1、Designation: D5246 15Standard Test Method forIsolation and Enumeration of Pseudomonas aeruginosa fromWater1This standard is issued under the fixed designation D5246; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last
2、revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 The test method covers the isolation and enumeration ofPseudomonas aeruginosa. Testing was performed on spikedsamples using
3、 reagent grade water as the diluent from surfacewaters; recreational waters; ground water, water supplies;especially rural nonchlorinated sources; waste water; andsaline waters. The detection limit of this test method is onemicroorganism per 100 mL.1.2 This test method was used successfully with rea
4、gentwater. It is the users responsibility to ensure the validity of thistest method for surface waters, recreational waters, groundwater, rural nonchlorinated sources; waste water; and salinewaters.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are inc
5、luded in thisstandard.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 standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior
6、to use. Specific hazardstatements are given in Section 10.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD2777 Practice for Determination of Precision and Bias ofApplicable Test Methods of Committee D19 on WaterD3370 Practices for S
7、ampling Water from Closed Conduits3. Terminology3.1 Definitions:3.1.1 For definitions of terms used in this standard, refer toTerminology D1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 Pseudomonas aeruginosa, nan aerobic, motile,gram negative rod that produces fluorescent pigments an
8、dpyocyanin.3.2.1.1 DiscussionIt is oxidase and caseinase positive, isable to grow at 42C, is relatively resistant to many antibiotics,and may utilize acetamide.3.2.2 refrigeration, nstorage at 2 to 8C.4. Summary of Test Method4.1 A water sample is passed through a 0.45 mm orequivalent membrane filte
9、r. The filter carrying the retainedorganisms is placed on a selective medium (M-PA-C)3and isincubated at 41.5 6 0.5C for 72 h. The resulting pink-brownto black colonies of Pseudomonas aeruginosa are counted andreported per 100 mL of the sample. Colonies may be verifiedon skim milk agar.5. Significan
10、ce 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 to a susceptible host.NOTE 1Fecal waste is 95 % E. coli which is found in humans andwarm bloodied animals.5.2
11、The membrane filtration procedure described 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 filterme
12、dium.This effect may be pronounced in this test method dueto the presence of antibiotics and the elevated incubationtemperature.1This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition appr
13、oved June 1, 2015. Published June 2015. Originallyapproved in 1992. Last previous edition approved in 2013 as D5246 13. DOI:10.1520/D5246-15.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volu
14、me 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.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, We
15、st Conshohocken, PA 19428-2959. United States16.2 The selection of an appropriate dilution volume isessential. Too small a dilution volume may fail to detect any P.aeruginosa organisms, while too large a volume may cause anoverabundance of colonies that would interfere with an accu-rate count.6.3 Ch
16、emicals or a combination of chemicals in certainsamples 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. aerugin
17、osa. Utilize the procedure defined inPractices D3370 to address chlorinated water samples.7. Apparatus7.1 Equipment for collection and transport of samples tolaboratory:7.1.1 Autoclavable sample containerUse sterile, non-toxic, glass or plastic containers with a leak-proof lid. Ensurethat the sample
18、 container is capable of holding a 1-L samplewith ample headspace to facilitate mixing of sample by shakingprior to analysis.7.1.2 Ice chest.7.1.3 Ice packs.7.2 Rinse water bottles, sterile, polypropylene or glass.7.3 Pipettes, sterile, plastic or autoclavable glass pipetteswith a 2.5 % tolerance wi
19、th pipette bulbs or automatic pipette.Automatic pipettors and associated sterile pipette tips capableof delivering up to 10 mL and 1 mL volumes (optional).7.4 Pipette container, autoclavable stainless steel, aluminumor borosilicate glass (if using glass pipettes).7.5 Inoculation loops, at least 3 mm
20、 diameter, and needles,nichrome or platinum wire, 26 B or 15 60 mm, glass or plastic, with loose-fittinglids; or 15 100 mm.7.10.2 Membrane filtration units (filter base and funnel),glass, plastic or stainless steel, wrapped with aluminum foil orkraft paper and sterilized. Purchased disposable plasti
21、c sterilefilters can also be used.7.10.3 Membrane filters, sterile, white, grid marked, 47 mmdiameter, with 0.45 6 0.02 m pore size.7.10.4 Ultraviolet unit for sanitization of the filter funnelbetween filtrations (optional).7.10.5 Line vacuum, electric vacuum pump, or aspirator foruse as a vacuum so
22、urce. In an emergency or in the field, a handpump or a syringe equipped with a check valve to prevent thereturn flow of air, can be used.7.10.6 Filter flask, vacuum, usually 1 L, with appropriatetubing.7.10.7 Flask for safety trap placed between the filter flaskand the vacuum source.7.10.8 Membrane
23、filters, sterile, white, grid marked, 47 mmdiameter, with 0.45 6 0.02 m pore size.7.10.9 Flame or electric incinerator for sterilizing metalinoculating loops and forceps.7.11 Forceps, straight or curved, with smooth tips to handlefilters.7.12 Incubator, hot air or water-jacketed microbiologicaltype
24、to maintain a temperature of 41.5 6 0.5C and 35.0 60.5C.7.13 Magnification of 1015 with a cool white fluorescentlight or optional stereoscopic microscope.7.14 Colony counting device, mechanical, electric or handtally (optional).7.15 365 nm UV lamp.8. Reagents and Materials8.1 Purity of ReagentsReage
25、nt 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 such specifications are available.4Other grades may beused, provided it is fir
26、st 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 mean reagent water conformingto Type II of Specification D1193.8.3 Buffered Wate
27、rDispense 1.25 mL of buffered waterstock solution (see 8.4) and 5.0 mL magnesium chloridesolution (see 8.5) and dilute to 1 L with water. Dispense inamount to provide 99 mL after sterilization. This can bepurchased or prepared in the laboratory. Add after stocksolution (see 8.4). Add after 99 mL or
28、volume as requiredbased on dilution.NOTE 2Sterile buffered water can be purchased or made in thelaboratory.4Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC, www.chemistry.org. For suggestions on thetesting of reagents not listed by the American C
29、hemical Society, see AnalarStandards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and theUnited States Pharmacopeia and National Formulary, U.S. PharmacopeialConvention, Inc. (USPC), Rockville, MD, http:/www.usp.org.D5246 1528.4 Buffered Water StockDissolve 34.0 g potassium dihy-drogen p
30、hosphate (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)Dissolve 5.6 gof potassiu
31、m hydroxide (KOH) in water and dilute to 1 L withwater.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 1.25 gLactose 1.25 gPhenol red 0.08 gFerric ammonium citrate, anhydrous 0.80 gSodium thiosulfate, anhydro
32、us 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 solubilizethe chemicals. Cool to 45 to 50C before dispensing. Pour oneplate of medium and measure the pH
33、 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 solution. (It isrecommended this should be purchased and not prepared fromindividual ingredients.)9.1.2 Asept
34、ically 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 AgarSkim milk powder is high grade skimmilk reduced to powder by a spraying process. Slowly add 100g of skim milk powde
35、r to 500 mLof 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 the skim milk sol
36、ution 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 (it is recommended this should be purchased and notprepared from
37、 individual ingredients):Pancreatic digest of casein 15.0 gPapaic digest of soybean meal 5.0 gSodium chloride 5.0 gAgar 15.0 g9.3.1 Soybean Casein Digest AgarPrepare the media ac-cording to manufacturers instructions and dispense it asepti-cally into sterile petri dishes.10. Hazards10.1 P. aeruginos
38、a is an opportunistic human pathogen; thushandle all culture material (plates, pipets, dilution tubes) usingaccepted microbiological techniques, including sterilization ofall discarded material.10.2 Hold TimeRecent EPA MUR (May 2012) indicateshold time to testing of 8 h.11. Sampling, Test Specimens,
39、 and Test Units11.1 Collect the sample according to Practices D3370,refrigerate, and analyze the sample within 8 h from time ofsampling to incubation.12. Preparation of Apparatus12.1 Washing and CleaningThoroughly clean all glass-ware and filtration equipment, using a suitable detergent in hotwater.
40、 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 20to 30 min
41、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, on the base of
42、 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 series of kn
43、own 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 dilution into
44、 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 times with 2
45、0 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 carefully rolling it
46、 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.5Difco or BBL Trypticase Soy Agar, available from BBL MicrobiologicalSystems, Division of Becton Dickinson and Co., C
47、ockeysville, MD 21030. Othersuppliers may be utilized if equivalent.D5246 15313.2 Incubation:13.2.1 Within 30 min after filtration, invert the petri dishesand place them in a humidified incubator at 41.5 6 0.5C. Toprovide a humid atmosphere, place the dishes in plasticcontainers lined with moistened
48、 paper towels and sealed witha lid.13.2.2 Incubate 72 6 2h.13.3 Counting Colonies:13.3.1 Typical P. aeruginosa colonies are flat and arepink-brown to black in color. The colony usually has a darkcenter with lighter colored edges. Slowly developing P. aerugi-nosa colonies may be almost clean with a s
49、mall dark center.13.3.2 Count bacterial colonies using a stereoscopic micro-scope with 10 magnification. The cool white fluorescent lightis set up to provide the best illumination.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. Incubate for 24 to 48 h at 35 6 0.5C.14.2 Aseptically transfer a ty
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