1、Designation: E979 09 (Reapproved 2015)Standard Practice forEvaluation of Antimicrobial Agents as Preservatives forInvert Emulsion and Other Water Containing HydraulicFluids1This standard is issued under the fixed designation E979; the number immediately following the designation indicates the year o
2、foriginal 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.INTRODUCTIONInvert emulsion hydraulic fluids typically contain 60 % mi
3、neral oil and 40 % water (by volume).These fluids routinely are prepared using proprietary, oil-soluble, emulsifying agents, as well as otheremulsifiable constituents. They are recommended for use where conditions indicate a low-cost, fireretardant product, compatible with water-based metal working
4、fluids.The high water content of these hydraulic fluids makes them susceptible to microbial attack.Uncontrolled microbial growth in these fluids can cause cartridge filter unit plugging, maladorousconditions, or general biodeterioration. Problem microorganisms associated with these fluids includebac
5、teria and fungi.The hydraulic system is essentially a closed one in which water of evaporation is added to maintaina fixed volume. The inclusion of an efficacious preservative in the water containing hydraulic fluidscan prevent microbial growth and the resulting problems that follow.1. Scope*1.1 Thi
6、s laboratory practice is designed to evaluate theutility and effectiveness of antimicrobial agents intended tocontrol microbial growth in invert emulsions and other watercontaining hydraulic fluids.NOTE 1Procedures for preparation of water soluble hydraulic fluidsand recovery of organisms appear in
7、Practice E2169.1.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3 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
8、 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 WaterD4454 Test Method for Simultaneous Enumeration of Totaland Respiring Bacteria inAquatic Systems by
9、 Microscopy(Withdrawn 2015)3D5465 Practice for Determining Microbial Colony Countsfrom Waters Analyzed by Plating MethodsE1326 Guide for Evaluating Non-culture MicrobiologicalTestsE2169 Practice for Selecting Antimicrobial Pesticides forUse in Water-Miscible Metalworking FluidsE2523 Terminology for
10、Metalworking Fluids and Opera-tions1This practice is under the jurisdiction of ASTM Committee E35 on Pesticides,Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2015. Published February 2016
11、. Originallyapproved in 1984. Last previous edition approved in 2009 as E979 09. DOI:10.1520/E0979-09R15.2For 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 standar
12、ds Document Summary page onthe ASTM website.3The last approved version of this historical standard is referenced onwww.astm.org.*A Summary of Changes section appears at the end of this standardCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United
13、States13. Terminology3.1 Terms used in this practice are defined in TerminologiesD1129 and E2523.4. Summary of Test Method4.1 The antimicrobial agent to be evaluated is incorporatedinto an emulsion system by (a) addition to the aqueous phaseemployed in the preparation of the emulsion, (b) in doses t
14、o theformulated system, or (c) by other methods suitable for the testcompound.4.2 A heavy bacterial or fungal inoculum, or both, is thenadded.4.3 The resulting mixture is aerated and passed over thesurface of a simulated filter system for a minimum period ofeight weeks either continuously or with sh
15、utdowns to simulateactual operations conditions.4.4 The degree of microbial control is determined byperiodically quantifying the bioburden in the emulsion bydirect microscopic count (Test Method D4454), plate count(Practice D5465), or other appropriate method (Guide E1326)and visual observations for
16、 microbial fouling of the simulatedfilter surface.NOTE 2A knowledge of standard microbiological techniques isrequired for this procedure. It is also required that good laboratorypractices be followed throughout these tests. This means appropriatecontainment for the microbiological systems being eval
17、uated.The systemsshould be maintained in an enclosure so that during the aeration processthe mists and aerosols generated do not contaminate the laboratoryenvironment.5. Significance and Use5.1 This procedure is designed to determine the effective-ness of antimicrobial agents intended for microbial
18、control ininvert emulsions and other water containing hydraulic fluids.6. Apparatus6.1 Air SupplyAny air source which is free from organicvapors, organic matter, or other objectionable material may beused.NOTE 3If desired, air may be sterilized as follows:Pack two 150-mm long drying tubes (bulb type
19、) loosely with glasswool in a series with neoprene stoppers, glass tubing, and neoprenetubing. Wrap loosely in aluminum foil and steam sterilize at 15 to 20 psifor 30 minutes. Cool to room temperature while still wrapped. In-linepre-sterilization air filters are available from most local laboratory
20、supplyhouses.Insert into air line with bulbs on upstream side. Average lifetime incontinuous use is two weeks. Discard sooner if upstream filter becomeswet or contaminated with oil.6.2 Colony CounterAny one of several types may beused.6.3 IncubatorAny cabinet capable of maintaining a tem-perature of
21、 35 6 1C may be used.6.4 Test CabinetA large cabinet capable of maintaining atemperature of 35 6 1C, able to house several two litrebeakers, and into which an air line can be introduced.6.5 SterilizerAny suitable steam sterilizer capable ofproducing the conditions of sterilization is acceptable.6.6
22、Simulated Filters:6.6.1 Strainer, 3-in. epoxy coated,14-in. mesh gutterstrainer.46.6.2 Screen, 16 by 18 in. fiberglass screening material.NOTE 4Fiberglass mesh screening material (16 by 18 in.) is availablefrom any local hardware dealer.6.6.3 Wire, 20-gage, galvanized or stainless steel.6.7 Tubing,1
23、4-in. ID Tygon.NOTE 5Tygon is available from most local laboratory supply houses.6.8 T-Connectors,14-in. polypropylene.6.9 Laboratory BlenderAny standard adjustable speedlaboratory blender having a 2-L capacity glass or metalcontainer is satisfactory.6.10 Hypodermic Needle, 16-gage needle.6.11 Micro
24、scope, Brightfield microscope equipped with 40and 100 objectives.6.12 Labware:6.12.1 Culture Dishes100 by 15 mm sterile culture dishesmade of glass or plastic are required for making standard platecounts.NOTE 6Presterilized and disposable plastic petri dishes are availablefrom most local laboratory
25、supply houses.6.12.2 Bacteriological Pipettes of 1.1 or 2.2-mL capacity.NOTE 7Presterilized and disposable 1.1-mL bacteriological pipettesare available from most local laboratory supply houses.6.12.3 Water Dilution BottlesAny sterilizable glass con-tainers having a 150 to 200-mLcapacity and tight cl
26、osures maybe used.NOTE 8Milk dilution bottles of 160-mL capacity having screw-capclosures are available from most local laboratory supply houses.6.12.4 Two-Liter Borosilicate Glass Beakers.6.12.5 Bent Glass Rod.6.12.6 Screw Cap Culture Tubes, autoclavable, 15 by 150mm.6.13 Water BathMaintain at 46 6
27、 2C to anneal agarbased microbiological media.6.14 Aluminum Foil.7. Reagents and Materials7.1 Invert Emulsion Emulsifier.57.2 Paraffnic Mineral Oil.7.3 Deionized or Distilled Water (2 MOHM quality)4The sole source of supply of the apparatus known to the committee at this timeis Billy Penn Corp., Phi
28、ladelphia, PA 19122. If you are aware of alternativesuppliers, please provide this information to ASTM International Headquarters.Your comments will receive careful consideration at a meeting of the responsibletechnical committee,1which you may attend.5The sole source of supply of a satisfactory emu
29、lsifier for the preparation ofinvert emulsion hydraulic fluids (Compound #5162) known to the committee at thistime is the Lubrizol Co., Wickliffe, OH. If you are aware of alternative suppliers,please provide this information to ASTM International Headquarters. Your com-ments will receive careful con
30、sideration at a meeting of the responsible technicalcommittee,1which you may attend.E979 09 (2015)27.4 Gentamicin Sulfate.67.5 Arlacel 80.77.6 Tween 60.77.7 Phosphate Buffer For serial dilutions.7.8 Mineral oil, sterile.7.9 Microbiological MediaGeneral retrieval media con-sistent with good microbiol
31、ogical practices are acceptable.Examples are as follows:7.9.1 Soybean-Casein Digest Agar, U.S.P. XIX, Medium II.NOTE 9Soybean-casein digest agar is available in dehydrated formfrom most laboratory media supply houses.7.9.2 Fluid Soybean-Casein Digest Medium, U.S.P. XIX,Medium III.87.9.3 Sabouraud De
32、xtrose Agar, U.S.P. XIX, Medium 20.87.9.4 Sabouraud Dextrose Broth, U.S.P. XIX, Medium 21.87.9.5 Sulfate American Petroleum Institute (API) Agar,7forenumeration of sulfate reducing bacteria.7.10 Inoculum:7.10.1 The inoculum may vary according to the usersrequirements. It may be either undefined or d
33、efined.7.10.1.1 An undefined inoculum may consist of microor-ganisms isolated from a “spoiled” invert emulsion hydraulicfluid which exhibits microbiologically induced phasegeneration, or which is known to have caused plugging of ahydraulic system filter due to microbial slime, and grown in anutrient
34、 medium.7.10.1.2 An undefined inoculum may consist of the follow-ing: (1) equal volumes of fluid soybean-casein digest and“spoiled” (see 7.10.1.1) hydraulic fluid aerated at 35C for 24 h(typically) until the bacterial count reaches 109CFU/mL, (2)equal volumes of sabouraud dextrose broth and “spoiled
35、” (see7.10.1.1) hydraulic fluid aerated at 35C for 24 h (typically) oruntil fungal count reaches 106to 107CFU/mL, or (3) equalvolumes of (1) and (2) if both bacteria and fungi are the desiredtest organisms.7.10.2 A defined inoculum consisting of a mixed culture ofspecific microorganisms may also be
36、used.7.10.2.1 The defined inoculum may be prepared by isolatingand identifying specific microorganisms from a “spoiled” (see7.10.1.1) hydraulic fluid emulsion and culturing the bacterialisolates in soybean-casein digest medium and the fungalisolates in sabouraud dextrose broth until there are 109CFU
37、bacteria or 106to 107CFU fungi, or both, per mL, respectively.7.10.2.2 Other microorganisms of particular interest (Ross-moore and Szlatky)9may be used such as: Pseudomonasfluorescens, Pseudomonas cepacia, Klebsiella pneumoniae,Proteus mirabilis, Desulfovibrio desulfuricans, Aspergillusniger, Cephal
38、osporium sp., Fusarium sp., Candida sp.7.10.2.3 Equal mixtures of any two of the above bacterialspecies or two of the above mold species, or both, plus theCandida species to provide a final titer of 109CFU bacteria, or106to 107CFU fungi, or both, per mL, should be used as aninoculum for the emulsion
39、 system.7.11 Antimicrobial AgentsThe chemical agents to beevaluated as preservatives.8. Preparation of Simulated Filters8.1 Cut the epoxy-coated,14-in. mesh gutter strainers 16 by18 in. mesh fiberglass screening material into 3 by 5 in.sections. Secure the screening to the strainers with 20-gagewire
40、 or with staples.8.2 Preparation of AeratorsCut tubing (see 6.7) into13-in. sections. Bend tubing in a circle and connect both endsusing a T connector (see 6.8). Connect third arm of T connectorto a 20-in. length of tygon tubing. This tubing will beconnected to the main air supply line. Using a hot
41、16-gageneedle, carefully punch a series of holes,12 in. apart, along theouter circumference of the tubing which forms the ring. Alsopunch similar holes12 in. apart on the upper and lower surfaceof the tubing, at right angles to the holes previously punched.These holes allow the air from the air sour
42、ce to bubble upthrough the hydraulic fluid producing a cascading effect overthe surface of the simulated filter.9. Preparation of Microbiological Medium9.1 Microbiological media should be prepared in accor-dance with manufacturers instructions. Media to be aug-mented with antibiotics should be annea
43、led in a 46 6 2Cwater bath before antibiotics are added. Antibiotics should beadded just before pouring. Use 100 g gentamicin sulfate permL to suppress bacterial growth on fungal recovery media.10. Microbiological Methods10.1 Solubilize the invert emulsion aliquot (see 7.1) accord-ing to the procedu
44、re of McConville, et al.,10,11as follows:10.1.1 Disperse 1 mL of the invert emulsion in 1 mL ofArlacel 80 and bring the volume up to 10 mLwith 10 % Tween60 solution.6The sole source of supply of gentamicin sulfate known to the committee at thistime is as Garamycin Reagent Solution, available in two
45、concentrations of 10 and50 mg/mL, from the Schering Corp., Kenilworth, NJ 07033 If you are aware ofalternative suppliers, please provide this information to ASTM InternationalHeadquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee,1which you
46、may attend.7The sole source of supply of the reagents (Arlacel 80, Tween 60, and SulfateAPIAgar) known to the committee at this time is SigmaAldrich Co., St. Louis, MO63178, http:/. If you are aware of alternative suppliers,please provide this information to ASTM International Headquarters. Your com
47、-ments will receive careful consideration at a meeting of the responsible technicalcommittee,1which you may attend.8The sole source of supply of the media, available in dehydrated form, knownto the committee at this time is Baltimore Biological Laboratories, Cockeysville,MD or Difco Laboratories, De
48、troit, MI. If you are aware of alternative suppliers,please provide this information to ASTM International Headquarters. Your com-ments will receive careful consideration at a meeting of the responsible technicalcommittee,1which you may attend.9Rossmore, H. W., and Szlatky, K. ,“Characterization of
49、the Microbial Flora ofInvert Emulsion Hydraulic Fluids,” Int. Biodetn. Bulletin, Vol 13, No. 4), 1977, pp.96100.10McConville, J. F., et al., “Method for Performing Aerobic Plate Counts ofAnhydrous Cosmetics Utilizing Tween 60 and Arlacel 80 as Dispersing Agents,”Applied Microbiology , Vol 27, 1974, pp. 57.11Hoffman, N. M., “Hydraulic Fluid of 95-Percent Water,” LubricationEngineering, Vol 35, No. 2, 1979, pp. 6571.E979 09 (2015)310.2 Enumerate the bacteria in the solubilized invert emul-sion samples (see Test Method D4
