1、Designation: D 5246 92 (Reapproved 2004)Standard Test Method forIsolation and Enumeration of Pseudomonas aeruginosa fromWater1This standard is issued under the fixed designation D 5246; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revisio
2、n, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the isolation and enumeration ofPseudomonas aeruginosa (P. aeruginosa) from su
3、rface waters;recreational waters; ground water, water supplies; especiallyrural nonchlorinated sources; waste water; and saline waters.The detection limit of this test method is one microorganismper 100 mL.1.2 This test method was used successfully with reagentwater and it is the users responsibilit
4、y to ensure the validity ofthis test method for waters of untested matrices.1.3 The values stated in SI units are to be regarded as thestandard.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standa
5、rd to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. Specific hazardstatements are given in Section 10.2. Referenced Documents2.1 ASTM Standards:2D 1129 Terminology Relating to WaterD 1193 Specification for Reagent WaterD 2
6、777 Practice for Determination of Precision and Bias ofApplicable Methods of Committee D19 on WaterD 3370 Practices for Sampling Water from Closed Conduits3. Terminology3.1 Definitions:3.1.1 For definitions of terms used in this test method, referto Terminology D 1129.3.2 Definitions of Terms Specif
7、ic to This Standard:3.2.1 Pseudomonas aeruginosaan aerobic, motile, gramnegative rod that produces fluorescent pigments and pyocya-nin. It is oxidase and caseinase positive, is able to grow at42C, is relatively resistant to many antibiotics, and may utilizeacetamide.3.2.2 refrigerationstorage at 2 t
8、o 8C.4. Summary of Test Method4.1 A water sample is passed through a 0.45 mm orequivalent membrane filter. The filter carrying the retainedorganisms is placed on a selective medium (M-PA-C)3and isincubated at 41.5 6 0.5C for 48 to 72 h. The resultingpink-brown to black colonies of Pseudomonas aerugi
9、nosa arecounted and reported per 100 mL of the sample. Colonies maybe verified on skim milk agar.5. Significance and Use5.1 Pseudomonas aeruginosa is an opportunistic pathogen,and has been linked as the causative agent of numerousinfections that may be transmitted through a contaminatedwater supply
10、to a susceptible host. In addition to its directpathogenicity, the association of P. aeruginosa with humanfecal waste indicates that where elevated levels of P. aerugi-nosa are found, a serious health hazard may exist due to thepresence of other pathogens.5.2 The membrane filtration procedure descri
11、bed is a rapidand reliable test method of detecting P. aeruginosa in water.6. Interferences6.1 For certain samples, bacterial cells may have beenexposed to adverse environmental factors that lower theirprobability for survival and growth on a membrane filtermedium. This effect may be pronounced in t
12、his test method dueto the presence of antibiotics and the elevated incubationtemperature.6.2 The selection of an appropriate dilution volume isessential. Too small a dilution volume may fail to detect any P.1This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct r
13、esponsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved June 1, 2004. Published June 2004. Originallyapproved in 1992. Last previous edition approved in 1998 as D 5246 92 (1998).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer
14、Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from BBL Microbiological Systems, Division of Becton Dickinsonand Co., Cockeysville, MD 21030. Other suppliers may be utilized if equivalent.1Co
15、pyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.aeruginosa organisms, while too large a volume may cause anoverabundance of colonies that would interfere with an accu-rate count.6.3 Chemicals or a combination of chemicals in certainsamp
16、les can have a toxic effect upon P. aeruginosa whenconcentrated.6.4 Turbidity in samples may clog filter or effect colordetection of organisms that develop on the filter.6.5 Water samples containing residual chlorine can bedetrimental to P. aeruginosa. Utilize the procedure defined inPractices D 337
17、0 to address chlorinated water samples.7. Apparatus7.1 Top-Loading Balance, sensitive to 0.1 g.7.2 pH Meter and Surface pH Electrode.7.3 Incubator, capable of maintaining temperature of 41.56 0.5C and 35 6 0.5C.7.4 Stereoscopic Microscope, with a cool white fluorescentlight.7.5 Colony Counter.7.6 Co
18、ntainers, with lids (for incubating test petri dishescontaining membrane filters under high humidity).7.7 Long-Wave Ultraviolet Light.7.8 Autoclave, or other sterilizing equipment.7.9 Petri Dishes, sterile, 50 by 9 or 60 by 15 mm and 100 by15 mm.7.10 Pipets, sterile, 1 and 10 mL, with 0.1-mL graduat
19、ionsand an accuracy of 65%.8. Reagents and Materials8.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents shall conform to the specifications of the Commit-tee on Analytical Reagents of the American Chemical Society,where
20、 such specifications are available.4Other grades may beused, provided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.8.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to m
21、ean reagent water conformingto Type II of Specification 1193.8.3 Buffered WaterDispense 1.25 mL of buffered waterstock solution and 5.0 mL magnesium chloride solution (see8.5) and dilute to 1 L with water. Dispense in amount toprovide 99 mL after sterilization.8.4 Buffered Water StockDissolve 34.0 g
22、 potassium dihy-drogen phosphate (KH2PO4) in 500 mL water, adjust to pH 7.2with KOH solution (5.6 g/L) and dilute to 1 L with water.8.5 Magnesium Chloride Solution (81.1 g/L)Dissolve81.1 g magnesium chloride (MgCl26H2O) in water and diluteto 1 L with water.8.6 Potassium Hydroxide Solution (5.6 g/L)D
23、issolve 5.6 gof potassium hydroxide (KOH) in water and dilute to 1 L withwater.8.7 Membrane Filters, sterile, 47 mm with grid (0.45 mpore size) or equivalent.9. Media Preparation9.1 M-PA-C Medium3Formula per litre of water:L-lysine 5.0 gSodium chloride 5.0 gYeast extract 2.0 gXylose 1.25 gSucrose 1.
24、25 gLactose 1.25 gPhenol red 0.08 gFerric ammonium citrate, anhydrous 0.80 gSodium thiosulfate, anhydrous 5.0 gAgar 12.0 gMagnesium sulfate, anhydrous 1.5 gKanamycin 0.008 gNalidixic acid 0.037 g9.1.1 M-PA-C Medium3or EquivalentDissolve mixture ofabove items into 1 L of water, boiling for 1 min to s
25、olubilizethe chemicals. Cool to 45 to 50C before dispensing. Pour oneplate of medium and measure the pH of the surface with asuitable pH electrode. The surface pH of the solidified mediumshould be 7.2 6 0.1. If it is not, adjust pH of the remainingsolution accordingly with potassium hydroxide soluti
26、on.9.1.2 Aseptically dispense 5 to 6 mL of media into eachsterile 50 or 60 mm petri dish. This medium should be storedunder refrigeration and used within one week after preparation.9.2 Skim Milk Agar Skim milk powder is high grade skimmilk reduced to powder by a spraying process. Slowly add 100g of
27、skim milk powder to 500 mL of water and stir without heatfor approximately 30 min. Prepare an agar solution by adding15.0 g of agar to 500 mL of water and heat at 90C for 10 to12 min. Autoclave the solutions separately at 121C for 12min. Cool, with stirring, until temperature reaches 50 to 55C.Add t
28、he skim milk solution to the agar solution, thoroughlymix, and dispense aseptically into sterile petri plates. Theplates may be stored in sealed containers in the refrigerator forup to two weeks.9.3 Soybean Casein Digest Agar5Formula per litre ofwater:Pancreatic digest of casein 15.0 gPapaic digest
29、of soybean meal 5.0 gSodium chloride 5.0 gAgar 15.0 g9.3.1 Soybean Casein Digest AgarPrepare the mediaaccording to manufacturers instructions and dispense it asep-tically into sterile petri dishes.10. Hazards10.1 P. aeruginosa is an opportunistic human pathogen;thus handle all culture material (plat
30、es, pipets, dilution tubes)using accepted microbiological techniques, including steriliza-tion of all discarded material.11. Sampling, Test Specimens, and Test Units11.1 Collect the sample according to Practices D 3370,refrigerate, and analyze the sample within 6 h.4Reagent Chemicals, American Chemi
31、cal Society Specifications. Am. Chem.Soc., Washington, D.C. For suggestions on the testing of reagents not listed by theAmerican Chemical Society, see “Analar Standards For Laboratory Chemicals,”BDH Ltd., Poole, Dorset, UK, and the“ United States Pharmacopeia.”5Difco or BBL Trypticase Soy Agar, avai
32、lable from BBL MicrobiologicalSystems, Division of Becton Dickinson and Co., Cockeysville, MD 21030. Othersuppliers may be utilized if equivalent.D 5246 92 (2004)212. Preparation of Apparatus12.1 Washing and CleaningThoroughly clean all glass-ware and filtration equipment, using a suitable detergent
33、 in hotwater. After rinsing first in hot tap water and then in water,thoroughly dry the equipment prior to sterilization.12.2 SterilizationFollow standard microbiological labora-tory practices for preparing glassware and filtration equipmentprior to sterilization. Autoclave the apparatus at 121C for
34、 20to 30 min or at 132C for 5 min. Sterilization times should beincreased in cases where large loads are sterilized at one time.13. Procedure13.1 Membrane Filtration:13.1.1 Using alcohol-flamed forceps, aseptically remove thepresterilized membrane filter from its package and place it, gridside up, o
35、n the base of the membrane filter unit. Connect thefiltration flask and vacuum trap to a vacuum source.13.1.2 If P. aeruginosa levels are high the sample must bediluted to obtain measurable levels. This is accomplished byserially diluting a known quantity of the sample (for example,1.0 mL) through a
36、 series of known volumes (for example, 99mL) of sterile buffered water until the desired bacterial densityis obtained.13.1.3 Thoroughly mix the sample prior to filtration.13.1.4 Pour appropriate volumes (for example, 100 to 200mL for natural waters, 500 mL for swimming pools) of thesample or sample
37、dilution into the filter funnel. If smallervolumes (for example, 1 to 10 mL) of the sample are to befiltered, add 20 to 30 mL of sterile buffered water to the filterfunnel before adding the sample to evenly disperse cells.Applyvacuum, filter the contents of the funnel, and then rinse thefunnel three
38、 times with 20 to 30 mL of sterile buffered water.Shut off the vacuum when all the liquid has been filtered.Remove the funnel and, with sterile forceps, carefully removethe membrane filter from the base.13.1.5 Place the membrane on the M-PA-C3medium byholding the filter at an angle of 45 and careful
39、ly rolling it ontothe agar surface. Ensure that there is no air trapped between themembrane and the surface of the medium. If an air bubble isobserved, raise the membrane and repeat the procedure.13.2 Incubation:13.2.1 Within 30 min after filtration, invert the petri dishesand place them in a humidi
40、fied incubator at 41.5 6 0.5C. Toprovide a humid atmosphere, place the dishes in plasticcontainers lined with moistened paper towels and sealed witha lid.13.2.2 Incubate 48 6 2h.13.3 Counting Colonies:13.3.1 Typical P. aeruginosa colonies are flat and arepink-brown to black in color. The colony usua
41、lly has a darkcenter with lighter colored edges. Slowly developing P. aerugi-nosa colonies may be almost clean with a small dark center.13.3.2 Count bacterial colonies using a stereoscopic micro-scope with 103 magnification. The cool white fluorescent lightis set up to provide the best illumination.
42、13.3.3 The density range for accurate counting should be 20to 80 P. aeruginosa colonies and not more than 150 totalcolonies.14. Interpretation of Results14.1 Pick a well isolated colony with a sterile loop or needleand streak onto a soybean casein digest agar plate to obtainisolated colonies. Incuba
43、te for 24 to 48 h at 35 6 0.5C.14.2 Aseptically transfer a typical colony from TSA onto askim milk agar plate, prepared as described in 9.2 and incubateat 35 6 0.5C for 24 to 48 h. Clearing of the medium(caseinase production), production of a diffusible blue-greenpyocyanin pigment, and a diffusible
44、yellow-green fluorescentpigment constitutes a positive result. Observe production of thefluorescent pigment in a darkened room shining long-wave(366-nm) ultraviolet light source on the plate.14.3 Adjust counts based on percentage of confirmed colo-nies.15. Report15.1 Report counts as number of organ
45、isms per 100 mL. Ifprecise counts are required, verify all colonies. As a qualitycontrol check, verify at least ten colonies per plate.16. Precision and Bias16.1 Because microbiological samples are not stable, it isnot possible to distribute environmental samples to multiplelaboratories and establis
46、h precision and bias for commonenvironmental samples.16.2 In the collaborative study, each of seven laboratorieswas asked to collect two environmental water samples foranalyses, expected to contain Pseudomonas aeruginosa.Afreeze-dried reference sample was shipped to all laboratoriesfor analyses at t
47、wo dilution levels, for a total of four samples.Three replicate analyses were performed on each sampledilution. Then colonies were picked from each countable plate(20 to 80 colonies) and verified to determine final counts. Allseven laboratories reported data for the freeze-dried samples,but one labo
48、ratory did not report any data for environmentalsamples and another laboratory only reported data for only oneenvironmental sample.16.3 The data analysis was performed utilizing the proce-dures defined in Practice D 2777.16.4 Arithmetic means, single operator precision (So) andrelative standard devi
49、ations were calculated for each environ-mental sample at each laboratory. Since the reference culturewas common to all laboratories, the grand mean, pooled Soandrelative standard deviations were calculated for each labora-tory, and the grand mean, overall precision, St, and the overallrelative standard deviation were calculated for the study. Thesedata and statistical estimates are reported in Table 1. The meansand standard deviations are stated in terms of colony formingunits.17. Keywords17.1 enumeration; membrane filtration; PseudomonasaeruginosaD 5
