NVK 2006 - Reykjavik
Rapid methods for detection of bacteria
Charlotte B. Corfitzen*1, Bettina Ø. Andersen*, Morten Miller**4, Christian Ursin***5, Erik Arvin*2, Hans-Jørgen Albrechtsen*3 * Institute of Environment & Resources, Bygningstorvet, Building 115, DK-2800 Kgs. Lyngby, Denmark; 1 2 3
[email protected];
[email protected];
[email protected] ** MycoMeter Aps, Lersø Parkalle 40, DK-2100 København Ø, Denmark;
[email protected] *** CU Test, Astilbehaven 154, DK-2830 Virum, Denmark;
[email protected] Abstract. Traditional methods for detection of bacteria in drinking water e.g. Heterotrophic Plate Counts (HPC) or Most Probable Number (MNP) take 48-72 hours to give the result. New rapid methods for detection of bacteria are needed to protect the consumers against contaminations. Two rapid methods: Measurements of Adosine Triphosphate and BactiQuantTM have shown promising results as new monitoring tools, which gives the result within minutes/hours.
Introduction Traditional methods for detection of bacteria in drinking water e.g. Heterotrophic Plate Counts (HPC) or Most Probable Number (MNP) take 48-72 hours to give the result. This means that elevated bacteria levels will have reached the consumer before the results are available. New methods, which can give the result within minutes or hours, will therefore be of great importance especially in connections with repair situations and contamination incidents. This paper introduces two rapid methods: Adenosine Triphosphate (ATP) and BactiQuantTM, and relate them to traditional methods.
Principles When working with a pure bacteria strain it is possible to determine the total bacteria number by HPC simply by offering the bacteria a media they can grow on (form colonies = colony forming unit: CFU). The general problem when determining bacteria numbers in drinking water with HPC is that no media will give growth and thereby detection of all the bacteria strains naturally occurring in drinking water. The media used in the standard methods (Yeast Extract agar – ISO 6222) only detects a very small fraction of the natural bacteria population (tip of the iceberg), and is thus chosen to indicate extreme changes (contaminations) in the population. R2A is a media especially developed to drinking water investigation, which with prolonged incubation (7-14 days) gives much higher bacteria counts (CFU) than the standard methods, but still only up to 10% of the total bacteria number can be expected to be detected. The two rapid methods descried in the following both include all active bacteria. ATP Adenosine Triphosphate (ATP - Figure 1) is an energy carrying molecule in all living cells and can as such be taken as indirect measure for cell density. ATP is measured as a bioassay, where a luciferase enzyme (from firefly Photinus pyralis) catalyses an oxidation by O2 of D-luceferin using ATP as energy source, thereby reducing Adenosine triphosphate to Adenosine diphosphate and releasing the freed energy as light: firefly luciferase + Mg 2+ ATP + D − luciferin + O2 ⎯⎯ ⎯ ⎯ ⎯ ⎯ ⎯⎯→ AMP + PPi + oxylucifer in + CO2 + light
The light is measured in a luminometer as Relative Light Units (rlu) and converted to ATP values by a calibration curve on ATP standard salt (Figure 2).
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ADP ATP
Figure 1 Molecular structure of adenosine triphosphate.
A number of commercial luminometers and reagent-kits exist on the marked. The can vary in suitability for different applications, but for all includes the following steps: Addition of an extraction reagent (e.g. 100 µl) to the sample (e.g. 100 µl) followed by an extraction period (e.g. 10 s). Addition of the luciferase/luciferin reagents (e.g. 100 µl) followed by an integration period (e.g. 10 s). Thus the measurement can be preformed within minutes. In water ATP can be released from the cells, and remain for shorter periods in the water phase. If only the intracellular fraction is of interest, the bacteria can be isolated by filtration, which also is a way of concentrating a sample to obtain higher sensitivity, though the sensitivity with most equipments and reagent-kits are good (0.5 pg ATP/ml). The ATP value can not be converted directly to number of bacteria, since the ATP content of the single cell depends of bacteria type and its growth phase. However, within a given environment correlation factors can generally be estimated. ATP calibration curve 16,000 2
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Figure 2 Example of ATP calibration curve. Measured with Lumin(EX)/Lumin(ATE)-kit on an Advance Coupe luminometer (both Celsis, Landgraaf, The Netherlands).
BactiQuantTM In the BactiQuantTM method a specific bacteria enzyme is used as indirect measure of cell density. A substrate containing the fluorescent compound 4-Methyl Umbelliferone (MU) binds with the specific enzyme, thereby releasing the MU-ion, which then can be detected in a flourometer. The result is given in fluorescence units (FE).
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A given sample (e.g. 250 ml) is filtrated through a 0.22 µm filter capturing the bacteria on the filter. The MU-marked substrate liquid is added to the filter for a given reaction time (e.g. 30 minutes). A subsample of the substrate liquid (e.g. 100 µl) is then measured in a flourometer. Thus the measurement can be performed within 40 minutes. Both equipment and substrate-reagent are patented and distributed by MycoMeter Aps, Denmark. As with the ATP method the result can not be directly transferred to a bacteria count, as the content of the specific enzyme depends on bacteria strain and its growth stages.
Figure 3 Molecular structure of fluorescent 4-methyl umbelliferone used in the BactiQuantTM method.
Results Both ATP and BactiQuantTM show good linearity with increasing cell numbers. Figure 4 gives examples of linearity measured with the two methods by serial dilution of a water samples. A
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Figure 4 Examples of the linearity of the measurement of ATP and BactiQuantTM. A: BactiQuantTM, dilution of hot water sample (Andersen, 2005); B: ATP, dilution of Pseudomonas fluorescence P17 culture (E&R DTU, unpublished data) measured with PCP-kit on an Advance Coupe luminometer (both Celsis, Landgraaf, The Netherlands).
Growth investigations have shown good correlation between ATP, BactiQuantTM and HPC. Growth of the bacteria strain Pseudomonas fluorescence P17 in water extracts of polymeric materials gives an example of measurements on pure bacteria cultures. Growth curves measured by HPC and ATP were comparable (Figure 5), and an average ATP content per cell over the growth period could be estimated to 1.5±0.2x10-16 g ATP/cell. The growth curves in Figure 6 are examples of growth investigations with natural drinking water bacteria populations. Drinking water with and without addition of yeast extract to obtain different bacteria levels was incubated at 20°C and the growth followed over time by ATPintracellular, BactiQuantTM and HPCR2A /ref/. A good correlation between HPC, BactiQuantTM and ATP was demonstrated, as high bacteria counts due to growth in the sample added yeast extract also resulted in high ATP- and BactiQuantTM values (max. 380 pg ATP/ml and 400 FE/ml), whereas the sample with limited growth had low ATP- and BactiQuantTM values (≤6 pg ATP/ml and ≤1 FE/mL).
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Pseudonomas fluorescens (P17)
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Figure 5 Growth of the bacteria strain P17 at 15°C in drinking water extracts of polymers (two PVC materials and a PEM material) and in blank (water alone) measured by HPC and ATP (Corfitzen et al., 2002). HPC was determined on R2A with 3 days incubation at 25°C; ATP was measured with Lumin(EX)/Lumin(ATE)-kit on an Advance Coupe luminometer (both Celsis, Landgraaf, The Netherlands). Incubation of drinking water at 20C +2 mg Yeast/l
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Figure 6 Incubation at 20°C of drinking water with and without addition of 2 mg/l yeast extract. Growth of TM
the natural bacteria population was followed over time by HPCR2A, BactiQuant (Andersen, 2005) and ATPintracellular (measured with Lumin(EX)/Lumin(ATE)-kit on an Advance Coupe luminometer both Celsis, Landgraaf, The Netherlands).
Implementations in drinking water application Water samples taken from Danish water works and distribution systems indicate that water meeting the guidelines and posses biostability has ATP content below 10 pg ATP/ml (Corfitzen, 2004). Thus ATP could be used as a routine control parameter for water quality at the waterworks and in the distribution system. In Denmark the company CU Test is currently working on developing equipment for online monitoring of ATP at the waterworks. Furthermore, ATP will be used as microbial parameter in the future EAS method for acceptance of materials to be in contact with drinking water (van der Kooij et al., 2003).
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Likewise BactiQuantTM shows potential as quality control parameter. Copenhagen Energy, Denmark has in cooperation with MycoMeter Asp. performed a screening of water at the waterworks and in the distribution system with BactiQuantTM. With filtration of 250 ml sample they have suggested the following general acceptance categories: Best quality 200 FE. Water keeping the guideline values will fall in the ‘Best quality’ category (Bjerregaarde et al., 2005). Copenhagen Energy will continue to include BactiQuantTM in their normal routine measurements.
Acknowledgement Elements of the work referred here are partly financed by DANVA.
References Andersen, B. (2005) Kvantificering af mikroorganismer i drikkevand og biofilm. M.Sc. Thesis, Environment & Resources, Tech. Univ. of Denmark Bjergaarde, N.E.; Hansen, H.; Lind, S; Miller, M. (2005) Ny hurtigmetode til måling af totalkim I drikkevand – Lovende testresultater hos KE Vand’s drftlaboratorium. Pressemeddelelse Københavns Energi/Mycometer Corfitzen, C.B. (2004) Investigation of aftergrowth potential of polymers for use in drinking water distribution, PhD. Thesis, Environment & Resources, Tech. Univ. of Denmark Corfitzen, C.B.; Albrechtsen, H.-J.; Arvin, E.; Jørgensen, C. (2002) Afgivelse af organisk stof fra polymere materialer – mikrobiel vækst. Miljøprojekt nr. 718, Miljøstyrelsen ISO 6222: 1999; Water quality - Enumeration of culturable micro-organisms - Colony count by inoculation in a nutrient agar culture medium
van der Kooij, D.; Albrechtsen, H.-J.; Corfitzen, C.B.; Ashworth, J.; Parry, I.: Enkiri, F.; Hambsch, B.; Hametner, C. Kloiber, R.; Veenendaal, H.R.; Verhamme, D.; Hoekstra, E.J. (2003) Assessment of the microbial growth support potential of construction product in contact with drinking water (CPDW) – Development of a harmonised test to be used in the European Acceptance Scheme concerning CPDW – EVK1-CT2000-00052
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