1、Designation: D 6974 04An American National StandardStandard Practice forEnumeration of Viable Bacteria and Fungi in Liquid FuelsFiltration and Culture Procedures1This standard is issued under the fixed designation D 6974; the number immediately following the designation indicates the year oforiginal
2、 adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice covers a membrane filter (MF) procedurefor the detec
3、tion and enumeration 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 D 5259.1.3 This test may be performed either in the field or in thelaboratory.1.4
4、 The ability of 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. Conse-quently, test results should not be interpreted as absolutevalues. Rathe
5、r they should be used as part of a diagnostic orcondition monitoring effort that includes other test parameters,in accordance with Guide D 6469.1.5 This practice offers alternative options for deliveringfuel sample microbes to the filter membrane, volumes ordilutions filtered, growth media used to c
6、ultivate fuel-bornemicrobes, 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 as thestandar
7、d.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 limitations prior to use.2. Referenced
8、 Documents2.1 ASTM Standards:2D 1129 Terminology Relating to WaterD 1193 Specification for Reagent WaterD 4057 Practice for Manual Sampling of Petroleum andPetroleum ProductsD 4175 Terminology Relating to Petroleum, PetroleumProducts, and LubricantsD 5259 Test Method for Isolation and Enumeration of
9、Enterococci from Water by the Membrane Filter ProcedureD 6426 Test Method for Determining Filterability of Distil-late Fuel OilsD 6469 Guide for Microbial Contamination in Fuels andFuel SystemsE 1326 Guide for Evaluating Nonconventional Microbio-logical Tests Used for Enumerating BacteriaF 1094 Test
10、 Methods for Microbiological Monitoring ofWater Used for Processing Electronic and MicroelectronicDevices by Direct Pressure Tap Sampling Valve and by thePresterilized Plastic Bag Method2.2 Energy Institute Standards:3IP 385 Viable aerobic microbial content of fuels and fuelcomponents boiling below
11、90CFiltration and culturemethod3. Terminology3.1 DefinitionsFor definition of terms used in this methodrefer to Terminologies D 1129 and D 4175, and Guide D 6469.3.1.1 aseptic, adjsterile, free from viable microbiologicalcontamination.3.2 Acronyms:1This practice is under the jurisdiction of ASTM Com
12、mittee 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, 2004. Published June 2004. Originallyapproved in 2003. Last previous edition approved in 2003 as D 697403.2For referenced
13、 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 website.3Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR,U.K.
14、1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.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
15、 Nnumber of CFU L-13.3.2 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
16、membrane filter aseptically 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
17、 of agar growth media, 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,b
18、ased on colony color, 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-
19、water interface (Guide D 6469). How-ever, 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,
20、fuel-phase organisms are oftenthe most readily available indicators of fuel and fuel systemmicrobial contamination.5.2 Growth Medium SelectivityGuide E 1326 discussesthe limitations of growth medium selection. Any mediumselected will favor colony formation by some species andsuppress colony formatio
21、n by others. As noted in 6.3, physical,chemical and physiological variables can affect viable cellenumeration test results.5.3 Since a wide range of sample sizes, or dilutions thereof,can be analyzed by the membrane filter technique (TestMethods D 5259 and F 1094), the test sensitivity can beadjuste
22、d for 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 the
23、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 The
24、 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 viable
25、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 ti
26、ps.7.4 Membrane Filter, mixed esters of cellulose, presteril-ized, preferably gridded, 47 mm diameter, nominal pore size0.45 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
27、Unit, one of:7.5.1 Unit, as described in Test Method D 6426, withpre-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
28、.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-
29、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.7
30、.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, 500mL nominal capacity.7.12 Culture Tu
31、bes, glass, 16 by 125 mm, screw cap.7.13 Autoclave, with capacity to hold 500 mL glass bottlesupright.D6974042NOTE 4Items 7.10-7.13 are not needed if using commercially pre-pared Petri dishes, as indicated in Note 3.8. Reagents and Materials8.1 Purity of ReagentsReagent grade chemicals shall beused
32、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 grade.
33、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 D 1193.8.4 Chlortetracycline, 0.1 % (w/v) aqueous. Dissolve 0.1 gchlortetracycline in water and dilute to 100
34、 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 Acid, 1 mo
35、l 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 extract
36、, 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. Cool and
37、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 0.2 m filter, see 8.4) per 100 mL MEA andmix by shaking. If the medium is required at pH 3.
38、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 approximately 4mm thick. Allow to cool and set.NOTE 5MEA
39、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 6Alternative
40、 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 not yeast
41、 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 capped cultu
42、re 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 Composition/Litre
43、: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 sterilized ag
44、ar ina water bath (7.10) at 47 6 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 approximately 4mm thick. Allow to cool and set.NOTE 9TSA is available fr
45、om 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:9.1.1
46、 Samples shall be drawn in accordance with PracticeD 4057 as amplified by Hill.69.1.1.1 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.9.1.1.2 It may not be
47、 possible to use aseptic technique underfield conditions. To reduce risk of cross-contaminatingsamples, sampling devices shall be rinsed with 70 % alcohol4“Reagent Chemicals, American Chemical Society Specifications,” AmericanChemical Society, Washington DC. For suggestions on the testing of reagent
48、s notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.5Tween 80 has been found to be suitable for this purpose. Twee
49、n is a registeredtrademark of ICI Americas, Inc. Wilmington, DE.6Hill, G., “Sampling Methods for Detecting Microbial Contamination in Fuelsand Fuel Systems,” Fuels and Fuel Systems Microbiology: Fundamentals, Diagno-sis, and Contamination Control. ASTM MNL 47, ASTM International, 2002, p.14.D6974043(ethanol, methanol, or isopropanol) to disinfect sample contactsurfaces before samples are drawn. All samples and devicesshould be handled in such manner as to minimize the likeli-hood of introducing microbial contaminants into the s
