1、Designation: D6974 09 (Reapproved 2013)2Standard Practice forEnumeration of Viable Bacteria and Fungi in Liquid FuelsFiltration and Culture Procedures1This standard is issued under the fixed designation D6974; the number immediately following the designation indicates the year oforiginal adoption or
2、, 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.1NOTESubsections 8.4 and 8.8.2 were editorially corrected in August 2014.2NOTEReferences in
3、 Section 9 and the title of IP 385 were editorially corrected in January 2015.1. Scope1.1 This practice covers a membrane filter (MF) procedurefor the detection and enumeration of Heterotrophic bacteria(HPC) and fungi in liquid fuels with kinematic viscosities24 mm2s-1at ambient temperature.1.2 This
4、 quantitative practice is drawn largely from IPMethod 385 and Test Method D5259.1.3 This test may be performed either in the field or in thelaboratory.1.4 The ability of individual microbes to form colonies onspecific growth media depends on the taxonomy and physi-ological state of the microbes to b
5、e enumerated, the chemistryof the growth medium, and incubation conditions.Consequently, test results should not be interpreted as absolutevalues. Rather they should be used as part of a diagnostic orcondition monitoring effort that includes other test parameters,in accordance with Guide D6469.1.5 T
6、his practice offers alternative options for deliveringfuel sample microbes to the filter membrane, volumes ordilutions filtered, growth media used to cultivate fuel-bornemicrobes, and incubation temperatures. This flexibility isoffered to facilitate diagnostic efforts. When this practice isused as p
7、art of a condition monitoring program, a singleprocedure should be used consistently.1.6 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.7 This standard does not purport to address all of thesafety concerns, if any, associated
8、 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 to use.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reag
9、ent WaterD4175 Terminology Relating to Petroleum, PetroleumProducts, and LubricantsD5259 Test Method for Isolation and Enumeration of En-terococci from Water by the Membrane Filter ProcedureD6426 Test Method for Determining Filterability of MiddleDistillate Fuel OilsD6469 Guide for Microbial Contami
10、nation in Fuels and FuelSystemsD7463 Test Method forAdenosine Triphosphate (ATP) Con-tent of Microorganisms in Fuel, Fuel/Water Mixtures, andFuel Associated WaterD7464 Practice for Manual Sampling of Liquid Fuels, As-sociated Materials and Fuel System Components forMicrobiological TestingE1326 Guide
11、 for Evaluating Nonconventional Microbiologi-cal Tests Used for Enumerating BacteriaF1094 Test Methods for Microbiological Monitoring ofWater Used for Processing Electron and MicroelectronicDevices by Direct Pressure Tap Sampling Valve and bythe Presterilized Plastic Bag Method2.2 Energy Institute S
12、tandards:3IP 385 Determination of the Viable Aerobic Microbial Con-tent of Fuels and Fuel Components Boiling Below390CFiltration and Culture Method3. Terminology3.1 Definitions:3.1.1 For definition of terms used in this method refer toTerminologies D1129 and D4175, and Guide D6469.1This practice is
13、under the jurisdiction of ASTM Committee D02 on PetroleumProducts, Liquid Fuels, and Lubricantsand is the direct responsibility of Subcom-mittee D02.14 on Stability and Cleanliness of Liquid Fuels.Current edition approved May 1, 2013. Published August 2013. Originallyapproved in 2003. Last previous
14、edition approved in 2009 as D6974 09. DOI:10.1520/D6974-09R13E02.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM webs
15、ite.3Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR,U.K., http:/www.energyinst.org.uk.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.2 aseptic, adjsterile, free from viable microbiologicalcontamination.3.2
16、 Acronyms:3.2.1 CFUcolony forming unit3.2.2 HPCheterotrophic plate count3.2.3 MFmembrane filter3.2.4 MEAmalt extract agar3.2.5 TNTCtoo numerous to count3.2.6 TSAtryptone soy agar3.3 Symbols:3.3.1 Nnumber of CFU L-13.3.2 CCnumber of colonies on membrane filter3.3.3 Vsample volume filtered, mL4. Summa
17、ry of Practice4.1 Any free water present in a fuel sample is removed bysettling in a separatory funnel. After the water has beenremoved, a known volume of the remaining fuel is filteredthrough a membrane filter aseptically by one of three methods.4.2 The filter membrane retains microbes present in t
18、he fuel.Filter replicate fuel samples through fresh membranes topermit replicate testing, growth on alternative nutrient media,or both.4.3 After filtration, place each membrane on one of twotypes of agar growth media, incubate at a designated tempera-ture for three days, and examine for the presence
19、 of CFU.4.4 Incubate the filter media on agar for two more days, thenreexamine.4.5 Count the colonies manually or by electronic counter.4.5.1 If practical, identify colonies on each agar medium,based on colony color, morphology, and microscopic exami-nation.4.5.2 Convert bacterial and fungal colony
20、counts to CFUper litre of fuel.5. Significance and Use5.1 Biodeteriogenic microbes infecting fuel systems typi-cally are most abundant within slime accumulations on systemsurfaces or at the fuel-water interface (Guide D6469).However, it is often impractical to obtain samples from theselocations with
21、in fuel systems. Although the numbers of viablebacteria and fungi recovered from fuel-phase samples arelikely to be several orders of magnitude smaller than thosefound in water-phase samples, fuel-phase organisms are oftenthe most readily available indicators of fuel and fuel systemmicrobial contami
22、nation.5.2 Growth Medium SelectivityGuide E1326 discusses thelimitations of growth medium selection. Any medium selectedwill favor colony formation by some species and suppresscolony formation by others.As noted in 6.3, physical, chemicaland physiological variables can affect viable cell enumeration
23、test results. Test Method D7463 provides a non-culture meansof quantifying microbial biomass in fuels and fuel associatedwater.5.3 Since a wide range of sample sizes, or dilutions thereof,can be analyzed by the membrane filter technique (TestMethods D5259 and F1094), the test sensitivity can be adju
24、stedfor the population density expected in the sample.5.4 Enumeration data should be used as part of diagnosticefforts or routine condition monitoring programs. Enumerationdata should not be used as fuel quality criteria.6. Interferences6.1 High non-biological particulate loads (sediment) canclog th
25、e membrane and prevent filtration.6.2 Each CFU is assumed to originate from a single micro-bial cell. In reality, microbes often form aggregates whichappear as a single colony. Consequently, viable count data arelikely to underestimate the total number of viable organisms inthe original sample.6.3 T
26、he metabolic state of individual microbes may beaffected by numerous physical-chemical variables in the fuel.Injured cells or cells that have relatively long generation timesmay not form colonies within the time allotted for testobservations. This results in an underestimation of the numbersof viabl
27、e microbes in the original fuel sample.7. Apparatus7.1 Separatory Funnels, glass, nominal capacity 500 mL.7.2 Measuring Cylinders, glass, nominal capacity 100 mLand1L.7.3 Pipettes, glass or sterile disposable plastic, nominalcapacity 10 mL, or adjustable volume pipette and steriledisposable plastic
28、tips.7.4 Membrane Filter, mixed esters of cellulose,presterilized, preferably gridded, 47 mm diameter, nominalpore size 0.45 m.NOTE 1While the recommended filter material is mixed esters ofcellulose, the selection of membrane material will depend on individualpreference and fuel type.7.5 Filtration
29、Unit, one of:7.5.1 Unit, as described in Test Method D6426, with pre-sterilized in-line filter housing, or7.5.2 Hypodermic Syringe, sterile, 100 mL, with pre-sterilized in-line filter housing, or7.5.3 Filter Holder Assembly, single or manifold, glass,stainless steel, or polypropylene, pre-sterilized
30、.NOTE 2If the vacuum filtration option (7.5.3) is chosen, a vacuumsource, not more than -66 kPa will also be needed.7.6 Forceps, blunt tipped.7.7 Filter Flask, of sufficient capacity to receive the entiresample being filtered plus washings.7.8 Petri Dishes, disposable plastic or glass, nominal diam-
31、eter 50 mm.NOTE 3Pre-poured Petri dishes, containing the growth media de-scribed below are available commercially and may be substituted for thedishes listed here.7.9 Incubator, capable of maintaining a temperature of 25 62C or any other temperature (within the rangeambient to60C), as appropriate.D6
32、974 09 (2013)227.10 Water Bath, capable of maintaining a temperature of 476 2C and receiving 500 mL bottles. Water bath capacityshould be sufficient to accommodate at least one bottle of eachtype of agar growth medium used.7.11 Glass Bottles, screw cap with gas-tight closures,500 mL nominal capacity
33、.7.12 Culture Tubes, glass, 16 by 125 mm, screw cap.7.13 Autoclave, with capacity to hold 500 mL glass bottlesupright.NOTE 4Items 7.10 7.13 are not needed if using commerciallyprepared Petri dishes, as indicated in Note 3.8. Reagents and Materials8.1 Purity of ReagentsReagent grade chemicals shall b
34、eused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.48.2 The agar used in preparation of culture media shall be ofmicrobiological g
35、rade. Whenever possible, use commercialculture media.8.3 Water PurityUnless otherwise indicated, references towater shall be understood to mean reagent water as defined byType III of Specification D1193.8.4 Chlorotetracycline, 0.1 % (w/v) aqueous. Dissolve 0.1 gchlorotetracycline in water and dilute
36、 to 100 mL. Sterilize bypassing through a 0.2 m filter.8.5 Detergent Solution 0.1 % (v v)Dissolve 10 mL ofpolyoxyethylene (20) sorbitan monooleate5in 990 mL water.Sterilize, either by passing through a 0.2 m membrane filterinto a sterile vessel, or autoclaving at 121C for 15 min.8.6 Hydrochloric Aci
37、d, 1 mol HCl L-1.8.7 Lactic Acid, 10 % (w/v) aqueous. Dissolve 10 g of lacticacid in water and dilute to 100 mL. Sterilize by passing througha 0.2 m filter.8.8 Malt Extract Agar (MEA):8.8.1 Composition/Litre:Malt Extract 30 gMycological Peptone 5 gAgar 15 gWater 1 L8.8.2 PreparationSuspend the malt
38、extract, mycologicalpeptone and agar in 1 L of water and boil to dissolve. Adjustthe pH to 5.4 6 0.2 using either 1 mL L-1hydrochloric acid(8.6) or sodium hydroxide 10 % w/v (8.10). Dispense 250 mLportions into 500 mL glass screw-cap bottles (7.11). Sterilizeby autoclaving at 121 6 2C for 10 min. Co
39、ol and maintain thesterilized agar in a water bath (7.10) at 47 6 2C. Optionally,after the agar has cooled to 47 6 2C, add 1 mL of a 0.1 %aqueous solution of chlorotetracycline (filter sterilized bypassing through a 0.2 m filter, see 8.4) per 100 mL MEA andmix by shaking. If the medium is required a
40、t pH 3.5, add 10 %lactic acid (filter sterilized by passing through a 0.2 m filter,see 8.7) to adjust pH. Once acidified, the MEA shall not bereheated. Make agar plates of the medium by pouring sufficientMEA into sterile petri dishes to give a layer approximately4 mm thick. Allow to cool and set.NOT
41、E 5MEA is available from various manufacturers in dehydratedform and in pre-poured plates with and without added antibiotic, either ofwhich may be used. When sterilizing MEA prepared from commercialdehydrated media, follow the manufacturers instructions for sterilization.Avoid overheating.NOTE 6Alte
42、rnative media to MEA may be used, providing the abilityof any alternative medium to support comparable growth of yeast andmolds that are likely to be encountered in test samples can be demon-strated.NOTE 7Alternative antibiotics may be used providing their ability toinhibit growth of bacteria but no
43、t yeast and molds has been validated.8.9 Ringers Solution, One-Quarter Strength:8.9.1 Composition/Litre:Sodium chloride 2.25 gPotassium chloride 0.105 gCalcium chloride 0.12 gSodium bicarbonate 0.05 gWater 1 L8.9.2 PreparationDissolve salts in 1 L of water anddispense 10 mL portions into screw cappe
44、d culture tubes(7.12). Sterilize by autoclaving at 121C for 15 min.NOTE 8One-quarter strength Ringers salts are available in tabletform from various manufacturers.8.10 Sodium Hydroxide, 10 % (w/v) aqueous. Dissolve 10 gNaOH in water and dilute to 100 mL.8.11 Tryptone Soy Agar (TSA):8.11.1 Compositio
45、n/Litre:Tryptone 15 gSoy protein 5 gSodium chloride 5 gAgar 15 gWater 1 L8.11.2 PreparationSuspend the dry ingredients in 1 L ofwater and boil to dissolve. Dispense 250 mL portions into 500mL glass screw-cap bottles (7.11). Sterilize by autoclaving at121 6 2C for 10 min. Cool and maintain the steril
46、ized agar ina water bath (7.10)at476 2C. Draw a sample and test thepH. If the pH 7.3 6 0.3, reject the batch and make a freshmixture. Make agar plates of the medium by pouring sufficientTSA into sterile petri dishes to give a layer approximately4 mm thick. Allow to cool and set.NOTE 9TSA is availabl
47、e from various manufacturers in dehydratedform and in pre-poured plates.NOTE 10Alternative media to TSA may be used, providing the abilityof any alternative medium to support comparable growth of bacteria thatare likely to be encountered in test samples can be demonstrated.9. Procedure9.1 Sampling:4
48、Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For Suggestions on the testing of reagents notlisted by the American Chemical Society, see Annual Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopei
49、aand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.5The sole source of supply of Tween 80 known to the committee at this time isSigma Aldrich Co., St. Louis, MO 63178, http:/. If you areaware of alternative suppliers, please provide this information to ASTM Interna-tional Headquarters. Your comments will receive careful consideration at a meetingof the responsible technical committee,1which you may attend.D6974 09 (2013)239.1.1 Samples shall be drawn in accor
