1、Designation: E 979 91 (Reapproved 2004)Standard Test Method 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 y
2、ear oforiginal 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.INTRODUCTIONInvert emulsion hydraulic fluids typically contain 6
3、0 % mineral 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 wo
4、rking 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 incl
5、udebacteria 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. Scope1.
6、1 This laboratory test method 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 ap
7、pear in Method E 686.1.2 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 t
8、o use.2. Referenced Documents2.1 ASTM Standards:2D 4454 Test Method for Simultaneous Enumeration of Totaland Respiring Bacteria in Aquatic Systems by MicroscopyE 686 Method for Evaluation of Antimicrobial Agents inAqueous Metal Working Fluids3. Summary of Test Method3.1 The antimicrobial agent to be
9、 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.3.2 A heavy bacterial or fungal inoculum, or both, is thenadded.3.3 The resul
10、ting 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.3.4 The degree of microbial control is determined byperiodic plate counts of the emulsion and visual observ
11、ationsfor microbial fouling of the simulated filter surface.NOTE 2A knowledge of standard microbiological techniques is re-quired for this procedure. It is also required that good laboratory practicesbe followed throughout these tests. This means appropriate containmentfor the microbiological system
12、s being evaluated. The systems should bemaintained in an enclosure so that during the aeration process the mistsand aerosols generated do not contaminate the laboratory environment.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternate Control Agents and is the
13、direct responsibility of Subcom-mittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2004. Published May 2004. Originallyapproved in 1984. Last previous edition approved in 1998 as E 979 91 (1998).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM
14、 Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.4. Significance and Use4.1 Thi
15、s procedure is designed to determine the effective-ness of antimicrobial agents intended for microbial control ininvert emulsions and other water containing hydraulic fluids.5. Apparatus5.1 Air SupplyAny air source which is free from organicvapors, organic matter, or other objectionable material may
16、 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 loosely in aluminum foil and steam sterilize at 15 to 20 psi for 30minutes. Cool to room temperat
17、ure 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 two weeks. Discard sooner if upstream filter becomeswet or contaminated with oil.5.2 Colony Coun
18、terAny one of several types may beused.5.3 IncubatorAny cabinet capable of maintaining a tem-perature of 35C 6 1C may be used.5.4 Test CabinetA large cabinet capable of maintaining atemperature of 35C 6 1C, able to house several two literbeakers, and into which an air line can be introduced.5.5 Ster
19、ilizerAny suitable steam sterilizer capable ofproducing the conditions of sterilization is acceptable.5.6 Simulated Filters:5.6.1 Strainer, 3-in. epoxy coated,14-in. mesh gutterstrainer.35.6.2 Screen, 16 by 18 in. fiberglass screening material.45.6.3 Wire, 20-gage, galvanized or stainless steel.5.7
20、Tubing,14-in. ID Tygon.55.8 T-Connectors,14-in. polypropylene.5.9 Laboratory BlenderAny standard adjustable speedlaboratory blender having a 2-L capacity glass or metalcontainer is satisfactory.5.10 Hypodermic Needle, 16-gage needle.5.11 Microscope, Brightfield microscope equipped with403 and 1003 o
21、bjectives.5.12 Labware:5.12.1 Culture Dishes100 mm by 15 mm sterile culturedishes made of glass or plastic are required for makingstandard plate counts.65.12.2 Bacteriological Pipettes of 1.1 or 2.2-mL Capacity.75.12.3 Water Dilution BottlesAny sterilizable glass con-tainers having a 150 to 200-mL c
22、apacity and tight closures maybe used.85.12.4 Two-Liter Borosilicate Glass Beakers.5.12.5 Bent Glass Rod.5.12.6 Screw Cap Culture Tubes, autoclavable, 15 by 150mm.5.13 Water BathMaintain at 46C 6 2C to anneal agarbased microbiological media.5.14 Aluminum Foil.6. Reagents and Materials6.1 Invert Emul
23、sion Emulsifier.96.2 Paraffnic Mineral Oil.6.3 Deionized or Distilled Water (2 MOHM quality)6.4 Gentamicin Sulfate.106.5 Arlacel 80.116.6 Tween 60.116.7 Phosphate BufferFor serial dilutions.6.8 Mineral oil, sterile.6.9 Microbiological MediaGeneral retrieval media con-sistent with good microbiologica
24、l practices are acceptable.Examples are as follows:6.9.1 Soybean-Casein Digest Agar, U.S.P. XIX, MediumII.126.9.2 Fluid Soybean-Casein Digest Medium, U.S.P. XIX,Medium III.126.9.3 Sabouraud Dextrose Agar, U.S.P. XIX, Medium 20.96.9.4 Sabouraud Dextrose Broth, U.S.P. XIX, Medium 21.96.9.5 American Pe
25、troleum Institute (API) agar,9for enu-meration of sulfate reducing bacteria.6.10 Inoculum:6.10.1 The inoculum may vary according to the usersrequirements. It may be either undefined or defined.6.10.1.1 An undefined inoculum may consist of microor-ganisms isolated from a “spoiled” invert emulsion hyd
26、raulicfluid 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.6.10.1.2 An undefined inoculum may consist of the follow-ing: (1) equal volumes of fluid soybean-casein
27、digest and“spoiled” (see section 6.1.1.1) hydraulic fluid aerated at 35Cfor 24 h (typically) until the bacterial count reaches 109CFU/mL, (2) equal volumes of sabouraud dextrose broth and“spoiled” (see 6.10.1.1) hydraulic fluid aerated at 35C for 243Gutter strainers available from Billy Penn Corp.,
28、Philadelphia, PA 19122, havebeen found suitable.4Fiberglass mesh screening material (18 by 16) is available from any localhardware dealer.5Tygon is available from most local laboratory supply houses.6Presterilized and disposable plastic petri dishes are available from most locallaboratory supply hou
29、ses.7Presterilized and disposable 1.1-mL bacteriological pipettes are available frommost local laboratory supply houses.8Milk dilution bottles of 160-mL capacity having screw-cap closures areavailable from Corning Glass Works, P.O. Box 5000, Corning, NY 14831, OwensIllinois Glass Co., P.O. Box 230,
30、Vineland, NJ 08360, or most laboratory supplyhouses.9A satisfactory emulsifier for the preparation of invert emulsion hydraulic fluidsis Compound #5162 available from the Lubrizol Co., Wickliffe, OH.10Gentamicin sulfate can be obtained as Garamycin Reagent Solution, availablein two concentrations of
31、 10 and 50 mg/mL, from the Schering Corp., Kenilworth,NJ 07033.11Arlacel 80 and Tween 60 are available from the Specialty Chemicals Division,ICI American Inc., Wilmington, DE 19897.12Available in dehydrated form from Baltimore Biological Laboratories, Cock-eysville, MD; Difco Laboratories, Detroit,
32、MI, or other laboratory media supplyhouses.E 979 91 (2004)2h (typically) or until fungal count reaches 106107CFU/mL, or(3) equal volumes of (1) and (2) if both bacteria and fungi arethe desired test organisms.6.10.2 A defined inoculum consisting of a mixed culture ofspecific microorganisms may also
33、be used.6.10.2.1 The defined inoculum may be prepared by isolatingand identifying specific microorganisms from a “spoiled” (see6.10.1.1) hydraulic fluid emulsion and culturing the bacterialisolates in soybean-casein digest medium and the fungalisolates in sabouraud dextrose broth until there are 109
34、CFUbacteria or 106107CFU fungi, or both, per mL, respectively.6.10.2.2 Other microorganisms of particular interest (1)13(Rossmoore and Szlathy) may be used such as: Pseudomonasfluorescens, Pseudomonas cepacia, Klebsiella pneumoniae,Proteus mirabilis, Desulfovibrio desulfuricans, Aspergillusniger, Ce
35、phalosporium sp., Fusarium sp., Candida sp.6.10.2.3 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, or106107CFU fungi, or both, per mL, should be used as aninoculum for the emulsio
36、n system.6.11 Antimicrobial AgentsThe chemical agents to beevaluated as preservatives.7. Preparation of Simulated Filters7.1 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-gagewir
37、e or with staples.7.2 Preparation of AeratorsCut tubing (see 5.7) into13-in. sections. Bend tubing in a circle and connect both endsusing a T connector (see 5.8). 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
38、 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 sou
39、rce to bubble upthrough the hydraulic fluid producing a cascading effect overthe surface of the simulated filter.8. Preparation of Microbiological Medium8.1 Microbiological media should be prepared in accor-dance with manufacturers instructions. Media to be aug-mented with antibiotics should be anne
40、aled in a 46C 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.9. Microbiological Methods9.1 Solubilize the invert emulsion aliquot (see 6.1) accord-ing to the procedu
41、re of McConville, et al., (2), (3) as follows:9.1.1 Disperse 1 mL of the invert emulsion in 1 mL ofArlacel 80 and bring the volume up to 10 mL with 10 % Tween60 solution.9.2 Enumerate the bacteria in the solubilized invert emul-sion samples (see Test Method D 4454) by a standard pourplate procedure
42、such as that described in Standard Methods forthe Analysis of Water and Wastewater (4) or a spread plateprocedure such as that described in the Manual of Methods forGeneral Bacteriology (5). Do not use these procedures inter-changably since a variation in results may occur. If a pour plateprocedure
43、is used, plate solubilized fluid as well as 1 mL of101to 106dilutions prepared in phosphate buffer. If thespread plate procedure is used, plate 0.1 mL of the solubilizedfluid as well as 0.1 mL of 101to 105dilutions prepared inphosphate buffer. Do not use these plating procedures inter-changeably sin
44、ce a variation in results may occur. Incubate allplates for three days at 35C.9.3 Enumerate the mold and yeast populations in the solu-bilized invert emulsion samples by using the same proceduresas in 9.2, but use sabouraud dextrose agar containing 100 g ofgentamicin sulfate per mL as the plating me
45、dium. Incubate allplates for five days at 35C.9.4 Enumerate sulfate reducing bacteria populations in thesolubilized invert emulsion samples by serially diluting 1.0 mLaliquots in 9.0 mL molten, API agar in 15 mm by 150 mmscrewcap culture tubes. Prepare a series of 101and 104dilutions. Gently tip tub
46、e back and forth several times to mixinoculum with API agar while minimizing aeration. Warmpipet gently over bunsen burner flame before transferring asample from one dilution tube to the next in a series. Onceinoculated and the API agar has gelled, fill each culture tubewith sterile mineral oil. Inc
47、ubate at 35 6 1C. Observe for theformation of black colonies weekly for four weeks. Recordfinal titer.10. Procedure10.1 Add 67.5 mL of emulsifier (see 6.1) to 832.5 mL ofparaffinic mineral oil (see 6.2).10.2 Transfer mixture intoa2Lblender cup and add 450mL of deionized water and 150 mL of a broth i
48、noculumprepared as described in 6.8.NOTE 4Add the water and inoculum very gradually with the blenderrunning at slow speed to avoid raising the temperature of the mixtureabove 24 to 38C.10.3 Continue mixing until stable water-in-oil emulsion isproduced. This emulsion will serve as the untreated contr
49、olsample.10.4 Prepare the treated emulsion samples as described in10.1-10.3, but add the antimicrobial to be tested to thedeionized water in a concentration which will provide thedesired antimicrobial test dose in the completed emulsion or ina manner consistent with industrial practice and proposedrecommendations. When preparing the treated emulsions,make sure to add the inoculum to the blender after all of thedeionized water containing the antimicrobial has been incor-porated.10.5 Add each test emulsion sample to a separate 2 L bea
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