1、Designation: D7464 08 (Reapproved 2013)Standard Practice forManual Sampling of Liquid Fuels, Associated Materials andFuel System Components for Microbiological Testing1This standard is issued under the fixed designation D7464; the number immediately following the designation indicates the year ofori
2、ginal 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 distinguish m
3、icrobiological 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, however, unlikei
4、nanimate 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 to the sample.
5、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-structure,bottom-wate
6、r-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 specific to the
7、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 also covers sam
8、ple handling consider-ations that reflect the perishability of samples collected formicrobiological testing.1.3 This practice supplements Practice D4057 by providingguidance specific to the manual sampling of fuels whensamples are to be tested for microbial contamination.1.4 The values stated in SI
9、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 safety and health p
10、ractices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D396 Specification for Fuel OilsD910 Specification for Aviation GasolinesD975 Specification for Diesel Fuel OilsD1129 Terminology Relating to WaterD1193 Specification for Reage
11、nt WaterD1655 Specification for Aviation Turbine FuelsD2069 Specification for Marine Fuels (Withdrawn 2003)3D2880 Specification for Gas Turbine Fuel OilsD3508 Method for Evaluating Water Testing MembraneFilters for Fecal Coliform Recovery (Withdrawn 1995)3D3699 Specification for KerosineD4057 Practi
12、ce for Manual Sampling of Petroleum andPetroleum Products1This practice is under 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 May 1, 2013. Publis
13、hed August 2013. Originallyapproved in 2008. Last previous edition approved in 2008 as D7464 08. DOI:10.1520/D7464-08R13.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refe
14、r to the standards Document Summary page onthe ASTM website.3The last approved version of this historical standard is referenced onwww.astm.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1D4814 Specification for Automotive Spark-I
15、gnition EngineFuelD5245 Practice for Cleaning Laboratory Glassware,Plasticware, and Equipment Used in MicrobiologicalAnalysesD6227 Specification for Unleaded Aviation Gasoline Con-taining a Non-hydrocarbon ComponentD6469 Guide for Microbial Contamination in Fuels and FuelSystemsD6751 Specification f
16、or Biodiesel Fuel Blend Stock (B100)for Middle Distillate FuelsD6974 Practice for Enumeration of Viable Bacteria andFungi in Liquid FuelsFiltration and Culture Procedures2.2 American Petroleum Institute (API) Standard:4Manual of Petroleum Measurement Standards Chapter3Tank Gauging, section 1AStandar
17、d 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. Terminology3.1 For definition of terms used in this method refer toTerminologies D1129 and D4175, Prac
18、tice D4057 and GuideD6469.3.2 Definitions:3.2.1 aseptic, adjsterile, free from viable microbiologicalcontamination.3.2.2 scrape sample, na portion of residue removed froma surface by forceful strokes of an instrument such as a spatula.4. Summary of Practices4.1 Liquid Sampling:4.1.1 Fuel and fuel-as
19、sociated bottom-water samples in-tended for microbiological testing are collected similarly toconventional samples as described in Practice D4057, howeverspecific measures are added to reduce the risk of samplecontamination.4.1.2 Sampling devices are disinfected before collectingmicrobiological samp
20、les.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 testing.4.2 Surface Sampling:4.2.1 Sterile swabs are used to collect surface samples formicrobi
21、ological 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 forliquid samples apply.4.3 Filter Media:4.3.1 Canister Elements:4.3.1.1 Filter elements are tr
22、ansferred 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 andtransferred to tared, sterile containers.4.3.2.2 The post-sampling chain of custody procedures forliqui
23、d 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 accuratelyassess the biodeterioration risk posed to the fuel, fuel-systemcomponents or both.
24、Uncontrolled microbial contaminationcan affect fuel specification properties adversely.6As discussedin Guide D6469, microbes can cause a variety of operationalproblems, including filter plugging and microbially influencedcorrosion (MIC), the latter of which causes valve failure, tankand pipeline fai
25、lure.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 sample does not change significantly betweenthe time of sampling and the time of testing.5.3 The ob
26、jective 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. Manual 477addresses therational for and design of microbial contamination programs.5.4 The ph
27、ysical, 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 are not normally designed to facilitateoptimal microbiological sampling. Consequently, the se
28、lectionof sampling device and sample source reflect compromisesbetween accessibility and suitability for meeting the samplecollection objective.5.6 The guidance provided in Practice D4057 generallyapplies to this practice as well. Consequently, this practice willaddress only those procedures that ap
29、ply uniquely to micro-biological sampling.6. Apparatus6.1 The general considerations provided in Practice D4057apply here. Sample containers come in a variety of shapes,4Available from American Petroleum Institute (API), 1220 L. St., NW,Washington, DC 20005-4070, http:/www.api.org.5Available from Pe
30、troleum Equipment Institute 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
31、 Detecting Microbial Contamination in Fuelsand Fuel Systems,” in Passman, F. J., Ed., ASTM Manual 47Fuel and Fuel SystemMicrobiology: Fundamentals, Diagnosis and Contamination Control, ASTMInternational, West Conshohocken, PA, 2003.D7464 08 (2013)2sizes and materials. To paraphrase D4057, Paragraph
32、6.1, inorder to be able to select the right container for a givenapplication one must ensure that there will be no interactionbetween the sampled material and the container which wouldaffect the integrity of the other. For general microbiologicaltesting, either glass or plastic containers are approp
33、riate.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 purposes of most microbiological testing,previously unused containers that are received in origi
34、nalmanufacturers packaging are sufficiently clean to substitutefor sterile containers.6.1.1.3 Practice D5245 provides details on cleaning previ-ously used glassware, plasticware and equipment.6.1.1.4 Method D3508 specifies the protocol for sterilizingcontainers and labware.6.2 Sampling DevicesSampli
35、ng 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 cleaning is not necessary be-tween repeated spot samples obtained either for the purpose offilling
36、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 differentparameters, when the contents of a single sample container areused for a single analysis (for example Practice D6974), forobtaining
37、 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-associated, free-water samples (see 8.2).6.3 Funnel20 to 25 cm diameter mouth; 1.9 cm diameteroutlet
38、(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 1The use of surgical gloves may create a static electricitydischarge risk that presents an explosion
39、 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 creating a skin contact hazard. Whereeither spark, product incompatibility or both types of risk exis
40、t, 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 microbes associated withhuman skin.6.6 SpatulaStainless steel; 1.5 by 10 cm for collectingsurface res
41、idue 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 (SpecificationD1193; Terminology D1129).8. Manual Sampling Considerations8.1 The considerations detailed in Practice D4057 Section 7apply.8.
42、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 points below 90C,procedures generally used to disinfect apparatus used formicrobiological sampling cannot
43、 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 previoussamples.8.2.1.2 Rinse device with alcohol (7.1) by filling the deviceapproximately14 to13 with alc
44、ohol 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 devicesurfaces.9. Special Precautions9.1 The precautions enumerated in Practice D4057 Section8 apply to sampling for
45、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 103bacteria/cm2. Precautions shall be taken to minimize the risk ofcontaminating samples with skin micro
46、flora. 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 samples. (SeeNote 1.)9.2.2 Sampling Devices can become contaminated withresidue from collected samples. The
47、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 airborne microbes. Allsurfaces with which the sample will come into contact shall bedisinfected. After collecting
48、 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.9.2.3 Drain and Tap SamplesMicrobiological testing mayalso be performed on drain samples. If sampling from a fl
49、uiddrain line or dispenser nozzle, clean the area around thedischarge orifice and wipe the area with alcohol (7.1). Followthe guidance provide in Practice D4057, Paragraph 13.6.9.2.4 Sample Containers 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.D7464 08 (2013)39.3 Sample PerishabilityMicrobes are living organisms.Consequently,
