ASTM E979 - 20

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Designation: E979 −20

Standard Practice for

Evaluation of Antimicrobial Agents as Preservatives for

Invert Emulsion and Other Water Containing Hydraulic

Fluids

This standard is issued under the ﬁxed designation E979; 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 (´) 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*

1.1 This laboratory practice 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 Practice E2169.

1.2 The values stated in SI units are to be regarded as

standard. No other units of measurement are included in this

standard.

1.3 This standard does not purport to address all of the

safety concerns, if any, associated with its use. It is the

responsibility of the user of this standard to establish appro-

priate safety, health, and environmental practices and deter-

mine the applicability of regulatory limitations prior to use.

1.4 This international standard was developed in accor-

dance with internationally recognized principles on standard-

ization established in the Decision on Principles for the

Development of International Standards, Guides and Recom-

mendations issued by the World Trade Organization Technical

Barriers to Trade (TBT) Committee.

2. Referenced Documents

2.1 ASTM Standards:

D1129 Terminology Relating to Water

D4454 Test Method for Simultaneous Enumeration of Total

and Respiring Bacteria in Aquatic Systems by Microscopy

(Withdrawn 2015)

D5465 Practices for Determining Microbial Colony Counts

from Waters Analyzed by Plating Methods

E1326 Guide for Evaluating Non-culture Microbiological

Tests

E2169 Practice for Selecting Antimicrobial Pesticides for

Use in Water-Miscible Metalworking Fluids

E2523 Terminology for Metalworking Fluids and Opera-

tions

E2694 Test Method for Measurement of Adenosine Triphos-

phate in Water-Miscible Metalworking Fluids

This practice is under the jurisdiction of ASTM Committee E35 on Pesticides,

Antimicrobials, and Alternative Control Agents and is the direct responsibility of

Subcommittee E35.15 on Antimicrobial Agents.

Current edition approved Dec. 1, 2020. Published December 2020. Originally

approved in 1984. Last previous edition approved in 2015 as E979 – 09(2015). DOI:

10.1520/E0979-20.

For referenced ASTM standards, visit the ASTM website, www.astm.org, or

contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM

Standards volume information, refer to the standard’s Document Summary page on

the ASTM website.

The last approved version of this historical standard is referenced on

www.astm.org.

*A Summary of Changes section appears at the end of this standard

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the

Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

3. Terminology

3.1 Terms used in this practice are deﬁned in Terminologies

D1129 and E2523.

4. Summary of Test Method

4.1 The antimicrobial agent to be evaluated is incorporated

into an emulsion system by (a) addition to the aqueous phase

employed in the preparation of the emulsion, (b) in doses to the

formulated system, or (c) by other methods suitable for the test

compound.

4.2 A heavy bacterial or fungal inoculum, or both, is then

added.

4.3 The resulting mixture is aerated and passed over the

surface of a simulated ﬁlter system for a minimum period of

eight weeks either continuously or with shutdowns to simulate

actual operations conditions.

4.4 The degree of microbial control is determined by

periodically quantifying the bioburden in the emulsion by

direct microscopic count (Test Method D4454), plate count

(Practice D5465), or other appropriate method (Guide E1326)

and visual observations for microbial fouling of the simulated

ﬁlter surface.

NOTE 2—A knowledge of standard microbiological techniques is

required 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 maintained in an enclosure so that during the aeration process

the mists and aerosols generated do not contaminate the laboratory

environment.

5. Signiﬁcance and Use

5.1 This procedure is designed to determine the effective-

ness of antimicrobial agents intended for microbial control in

invert emulsions and other water containing hydraulic ﬂuids.

6. Apparatus

6.1 Air Supply—Any air source which is free from organic

vapors, organic matter, or other objectionable material may be

used.

NOTE 3—If desired, air may be sterilized as follows:

Pack two 150-mm long drying tubes (bulb type) loosely with glass

wool in a series with neoprene stoppers, glass tubing, and neoprene

tubing. Wrap loosely in aluminum foil and steam sterilize at 15 to 20 psi

for 30 minutes. Cool to room temperature while still wrapped. In-line

pre-sterilization air ﬁlters are available from most local laboratory supply

houses.

Insert into air line with bulbs on upstream side. Average lifetime in

continuous use is two weeks. Discard sooner if upstream ﬁlter becomes

wet or contaminated with oil.

6.2 Colony Counter—Any one of several types may be

used.

6.3 Incubator—Any cabinet capable of maintaining a tem-

perature of 35 61 °C may be used.

6.4 Test Cabinet—A large cabinet capable of maintaining a

temperature of 35 61 °C, able to house several two litre

beakers, and into which an air line can be introduced.

6.5 Sterilizer—Any suitable steam sterilizer capable of

producing the conditions of sterilization is acceptable.

6.6 Simulated Filters:

6.6.1 Strainer, 3-in. epoxy coated,

⁄

-in. mesh gutter

strainer.

6.6.2 Screen, 16 by 18 in. ﬁberglass screening material.

NOTE 4—Fiberglass mesh screening material (16 by 18 in.) is available

from any local hardware dealer.

6.6.3 Wire, 20-gauge, galvanized or stainless steel.

6.7 Tubing,

⁄

-in. ID Tygon.

NOTE 5—Tygon is available from most local laboratory supply houses.

6.8 T-Connectors,

⁄

-in. polypropylene.

6.9 Laboratory Blender—Any standard adjustable speed

laboratory blender having a 2-L capacity glass or metal

container is satisfactory.

6.10 Hypodermic Needle, 16-gauge needle.

6.11 Microscope, Brightﬁeld microscope equipped with 40×

and 100× objectives.

6.12 Labware:

6.12.1 Culture Dishes—100 by 15 mm sterile culture dishes

made of glass or plastic are required for making standard plate

counts.

NOTE 6—Presterilized and disposable plastic petri dishes are available

from most local laboratory supply houses.

6.12.2 Bacteriological Pipettes of 1.1 or 2.2-mL capacity.

NOTE 7—Presterilized and disposable 1.1-mL bacteriological pipettes

are available from most local laboratory supply houses.

6.12.3 Water Dilution Bottles—Any sterilizable glass con-

tainers having a 150 to 200-mL capacity and tight closures may

be used.

NOTE 8—Milk dilution bottles of 160-mL capacity having screw-cap

closures 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 150

mm.

6.13 Water Bath—Maintain at 46 62 °C to anneal agar

based microbiological media.

6.14 Aluminum Foil.

7. Reagents and Materials

7.1 Invert Emulsion Emulsiﬁer.

7.2 Paraffınic Mineral Oil.

7.3 Deionized or Distilled Water (>2 MOHM quality)

The sole source of supply of the apparatus known to the committee at this time

is Billy Penn Corp., Philadelphia, PA 19122. If you are aware of alternative

suppliers, please provide this information to ASTM International Headquarters.

Your comments will receive careful consideration at a meeting of the responsible

technical committee,

which you may attend.

The sole source of supply of a satisfactory emulsiﬁer for the preparation of

invert emulsion hydraulic ﬂuids (Compound #5162) known to the committee at this

time 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 technical

committee,

which you may attend.

E979 − 20

7.4 Gentamicin Sulfate.

7.5 Arlacel 80.

7.6 Tween 60.

7.7 Phosphate Buffer— For serial dilutions.

7.8 Mineral oil, sterile.

7.9 Microbiological Media—General 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, Medium II.

NOTE 9—Soybean-casein digest agar is available in dehydrated form

from most laboratory media supply houses.

7.9.2 Fluid Soybean-Casein Digest Medium, U.S.P. XIX,

Medium III.

7.9.3 Sabouraud Dextrose Agar, U.S.P. XIX, Medium 20.

7.9.4 Sabouraud Dextrose Broth, U.S.P. XIX, Medium 21.

7.9.5 Sulfate American Petroleum Institute (API) Agar,

for

enumeration of sulfate reducing bacteria.

7.10 Inoculum:

7.10.1 The inoculum may vary according to the users’

requirements. It may be either undeﬁned or deﬁned.

7.10.1.1 An undeﬁned inoculum may consist of microor-

ganisms isolated from a “spoiled” invert emulsion hydraulic

ﬂuid which exhibits microbiologically induced phase

generation, or which is known to have caused plugging of a

hydraulic system ﬁlter due to microbial slime, and grown in a

nutrient 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 35 °C for

24 h (typically) until the bacterial count reaches 10

CFU/mL,

(2) equal volumes of sabouraud dextrose broth and “spoiled”

(see 7.10.1.1) hydraulic ﬂuid aerated at 35 °C for 24 h (typi-

cally) or until fungal count reaches 10

to 10

CFU/mL, or (3)

equal volumes of (1) and (2) if both bacteria and fungi are the

desired test organisms.

7.10.2 A deﬁned inoculum consisting of a mixed culture of

speciﬁc microorganisms may also be used.

7.10.2.1 The deﬁned inoculum may be prepared by isolating

and identifying speciﬁc microorganisms from a “spoiled” (see

7.10.1.1) hydraulic ﬂuid emulsion and culturing the bacterial

isolates in soybean-casein digest medium and the fungal

isolates in sabouraud dextrose broth until there are 10

CFU

bacteria or 10

to 10

CFU fungi, or both, per mL, respectively.

7.10.2.2 Other microorganisms of particular interest (Ross-

moore and Szlatky)

may be used such as: Pseudomonas

ﬂuorescens,Pseudomonas cepacia,Klebsiella pneumoniae,

Proteus mirabilis,Desulfovibrio desulfuricans,Aspergillus

niger,Cephalosporium sp., Fusarium sp., Candida sp.

7.10.2.3 Equal mixtures of any two of the above bacterial

species or two of the above mold species, or both, plus the

Candida species to provide a ﬁnal titer of 10

CFU bacteria, or

to 10

CFU fungi, or both, per mL, should be used as an

inoculum for the emulsion system.

7.11 Antimicrobial Agents—The chemical agents to be

evaluated as preservatives.

8. Preparation of Simulated Filters

8.1 Cut the epoxy-coated,

⁄

-in. mesh gutter strainers 16 by

18 in. mesh ﬁberglass screening material into 3 by 5 in.

sections. Secure the screening to the strainers with 20-gauge

wire or with staples.

8.2 Preparation of Aerators—Cut tubing (see 6.7) into

13-in. sections. Bend tubing in a circle and connect both ends

using a T connector (see 6.8). Connect third arm of T connector

to a 20-in. length of tygon tubing. This tubing will be

connected to the main air supply line. Using a hot 16-gauge

needle, carefully punch a series of holes,

⁄

in. apart, along the

outer circumference of the tubing which forms the ring. Also

punch similar holes

⁄

in. apart on the upper and lower surface

of the tubing, at right angles to the holes previously punched.

These holes allow the air from the air source to bubble up

through the hydraulic ﬂuid producing a cascading effect over

the surface of the simulated ﬁlter.

9. Preparation of Microbiological Medium

9.1 Microbiological media should be prepared in accor-

dance with manufacturer’s instructions. Media to be aug-

mented with antibiotics should be annealed in a 46 62°C

water bath before antibiotics are added. Antibiotics should be

added just before pouring. Use 100 g gentamicin sulfate per

mL to suppress bacterial growth on fungal recovery media.

10. Microbiological Methods

10.1 Solubilize the invert emulsion aliquot (see 7.1) accord-

ing to the procedure of McConville, et al.,

10,11

as follows:

10.1.1 Disperse 1 mL of the invert emulsion in 1 mL of

Arlacel 80 and bring the volume up to 10 mL with 10 % Tween

60 solution.

The sole source of supply of gentamicin sulfate known to the committee at this

time is as Garamycin Reagent Solution, available in two concentrations of 10 and

50 mg/mL, from the Schering Corp., Kenilworth, NJ 07033.. If you are aware of

alternative suppliers, please provide this information to ASTM International

Headquarters. Your comments will receive careful consideration at a meeting of the

responsible technical committee,

which you may attend.

The sole source of supply of the reagents (Arlacel 80, Tween 60, and Sulfate

API Agar) known to the committee at this time is Sigma Aldrich Co., St. Louis, MO

63178, http://www.sigmaaldrich.com. 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 technical

committee,

which you may attend.

The sole source of supply of the media, available in dehydrated form, known

to the committee at this time is Baltimore Biological Laboratories, Cockeysville,

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 technical

committee,

which you may attend.

Rossmore, H. W., and Szlatky, K. ,“Characterization of the Microbial Flora of

Invert Emulsion Hydraulic Fluids,” Int. Biodetn. Bulletin, Vol 13, No. 4), 1977, pp.

96–100.

McConville, J. F., et al., “Method for Performing Aerobic Plate Counts of

Anhydrous Cosmetics Utilizing Tween 60 and Arlacel 80 as Dispersing Agents,”

Applied Microbiology , Vol 27, 1974, pp. 5–7.

Hoffman, N. M., “Hydraulic Fluid of 95-Percent Water,” Lubrication

Engineering, Vol 35, No. 2, 1979, pp. 65–71.

E979 − 20

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摘要：

ASTM E979 - 20 是美国材料与试验协会发布的一项标准规范，主要涵盖用于检测病原微生物对抗菌药物敏感性（即耐药性）的测试方法标准。该标准为医疗、制药及公共卫生领域提供了统一的实验操作流程与结果判读依据，旨在提高耐药性检测的准确性与可重复性。通过规范培养基选择、接种量、孵育条件及最小抑菌浓度（MIC）测定等关键环节，ASTM E979 - 20 有助于确保不同实验室之间数据的可比性，从而支持有效治疗方案的制定与抗微生物耐药性的监测与防控。

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