1、Designation: E 979 09Standard Practice forEvaluation of Antimicrobial Agents as Preservatives forInvert Emulsion and Other Water Containing HydraulicFluids1This standard is issued under the fixed designation E 979; the number immediately following the designation indicates the year oforiginal adopti
2、on 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 % mineral oil and 40
3、 % 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 fluids.The high
4、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 includebacteria and fungi.
5、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 This laboratory pra
6、ctice 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 Practice E 2169.
7、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 establish appr
8、o-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 1129 Terminology Relating to WaterD 4454 Test Method for Simultaneous Enumeration ofTotaland Respiring Bacteria inAquatic Systems by MicroscopyD 5
9、465 Practice for Determining Microbial Colony Countsfrom Waters Analyzed by Plating MethodsE 1326 Guide for Evaluating Nonconventional Microbio-logical Tests Used for Enumerating BacteriaE 2169 Practice for Selecting Antimicrobial Pesticides forUse in Water-Miscible Metalworking FluidsE 2523 Termino
10、logy for Metalworking Fluids and Opera-tions3. Terminology3.1 Terms used in this practice are defined in TerminologiesD 1129 and E 2523.1This practice is under the jurisdiction of ASTM Committee E35 on Pesticidesand Alternative Control Agents and is the direct responsibility of SubcommitteeE35.15 on
11、 Antimicrobial Agents.Current edition approved April 1, 2009. Published April 2009. Originallyapproved in 1984. Last previous edition approved in 2004 as E 979 91 (2004).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annu
12、al Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1*A Summary of Changes section appears at the end of this standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.4. Summary
13、 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 to theformulated system, or (c) by other methods suitable for the testcompound.4.2 A heavy bacterial or fung
14、al 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 shutdowns to simulateactual operations conditions.4.4 The degree of microbial control is determined byperiodi
15、cally quantifying the bioburden in the emulsion bydirect microscopic count (Test Method D 4454), plate count(Practice D 5465), or other appropriate method (Guide E 1326)and visual observations for microbial fouling of the simulatedfilter surface.NOTE 2A knowledge of standard microbiological techniqu
16、es is re-quired for this procedure. It is also required that good laboratory practicesbe followed throughout these tests. This means appropriate containmentfor the microbiological systems being evaluated. The systems should bemaintained in an enclosure so that during the aeration process the mistsan
17、d aerosols generated do not contaminate the laboratory environment.5. Significance and Use5.1 This procedure is designed to determine the effective-ness of antimicrobial agents intended for microbial control ininvert emulsions and other water containing hydraulic fluids.6. Apparatus6.1 Air SupplyAny
18、 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) loosely with glass woolin a series with neoprene stoppers, glass tubing, and neoprene tubing.Wrap
19、loosely in aluminum foil and steam sterilize at 15 to 20 psi for 30minutes. Cool to room temperature while still wrapped. In-line pre-sterilization air filters are available from most local laboratory supplyhouses.Insert into air line with bulbs on upstream side. Average lifetime incontinuous use is
20、 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 35 6 1C may be used.6.4 Test CabinetA large cabinet capable of maintaining atemperature of 35 6 1C
21、, 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 Simulated Filters:6.6.1 Strainer, 3-in. epoxy coated,14-in. mesh gutterstrainer.36.6.2 Screen, 16 b
22、y 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,14-in. ID Tygon.NOTE 5Tygon is available from most local laboratory supply houses.6.8 T-Connectors,1
23、4-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 Microscope, Brightfield microscope equipped with403 and 1003 objectives.6.12 Labware:6.12.1 Culture Dish
24、es100 mm by 15 mm sterile culturedishes made of glass or plastic are required for makingstandard plate counts.NOTE 6Presterilized and disposable plastic petri dishes are availablefrom most local laboratory supply houses.6.12.2 Bacteriological Pipettes of 1.1 or 2.2-mL capacity.NOTE 7Presterilized an
25、d 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 closures maybe used.NOTE 8Milk dilution bottles of 160-mL capacity having screw-capclosures are
26、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 46C 6 2C to anneal agarbased microbiological media.6.14 Aluminum Foil.7. Reagents and Materials7.1
27、 Invert Emulsion Emulsifier.47.2 Paraffnic Mineral Oil.7.3 Deionized or Distilled Water (2 MOHM quality)3The sole source of supply of the apparatus known to the committee at this timeis Billy Penn Corp., Philadelphia, PA 19122. If you are aware of alternativesuppliers, please provide this informatio
28、n to ASTM International Headquarters.Your comments will receive careful consideration at a meeting of the responsibletechnical committee,1which you may attend.4The sole source of supply of a satisfactory emulsifier for the preparation ofinvert emulsion hydraulic fluids (Compound #5162) known to the
29、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 consideration at a meeting of the responsible technicalcommittee,1which you may attend.E9790927.
30、4 Gentamicin Sulfate.57.5 Arlacel 80.67.6 Tween 60.67.7 Phosphate BufferFor serial dilutions.7.8 Mineral oil, sterile.7.9 Microbiological MediaGeneral retrieval media con-sistent with good microbiological practices are acceptable.Examples are as follows:7.9.1 Soybean-Casein Digest Agar, U.S.P. XIX,
31、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.77.9.3 Sabouraud Dextrose Agar, U.S.P. XIX, Medium 20.77.9.4 Sabouraud Dextrose Broth, U.S.P. XIX, Medium 21.77.9.5 Sulfa
32、te American Petroleum Institute (API) Agar,6forenumeration of sulfate reducing bacteria.7.10 Inoculum:7.10.1 The inoculum may vary according to the usersrequirements. It may be either undefined or defined.7.10.1.1 An undefined inoculum may consist of microor-ganisms isolated from a “spoiled” invert
33、emulsion hydraulicfluid which exhibits microbiologically induced phase genera-tion, or which is known to have caused plugging of a hydraulicsystem filter due to microbial slime, and grown in a nutrientmedium.7.10.1.2 An undefined inoculum may consist of the follow-ing: (1) equal volumes of fluid soy
34、bean-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” (see7.10.1.1) hydraulic fluid aerated at 35C for 24 h (typically) oruntil fungal count reaches 106
35、to 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 used.7.10.2.1 The defined inoculum may be prepared by isolatingand identifying specific microorganis
36、ms 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 109CFUbacteria or 106to 107CFU fungi, or both, per mL, respectively.7.10.2.2 Other microorganisms of parti
37、cular interest (Ross-moore and Szlatky)8may be used such as: Pseudomonasfluorescens, Pseudomonas cepacia, Klebsiella pneumoniae,Proteus mirabilis, Desulfovibrio desulfuricans, Aspergillusniger, Cephalosporium sp., Fusarium sp., Candida sp.7.10.2.3 Equal mixtures of any two of the above bacterialspec
38、ies 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 system.7.11 Antimicrobial AgentsThe chemical agents to beevaluated as preservatives.8. Preparation
39、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 or with staples.8.2 Preparation of AeratorsCut tubing (see 6.7) into13-in. sections. Bend tubing in
40、 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 16-gageneedle, carefully punch a series of holes,12 in. apart, along theouter circumference of the t
41、ubing 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 source to bubble upthrough the hydraulic fluid producing a cascading effect overthe surface of the simul
42、ated 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 annealed in a 46 6 2Cwater bath before antibiotics are added. Antibiotics should beadded just before pour
43、ing. 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 procedure of McConville, et al.,9, 10as follows:10.1.1 Disperse 1 mL of the invert emulsion in 1 mL ofArlac
44、el 80 and bring the volume up to 10 mLwith 10 % Tween60 solution.10.2 Enumerate the bacteria in the solubilized invert emul-sion samples (see Test Method D 4454, Practice D 5465,orGuide E 1326)5The sole source of supply of gentamicin sulfate known to the committee at thistime is as Garamycin Reagent
45、 Solution, available in two 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 tec
46、hnical committee,1which you may attend.6The 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 Internat
47、ional Headquarters. Your com-ments will receive careful consideration at a meeting of the responsible technicalcommittee,1which you may attend.7The sole source of supply of the media, available in dehydrated form, knownto the committee at this time is Baltimore Biological Laboratories, Cockeysville,
48、MD or Difco Laboratories, Detroit, 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.8Rossmore, H. W., and Szlatk
49、y, K. ,“Characterization of the Microbial Flora ofInvert Emulsion Hydraulic Fluids,” Int. Biodetn. Bulletin ,Vol 13, No. 4), 1977, pp.96100.9McConville, 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. 57.10Hoffman, N. M., “Hydraulic Fluid of 95-Percent Water,” LubricationEngineering , Vol 35, No. 2, 1979, pp. 6571.E979093NOTE 10Do not use enumeration procedures interchangably sinceeach bioburd
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