1、Designation: D 7464 08An American National StandardStandard Practice forManual Sampling of Liquid Fuels, Associated Materials andFuel System Components for Microbiological Testing1This standard is issued under the fixed designation D 7464; the number immediately following the designation indicates t
2、he year oforiginal adoption or, in the case of revision, 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.INTRODUCTIONThere are several important characteristics that
3、distinguish microbiological parameters from otherparameters for which manually collected fuel samples are tested.Microbes, when present in fuels or fuel systems are invariably present as contaminants. Similarlyto particulates, microbes are discrete entities rather than dissolved solutes in fuel, how
4、ever, unlikeinanimate particles; microbes can proliferate or die during the interval between sampling and testing.An important consequence of this is that microbes introduced into the sample from sources otherthan the sample itself, can proliferate and potentially eclipse the population indigenous t
5、o the sample.Although microbes can be transported in fuel, they require free-water in order to grow andproliferate. Consequently, microbes tend to form colonies that are embedded in hydrophilic matrices.These matrices are most likely to form at system interfaces, including: fuel-water, fuel-structur
6、e,bottom-water-structure and air and fuel-vapor to structure. Microbes growing within these coloniesproduce chemicals (metabolites and biomolecular detritus) that are deteriogenic (can degrade fuel andfuel system components) and diffuse into fuel.These factors combine to require unique practices spe
7、cific to the collection of samples that areintended for microbiological testing.1. Scope1.1 This practice covers aspects of sample device prepara-tion and sample handling that prevent samples from becomingcontaminated with microorganisms not originally containedwithin the sample.1.2 This practice al
8、so covers sample handling consider-ations that reflect the perishability of samples collected formicrobiological testing.1.3 This practice supplements Practice D 4057 by providingguidance specific to the manual sampling of fuels whensamples are to be tested for microbial contamination.1.4 The values
9、 stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.5 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 safet
10、y and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 396 Specification for Fuel OilsD 910 Specification for Aviation GasolinesD 975 Specification for Diesel Fuel OilsD 1129 Terminology Relating to WaterD 1193 Spec
11、ification for Reagent WaterD 1655 Specification for Aviation Turbine FuelsD 2069 Specification for Marine Fuels3D 2880 Specification for Gas Turbine Fuel OilsD 3508 Method for Evaluating Water Testing MembraneFilters for Fecal Coliform Recovery3D 3699 Specification for Kerosine1This practice is unde
12、r the jurisdiction of ASTM Committee D02 on PetroleumProducts and Lubricants and is the direct responsibility of Subcommittee D02.14 onStability and Cleanliness of Liquid Fuels.Current edition approved July 1, 2008. Published August 2008.2For referenced ASTM standards, visit the ASTM website, www.as
13、tm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Withdrawn.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United Stat
14、es.D 4057 Practice for Manual Sampling of Petroleum andPetroleum ProductsD 4814 Specification forAutomotive Spark-Ignition EngineFuelD 5245 Practice for Cleaning Laboratory Glassware, Plas-ticware, and Equipment Used in Microbiological AnalysesD 6227 Specification for Grade 82 Unleaded AviationGasol
15、ineD 6469 Guide for Microbial Contamination in Fuels andFuel SystemsD 6751 Specification for Biodiesel Fuel Blend Stock(B100) for Middle Distillate FuelsD 6974 Practice for Enumeration of Viable Bacteria andFungi in Liquid FuelsFiltration and Culture Procedures2.2 American Petroleum Institute (API)
16、Standard:4Manual of Petroleum Measurement Standards Chapter3Tank Gauging, section 1AStandard Practice for theManual Gauging of Petroleum and Petroleum Products2.3 Petroleum Equipment Institute (PEI) Standard:5900-08 Recommended Practices for the Inspection andMaintenance of UST Systems3. Terminology
17、3.1 DefinitionsFor definition of terms used in this methodrefer to Terminologies D 1129 and D 4175, Practice D 4057and Guide D 6469.3.1.1 aseptic, adjsterile, free from viable microbiologicalcontamination.3.1.2 scrape sample, na portion of residue removed froma surface by forceful strokes of an inst
18、rument such as a spatula.4. Summary of Practices4.1 Liquid Sampling:4.1.1 Fuel and fuel-associated bottom-water samples in-tended for microbiological testing are collected similarly toconventional samples as described in Practice D 4057, how-ever specific measures are added to reduce the risk of sam
19、plecontamination.4.1.2 Sampling devices are disinfected before collectingmicrobiological samples.4.1.3 Sterile sample containers are used.4.1.4 Unique chain of custody procedures are used tominimize the potential qualitative, quantitative or both types ofchanges in the sample between sampling and te
20、sting.4.2 Surface Sampling:4.2.1 Sterile swabs are used to collect surface samples formicrobiological testing.4.2.2 Swabbed areas are measured to facilitate test resultnormalization into parameter units per unit surface area (forexample CFU/cm2).4.2.3 The post-sampling chain of custody procedures fo
21、rliquid samples apply.4.3 Filter Media:4.3.1 Canister Elements:4.3.1.1 Filter elements are transferred aseptically to sterileplastic bags.4.3.1.2 The post-sampling chain of custody procedures forliquid samples apply.4.3.2 Depth Media:4.3.2.1 Media core-samples are collected aseptically andtransferre
22、d to tared, sterile containers.4.3.2.2 The post-sampling chain of custody procedures forliquid samples apply.5. Significance and Use5.1 Representative samples of fuel products and associatedsubstances are required for the determination of microbialcontamination in fuels and fuel systems in order to
23、accuratelyassess the biodeterioration risk posed to the fuel, fuel-systemcomponents or both. Uncontrolled microbial contaminationcan affect fuel specification properties adversely.6As discussedin Guide D 6469, microbes can cause a variety of operationalproblems, including filter plugging and microbi
24、ally influencedcorrosion (MIC), the latter of which causes valve failure, tankand pipeline failure.5.2 These practices for microbiological sampling decreasethe risk of contaminating samples with extraneous microbes,thereby increasing the probability that the original microbialpopulation in the sampl
25、e does not change significantly betweenthe time of sampling and the time of testing.5.3 The objective of sampling for microbiological testing isto obtain a representative sample that is likely to reflect thedegree and nature of microbial contamination in the systemfrom which the sample is collected.
26、 Manual 477addresses therational for and design of microbial contamination programs.5.4 The physical, chemical and microbiological propertytests to be performed on a sample will dictate the samplingprocedures, the sample quantity required, and many of thesample handling requirements.5.5 Fuel systems
27、 are not normally designed to facilitateoptimal microbiological sampling. Consequently, the selectionof sampling device and sample source reflect compromisesbetween accessibility and suitability for meeting the samplecollection objective.5.6 The guidance provided in Practice D 4057 generallyapplies
28、to this practice as well. Consequently, this practice willaddress only those procedures that apply uniquely to micro-biological sampling.6. Apparatus6.1 The general considerations provided in Practice D 4057apply here. Sample containers come in a variety of shapes,sizes and materials. To paraphrase
29、D 4057, Paragraph 6.1, inorder to be able to select the right container for a givenapplication one must ensure that there will be no interaction4Available from American Petroleum Institute (API), 1220 L. St., NW, Wash-ington, DC 20005-4070, http:/www.api.org.5Available from Petroleum Equipment Insti
30、tute website, www.pei.org.6Passman, F. J., McFarland, B. L., and Hillyer, M. J., “Oxygenated GasolineBiodeterioration and its Control in Laboratory Microcosms,” International Biode-terioration and Biodegradation, Vol 47, No. 2, 2001, pp. 95-106.7Hill, G., “Sampling Methods for Detecting Microbial Co
31、ntamination in Fuelsand Fuel Systems,” in Passman, F. J., Ed., ASTM Manual 47Fuel and Fuel SystemMicrobiology: Fundamentals, Diagnosis and Contamination Control, ASTM Inter-national, West Conshohocken, PA, 2003.D7464082between the sampled material and the container which wouldaffect the integrity of
32、 the other. For general microbiologicaltesting, either glass or plastic containers are appropriate.However, containers should be appropriate for the specificmethod of analysis intended.6.1.1 Sample Container Cleanliness:6.1.1.1 Sample containers must be clean and should besterile.6.1.1.2 For the pur
33、poses of most microbiological testing,previously unused containers that are received in originalmanufacturers packaging are sufficiently clean to substitutefor sterile containers.6.1.1.3 Practice D 5245 provides details on cleaning previ-ously used glassware, plasticware and equipment.6.1.1.4 Method
34、 D 3508 specifies the protocol for sterilizingcontainers and labware.6.2 Sampling DevicesSampling devices are described indetail under each of the specific sampling procedures.6.2.1 Sampling Device Cleanliness:6.2.1.1 Sampling devices shall be cleaned between use inaccordance with 8.2.1, except clea
35、ning is not necessary be-tween repeated spot samples obtained either for the purpose offilling a single sample container or filling multiple samplecontainers intended to be used as replicate spot samples. Suchreplicates may be used to test the sample for different param-eters, when the contents of a
36、 single sample container are usedfor a single analysis (for example Practice D 6974), forobtaining replicate data in order to determine parametervariability, or both.6.2.1.2 It can be impractical to sterilize some types ofsampling devices used to obtain liquid petroleum, petroleumproduct or fuel-ass
37、ociated, free-water samples (see 8.2).6.3 Funnel20 to 25 cm diameter mouth; #1.9 cm diam-eter outlet (diameter small enough to fit into mouth of samplecontainer).6.4 Absorbent Spill Pads.6.5 Gloves; SurgicalUsed to prevent the contamination ofsamples with microorganisms indigenous to human skin.NOTE
38、 1The use of surgical gloves may create a static electricitydischarge risk that presents an explosion hazard when handling certainfuels. Additionally, polymers from which some surgical gloves aremanufactured are incompatible with certain fuels, and can disintegrate oncontact with such fuel, thereby
39、creating a skin contact hazard. Whereeither spark, product incompatibility or both types of risk exist, use analternative, clean, non-porous glove that has been disinfected in accor-dance with 8.2 in order to address the explosion hazard risk and stillminimize the risk of contaminating samples with
40、microbes associated withhuman skin.6.6 SpatulaStainless steel; 1.5 by 10 cm for collectingsurface residue samples.6.7 SwabsSterile, ATP-free.7. Reagents7.1 Alcohol,$70 % methanol, ethanol or isopropanol, tech-nical grade.7.2 WaterType I Reagent Grade or better (SpecificationD 1193; Terminology D 112
41、9).8. Manual Sampling Considerations8.1 The considerations detailed in Practice D 4057 Section7 apply.8.2 Sampling Device Disinfection:8.2.1 Before collecting a sample, the sampling device shallbe cleaned and disinfected. Due to the risk of fire and explosionwhen handling liquid fuels with boiling p
42、oints below 90C,procedures generally used to disinfect apparatus used formicrobiological sampling cannot be used in the liquid fuelenvironment. The following procedure shall be used instead:8.2.1.1 Clean the device, taking particular care to removeany liquid and particulate residue remaining from pr
43、evioussamples.8.2.1.2 Rinse device with alcohol (7.1) by filling the deviceapproximately14 to13 with alcohol and shaking the closeddevice for 30 s.8.2.1.3 Drain the alcohol thoroughly from the device into asuitable disposal container.8.2.1.4 Allow all residual alcohol to evaporate from devicesurface
44、s.9. Special Precautions9.1 The precautions enumerated in Practice D 4057 Section8 apply to sampling for microbiological testing.9.2 Contamination ControlAdditional caution is requiredto prevent the contamination of samples with non-indigenousmicrobes.9.2.1 The normal microflora of healthy skin is 1
45、 3 103bacteria/cm2. Precautions shall be taken to minimize the risk ofcontaminating samples with skin microflora. Wearing surgicalgloves provides an adequate barrier between the skin, samplingdevices and sample containers. Gloves should either be re-placed or rinsed with 70 % alcohol (7.1) between s
46、amples. (SeeNote 1.)9.2.2 Sampling Devices can become contaminated withresidue from collected samples. The procedure described in 8.2minimizes the risk of cross-contamination. Device disinfectionshould be completed just before sample collection in order toreduce the risk of contamination from airbor
47、ne microbes. Allsurfaces with which the sample will come into contact shall bedisinfected. After collecting sample and before dispensingsample into sample container, wipe any debris from thesamplers external surfaces and use alcohol to disinfect thefunnel surface over which the sample will flow.9.2.
48、3 Drain and Tap SamplesMicrobiological testing mayalso be performed on drain samples. If sampling from a fluiddrain line or dispenser nozzle, clean the area around thedischarge orifice and wipe the area with alcohol (7.1). Followthe guidance provide in Practice D 4057, Paragraph 13.6.9.2.4 Sample Co
49、ntainers should remain closed until justbefore the sample is dispensed from the sampling device intothe container, and should be re-closed immediately after thesample has been dispensed. This reduces the risk of contami-nation from airborne particles or during sample containerhandling.9.3 Sample PerishabilityMicrobes are living organisms.Consequently, samples for microbiological testing are highlyperishable. Optimally, microbiological tests are initiated withinD74640834 h after sample collection. If testing is to be performed within4 h, samples may be stored at
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