ASTM E979-91(1998)
(Test Method)Standard Test Method for Evaluation of Antimicrobial Agents as Preservatives for Invert Emulsion and Other Water Containing Hydraulic Fluids
Standard Test Method for Evaluation of Antimicrobial Agents as Preservatives for Invert Emulsion and Other Water Containing Hydraulic Fluids
SCOPE
1.1 This laboratory test method is designed to evaluate the utility and effectiveness of antimicrobial agents intended to control microbial growth in invert emulsions and other water containing hydraulic fluids.
Note 1--Procedures for preparation of water soluble hydraulic fluids and recovery of organisms appear in Method E686.
1.2 This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: E 979 – 91 (Reapproved 1998)
Standard Test Method for
Evaluation of Antimicrobial Agents as Preservatives for
Invert Emulsion and Other Water Containing Hydraulic
Fluids
This standard is issued under the fixed designation E 979; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Invert emulsion hydraulic fluids typically contain 60 % mineral oil and 40 % water (by volume).
These fluids routinely are prepared using proprietary, oil-soluble, emulsifying agents, as well as other
emulsifiable constituents. They are recommended for use where conditions indicate a low-cost, fire
retardant product, compatible with water-based metal working 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, maladorous
conditions, or general biodeterioration. Problem microorganisms associated with these fluids include
bacteria and fungi.
The hydraulic system is essentially a closed one in which water of evaporation is added to maintain
a fixed volume. The inclusion of an efficacious preservative in the water containing hydraulic fluids
can prevent microbial growth and the resulting problems that follow.
1. Scope E 686 Method for Evaluation of Antimicrobial Agents in
Aqueous Metal Working Fluids
1.1 This laboratory test method is designed to evaluate the
utility and effectiveness of antimicrobial agents intended to
3. Summary of Test Method
control microbial growth in invert emulsions and other water
3.1 The antimicrobial agent to be evaluated is incorporated
containing hydraulic fluids.
into an emulsion system by (a) addition to the aqueous phase
NOTE 1—Procedures for preparation of water soluble hydraulic fluids
employed in the preparation of the emulsion, (b) in doses to the
and recovery of organisms appear in Method E 686.
formulated system, or (c) by other methods suitable for the test
1.2 This standard does not purport to address all of the
compound.
safety concerns, if any, associated with its use. It is the
3.2 A heavy bacterial or fungal inoculum, or both, is then
responsibility of the user of this standard to establish appro-
added.
priate safety and health practices and determine the applica-
3.3 The resulting mixture is aerated and passed over the
bility of regulatory limitations prior to use.
surface of a simulated filter system for a minimum period of
eight weeks either continuously or with shutdowns to simulate
2. Referenced Documents
actual operations conditions.
2.1 ASTM Standards:
3.4 The degree of microbial control is determined by
D 4454 Test Method for Simultaneous Enumeration of Total
periodic plate counts of the emulsion and visual observations
and Respiring Bacteria in Aquatic Systems by Micros-
for microbial fouling of the simulated filter surface.
copy
NOTE 2—A knowledge of standard microbiological techniques is re-
quired for this procedure. It is also required that good laboratory practices
be followed throughout these tests. This means appropriate containment
for the microbiological systems being evaluated. The systems should be
This test method is under the jurisdiction of ASTM Committee E-35 on
maintained in an enclosure so that during the aeration process the mists
Pesticides and is the direct responsibility of Subcommittee E35.15 on Antibacterial
and aerosols generated do not contaminate the laboratory environment.
and Antiviral Agents.
Current edition approved July 15, 1991. Published September 1991. Originally
published as E 979 – 84. Last previous edition E 979 – 84.
2 3
Annual Book of ASTM Standards, Vol 11.02. Annual Book of ASTM Standards, Vol 11.05.
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E 979
4. Significance and Use 5.12.3 Water Dilution Bottles—Any sterilizable glass con-
tainers having a 150 to 200-mL capacity and tight closures may
4.1 This procedure is designed to determine the effective-
be used.
ness of antimicrobial agents intended for microbial control in
5.12.4 Two-Liter Borosilicate Glass Beakers.
invert emulsions and other water containing hydraulic fluids.
5.12.5 Bent Glass Rod.
5.12.6 Screw Cap Culture Tubes, autoclavable, 15 by 150
5. Apparatus
mm.
5.1 Air Supply—Any air source which is free from organic
5.13 Water Bath—Maintain at 46°C6 2°C to anneal agar
vapors, organic matter, or other objectionable material may be
based microbiological media.
used.
5.14 Aluminum Foil.
NOTE 3—If desired, air may be sterilized as follows:
6. Reagents and Materials
Pack two 150-mm long drying tubes (bulb type) loosely with glass wool
6.1 Invert Emulsion Emulsifier.
in a series with neoprene stoppers, glass tubing, and neoprene tubing.
6.2 Paraffınic Mineral Oil.
Wrap loosely in aluminum foil and steam sterilize at 15 to 20 psi for 30
6.3 Deionized or Distilled Water (>2 MOHM quality)
minutes. Cool to room temperature while still wrapped. In-line pre-
sterilization air filters are available from most local laboratory supply
6.4 Gentamicin Sulfate.
houses.
6.5 Arlacel 80.
Insert into air line with bulbs on upstream side. Average lifetime in
6.6 Tween 60.
continuous use is two weeks. Discard sooner if upstream filter becomes
6.7 Phosphate Buffer—For serial dilutions.
wet or contaminated with oil.
6.8 Mineral oil, sterile.
6.9 Microbiological Media—General retrieval media con-
5.2 Colony Counter—Any one of several types may be
used. sistent with good microbiological practices are acceptable.
Examples are as follows:
5.3 Incubator—Any cabinet capable of maintaining a tem-
6.9.1 Soybean-Casein Digest Agar, U.S.P. XIX, Medium
perature of 35°C 6 1°C may be used.
II.
5.4 Test Cabinet—A large cabinet capable of maintaining a
6.9.2 Fluid Soybean-Casein Digest Medium, U.S.P. XIX,
temperature of 35°C 6 1°C, able to house several two liter
Medium III.
beakers, and into which an air line can be introduced.
6.9.3 Sabouraud Dextrose Agar, U.S.P. XIX, Medium 20.
5.5 Sterilizer—Any suitable steam sterilizer capable of
6.9.4 Sabouraud Dextrose Broth, U.S.P. XIX, Medium 21.
producing the conditions of sterilization is acceptable.
6.9.5 American Petroleum Institute (API) agar, for enu-
5.6 Simulated Filters:
meration of sulfate reducing bacteria.
5.6.1 Strainer, 3-in. epoxy coated, ⁄4-in. mesh gutter
6.10 Inoculum:
strainer.
6.10.1 The inoculum may vary according to the users’
5.6.2 Screen, 16 by 18 in. fiberglass screening material.
requirements. It may be either undefined or defined.
5.6.3 Wire, 20-gage, galvanized or stainless steel.
6.10.1.1 An undefined inoculum may consist of microor-
ganisms isolated from a “spoiled” invert emulsion hydraulic
5.7 Tubing, ⁄4-in. ID Tygon.
fluid which exhibits microbiologically induced phase genera-
5.8 T-Connectors, ⁄4-in. polypropylene.
tion, or which is known to have caused plugging of a hydraulic
5.9 Laboratory Blender—Any standard adjustable speed
system filter due to microbial slime, and grown in a nutrient
laboratory blender having a 2-L capacity glass or metal
medium.
container is satisfactory.
6.10.1.2 An undefined inoculum may consist of the follow-
5.10 Hypodermic Needle, 16-gage needle.
ing: (1) equal volumes of fluid soybean-casein digest and
5.11 Microscope, Brightfield microscope equipped with
“spoiled” (see section 6.1.1.1) hydraulic fluid aerated at 35°C
403 and 1003 objectives.
for 24 h (typically) until the bacterial count reaches 10
5.12 Labware:
CFU/mL, (2) equal volumes of sabouraud dextrose broth and
5.12.1 Culture Dishes—100 mm by 15 mm sterile culture
“spoiled” (see 6.10.1.1) hydraulic fluid aerated at 35°C for 24
dishes made of glass or plastic are required for making
standard plate counts.
Milk dilution bottles of 160-mL capacity having screw-cap closures are
5.12.2 Bacteriological Pipettes of 1.1 or 2.2-mL Capacity.
available from Corning Glass Works, P.O. Box 5000, Corning, NY 14831, Owens
Illinois Glass Co., P.O. Box 230, Vineland, NJ 08360, or most laboratory supply
houses.
A satisfactory emulsifier for the preparation of invert emulsion hydraulic fluids
Gutter strainers available from Billy Penn Corp., Philadelphia, PA 19122, have is Compound #5162 available from the Lubrizol Co., Wickliffe, OH.
been found suitable. Gentamicin sulfate can be obtained as Garamycin Reagent Solution, available
Fiberglass mesh screening material (18 by 16) is available from any local in two concentrations of 10 and 50 mg/mL, from the Schering Corp., Kenilworth,
hardware dealer. NJ 07033.
6 12
Tygon is available from most local laboratory supply houses. Arlacel 80 and Tween 60 are available from the Specialty Chemicals Division,
Presterilized and disposable plastic petri dishes are available from most local ICI American Inc., Wilmington, DE 19897.
laboratory supply houses. Available in dehydrated form from Baltimore Biological Laboratories, Cock-
Presterilized and disposable 1.1-mL bacteriological pipettes are available from eysville, MD; Difco Laboratories, Detroit, MI, or other laboratory media supply
most local laboratory supply houses. houses.
E 979
6 7
h (typically) or until fungal count reaches 10 –10 CFU/mL, or 9.2 Enumerate the bacteria in the solubilized invert emul-
(3) equal volumes of (1) and (2) if both bacteria and fungi are sion samples (see Test Method D 4454) by a standard pour
the desired test organisms. plate procedure such as that described in Standard Methods for
6.10.2 A defined inoculum consisting of a mixed culture of the Analysis of Water and Wastewater (4) or a spread plate
specific microorganisms may also be used. procedure such as that described in the Manual of Methods for
6.10.2.1 The defined inoculum may be prepared by isolating General Bacteriology (5). Do not use these procedures inter-
and identifying specific microorganisms from a “spoiled” (see changably since a variation in results may occur. If a pour plate
6.10.1.1) hydraulic fluid emulsion and culturing the bacterial procedure is used, plate solubilized fluid as well as 1 mL of
−1 −6
isolates in soybean-casein digest medium and the fungal 10 to 10 dilutions prepared in phosphate bu
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