飲用水中細菌的快速檢測(英文文獻)

1、rapid detection of bacteria indrinkingwaterr.a. deininger and j. lee school of public health, the university of michigan, ann arbor, michigan, usaabstracta heterotrophic plate count (hpc) is a measure of the total number of heterotrophic bacteria in a water sample. it does not indicate whether the b

2、acteria are harmful or harmless to humans, but serves as an indicator of the cleanliness of the raw water and treatment processes. the most sensitive test uses the r2a agar and an incubation time of 7 days. other media and shorterincubation times lead to lower counts. whichever test is used, an incu

3、bation of 2-7 days makes the test clearly useless for operational decisions and intervention. intentional or accidental contamination of a water supply must be determinable in minutes.we have studied over the past three years several water sources, such as municipal water, bottled water, beach and p

4、ool water, and ballast water on ships. using portable equipment, we have determined the atp inthe water sample by lysing the bacteria, adding luciferine/luciferase to develop light, and measuring the light emission in a luminometer. the test isnot new and is described in standard methods. what is ne

5、w is that thepresent test is more sensitive, the instrumentation fits on a clipboard, and the test can be done in the field at the location where the sample is taken. if theestimated hpc is high, a second step may be necessary to identify if there are pathogens in the water sample. a rapid test fore

6、.colio157:h7,pseudomonas , and legionella pneumophila has been developed usingimmunomagnetic separation. these tests take longer, but are still under anhour for each target bacterium. the detection of spores in the water sampleis also possible. the essence of the method is that a contamination of bo

7、ttled water with bacteria can be detectedwhile the batch of bottled water is stillon the shipping dock. 71a. omelchenko et al (eds.),modern tools and methods of water treatment for improving living standards, 7178.? 2005 springer. printed in the netherlands. 72introductionthe current microbiological

8、 standards focus on a single group of indicator organisms for the bacteriological safety of drinking water (federalregister 1996). although the current standards of water quality have eliminated massive outbreaks of waterborne disease, a question has been raised about the adequacy of the standards o

9、f drinking water quality to prevent water-borne illnesses (payment et al. 1991). cases of gastrointestinalillnesses were reported among individuals drinking tap water that had met microbiological and physico-chemical water quality criteria. thedetermination of the total number of heterotrophic bacte

10、ria has been knownas a better indicator of water quality than the coliform test because many opportunistic pathogens are not in the coliform group (geldreich et al. 1972) and a high hpc has shown to interfere with the determination of thecoliforms (lechevallier et al. 1985).the present hpc method us

11、ing r2a agar is known to be the most sensitive test for enumerating bacteria from treated water (reasoner et al. 1985). the disadvantage of the test is that it takes seven days to complete and when the results are known, the water has long been consumed. a test is needed that determines the total ba

12、cterial populations in a very short timeso that corrective actions can be taken in a timely manner. the atp bioluminescence assay allows an estimation of bacterial populations within minutes and it can be applied on-site also. the estimationof the bacterial count based on the atp of the water is not

13、 new. standardmethods (apha-awwa-wef 1995) indicate that the test requires one hour, one liter of water, and has a sensitivity of 100,000 cells. what is new is that our method is over 100 times more sensitive, requires one-hundredth of the sample volume, and is over 10 times faster. the test is also

14、 described and specified in (astm d 4012-81).thus the purpose of this study was to determine if a rapid atp assay could estimate the bacterial populations in a practical and timely manner. for quality control purposes and to test the accuracy of both the atp and hpc test, a direct enumeration of the

15、 bacteria in a water sample was done using two epifluorescence methods. one was the aodc method to enumerate the total number of bacteria, which include both the viable cellsand the nonviable cells. the other was the dvc method that selectively enumeratesviable bacteria.73materials and methodswater

16、sampleswater samples were obtained from drinking water fountains or distribution system sampling locations in the united states and abroad. thesamples in the united states were taken from locations in california,colorado, florida, georgia, illinois, kentucky, maryland, michigan, newyork, ohio, orego

17、n, tennessee, texas, washington, and washington, d.c. some of the samples were obtained from airports (california, illinois, kentucky, maryland, new york, oregon, tennessee, texas, and washington), and some were obtained from cooperating utilities (california, colorado, florida, georgia, michigan, a

18、nd ohio). a number of sampleswere obtained from argentina, australia, austria, brazil, egypt, france,germany, hungary, japan, korea, lithuania, netherlands, panama, peru,saudi arabia, switzerland, ukraine, and the united kingdom. in august of 1999, a number of public drinking fountains were sampled

19、in paris, france. samples were taken at the charles degaulleairport, the eiffel tower, hospital pitie, the louvre, notre dame, palais chaillot, sacre coeur, and the unesco headquarters. the water samples were also analyzed for their metal content by icp/ms. filtration of samplesa filtravettetm, whic

20、h is a combination of a filter and a cuvette withpore size of 0.45 microns, was placed into a swinex filter holder (13mm,millipore corporation, bedford, ma). a sterile syringe was used fordrawing the water samples. the testing volumes of the water were between0.1 and 10 ml, based on the expected num

21、ber of bacteria in the sample. the filter holder was screwed onto the syringe and the water sample was pushed through the filter. the filtravettetmwas taken out from the filter holder andplaced onto a sterile blotting paper. the remaining water inside thefiltravette was removed with a specially conv

22、erted 3 ml syringe by applyinggentle air pressure. atp bioluminescencea somaticcell releasingagent(new horizons diagnostic corporation (nhd), columbia, md) was used to lyse all non-bacterial cells and release their atp. air pressure was used to remove the non-bacterial atp throughthe filter. at this

23、 stage, the filtravettetm retains bacteria on its surface, and 74the bacterial atp remained within the bacterial cell membranes through this step of the procedure. the filtravettetmwas inserted into themicroluminometer (model 3550, nhd, columbia, md) (fig. 1) and the bacterial cell-releasing agent w

24、as then added to lyse the bacterial cells retained on the surface of the filter. the released bacterial atp was mixedwith 50 l of luciferin-luciferase (nhd, columbia, md) and the drawer of the microluminometer was closed. the light emission was recorded after 10-second integration of the light impul

25、ses and the unit was relative light unit (rlu). the result was expressed as rlu/ml by dividing the rluvalues by the filtered water volume. the detection limit and sensitivity of theluminometer was tested with serially diluted atp solutions (nhd, columbia, md). distilled deionized water was used for

26、the dilution and theph was 7.8. the activity of the luciferin-luciferase was checked by using an atp standard (nhd, columbia, md). the rlus are proportional to theamount of atp, and the amount of atp is proportional to the number of viable bacteria.figure 1. portable microluminometer with accessorie

27、s.bacterial enumeration: aodc, dvc, hpc the total (nonviable and viable) bacterial cells were determined fromformaldehyde fixed (2%, v/v, final concentration) samples with the aodc method (hobbie et al. 1977). the bacterial cells were stained with acridine orange (0.01%, w/v, fluka, switzerland) aft

28、er filtration onto a 0.2 micron pore-size black polycarbonate membrane filters (poretics, livermore, ca). cells were enumerated at a magnification of x1000 with an olympus provisepifluorescence microscope (olympus optical co., japan) equipped with a mercury arc lamp and a 460 - 490 nm excitation fil

29、ter. the number of bacteriawas countedin 10 microscopic fields using three subsamples and was75then averaged. the number of bacteria per milliliter of sample was calculated using the equation in standard methods (apha-awwa-wef, 1995).the viable cells were counted by the dvc method (coallier et al. 1

30、994) with some modifications. the samples were incubated with yeast extract (0.005%, w/v, difco, detroit, mi) and nalidixic acid (10mg/l,sigma, st. louis, mo) without dilution for 24 hours at 20oc. themodifications were: use of a lower concentration of yeast extract and no dilution. after incubation

31、, the fixation, counting, and calculation of elongated bacteria were done following the aodc method.the hpc was determined for each water sample in triplicate usingr2a medium (difco, detroit, michigan). the bacterial colonies were counted after an incubation period of 7 days at 28oc.results and disc

32、ussionthe detection limit of atp was determined with high accuracy(r=0.999). it showed that the microluminometer was able to determine atpconcentrations as low as 0.2 picogram. it is known that the average atpcontent in one bacterial cell is about 10-15g (i.e., 1 femtogram) (crombrugge et al. 1991).

33、 thus the 0.2 picograms correspond to 200 bacterial cells.about 120 water samples were analyzed with atp bioluminescence,hpc, dvc and aodc methods, each in triplicate. a statistical analysis of the data shows that there are high correlation coefficients between atp,hpc, dvc, and aodc, the correlatio

34、n coefficient between atp and hpcwas 0.84, and the correlation coefficient between atp and dvc was 0.8.both are statistically highly significant. the prediction of the hpc can beaccomplished by the following equation: hpc (cfu/ml) = rlu1.47the paris drinking water samples were analyzed using 3 metho

35、ds:the atp assay, the hpc using r2a agar, and the plate count according tofrench standards (casein peptone 5 g, yeast extract 2.5 g, glucose 1 g, agar15 g/l of distilled water; incubate at 22c for 3 days). since the watersamples were in transit for about two days one should not place great emphasis

36、on the absolute numbers, but rather the relative relations. the r2acounts were about 30 times higher than the counts obtained by using the french method. the water quality varied significantly from location to location, and the visual observation of the plates showed quite different colonies in size

37、 and color. subsequent analysis of the water for its metalcontent showed that the water had come from quite different sources. the metal concentrations are shown in table 1, and the bacterial concentrations areshown in table 2.a recently published paper (delahaye 2003) confirms the wide variations i

38、n the drinking water quality due to its many different sources. it 76also shows that the french (and european) methods grossly underestimatethe bacterial counts.table 1. concentrations of metals (pg/l)location pb li mg sr ba al palais chaillot 0.2 0.8 2,659 142 14 1.1 eiffel tower 3.1 1.2 1,454 135

39、24 1.5 hotel st andre 5.0 1.5 1,421 125 28 1.2notre dame 3.6 1.0 1,365 128 30 1.2 parc viviani 91 1.5 1,665 135 31 2.6 near louvre 2.4 1.4 1,647 133 29 1.9 hospital pitie 0.6 2 2,400 225 26 13 unesco 5.5 1.2 1,930 151 26 1.2sacre coeur 0.3 2.1 4,570 397 23 6.0cdg airport 0.02 2 5,370 463 17 20 bottl

40、ed water 0.02 12 16,000 1,100 19 0.7h2o dispenser 0.1 0.7 4,781 224 27 0.8table 2. bacteria in paris drinking water (no/ml).location rlu* hpc* fm*palais chaillot 728 1735 250 eiffel tower 435 10,920 2,300 hotel st andre 952 45,000 18,220 notre dame 4 65 10 parc viviani 296 16,310 1,000 near louvre 1

41、0 80 15hospital pitie 539 52,000 3,610 unesco 47,600 1760,000 2,000sacre coeur 27 33 5 cdg airport 173 4,670 170bottled water 13 108 2h2o dispenser 813 57,400 4,350*rlu = relative light units, *hpc = heterotrophic plate count, *fm =french method.conclusionsthis is the first study where a miniaturize

42、d atp bioluminescence method has been validated against the conventional plate method, the direct viable count, and the acridine orange direct count for the determination of heterotrophic bacteria in the drinking water samples. the atp assay was77found to be rapid and sensitive. the procedure is sim

43、ple and can be doneon-site with a portable power supply. the volume of water needed forfiltration is small (0.1-10 ml). a traveler wishing to determine the bacterial water quality in a foreign country will be able to do this in minutes. furtherstudiesin water with a high hpc, it is advisable to look

44、 for pathogens.specific bacteria can be isolated from the water using immunomagneticseparation (ims). studies have been done to estimate the number of e.colio157:h7, pseudomonas and legionella. after separation, the target cells are lysed and their number is estimated. this has been reported in a re

45、cent paper(lee, 2001).referencesapha-awwa-wef. 1995: standard methods for the examination of water and wastewater.19th ed. american public health association, inc., washington, d. c.astm d 401281. standard test method for adenosine triphospate(atp) content ofmicroorganisms in water. coallier et al. 1994: the optimization and applicationof two direct viable count methods forbacteria in distributed drinking water. can. j. microbiology . 40:830-836.crombrugge j and g waes. 1991: atp method. in:methods for assessing the bacteriologicalquality of raw milk fro