1、Designation: D6974 09 (Reapproved 2013)2D6974 16Standard 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 ado
2、ption 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.1 NOTESubsections 8.4 and 8.8.2 were editorially corrected in August 2014.2 NOTERef
3、erences in Section 9 and the title of IP 385 were editorially corrected in January 2015.1. Scope Scope*1.1 This practice covers a membrane filter (MF) procedure for the detection and enumeration of Heterotrophic bacteria (HPC)and fungi in liquid fuels with kinematic viscosities 24 mm2 s-1 at ambient
4、 temperature.1.2 This quantitative practice is drawn largely from IP Method 385 and Test Method D5259.1.3 This test may be performed either in the field or in the laboratory.1.4 The ability of individual microbes to form colonies on specific growth media depends on the taxonomy and physiologicalstat
5、e of the microbes to be enumerated, the chemistry of the growth medium, and incubation conditions. Consequently, test resultsshould not be interpreted as absolute values. Rather they should be used as part of a diagnostic or condition monitoring effort thatincludes other test parameters, in accordan
6、ce with Guide D6469.1.5 This practice offers alternative options for delivering fuel sample microbes to the filter membrane, volumes or dilutionsfiltered, growth media used to cultivate fuel-borne microbes, and incubation temperatures. This flexibility is offered to facilitatediagnostic efforts. Whe
7、n this practice is used as part of a condition monitoring program, a single procedure should be usedconsistently.1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.7 This standard does not purport to address all of the safe
8、ty concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1129 Terminology Relating to Water
9、D1193 Specification for Reagent WaterD4175 Terminology Relating to Petroleum Products, Liquid Fuels, and LubricantsD5259 Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter ProcedureD6426 Test Method for Determining Filterability of Middle Distillate Fuel OilsD
10、6469 Guide for Microbial Contamination in Fuels and Fuel SystemsD7463 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and FuelAssociated WaterD7464 Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for M
11、icrobiologicalTestingE1326 Guide for Evaluating Non-culture Microbiological TestsF1094 Test Methods for Microbiological Monitoring of Water Used for Processing Electron and Microelectronic Devices byDirect Pressure Tap Sampling Valve and by the Presterilized Plastic Bag Method1 This practice is unde
12、r the jurisdiction of ASTM Committee D02 on Petroleum Products, Liquid Fuels, and Lubricantsand is the direct responsibility of SubcommitteeD02.14 on Stability and Cleanliness of Liquid Fuels.Current edition approved May 1, 2013July 1, 2016. Published August 2013July 2016. Originally approved in 200
13、3. Last previous edition approved in 20092013 asD6974 09.D6974 09 (2013)2. DOI: 10.1520/D6974-09R13E02.10.1520/D6974-16.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. ForAnnual Book ofASTM Standardsvolume information, refer to
14、 the standards Document Summary page on the ASTM website.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes
15、 accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.*A Summary of Changes section appears at the end of this standardCopyright ASTM International, 100 Barr H
16、arbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States12.2 Energy Institute Standards:3IP 385 Determination of the Viable Aerobic Microbial Content of Fuels and Fuel Components Boiling Below 390CFiltration390 CFiltration and Culture Method3. Terminology3.1 Definitions:3.1.1 For de
17、finition of terms used in this method refer to Terminologies D1129 and D4175, and Guide D6469.3.1.2 aseptic, adjsterile, free from viable microbiological contamination.3.2 Acronyms:3.2.1 CFUcolony forming unit3.2.2 HPCheterotrophic plate count3.2.3 MFmembrane filter3.2.4 MEAmalt extract agar3.2.5 TN
18、TCtoo 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. Summary of Practice4.1 Any free water present in a fuel sample is removed by settling in a separatory funnel. After the water has been
19、 removed,a known volume of the remaining fuel is filtered through a 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 to permitreplicate testing, growth on alternative nutrient media
20、, or both.4.3 After filtration, place each membrane on one of two types of agar growth media, incubate at a designated temperature forthree days, and examine for the presence of CFU.4.4 Incubate the filter media on agar for two more days, then reexamine.4.5 Count the colonies manually or by electron
21、ic counter.4.5.1 If practical, identify colonies on each agar medium, based on colony color, morphology, and microscopic examination.4.5.2 Convert bacterial and fungal colony counts to CFU per litre of fuel.5. Significance and Use5.1 Biodeteriogenic microbes infecting fuel systems typically are most
22、 abundant within slime accumulations on system surfacesor at the fuel-water interface (Guide D6469). However, it is often impractical to obtain samples from these locations within fuelsystems. Although the numbers of viable bacteria and fungi recovered from fuel-phase samples are likely to be severa
23、l orders ofmagnitude smaller than those found in water-phase samples, fuel-phase organisms are often the most readily available indicatorsof fuel and fuel system microbial contamination.5.2 Growth Medium SelectivityGuide E1326 discusses the limitations of growth medium selection.Any medium selected
24、willfavor colony formation by some species and suppress colony formation by others. As noted in 6.3, physical, chemical andphysiological variables can affect viable cell enumeration test results. Test Method D7463 provides a non-culture means ofquantifying microbial biomass in fuels and fuel associa
25、ted water.5.3 Since a wide range of sample sizes, or dilutions thereof, can be analyzed by the membrane filter technique (Test MethodsD5259 and F1094), the test sensitivity can be adjusted for the population density expected in the sample.5.4 Enumeration data should be used as part of diagnostic eff
26、orts or routine condition monitoring programs. Enumeration datashould not be used as fuel quality criteria.6. Interferences6.1 High non-biological particulate loads (sediment) can clog the membrane and prevent filtration.3 Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR, U.K.,
27、 http:/www.energyinst.org.uk.D6974 1626.2 Each CFU is assumed to originate from a single microbial cell. In reality, microbes often form aggregates which appear asa single colony. Consequently, viable count data are likely to underestimate the total number of viable organisms in the originalsample.6
28、.3 The metabolic state of individual microbes may be affected by numerous physical-chemical variables in the fuel. Injuredcells or cells that have relatively long generation times may not form colonies within the time allotted for test observations. Thisresults in an underestimation of the numbers o
29、f viable microbes in the original fuel sample.7. Apparatus7.1 Separatory Funnels, glass, nominal capacity 500 mL.500 mL.7.2 Measuring Cylinders, glass, nominal capacity 100 mL and 1 L.100 mL and 1 L.7.3 Pipettes, glass or sterile disposable plastic, nominal capacity 10 mL, 10 mL, or adjustable volum
30、e pipette and steriledisposable plastic tips.7.4 Membrane Filter, mixed esters of cellulose, presterilized, preferably gridded, 47 mm 47 mm diameter, nominal pore size0.45 m.0.45 m.NOTE 1While the recommended filter material is mixed esters of cellulose, the selection of membrane material will depen
31、d on individual preferenceand 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, 100 mL, with pre-sterilized in-line filter housing, or7.5.3 Filter Holder Assembly, single or ma
32、nifold, glass, stainless steel, or polypropylene, pre-sterilized.NOTE 2If the vacuum filtration option (7.5.3) is chosen, a vacuum source, not more than -66 kPa 66 kPa will also be needed.7.6 Forceps, blunt tipped.7.7 Filter Flask, of sufficient capacity to receive the entire sample being filtered p
33、lus washings.7.8 Petri Dishes, disposable plastic or glass, nominal diameter 50 mm.50 mm.NOTE 3Pre-poured Petri dishes, containing the growth media described below are available commercially and may be substituted for the dishes listedhere.7.9 Incubator, capable of maintaining a temperature of 2525
34、C62C2 C or any other temperature (within the rangeambientto 60C),60 C), as appropriate.7.10 Water Bath, capable of maintaining a temperature of 4747 C62C2 C and receiving 500 mL500 mLbottles. Water bathcapacity should be sufficient to accommodate at least one bottle of each type of agar growth mediu
35、m used.7.11 Glass Bottles, screw cap with gas-tight closures, 500 mL nominal capacity.7.12 Culture Tubes, glass, 1616 mm by 125 mm, 125 mm, screw cap.7.13 Autoclave, with capacity to hold 500 mL 500 mL glass bottles upright.NOTE 4Items 7.10 7.13 are not needed if using commercially prepared Petri di
36、shes, as indicated in Note 3.8. Reagents and Materials8.1 Purity of ReagentsReagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that allreagents conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society where suc
37、hspecifications are available.48.2 The agar used in preparation of culture media shall be of microbiological grade. Whenever possible, use commercial culturemedia.8.3 Water PurityUnless otherwise indicated, references to water shall be understood to mean reagent water as defined by TypeIII of Specif
38、ication D1193.8.4 Chlorotetracycline, 0.1 % (w/v) aqueous. Dissolve 0.1 g 0.1 g chlorotetracycline in water and dilute to 100 mL. 100 mL.Sterilize by passing through a 0.2 m 0.2 m filter.8.5 Detergent Solution 0.1 % (vbyv) volumeDissolve 10 mL1.0 mLof polyoxyethylene (20) sorbitan monooleate5 in 990
39、 mL999 mL water. Sterilize, either by passing through a 0.2 m 0.2 m membrane filter into a sterile vessel, or autoclaving at121C121 C for 15 min.15 min.4 Reagent Chemicals, American Chemical Society Specifications, American Chemical Society, Washington, DC. For Suggestions on the testing of reagents
40、 not listed bythe American Chemical Society, see Annual Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and NationalFormulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.5 The sole source of supply of Tween 80 known to the committe
41、e at this time is Sigma Aldrich Co., St. Louis, MO 63178, http:/. If you are awareof alternative suppliers, please provide this information toASTM International Headquarters.Your comments will receive careful consideration at a meeting of the responsibletechnical committee,1 which you may attend.D69
42、74 1638.6 Hydrochloric Acid, 1 mol 1 mol HCl L-1.8.7 Lactic Acid, 10 % (w/v) aqueous. Dissolve 10 g 10 g of lactic acid in water and dilute to 100 mL. 100 mL. Sterilize bypassing through a 0.2 m 0.2 m filter.8.8 Malt Extract Agar (MEA):8.8.1 Composition/Litre:Malt Extract 30 gMycological Peptone 5 g
43、Agar 15 gWater 1 L8.8.2 PreparationSuspend the malt extract, mycological peptone and agar in 1 L of water and boil to dissolve. Adjust the pHto 5.460.2 using either 1 mL 1 mL L-1 hydrochloric acid (8.6) or sodium hydroxide 10 % w/v (8.10). Dispense 250 mL 250 mLportions into 500 mL 500 mL glass scre
44、w-cap bottles (7.11). Sterilize by autoclaving at 121121 C62C2 C for 10 min. 10 min.Cool and maintain the sterilized agar in a water bath (7.10)(7.10) at 4747 C 6 2C.2 C. Optionally, after the agar has cooled to4747 C 6 2C,2 C, add 1 mL 1 mL of a 0.1 % aqueous solution of chlorotetracycline (filter
45、sterilized by passing through a 0.2m 0.2 m filter, see 8.4) per 100 mL 100 mL MEAand mix by shaking. If the medium is required at pH 3.5, add 10 % lactic acid(filter sterilized by passing through a 0.2 m 0.2 m filter, see 8.7) to adjust pH. Once acidified, the MEA shall not be reheated.Make agar pla
46、tes of the medium by pouring sufficient MEA into sterile petri dishes to give a layer approximately 4 mm thick.Allow to cool and set.NOTE 5MEAis available from various manufacturers in dehydrated form and in pre-poured plates with and without added antibiotic, either of whichmay be used. When steril
47、izing MEA prepared from commercial dehydrated media, follow the manufacturers instructions for sterilization. Avoidoverheating.NOTE 6Alternative media to MEA may be used, providing the ability of any alternative medium to support comparable growth of yeast and moldsthat are likely to be encountered
48、in test samples can be demonstrated.NOTE 7Alternative antibiotics may be used providing their ability to inhibit 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 ch
49、loride 0.12 gSodium bicarbonate 0.05 gWater 1 L8.9.2 PreparationDissolve salts in 1 L 1 L of water and dispense 10 mL 10 mL portions into screw capped culture tubes(7.12). Sterilize by autoclaving at 121C121 C for 15 min.15 min.NOTE 8One-quarter strength Ringers salts are available in tablet form from various manufacturers.8.10 Sodium Hydroxide, 10 % (w/v) aqueous. Dissolve 10 g 10 g NaOH in water and dilute to 100 mL.100 mL.8.11 Tryptone Soy Agar (TSA):8.11.1 Compositi
