ASTM E2694-09

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An American National Standard
Designation:E2694–09
Standard Test Method for
Measurement of Adenosine Triphosphate in Water-Miscible
Metalworking Fluids
This standard is issued under the fixed designation E2694; 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.
1. Scope D6161 Terminology Used for Microfiltration, Ultrafiltra-
tion, Nanofiltration and Reverse Osmosis Membrane Pro-
1.1 The method provides a protocol for capturing, extract-
cesses
ing and quantifying the adenosine triphosphate (ATP) content
E1326 Guide for Evaluating Nonconventional Microbio-
associated with microorganisms found in water-miscible met-
logical Tests Used for Enumerating Bacteria
alworking fluids (MWF).
E1497 Practice for Selection and Safe Use of Water-
1.2 The ATP is measured using a bioluminescence enzyme
Miscible and Straight Oil Metal Removal Fluids
assay, whereby light is generated in amounts proportional to
E2523 Terminology for Metalworking Fluids and Opera-
the concentration ofATP in the samples. The light is produced
tions
and measured quantitatively as relative light units (RLU)
2.2 Government Standards:
which are converted by comparison with an ATP standard and
29 CFR 1910.1000 Occupational Safety and Health Stan-
computation to pg ATP/mL.
dards; Air contaminants
1.3 This method is equally suitable for use in the laboratory
29 CFR 1910.1450 Occupational Exposure to Hazardous
or field.
Chemicals in Laboratories
1.4 The method detects ATP concentrations in the range of
4.0 pg ATP/mL to 400,000 pg ATP/mL.
3. Terminology
1.5 Providing interferences can be overcome, biolumines-
3.1 Definitions:
cence is a reliable and proven method for qualifying and
For definition of terms used in this method, refer to Termi-
quantifying ATP. The method does not differentiate between
nology standards D1129, D6161, and E2523.
ATP from different sources, for example, from different types
3.2 adenosine triphosphate (ATP), n—a molecule com-
of microorganisms, such as bacteria and fungi.
prised of a purine and three phosphate groups that serves as the
1.6 The values stated in SI are to be regarded as standard.
primary energy transport molecule in all biological cells.
1.7 This standard does not purport to address all of the
3.3 adenosine monophosphate (AMP), n—the molecule
safety concerns, if any, associated with its use. It is the
formed by the removal of two molecules of phosphate (one
responsibility of the user of this standard to establish appro-
pyrophosphate molecule) from ATP.
priate safety and health practices and determine the applica-
3.4 aseptic, adj—sterile, free from viable microbial con-
bility of regulatory limitations prior to use.
tamination.
2. Referenced Documents 3.5 bioluminescence, n—the production and emission of
light by a living organism as the result of a chemical reaction
2.1 ASTM Standards:
during which chemical energy is converted to light energy.
D1129 Terminology Relating to Water
3.6 biomass, n—any matter which is or was a living
D4012 Test Method for Adenosine Triphosphate (ATP)
organism or excreted from a microorganism (D6161).
Content of Microorganisms in Water
3.7 culturable, adj—microorganisms that proliferate as in-
D4840 Guide for Sample Chain-of-Custody Procedures
dicated by the formation of colonies on solid growth media or
the development of turbidity in liquid growth media under
This test method is under the jurisdiction of ASTM Committee E34 on
specific growth conditions.
Occupational Health and Safety and is the direct responsibility of Subcommittee
3.8 Luciferase, n—a general term for a class of enzymes
E34.50 on Health and Safety Standards for Metal Working Fluids.
that catalyze bioluminescent reactions.
Current edition approved May 1, 2009. Published June 2009. DOI: 10.1520/
E2694-09.
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 AvailablefromU.S.GovernmentPrintingOfficeSuperintendentofDocuments,
Standards volume information, refer to the standard’s Document Summary page on 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
the ASTM website. www.access.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E2694–09
3.9 Luciferin, n—a general term for a class of light-emitting (or longer) required for culturable colonies to become visible,
biological pigments found in organisms capable of biolumi- to approximately five minutes.
nescence. 5.5 Although ATP data covary strongly with culture data in
3.10 luminometer, n—an instrument capable of measuring MWF , different factors affect ATP concentration than those
light emitted as a result of non-thermal excitation. that affect culturability.
3.11 relative light unit (RLU), n—an instrument-specific 5.5.1 Culturability is affected primarily by the ability of
unit of measurement reflecting the number of photons emitted captured microbes to proliferate on the growth medium pro-
by the Luciferin-Luciferase driven hydrolysis of ATP to AMP vided, under specific growth conditions. It have been estimated
plus pyrophosphate. that less than 1 % of the species present in an environmental
3.11.1 Discussion—RLU is not an SI unit, however, RLU sample will form colonies under any given set of growth
are proportional to ATP concentration. conditions.
3.12 viable microbial biomass, n—metabolicallyactive(liv- 5.5.2 ATP concentration is affected by: the microbial spe-
ing) microorganisms cies present, the physiological states of those species, and the
3.13 Acronyms: total bioburden (See Appendix X1).
5.5.2.1 One example of the species effect is that the amount
3.13.1 AMP—adenosine monophosphate
of ATP per cell is substantially greater for fungi than bacteria.
3.13.2 ATP—adenosine triphosphate
5.5.2.2 Within a species, cells that are more metabolically
3.13.3 HDPE—high density polyethylene
active will have more ATP per cell than dormant cells.
3.13.4 MWF—metalworking fluid
5.5.2.3 The greater the total bioburden, the greater the ATP
3.13.5 PP—polypropylene
concentration in a sample.
3.13.6 RLU—relative light unit
5.5.3 The possibility exists that the rinse step (11.15) may
noteliminateallchemicalsubstancesthatcaninterferewiththe
4. Summary of Test Method
bioluminescence reaction (11.39).
4.1 A control assay is performed using 100 µL of 1.0 ng
5.5.3.1 The presence of any such interferences can be
ATP/mL standard.
evaluated by performing a standard addition test series as
4.2 A 5.0 mL sample of MWF is placed into a syringe and
described in Appendix X3.
then pressure- filtered through a 0.7 µm, glass-fiber, in-line
5.5.3.2 Anyimpactofinterferingchemicalswillbereflected
depth filter.
asbiasrelativetodataobtainedfromfluidthatdoesnotcontain
4.3 The retentate is then washed with a reagent to remove
interfering chemicals.
extra-cellularATPandothercontaminantsthatmightotherwise
interfere with the ATP assay.
6. Apparatus
4.4 The filter is air-dried.
6.1 Culture tube, PP, 12 by 55 mm.
4.5 A lysing reagent is used to release ATP from microbial
6.2 Culture tube, PP, 17 by 100 mm with caps.
cells that have been captured on the glass-fiber filter, and the
6.3 Filter, 25 mm, sterile, disposable, in- line, 0.7 µm
filtrate is dispensed into an unused culture tube.
pore-size, glass-fiber, depth-type with Luer-Lok inlet.
4.6 The filtrate is diluted 1+9 with a buffer solution.
6.4 Luminometer, using photomultiplier tube, capable of
4.7 A100-µL volume of diluted filtrate is transferred to an
detecting light emission at 420 nm and with a cuvette chamber
unused culture tube into which 100 µLof Luciferin-Luciferase
that can hold a 12 by 55-mm culture tube.
reagent has previously been dispensed.
6.5 Macropipeter, adjustable, 1.0 to 5.0 mL.
4.8 The culture tube is placed into a luminometer and the
6.6 Micropipeter, adjustable, 100 to 1000 µL.
light intensity is read in RLU.
6.7 Pipet tips, sterile, disposable, PP, 100 to 1000 µL.
4.9 RLU are converted to Log [pg ATP/mL] of sample by
6.8 Pipet tips, sterile, disposable, PP, 1.0 to 5.0 mL.
computation.
6.9 Sample collection container, sterile, wide-mouth bottle,
100 mL.
5. Significance and Use
NOTE 1—ATP can adsorb onto glass surfaces. Consequently, PP or
5.1 This method measures the concentration ofATPpresent
HDPE containers are strongly preferred.
in the sample.ATPis a constituent of all living cells, including
6.10 Syringe, Luer-Lok , 20 mL, PP, sterile, disposable.
bacteria and fungi. Consequently, the presence of ATP is an
6.11 Syringe, Luer-Lok, 60 mL, PP, sterile disposable.
indicator of total microbial contamination in metalworking
6.12 Test tube rack,12mm.
fluids. ATP is not associated with matter of non-biological
6.13 Test tube rack,17mm.
origin.
6.14 Waste receptacle, any container suitable for receiving
5.2 Method D4012 validated ATP as a surrogate for cultur-
and retaining filtrate fluid for ultimate disposal.
able bacterial data (Guide E1326).
5.3 This method differs from Method D4012 in that it
eliminates interferences that have historically rendered ATP
Passman et al. “Real-time Testing of Bioburdens in Metalworking Fluids using
testing unusable with complex organic fluids such as MWF.
Adenosine Triphosphate as a Biomass Indicator,” 2009 STLE Annual Meeting,
5.4 The ATP test provides rapid test results that reflect the
Orlando, FL.
total bioburden in the sample. It thereby reduces the delay
Sloan, W. T., C. Quince and Curtis, T. P., “The Uncountables,” Accessing
between test initiation and data capture, from the 36 h to 48 h Uncultivated Microorganisms, ASM Press, Washington, DC, 2008, p. 35.
E2694–09
7. Reagents and Materials 9.3.2 Optimally samples should be tested on-site as soon as
possible (<4 h) after testing.
7.1 ATP standard, 1 ng ATP/mL
6 9.3.3 If testing is to be delayed for longer than 4 h, or to be
7.1.1 Commercially available;or
performed by an outside testing facility, samples may be stored
7.1.2 Dilute 1 mgATP into 1000 mLATP dilution buffer to
on ice or in a refrigerator for up to 24 h. Samples older than 24
get a 1000-ng ATP/mL stock solution. Then, dilute 1.0 mL of
h are unlikely to microbiologically representative of the MWF
1000 ng ATP/mL stock solution into 999.0 mL ATP dilution
at the time it was collected.
buffertogeta1ngATP/mL ATP standard.
7.2 ATP extract dilution buffer (proprietary)
10. Calibration and Standardization
7.3 ATP extraction reagent (proprietary)
6 10.1 Turn on power to luminometer (6.4) and allow instru-
7.4 Filter wash reagent (proprietary)
6 ment to warm-up, in accordance with manufacturer’s recom-
7.5 Luciferin-Luciferase reagent (proprietary); store be-
mendations.
tween -20°C and 4°C; allow to equilibrate to ambient tempera-
10.2 Ensure that all reagents have equilibrated to ambient
ture before using.
temperature before running any tests.
10.3 Useamicropipeter(6.6)withanew100to1000-µLtip
8. Hazards
(6.7) to dispense 100 µL Luciferin- Luciferase reagent (7.5)to
8.1 The analyst must know and observe good laboratory
an unused 12 by 55-mm culture tube (6.1).
safety practice in accordance with 29 CFR 1910.1450.
10.4 Replace the micropipeter tip with a fresh tip.
8.2 Inhalation or dermal exposure to MWF can pose health
10.5 Dispense 100 µL of 1 ng ATP/mL standard solution
problems for personnel involved with MWF sampling. Provi-
(7.1) into the culture tube.
sion of personal protective equipment (PPE) in the form of
10.6 Swirl gently for five times.
respirators, protective clothing or both may be indicated (see
10.7 Place the culture tube into the luminometer.
Practice E1497).
10.8 Read and record RLU (RLU ).
ctrl
8.3 Reviewmaterialsafetydatasheetsformaterialsinuseat
the facility to identify potential hazards in order to determine
11. Procedure
appropriate PPE (see 29 CFR 1910.1000).
11.1 Use aseptic procedure while performing this test
method; ATP from analyst’s hands, sputum, etc. can contami-
9. Sampling and Test Specs and Units
nate the sample with ATP from sources other than the sample
9.1 Sampling Site:
itself.
9.1.1 Select sampling site that will yield a representative
11.2 Remove plunger from a new 20-mLsyringe (6.10) and
MWF sample.
place onto a 17-mm test tube rack so that plunger tip does not
9.1.2 For routine condition monitoring, select individual
contact any surfaces.
sump(s) or central systems that have actively circulating fluid.
11.3 Affix filter (6.3) onto the 20-mL syringe.
9.1.3 For diagnostic testing, select zones of pooled or
11.4 Place a fresh 1.0 to 5.0-mL tip (6.8) onto the macropi-
stagnant MWF.
peter (6.5).
9.2 Sampling:
11.5 Shake sample for 15 seconds to ensure homogeneity.
9.2.1 If practical, draw sample from the mid-point of the
11.6 With minimal delay, remove lid from sample container
fluid reservoir, otherwise draw sample from below surface of
and, using the macropipeter, transfer 5.0 mL of sample to the
the MWF at an accessible location.
20-mL syringe barrel.
9.2.1.1 Microbial contamination will vary considerably
11.7 While holding the barrel over the waste receptacle
within the fluid system and it is important to be consistent in
(6.14), replace the plunger into the 20-mL syringe.
selecting the sampling location; this should be appropriate for
11.8 Apply even pressure to the 20-mL syringe plunger to
the analysis objectives.
pressure filter MWF sample, having filtrate discharge into the
9.2.2 Collect sample by removing lid from sample con-
waste receptacle.
tainer, immersing the open container (6.9), opening-down,
11.9 Remove filter from the 20-mLsyringe and place onto a
below the fluid surface and inverting the container to allow it
17-mm test tube rack so that filter outlet does not contact any
to fill with the sampled fluid.
surfaces.
9.2.3 If the fluid depth is insufficient to permit 9.2.1, use a
11.10 Remove plunger from the 20-mL syringe (6.10) and
sterile pipet to draw sample from the fluid and dispense it into
place onto a 17-mm test tube rack so that the plunger tip does
the sample container; collecting at least 25 mL of sample.
not contact any surfaces.
9.3 Sample Storage/Shipment:
11.11 Replace filter onto the end of the syringe barrel.
9.3.1 Labelthesamplecontainerandfollowacceptedchain-
11.12 Place a fresh 1.0 to 5.0 mLtip onto the macropipeter.
of-custody procedures (Guide D4840).
11.13 Transfer 5 mL of filter wash reagent (7.4) into the
syringe barrel.
11.14 While holding the barrel over the waste receptacle
ThesolesourceofsupplyoftheproprietaryATPdilutionbuf
...