1、Designation: D6974 09 (Reapproved 2013)Standard 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.1. Scope1.1 This practice covers a membrane filter (MF) procedurefor the detection and enume
3、ration of Heterotrophic bacteria(HPC) and fungi in liquid fuels with kinematic viscosities 24mm2s-1at ambient temperature.1.2 This 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
4、individual microbes to form colonies onspecific growth media depends on the taxonomy and physi-ological state of the microbes to be enumerated, the chemistryof the growth medium, and incubation conditions.Consequently, test results should not be interpreted as absolutevalues. Rather they should be u
5、sed as part of a diagnostic orcondition monitoring effort that includes other test parameters,in accordance with Guide D6469.1.5 This practice offers alternative options for deliveringfuel sample microbes to the filter membrane, volumes ordilutions filtered, growth media used to cultivate fuel-borne
6、microbes, and incubation temperatures. This flexibility isoffered to facilitate diagnostic efforts. When this practice isused as part 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 me
7、asurement are included in thisstandard.1.7 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 l
8、imitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD4175 Terminology Relating to Petroleum, PetroleumProducts, and LubricantsD5259 Test Method for Isolation and Enumeration of En-terococci from Water by the Membr
9、ane Filter ProcedureD6426 Test Method for Determining Filterability of MiddleDistillate Fuel OilsD6469 Guide for Microbial Contamination in Fuels and FuelSystemsD7463 Test Method forAdenosine Triphosphate (ATP) Con-tent of Microorganisms in Fuel, Fuel/Water Mixtures andFuel Associated WaterD7464 Pra
10、ctice for Manual Sampling of Liquid Fuels, As-sociated Materials and Fuel System Components forMicrobiological TestingE1326 Guide for Evaluating Nonconventional Microbiologi-cal Tests Used for Enumerating BacteriaF1094 Test Methods for Microbiological Monitoring ofWater Used for Processing Electron
11、and MicroelectronicDevices by Direct Pressure Tap Sampling Valve and bythe Presterilized Plastic Bag Method2.2 Energy Institute Standards:3IP 385 Viable aerobic microbial content of fuels and fuelcomponents boiling below 90CFiltration and culturemethod3. Terminology3.1 Definitions:3.1.1 For definiti
12、on of terms used in this method refer toTerminologies D1129 and D4175, and Guide D6469.3.1.2 aseptic, adjsterile, free from viable microbiologicalcontamination.1This practice is under the jurisdiction of ASTM Committee D02 on PetroleumProducts and Lubricantsand is the direct responsibility of Subcom
13、mittee D02.14 onStability and Cleanliness of Liquid Fuels.Current edition approved May 1, 2013. Published August 2013. Originallyapproved in 2003. Last previous edition approved in 2009 as D6974 09. DOI:10.1520/D6974-09R13.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orconta
14、ct ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR,U.K., http:/www.energyinst.org.uk.Copyright ASTM International, 10
15、0 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.2 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
16、.3.2 CCnumber of colonies on membrane filter3.3.3 Vsample volume filtered, mL4. Summary 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 asep
17、tically by one of three methods.4.2 The filter membrane retains microbes present in the 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 medi
18、a, incubate at a designated tempera-ture for three days, and examine for the presence 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
19、, morphology, and microscopic exami-nation.4.5.2 Convert bacterial and fungal colony 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 (Gui
20、de D6469).However, it is often impractical to obtain samples from theselocations within 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 ar
21、e oftenthe most readily available indicators of fuel and fuel systemmicrobial contamination.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
22、6.3, physical, chemicaland physiological variables can affect viable cell enumerationtest 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 memb
23、rane filter technique (TestMethods D5259 and F1094), the test sensitivity can be adjustedfor 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 cri
24、teria.6. Interferences6.1 High non-biological particulate loads (sediment) canclog the 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 arelik
25、ely to underestimate the total number of viable organisms inthe original sample.6.3 The 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 all
26、otted for testobservations. This results in an underestimation of the numbersof viable 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 plasti
27、c, nominalcapacity 10 mL, or adjustable volume pipette and steriledisposable plastic 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 o
28、f membrane material will depend on individualpreference and fuel type.7.5 Filtration 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
29、Assembly, single or manifold, glass,stainless steel, or polypropylene, pre-sterilized.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 bein
30、g filtered plus washings.7.8 Petri Dishes, disposable plastic or glass, nominal diam-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
31、of 25 62C or any other temperature (within the rangeambient to60C), as appropriate.D6974 09 (2013)27.10 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 mediu
32、m used.7.11 Glass Bottles, screw cap with gas-tight closures, 500mL nominal capacity.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 commercially pre-pared Petri dishes, as indicated in
33、 Note 3.8. Reagents and Materials8.1 Purity of ReagentsReagent grade chemicals shall beused 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 ava
34、ilable.48.2 The agar used in preparation of culture media shall be ofmicrobiological grade. 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 Chlorte
35、tracycline, 0.1 % (w/v) aqueous. Dissolve 0.1 gchlortetracycline in water and dilute 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
36、 filterinto a sterile vessel, or autoclaving at 121C for 15 min.8.6 Hydrochloric Acid, 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 Ext
37、ract 30 gMycological Peptone 5 gAgar 15 gWater 1 L8.8.2 PreparationSuspend the malt 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 m
38、L glass screw-cap bottles (7.11). Sterilizeby autoclaving at 121 6 2C for 10 min. Cool 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 1.0 %aqueous solution of chlorotetracycline (filter sterilized bypassing through a
39、0.2 m filter, see 8.4) per 100 mL MEA andmix by shaking. If the medium is required at 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 ste
40、rile petri dishes to give a layer approximately 4mm thick. Allow to cool and set.NOTE 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,
41、follow the manufacturers instructions for sterilization.Avoid overheating.NOTE 6Alternative 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
42、 antibiotics may be used providing their ability toinhibit growth of bacteria but not 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 P
43、reparationDissolve salts in 1 L of water anddispense 10 mL portions into screw capped 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 1
44、0 gNaOH in water and dilute to 100 mL.8.11 Tryptone Soy Agar (TSA):8.11.1 Composition/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
45、(7.11). Sterilize by autoclaving at121 6 2C for 10 min. Cool and maintain the sterilized 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
46、to give a layer approximately 4mm thick. Allow to cool and set.NOTE 9TSA is available 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 lik
47、ely to be encountered in test samples can be demonstrated.9. Procedure9.1 Sampling:4Reagent 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 L
48、aboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand 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 ar
49、eaware 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)39.1.1 Samples shall be drawn in accordance with PracticeD7464.9.1.1.1 To reduce the risk of accidental contamination,samples intended for viable microbial enumeration shall not beused for other tests until after they are no longer needed forenumeration testing.9.1
