. An ultra-rapid chemical detection method is the Lumac technique based on the detection of bacterial ATP via bioluminescence. The m a x i m u m test rate is up to 200 samples per hour when no sample pretreatment is required. Optimisation of the approach depends on the sample type being examined, e.g. minimising light quenching by extraction chemicals, buffers or other substances. A new instrument for the real-time estimation of biomass in
Ultra-rapid detection of microbial contamination
139
fermenters, the 'Bugmeter' (Dulas Engineering Ltd, Machynlleth, Powys) based on the measurement of dielectric permittivity, has recently been launched. Due to the p h e n o m e n o n of dielectric dispersion the dielectric permittivity of a suspension of micro-organisms is greater than that of the supporting medium, or indeed non-cellular particles (Harris et al., 1987). This instrument is useful for high concentrations of biomass, and it does provide a non-invasive real-time method of monitoring changes in biomass. The Biocheck offers a new electrochemical method for monitoring microbial activity. It is ultra-rapid, fully portable, user-friendly and relatively inexpensive (under £2000). The technique is based on a proprietary cocktail of chemical mediators which divert electrons from the respiratory chains of micro-organisms to an amperometric detector. The mediators are reduced by the electron transport chain and are subsequently re-oxidised at an electrode which is maintained at a constant preset potential. The current produced during a 2-min test period is proportional to the n u m b e r of bacteria present or more specifically to the level of microbial activity.
METHODS
Preparation of standard cell suspensions and spiked samples The bacteria examined using the Biocheck were grown overnight at 30 °C in a shake flask containing nutrient broth. They were harvested by centrifugation and resuspended either in 0.9% saline to give different cell concentrations, or resuspended directly into a real sample (for example raw milk, cutting fluid, process water). Viable counts were performed on these dilutions and spiked samples, using nutrient agar plates incubated overnight at 30°C.
Assessment of sewage samples Samples of raw sewage were taken from a local treatment plant both at the input and output end of the process. Samples were prepared by a double filtration method, first through a glass microfibre filter (Whatman Ltd) and subsequently through a Whatman's No. 1 filter. During preparation the filter cake was resuspended into rehydrated freeze-dried reagents (containing buffer, salt and glucose). This allowed the sewage to be examined directly in the Biocheck. Non-bacterial interference was
140
A. P F. Turner, M. Allen, B. H. Schneider, A. Swain & F. Taylor
assessed by heating samples at 80°C for l0 min. Viable counts were performed on the raw sewage, filtered and heat-treated samples.
Characteristics of the Biocheck and methodology The Biocheck prototype instrument comprises a single board microprocessor with appropriate software which controls a magnetic stirrer, applies a preset potential across the electrodes and records the current that flows. A disposable bioelectrochemical cell (BEC) was developed based on a graphite working electrode and a silver/silver chloride reference electrode (Turner et al., 1987). After introduction of the sample into the BEC the electrode assembly is inserted, and the completed unit placed into the instrument. Location of the BEC in the Biocheck completes an electric circuit which turns on the stirrer and applies the potential. After a settling down period (generally <5 min) the increase in current can be monitored. The change in current over a 2-rain period is measured, and at present the maximum slope in/~A/min is shown at the end of the test. Changes in current occurring during the test were also monitored with a JJ PL1000 xy/t chart recorder (JJ Lloyd Instruments, Warsash, Southampton, UK).
RESULTS A N D DISCUSSION
Range of micro-organisms detected The bioelectrochemical technique can be applied to a n u m b e r of different micro-organisms. A wide range of bacteria have been detected, Gram-negative and -positive organisms, aerobes and facultative anaerobes and bacteria of differing morphology. Table 1 lists some of the species examined with a rough guide to their relevance to real samples. From Table 1 it can be seen that the Biocheck is appropriate for use on industrial, clinical and food samples. The bioelectrochemical technique has also been found to work with yeasts (e.g. Saccharomyces cerevisiae) and fungi (e.g. Cladosporium resinae). Preliminary work has shown that fungal spoilage of grain may be monitored using the Biocheck.
Detection of bacteria in real samples by spiking with standard suspensions A range of real samples have been studied using the Biocheck. The levels of background interference have varied considerably, from effectively
Ultra-rapid
detection
~
~
of microbial
~
~
~
contamination
" ~:z~ ~ ,.-~ d ~
=- .~=
ca)
"r~
°~ ~
141
e~
142
A. P F. Turner, M. Allen, B. H. Schneider, A. Swain & F. Taylor TABLE 2 Real Samples Tested in the Biocheck, and Successful Detection of Different Bacteria when Spiked into these Samples
Sample
Bacterium detected
Sterilised milk Raw milk Raw milk Raw milk Cooling water Cooling water Meat washings Beer Swimming pool water Cutting fluid Vegetable washings
Streptococcus faecalis Escherichia coli Pseudomonas aeruginosa Lactobacillus bulgaricus Enterobacter cloacae Pseudomonas aeruginosa Salmonella typhimurium Escheriehia coli Klebsiella aerogenes" Escheriehia coli Escherichia coli
zero in a n u m b e r of industrial cooling a n d process water samples, to a high a n d varied b a c k g r o u n d signal with urine. Milk, meat a n d vegetable washings presented a relatively constant level of electrochemical interference but the levels of bacteria present were below the detection TABLE 3 Monitoring of Sewage Samples over 2-week Period
Date
Viable counts' (cfu/ml) Raw sample
Average calculated Biocheck reading"
After pretreatment
Inlet sample Day 1 1.9 × 106 4.1 X 106 Day 3 2.0 × 106 4.7 × 106 Day 8 2.3 × 106 7.0 × 106 Day 15 1.5 x 106 3.6 x 106 Heat-treated sample: viable counts = l02 cfu/ml Biocheck reading = 178 (12 samples)
653 756 693 713
Outlet sample Day 1 4-1 Day 3 4.8 Day 8 8-7 Day 15 2.6 Heat-treated sample:
X 105 1.7 x 106 X 105 2.4 x 106 X 105 1-1 X 106 X 105 9.8 X 105 viable counts = 102 cfu/ml Biocheck reading = 15 (12 samples)
aEach value is an average of three replicates.
90 272 223 253
Ultra-rapid detection of microbial contamination
143
limits of the Biocheck (approximately 105-106 cfu/ml). These samples were therefore spiked with a range of bacteria. Some of the combinations detected w h e n added to real samples are given in Table 2. They demonstrated that various bacteria could be detected in a range of complex liquid matrices.
Monitoring of micro-organisms in sewage Using the prototype instrument, coupled to an appropriate filtration/ preconcentration regime, it was possible to monitor the level of microbial activity of sewage from a local sewage works. This provided a constant level of viable organisms in the range 105-106 cfu/ml. Samples were taken from the inlet and outlet stages on a n u m b e r of separate occasions. The level of organic matter present was, as expected, considerably lower by the time the sewage was discharged. Table 3 lists the results obtained from these tests. The level of viable counts in both the inlet and outlet samples remained fairly constant over the test period. Similarly the calculated readings obtained with the Biocheck showed a good degree of uniformity. The prefiltration/ preconcentration step showed, with one exception, good repeatability. W h e n the treated sewage samples were heat-treated the Biocheck response was reduced to about 25% for the inlet sample, and almost completely eliminated in the outlet sample Although it was possible to detect the bacteria in the raw samples directly, the level of background interference was considerably higher than for the pretreated samples (61 and 76% of the total for inlet and outlet samples, respectively). On this basis it was possible to obtain a positive response as low as 2 × 105 cfu/ml directly in the outlet sample, i.e. without prefiltering. Results shown in Fig. 1 as a calibration plot of log[viable counts] against log[Biocheck reading] include treated and untreated samples. Each point is the averaged response of three samples with the averaged response of three heat-treated controls subtracted. A positive correlation was obtained with a regression coefficient of 0.8418, which relates to about 30% error in the averaged readings. On a single point analysis the scatter would have been larger. The results confirm that the Biocheck could be used to detect a mixture of bacteria in a real sample, was able to distinguish a three-fold difference in the n u m b e r of bacteria present, and could detect levels of bacteria down to as low as 105 cfu/ml. It could, with suitable sample pretreatment, provide an inexpensive on-site method for real time monitoring of the microbial activity in samples from the water industry, e.g. to indicate gross pollution upstream of the sewage plant resulting
144
A. P. F. Turner, M. Allen, B. H. Schneider, A. Swain & F. Taylor
IDa
A
I0 2
z~
]O
i
i
i
.
.
.
lO s
.
.
[
i
i0 e LOG
[VIABLE
i
i
i
i
i
i
IO T COUNTS]
Fig. 1. Calibration curve of log[viable counts] against the log of the averaged, background-corrected Biocheck reading based on the data from Table 3 and on data from raw samples which had not been filtered prior to testing in the Biocheck.
from industrial or agricultural wastes. Within the sewage treatment plant it could provide an indication of the treatability of wastes by monitoring their effect on the activity of the microbial flora. By testing the effluent of the plant the Biocheck could also furnish a guide to its efficiency. The technology could also be adapted to an in-line monitor, which would be of wide use within the water treatment industry. This paper has focused on the sewage treatment industry, but the Biocheck instrument should be widely applicable to a range of contaminated industrial fluids.
ACKNOWLEDGEMENTS The financial support of de la Pena Biotechnology Ltd is gratefully acknowledged. AS and BS are DTI/SERC Teaching Company Associates, and A P F T is a Senior Fellow of the British Diabetic Association. The authors would also like to thank Dr A. D. Wheatley for his invaluable advice.
Ultra-rapid detection of microbial contamination
145
REFERENCES Dziezak, J. D. (1987). Rapid methods for microbiological analysis of food. Food Technology, July, 54-73. Harris, C. M. & Kell, D. B. (1985). The estimation of microbial biomass. Biosensors, 1, 17-84. Harris, C. M., Todd, R. W., Bungard, S. J., Lovitt, R. W., Morris, J. G. & Kell, D. B. (1987). Dielectric permittivity of microbial suspension at radio frequencies: a novel method for the real-time estimation of microbial biomass. Enzyme Microb. Technol., 9, 181-6. Ramsay, G., Turner, A. P. F., Franklin, A. & Higgins, I. J. (1987). Rapid methods for the detection of living microorganisms. Proceedings of the 1st IFAC Conference on the Modelling and Control of Biotechnological Processes, Nordwikjerhout, December 1985, ed. A. Johnson. PergamonPress, Oxford. Turner, A. P. F., Cardosi, M. F., Ramsay, G., Schneider, B. H. & Swain, A. (1987). Biosensors for use in the food industry: a new rapid bioactivity monitor. In Biotechnology in the Food Industry. Online Publications, Pinner.
An Inexpensive Method for Ultra-Rapid Detection of Microbial Contamination in Industrial Fluids
A. P. F. Turner, M. Allen, B. H. Schneider, A. Swain & F. Taylor Bioelectronics Division, Biotechnology Centre, Cranfield Institute of Technology, Cranfield, Bedfordshire, MK43 0AL, UK
ABSTRACT Rapid methods for the detection of microbial contamination have received increasing attention in recent yearsfor applications in clinical microbiology, the food industry and for monitoring a diverse range of biodeterioration problems. The term 'rapid method' is generally accepted to mean a technique which offers substantial savings in labour and~or time over traditional microbiological practice," tests taking O.5-24 h fall into this category. A new generation of ultra-rapid methods, however, is emerging which permit realtime monitoring of microbiological contamination. This paper briefly reviews recent developments in ultra-rapid methodology, and describes in detail a new electrochemical method for monitoring microbial activity in a variety of water-based fluids, such as process waters, cutting fluids and sewage. A prototype instrument, the Biocheck, has been developedfor more in-depth testing of this electrochemical approach. Results obtained with a range of bacterial genera often associated with contamination are discussed, and a more detailed assessment of sewage taken from a local sewage treatment plant is presented.
INTRODUCTION
Traditional m e t h o d s for evaluating the microbiological status o f a given s a m p l e have relied heavily on culturing techniques, which entail 137 International Biodeterioration 0265-3036/89/$03.50© 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain.
138
A. P F. Turner, M. Allen, B. H. Schneider, A. Swain & F. Taylor
extended periods of incubation of two, three or more days. Such times are undesirable to manufacturers who have to pay storage costs on products awaiting despatch, to clinicians who need to prescribe antibiotics quickly and in industrial biodeterioration where action is required in real time. The trend has been towards more rapid methods of detecting microbial contamination, which enables savings in labour costs and in time. The methods available have been summarised by Harris & Kell (1985), Dziezak (1987) and Ramsay et al. (1987), and in general include any method taking less than 24 h. However, it is necessary to introduce the concept of ~ultra-rapid" technology, which, by providing an assessment of microbiological contamination in 5 min or less, approximates to real-time analysis. It is therefore necessary to measure a direct function of the bacterium, e.g. a direct count, assay of a particular chemical or to tap in directly to central metabolism. This excludes techniques such as impedance and conductance which require sample monitoring over extended periods of time. Examples of fully automated ultra-rapid systems are the Orbec urine screening system (Orbec Ltd, Sanderstead, Surrey), the Direct Epifluorescence Filter Technique (DEFT) which was developed by the National Institute for Research in Dairying, and the Lumac microbiological testing technique (Lumac, B. V., 6370 AC Schaesberg, the Netherlands). They all require sophisticated instruments costing in excess of £20 000. The Orbec system is a Coulter-counter type method. About 100 samples per hour can be analysed. One drawback is its inability to distinguish viable from non-viable micro-organisms. The D E F T technique involves filtration through a polycarbonate filter, where the bacteria are stained with acridine orange prior to counting using a fluorescence microscope. Generally, viable cells stain orange, and non-viable cells green. The introduction of semi-automatic counting by image analysis has overcome microscope operator fatigue as well as speeding up the analysis time. D E F T is not without its disadvantages, m a n y of which are dependent on the operator. A particular difficulty is experienced with heat-treated products containing Streptococci and Micrococci as these organisms tend to stain orange even after heat treatment. An ultra-rapid chemical detection method is the Lumac technique based on the detection of bacterial ATP via bioluminescence. The m a x i m u m test rate is up to 200 samples per hour when no sample pretreatment is required. Optimisation of the approach depends on the sample type being examined, e.g. minimising light quenching by extraction chemicals, buffers or other substances. A new instrument for the real-time estimation of biomass in
Ultra-rapid detection of microbial contamination
139
fermenters, the 'Bugmeter' (Dulas Engineering Ltd, Machynlleth, Powys) based on the measurement of dielectric permittivity, has recently been launched. Due to the p h e n o m e n o n of dielectric dispersion the dielectric permittivity of a suspension of micro-organisms is greater than that of the supporting medium, or indeed non-cellular particles (Harris et al., 1987). This instrument is useful for high concentrations of biomass, and it does provide a non-invasive real-time method of monitoring changes in biomass. The Biocheck offers a new electrochemical method for monitoring microbial activity. It is ultra-rapid, fully portable, user-friendly and relatively inexpensive (under £2000). The technique is based on a proprietary cocktail of chemical mediators which divert electrons from the respiratory chains of micro-organisms to an amperometric detector. The mediators are reduced by the electron transport chain and are subsequently re-oxidised at an electrode which is maintained at a constant preset potential. The current produced during a 2-min test period is proportional to the n u m b e r of bacteria present or more specifically to the level of microbial activity.
METHODS
Preparation of standard cell suspensions and spiked samples The bacteria examined using the Biocheck were grown overnight at 30 °C in a shake flask containing nutrient broth. They were harvested by centrifugation and resuspended either in 0.9% saline to give different cell concentrations, or resuspended directly into a real sample (for example raw milk, cutting fluid, process water). Viable counts were performed on these dilutions and spiked samples, using nutrient agar plates incubated overnight at 30°C.
Assessment of sewage samples Samples of raw sewage were taken from a local treatment plant both at the input and output end of the process. Samples were prepared by a double filtration method, first through a glass microfibre filter (Whatman Ltd) and subsequently through a Whatman's No. 1 filter. During preparation the filter cake was resuspended into rehydrated freeze-dried reagents (containing buffer, salt and glucose). This allowed the sewage to be examined directly in the Biocheck. Non-bacterial interference was
140
A. P F. Turner, M. Allen, B. H. Schneider, A. Swain & F. Taylor
assessed by heating samples at 80°C for l0 min. Viable counts were performed on the raw sewage, filtered and heat-treated samples.
Characteristics of the Biocheck and methodology The Biocheck prototype instrument comprises a single board microprocessor with appropriate software which controls a magnetic stirrer, applies a preset potential across the electrodes and records the current that flows. A disposable bioelectrochemical cell (BEC) was developed based on a graphite working electrode and a silver/silver chloride reference electrode (Turner et al., 1987). After introduction of the sample into the BEC the electrode assembly is inserted, and the completed unit placed into the instrument. Location of the BEC in the Biocheck completes an electric circuit which turns on the stirrer and applies the potential. After a settling down period (generally <5 min) the increase in current can be monitored. The change in current over a 2-rain period is measured, and at present the maximum slope in/~A/min is shown at the end of the test. Changes in current occurring during the test were also monitored with a JJ PL1000 xy/t chart recorder (JJ Lloyd Instruments, Warsash, Southampton, UK).
RESULTS A N D DISCUSSION
Range of micro-organisms detected The bioelectrochemical technique can be applied to a n u m b e r of different micro-organisms. A wide range of bacteria have been detected, Gram-negative and -positive organisms, aerobes and facultative anaerobes and bacteria of differing morphology. Table 1 lists some of the species examined with a rough guide to their relevance to real samples. From Table 1 it can be seen that the Biocheck is appropriate for use on industrial, clinical and food samples. The bioelectrochemical technique has also been found to work with yeasts (e.g. Saccharomyces cerevisiae) and fungi (e.g. Cladosporium resinae). Preliminary work has shown that fungal spoilage of grain may be monitored using the Biocheck.
Detection of bacteria in real samples by spiking with standard suspensions A range of real samples have been studied using the Biocheck. The levels of background interference have varied considerably, from effectively
Ultra-rapid
detection
~
~
of microbial
~
~
~
contamination
" ~:z~ ~ ,.-~ d ~
=- .~=
ca)
"r~
°~ ~
141
e~
142
A. P F. Turner, M. Allen, B. H. Schneider, A. Swain & F. Taylor TABLE 2 Real Samples Tested in the Biocheck, and Successful Detection of Different Bacteria when Spiked into these Samples
Sample
Bacterium detected
Sterilised milk Raw milk Raw milk Raw milk Cooling water Cooling water Meat washings Beer Swimming pool water Cutting fluid Vegetable washings
Streptococcus faecalis Escherichia coli Pseudomonas aeruginosa Lactobacillus bulgaricus Enterobacter cloacae Pseudomonas aeruginosa Salmonella typhimurium Escheriehia coli Klebsiella aerogenes" Escheriehia coli Escherichia coli
zero in a n u m b e r of industrial cooling a n d process water samples, to a high a n d varied b a c k g r o u n d signal with urine. Milk, meat a n d vegetable washings presented a relatively constant level of electrochemical interference but the levels of bacteria present were below the detection TABLE 3 Monitoring of Sewage Samples over 2-week Period
Date
Viable counts' (cfu/ml) Raw sample
Average calculated Biocheck reading"
After pretreatment
Inlet sample Day 1 1.9 × 106 4.1 X 106 Day 3 2.0 × 106 4.7 × 106 Day 8 2.3 × 106 7.0 × 106 Day 15 1.5 x 106 3.6 x 106 Heat-treated sample: viable counts = l02 cfu/ml Biocheck reading = 178 (12 samples)
653 756 693 713
Outlet sample Day 1 4-1 Day 3 4.8 Day 8 8-7 Day 15 2.6 Heat-treated sample:
X 105 1.7 x 106 X 105 2.4 x 106 X 105 1-1 X 106 X 105 9.8 X 105 viable counts = 102 cfu/ml Biocheck reading = 15 (12 samples)
aEach value is an average of three replicates.
90 272 223 253
Ultra-rapid detection of microbial contamination
143
limits of the Biocheck (approximately 105-106 cfu/ml). These samples were therefore spiked with a range of bacteria. Some of the combinations detected w h e n added to real samples are given in Table 2. They demonstrated that various bacteria could be detected in a range of complex liquid matrices.
Monitoring of micro-organisms in sewage Using the prototype instrument, coupled to an appropriate filtration/ preconcentration regime, it was possible to monitor the level of microbial activity of sewage from a local sewage works. This provided a constant level of viable organisms in the range 105-106 cfu/ml. Samples were taken from the inlet and outlet stages on a n u m b e r of separate occasions. The level of organic matter present was, as expected, considerably lower by the time the sewage was discharged. Table 3 lists the results obtained from these tests. The level of viable counts in both the inlet and outlet samples remained fairly constant over the test period. Similarly the calculated readings obtained with the Biocheck showed a good degree of uniformity. The prefiltration/ preconcentration step showed, with one exception, good repeatability. W h e n the treated sewage samples were heat-treated the Biocheck response was reduced to about 25% for the inlet sample, and almost completely eliminated in the outlet sample Although it was possible to detect the bacteria in the raw samples directly, the level of background interference was considerably higher than for the pretreated samples (61 and 76% of the total for inlet and outlet samples, respectively). On this basis it was possible to obtain a positive response as low as 2 × 105 cfu/ml directly in the outlet sample, i.e. without prefiltering. Results shown in Fig. 1 as a calibration plot of log[viable counts] against log[Biocheck reading] include treated and untreated samples. Each point is the averaged response of three samples with the averaged response of three heat-treated controls subtracted. A positive correlation was obtained with a regression coefficient of 0.8418, which relates to about 30% error in the averaged readings. On a single point analysis the scatter would have been larger. The results confirm that the Biocheck could be used to detect a mixture of bacteria in a real sample, was able to distinguish a three-fold difference in the n u m b e r of bacteria present, and could detect levels of bacteria down to as low as 105 cfu/ml. It could, with suitable sample pretreatment, provide an inexpensive on-site method for real time monitoring of the microbial activity in samples from the water industry, e.g. to indicate gross pollution upstream of the sewage plant resulting
144
A. P. F. Turner, M. Allen, B. H. Schneider, A. Swain & F. Taylor
IDa
A
I0 2
z~
]O
i
i
i
.
.
.
lO s
.
.
[
i
i0 e LOG
[VIABLE
i
i
i
i
i
i
IO T COUNTS]
Fig. 1. Calibration curve of log[viable counts] against the log of the averaged, background-corrected Biocheck reading based on the data from Table 3 and on data from raw samples which had not been filtered prior to testing in the Biocheck.
from industrial or agricultural wastes. Within the sewage treatment plant it could provide an indication of the treatability of wastes by monitoring their effect on the activity of the microbial flora. By testing the effluent of the plant the Biocheck could also furnish a guide to its efficiency. The technology could also be adapted to an in-line monitor, which would be of wide use within the water treatment industry. This paper has focused on the sewage treatment industry, but the Biocheck instrument should be widely applicable to a range of contaminated industrial fluids.
ACKNOWLEDGEMENTS The financial support of de la Pena Biotechnology Ltd is gratefully acknowledged. AS and BS are DTI/SERC Teaching Company Associates, and A P F T is a Senior Fellow of the British Diabetic Association. The authors would also like to thank Dr A. D. Wheatley for his invaluable advice.
Ultra-rapid detection of microbial contamination
145
REFERENCES Dziezak, J. D. (1987). Rapid methods for microbiological analysis of food. Food Technology, July, 54-73. Harris, C. M. & Kell, D. B. (1985). The estimation of microbial biomass. Biosensors, 1, 17-84. Harris, C. M., Todd, R. W., Bungard, S. J., Lovitt, R. W., Morris, J. G. & Kell, D. B. (1987). Dielectric permittivity of microbial suspension at radio frequencies: a novel method for the real-time estimation of microbial biomass. Enzyme Microb. Technol., 9, 181-6. Ramsay, G., Turner, A. P. F., Franklin, A. & Higgins, I. J. (1987). Rapid methods for the detection of living microorganisms. Proceedings of the 1st IFAC Conference on the Modelling and Control of Biotechnological Processes, Nordwikjerhout, December 1985, ed. A. Johnson. PergamonPress, Oxford. Turner, A. P. F., Cardosi, M. F., Ramsay, G., Schneider, B. H. & Swain, A. (1987). Biosensors for use in the food industry: a new rapid bioactivity monitor. In Biotechnology in the Food Industry. Online Publications, Pinner.