1、Designation: D5246 92 (Reapproved 2004)D5246 13Standard 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 r
2、evision, the year of last 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 ThisThe test method covers the isolation and enumeration of Pseudomonas aeruginosaaeruginosa. (Te
3、stingP. aeruginosawas) from surface waters; recreational waters; ground water, water supplies; especially rural nonchlorinated sources; waste water;and saline waters. The detection limit of this test method is one microorganism per 100 mL. performed on spiked reagent gradewater samples.1.2 This test
4、 method was used successfully with reagent water and it is the usersIt is the users responsibility to ensure thevalidity of this test method for waters of untested matrices.surface waters, ground waters, recreational waters fresh and marine),wastewaters.1.3 The values stated in SI units are to be re
5、garded as the standard. No other units of measurement are included in this standard.1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and
6、 determine the applicability of regulatorylimitations prior to use. Specific hazard statements 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 of Applicab
7、le Test Methods of Committee D19 on WaterD3370 Practices for Sampling Water from Closed Conduits3. Terminology3.1 Definitions:3.1.1 For definitions of terms used in this test method, refer to Terminology D1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 Pseudomonas aeruginosaan aerobic,
8、 motile, gram negative rod that produces fluorescent pigments and pyocyanin. It isoxidase and caseinase positive, is able to grow at 42C, is relatively resistant to many antibiotics, and may utilize acetamide.3.2.2 refrigerationstorage at 2 to 8C.4. Summary of Test Method4.1 A water sample is passed
9、 through a 0.45 mm or equivalent membrane filter. The filter carrying the retained organisms isplaced on a selective medium (M-PA-C)3 and is incubated at 41.5 6 0.5C for 48 to 72 h. The resulting pink-brown to blackcolonies of Pseudomonas aeruginosa are counted and reported per 100 mL of the sample.
10、 Colonies may be verified on skim milkagar.5. Significance and Use5.1 Pseudomonas aeruginosa is an opportunistic pathogen, and has been linked as the causative agent of numerous infectionsthat may be transmitted through a contaminated water supply to a susceptible host. In addition to its direct pat
11、hogenicity, the1 This test method is under the jurisdiction of ASTM Committee D19 on Water and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved June 1, 2004May 1, 2013. Published June 2004July 2013. Originally approved in 1992. Last previous edition
12、approved in 19982004 asD5246 92 (1998).(2004). DOI: 10.1520/D5246-92R04.10.1520/D5246-13.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summa
13、ry page on the ASTM website.3 Available from BBL Microbiological Systems, Division of Becton Dickinson and Co., Cockeysville, MD 21030. Other suppliers may be utilized if equivalent.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of wh
14、at changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered th
15、e official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1association of P. aeruginosa with human fecal waste indicates that where elevated levels of P. aeruginosa are found, a serious healthhazard may exist due to the prese
16、nce of other pathogens.NOTE 1Fecal waste is 95 % E. coli which is found in humans and warm bloodied animals.5.2 The membrane filtration procedure described is a rapid and reliable test method of detecting P. aeruginosa in water.6. Interferences6.1 For certain samples, bacterial cells may have been e
17、xposed to adverse environmental factors that lower their probability forsurvival and growth on a membrane filter medium. This effect may be pronounced in this test method due to the presence ofantibiotics and the elevated incubation temperature.6.2 The selection of an appropriate dilution volume is
18、essential.Too small a dilution volume may fail to detect any P. aeruginosaorganisms, while too large a volume may cause an overabundance of colonies that would interfere with an accurate count.6.3 Chemicals or a combination of chemicals in certain samples can have a toxic effect upon P. aeruginosa w
19、hen concentrated.6.4 Turbidity in samples may clog filter or effect color detection of organisms that develop on the filter.6.5 Water samples containing residual chlorine can be detrimental to P. aeruginosa. Utilize the procedure defined in PracticesD3370 to address chlorinated water samples.7. Appa
20、ratus7.1 Equipment for collection and transport of samples to laboratory:7.1.1 Autoclavable sample container; Use sterile, non-toxic, glass or plastic containers with a leak-proof lid. Ensure that thesample container is capable of holding a 1-L sample with ample headspace to facilitate mixing of sam
21、ple by shaking prior toanalysis.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 pipettes with a 2.5 % tolerance with pipette bulbs or automatic pipette.7.4 Automatic pipettors and associated sterile pipette
22、 tips capable of delivering up to 10 mL and 1 mL volumes (optional).7.5 Pipette container, autoclavable stainless steel, aluminum or borosilicate glass (if using glass pipettes).7.6 Inoculation loops, at least 3 mm diameter, and needles, nichrome or platinum wire, 26 B or 15 60 mm, glass or plastic,
23、 with loose-fitting lids;or 15 100 mm.7.11.2 Membrane filtration units (filter base and funnel), glass, plastic or stainless steel, wrapped with aluminum foil or kraftpaper and sterilized. Purchased disposable plastic sterile filters can also be used.7.11.3 Membrane filters, sterile, white, grid mar
24、ked, 47 mm diameter, with 0.45 6 0.02 m pore size.7.11.4 Ultraviolet unit for sanitization of the filter funnel between filtrations (optional).7.11.5 Line vacuum, electric vacuum pump, or aspirator for use as a vacuum source. In an emergency or in the field, a handpump or a syringe equipped with a c
25、heck valve to prevent the return flow of air, can be used.7.11.6 Filter flask, vacuum, usually 1 L, with appropriate tubing.7.11.7 Flask for safety trap placed between the filter flask and the vacuum source.7.11.8 Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45 6 0.02 m pore
26、 size.7.11.9 Flame or electric incinerator for sterilizing metal inoculating loops and forceps.7.12 Forceps, straight or curved, with smooth tips to handle filters.7.13 Incubator, capable of maintaining Incubator, hot air or water-jacketed microbiological type to maintain a temperature of41.5 6 0.5C
27、 and 3535.0 6 0.5C.7.14 Stereoscopic Microscope, Magnification of 1015 with a cool white fluorescent light.light or optional stereoscopicmicroscope.D5246 1327.15 Colony Counter. Colony counting device, mechanical, electric or hand tally (optional).7.6 Containers, with lids (for incubating test petri
28、 dishes containing membrane filters under high humidity).7.7 Long-Wave Ultraviolet Light.7.8 Autoclave, or other sterilizing equipment.7.16 Petri Dishes, sterile, 50 by 9 or 60 by 15 mm and 100 by 15 mm.365 nm UV lamp.7.10 Pipets, sterile, 1 and 10 mL, with 0.1-mL graduations and an accuracy of 65 %
29、.8. Reagents and Materials8.1 Purity of ReagentsReagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that allreagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, wheresuch specifications are a
30、vailable.4 Other grades may be used, provided it is first ascertained that the reagent is of sufficiently highpurity to permit its use without lessening the accuracy of the determination.8.2 Purity of WaterUnless otherwise indicated, references to water shall be understood to mean reagent water conf
31、orming toType II of Specification D1193.8.3 Buffered WaterDispense 1.25 mL of buffered water stock solution and 5.0 mL magnesium chloride solution (see 8.5) anddilute to 1 L with water. Dispense in amount to provide 99 mL after sterilization. This can be purchased or prepared in thelaboratory. Add a
32、fter stock solution (see 8.4). Add after 99 mL or volume as required based on dilution.8.4 Buffered Water StockDissolve 34.0 g potassium dihydrogen phosphate (KH2PO4) in 500 mL water, adjust to pH 7.2 withKOH solution (5.6 g/L) and dilute to 1 L with water.8.5 Magnesium Chloride Solution (81.1 g/L)D
33、issolve 81.1 g magnesium chloride (MgCl2 6H2O) in water and dilute to 1 Lwith water.8.6 Potassium Hydroxide Solution (5.6 g/L)Dissolve 5.6 g of potassium hydroxide (KOH) in water and dilute to 1 L withwater.8.7 Membrane Filters, sterile, 47 mm with grid (0.45 m pore size) or equivalent.9. Media Prep
34、aration9.1 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, anhydrous 5.0 gAgar 12.0 gMagnesium sulfate, anhydrous 1.5 gKanamycin 0.008
35、 gNalidixic acid 0.037 g9.1.1 M-PA-C Medium3 or EquivalentDissolve mixture of above items into 1 L of water, boiling for 1 min to solubilize thechemicals. Cool to 45 to 50C before dispensing. Pour one plate of medium and measure the pH of the surface with a suitable pHelectrode. The surface pH of th
36、e solidified medium should be 7.260.1. If it is not, adjust pH of the remaining solution accordinglywith potassium hydroxide solution. (It is recommended this should be purchased and not prepared from individual ingredients.)9.1.2 Aseptically dispense 5 to 6 mL of media into each sterile 50 or 60 mm
37、 petri dish. This medium should be stored underrefrigeration and used within one week after preparation.9.2 Skim Milk Agar Skim milk powder is high grade skim milk reduced to powder by a spraying process. Slowly add 100g of skim milk powder to 500 mL of water and stir without heat for approximately
38、30 min. Prepare an agar solution by adding15.0 g of agar to 500 mLof water and heat at 90C for 10 to 12 min.Autoclave the solutions separately at 121C for 12 min. Cool,with stirring, until temperature reaches 50 to 55C. Add the skim milk solution to the agar solution, thoroughly mix, and dispensease
39、ptically into sterile petri plates. The plates may be stored in sealed containers in the refrigerator for up to two weeks.4 Reagent Chemicals, American Chemical Society Specifications.Specifications, Am. Chem. Soc., Washington, D.C. American Chemical Society, Washington, DC,www.chemistry.org. For su
40、ggestions on the testing of reagents not listed by the American Chemical Society, see “AnalarAnalar Standards Forfor LaboratoryChemicals,”Chemicals, BDH Ltd., Poole, Dorset, UK,U.K., and the“the United States Pharmacopeia.”Pharmacopeia and National Formulary, U.S. PharmacopeialConvention, Inc. (USPC
41、), Rockville, MD, http:/www.usp.org.D5246 1339.3 Soybean Casein Digest Agar5Formula per litre of water:water (it is recommended this should be purchased and notprepared from individual ingredients):Pancreatic digest of casein 15.0 gPapaic digest of soybean meal 5.0 gSodium chloride 5.0 gAgar 15.0 g9
42、.3.1 Soybean Casein Digest AgarPrepare the media according to manufacturers instructions and dispense it aseptically intosterile petri dishes.10. Hazards10.1 P. aeruginosa is an opportunistic human pathogen; thus handle all culture material (plates, pipets, dilution tubes) usingaccepted microbiologi
43、cal techniques, including sterilization of all discarded material.10.2 Hold TimeRecent EPA MUR (May 2012) indicates hold time to testing of 8 h.11. Sampling, Test Specimens, and Test Units11.1 Collect the sample according to Practices D3370, refrigerate, and analyze the sample within 6 h.12. Prepara
44、tion of Apparatus12.1 Washing and CleaningThoroughly clean all glassware and filtration equipment, using a suitable detergent in hot water.After rinsing first in hot tap water and then in water, thoroughly dry the equipment prior to sterilization.12.2 SterilizationFollow standard microbiological lab
45、oratory practices for preparing glassware and filtration equipment priorto sterilization. Autoclave the apparatus at 121C for 20 to 30 min or at 132C for 5 min. Sterilization times should be increasedin cases where large loads are sterilized at one time.13. Procedure13.1 Membrane Filtration:13.1.1 U
46、sing alcohol-flamed forceps, aseptically remove the presterilized membrane filter from its package and place it, gridside up, on the base of the membrane filter unit. Connect the filtration flask and vacuum trap to a vacuum source.13.1.2 If P. aeruginosa levels are high the sample must be diluted to
47、 obtain measurable levels. This is accomplished by seriallydiluting a known quantity of the sample (for example, 1.0 mL) through a series of known volumes (for example, 99 mL) of sterilebuffered water until the desired bacterial density is obtained.13.1.3 Thoroughly mix the sample prior to filtratio
48、n.13.1.4 Pour appropriate volumes (for example, 100 to 200 mL for natural waters, 500 mL for swimming pools) of the sampleor sample dilution into the filter funnel. If smaller volumes (for example, 1 to 10 mL) of the sample are to be filtered, add 20 to30 mL of sterile buffered water to the filter f
49、unnel before adding the sample to evenly disperse cells. Apply vacuum, filter thecontents of the funnel, and then rinse the funnel three times with 20 to 30 mL of sterile buffered water. Shut off the vacuum whenall the liquid has been filtered. Remove the funnel and, with sterile forceps, carefully remove the membrane filter from the base.13.1.5 Place the membrane on the M-PA-C3 medium by holding the filter at an angle of 45 and carefully rolling it onto theagar surface. Ensure that there is no air trapped between the membrane and the su
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