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Designation: D6974 − 09(Reapproved 2013)

Standard Practice for

Enumeration of Viable Bacteria and Fungi in Liquid Fuels—
Filtration and Culture Procedures1
This standard is issued under the fixed designation D6974; 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 2. Referenced Documents

1.1 This practice covers a membrane filter (MF) procedure 2.1 ASTM Standards:2
for the detection and enumeration of Heterotrophic bacteria D1129 Terminology Relating to Water
(HPC) and fungi in liquid fuels with kinematic viscosities ≤24 D1193 Specification for Reagent Water
mm2 · s-1 at ambient temperature. D4175 Terminology Relating to Petroleum, Petroleum
Products, and Lubricants
1.2 This quantitative practice is drawn largely from IP D5259 Test Method for Isolation and Enumeration of En-
Method 385 and Test Method D5259. terococci from Water by the Membrane Filter Procedure
1.3 This test may be performed either in the field or in the D6426 Test Method for Determining Filterability of Middle
laboratory. Distillate Fuel Oils
D6469 Guide for Microbial Contamination in Fuels and Fuel
1.4 The ability of individual microbes to form colonies on Systems
specific growth media depends on the taxonomy and physi- D7463 Test Method for Adenosine Triphosphate (ATP) Con-
ological state of the microbes to be enumerated, the chemistry tent of Microorganisms in Fuel, Fuel/Water Mixtures and
of the growth medium, and incubation conditions. Fuel Associated Water
Consequently, test results should not be interpreted as absolute D7464 Practice for Manual Sampling of Liquid Fuels, As-
values. Rather they should be used as part of a diagnostic or sociated Materials and Fuel System Components for
condition monitoring effort that includes other test parameters, Microbiological Testing
in accordance with Guide D6469. E1326 Guide for Evaluating Nonconventional Microbiologi-
1.5 This practice offers alternative options for delivering cal Tests Used for Enumerating Bacteria
fuel sample microbes to the filter membrane, volumes or F1094 Test Methods for Microbiological Monitoring of
dilutions filtered, growth media used to cultivate fuel-borne Water Used for Processing Electron and Microelectronic
microbes, and incubation temperatures. This flexibility is Devices by Direct Pressure Tap Sampling Valve and by
offered to facilitate diagnostic efforts. When this practice is the Presterilized Plastic Bag Method
used as part of a condition monitoring program, a single 2.2 Energy Institute Standards:3
procedure should be used consistently. IP 385 Viable aerobic microbial content of fuels and fuel
components boiling below 90°C—Filtration and culture
1.6 The values stated in SI units are to be regarded as method
standard. No other units of measurement are included in this
standard. 3. Terminology
1.7 This standard does not purport to address all of the 3.1 Definitions:
safety concerns, if any, associated with its use. It is the 3.1.1 For definition of terms used in this method refer to
responsibility of the user of this standard to establish appro- Terminologies D1129 and D4175, and Guide D6469.
priate safety and health practices and determine the applica- 3.1.2 aseptic, adj—sterile, free from viable microbiological
bility of regulatory limitations prior to use. contamination.

1 2
This practice is under the jurisdiction of ASTM Committee D02 on Petroleum For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Products and Lubricantsand is the direct responsibility of Subcommittee D02.14 on contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Stability and Cleanliness of Liquid Fuels. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved May 1, 2013. Published August 2013. Originally the ASTM website.
3
approved in 2003. Last previous edition approved in 2009 as D6974 – 09. DOI: Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR,
10.1520/D6974-09R13. U.K., http://www.energyinst.org.uk.

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 D6974 − 09 (2013)
3.2 Acronyms: Methods D5259 and F1094), the test sensitivity can be adjusted
3.2.1 CFU—colony forming unit for the population density expected in the sample.
3.2.2 HPC—heterotrophic plate count 5.4 Enumeration data should be used as part of diagnostic
3.2.3 MF—membrane filter efforts or routine condition monitoring programs. Enumeration
3.2.4 MEA—malt extract agar data should not be used as fuel quality criteria.
3.2.5 TNTC—too numerous to count 6. Interferences
3.2.6 TSA—tryptone soy agar
6.1 High non-biological particulate loads (sediment) can
3.3 Symbols: clog the membrane and prevent filtration.
3.3.1 N—number of CFU · L-1
6.2 Each CFU is assumed to originate from a single micro-
3.3.2 CC—number of colonies on membrane filter bial cell. In reality, microbes often form aggregates which
3.3.3 V—sample volume filtered, mL appear as a single colony. Consequently, viable count data are
likely to underestimate the total number of viable organisms in
4. Summary of Practice the original sample.
4.1 Any free water present in a fuel sample is removed by 6.3 The metabolic state of individual microbes may be
settling in a separatory funnel. After the water has been affected by numerous physical-chemical variables in the fuel.
removed, a known volume of the remaining fuel is filtered Injured cells or cells that have relatively long generation times
through a membrane filter aseptically by one of three methods. may not form colonies within the time allotted for test
4.2 The filter membrane retains microbes present in the fuel. observations. This results in an underestimation of the numbers
Filter replicate fuel samples through fresh membranes to of viable microbes in the original fuel sample.
permit replicate testing, growth on alternative nutrient media,
or both. 7. Apparatus
4.3 After filtration, place each membrane on one of two 7.1 Separatory Funnels, glass, nominal capacity 500 mL.
types of agar growth media, incubate at a designated tempera- 7.2 Measuring Cylinders, glass, nominal capacity 100 mL
ture for three days, and examine for the presence of CFU. and 1 L.
4.4 Incubate the filter media on agar for two more days, then 7.3 Pipettes, glass or sterile disposable plastic, nominal
reexamine. capacity 10 mL, or adjustable volume pipette and sterile
4.5 Count the colonies manually or by electronic counter. disposable plastic tips.
4.5.1 If practical, identify colonies on each agar medium,
7.4 Membrane Filter, mixed esters of cellulose,
based on colony color, morphology, and microscopic exami-
presterilized, preferably gridded, 47 mm diameter, nominal
nation.
pore size 0.45 µm.
4.5.2 Convert bacterial and fungal colony counts to CFU NOTE 1—While the recommended filter material is mixed esters of
per litre of fuel. cellulose, the selection of membrane material will depend on individual
preference and fuel type.
5. Significance and Use
7.5 Filtration Unit, one of:
5.1 Biodeteriogenic microbes infecting fuel systems typi- 7.5.1 Unit, as described in Test Method D6426, with pre-
cally are most abundant within slime accumulations on system sterilized in-line filter housing, or
surfaces or at the fuel-water interface (Guide D6469). 7.5.2 Hypodermic Syringe, sterile, 100 mL, with pre-
However, it is often impractical to obtain samples from these sterilized in-line filter housing, or
locations within fuel systems. Although the numbers of viable 7.5.3 Filter Holder Assembly, single or manifold, glass,
bacteria and fungi recovered from fuel-phase samples are stainless steel, or polypropylene, pre-sterilized.
likely to be several orders of magnitude smaller than those
found in water-phase samples, fuel-phase organisms are often NOTE 2—If the vacuum filtration option (7.5.3) is chosen, a vacuum
source, not more than -66 kPa will also be needed.
the most readily available indicators of fuel and fuel system
microbial contamination. 7.6 Forceps, blunt tipped.
5.2 Growth Medium Selectivity—Guide E1326 discusses the 7.7 Filter Flask, of sufficient capacity to receive the entire
limitations of growth medium selection. Any medium selected sample being filtered plus washings.
will favor colony formation by some species and suppress 7.8 Petri Dishes, disposable plastic or glass, nominal diam-
colony formation by others. As noted in 6.3, physical, chemical eter ≥50 mm.
and physiological variables can affect viable cell enumeration NOTE 3—Pre-poured Petri dishes, containing the growth media de-
test results. Test Method D7463 provides a non-culture means scribed below are available commercially and may be substituted for the
of quantifying microbial biomass in fuels and fuel associated dishes listed here.
water. 7.9 Incubator, capable of maintaining a temperature of 25 6
5.3 Since a wide range of sample sizes, or dilutions thereof, 2°C or any other temperature (within the range–ambient to
can be analyzed by the membrane filter technique (Test 60°C), as appropriate.

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 D6974 − 09 (2013)
7.10 Water Bath, capable of maintaining a temperature of 47 portions into 500 mL glass screw-cap bottles (7.11). Sterilize
6 2°C and receiving 500 mL bottles. Water bath capacity by autoclaving at 121 6 2°C for 10 min. Cool and maintain the
should be sufficient to accommodate at least one bottle of each sterilized agar in a water bath (7.10) at 47 6 2°C. Optionally,
type of agar growth medium used. after the agar has cooled to 47 6 2°C, add 1 mL of a 1.0 %
7.11 Glass Bottles, screw cap with gas-tight closures, 500 aqueous solution of chlorotetracycline (filter sterilized by
mL nominal capacity. passing through a 0.2 µm filter, see 8.4) per 100 mL MEA and
mix by shaking. If the medium is required at pH 3.5, add 10 %
7.12 Culture Tubes, glass, 16 by 125 mm, screw cap. lactic acid (filter sterilized by passing through a 0.2 µm filter,
7.13 Autoclave, with capacity to hold 500 mL glass bottles see 8.7) to adjust pH. Once acidified, the MEA shall not be
upright. reheated. Make agar plates of the medium by pouring sufficient
NOTE 4—Items 7.10-7.13 are not needed if using commercially pre- MEA into sterile petri dishes to give a layer approximately 4
pared Petri dishes, as indicated in Note 3. mm thick. Allow to cool and set.
8. Reagents and Materials NOTE 5—MEA is available from various manufacturers in dehydrated
form and in pre-poured plates with and without added antibiotic, either of
8.1 Purity of Reagents—Reagent grade chemicals shall be which may be used. When sterilizing MEA prepared from commercial
used in all tests. Unless otherwise indicated, it is intended that dehydrated media, follow the manufacturer’s instructions for sterilization.
all reagents conform to the specifications of the Committee on Avoid overheating.
NOTE 6—Alternative media to MEA may be used, providing the ability
Analytical Reagents of the American Chemical Society where
of any alternative medium to support comparable growth of yeast and
such specifications are available.4 molds that are likely to be encountered in test samples can be demon-
8.2 The agar used in preparation of culture media shall be of strated.
NOTE 7—Alternative antibiotics may be used providing their ability to
microbiological grade. Whenever possible, use commercial inhibit growth of bacteria but not yeast and molds has been validated.
culture media.
8.9 Ringer’s Solution, One-Quarter Strength:
8.3 Water Purity—Unless otherwise indicated, references to 8.9.1 Composition/Litre:
water shall be understood to mean reagent water as defined by Sodium chloride 2.25 g
Type III of Specification D1193. Potassium chloride 0.105 g
Calcium chloride 0.12 g
8.4 Chlortetracycline, 0.1 % (w/v) aqueous. Dissolve 0.1 g Sodium bicarbonate 0.05 g
chlortetracycline in water and dilute to 100 mL. Sterilize by Water 1L
passing through a 0.2 µm filter. 8.9.2 Preparation—Dissolve salts in 1 L of water and
8.5 Detergent Solution 0.1 % (v/v)—Dissolve 10 mL of dispense 10 mL portions into screw capped culture tubes
polyoxyethylene (20) sorbitan monooleate5 in 990 mL water. (7.12). Sterilize by autoclaving at 121°C for 15 min.
Sterilize, either by passing through a 0.2 µm membrane filter NOTE 8—One-quarter strength Ringer’s salts are available in tablet
into a sterile vessel, or autoclaving at 121°C for 15 min. form from various manufacturers.
8.6 Hydrochloric Acid, 1 mol HCl · L-1. 8.10 Sodium Hydroxide, 10 % (w/v) aqueous. Dissolve 10 g
8.7 Lactic Acid, 10 % (w/v) aqueous. Dissolve 10 g of lactic NaOH in water and dilute to 100 mL.
acid in water and dilute to 100 mL. Sterilize by passing through 8.11 Tryptone Soy Agar (TSA):
a 0.2 µm filter. 8.11.1 Composition/Litre:
8.8 Malt Extract Agar (MEA): Tryptone 15 g
Soy protein 5g
8.8.1 Composition/Litre: Sodium chloride 5g
Malt Extract 30 g Agar 15 g
Mycological Peptone 5g Water 1L
Agar 15 g 8.11.2 Preparation—Suspend the dry ingredients in 1 L of
Water 1L
water and boil to dissolve. Dispense 250 mL portions into 500
8.8.2 Preparation—Suspend the malt extract, mycological mL glass screw-cap bottles (7.11). Sterilize by autoclaving at
peptone and agar in 1 L of water and boil to dissolve. Adjust 121 6 2°C for 10 min. Cool and maintain the sterilized agar in
the pH to 5.4 6 0.2 using either 1 mL · L-1 hydrochloric acid a water bath (7.10) at 47 6 2°C. Draw a sample and test the
(8.6) or sodium hydroxide 10 % w/v (8.10). Dispense 250 mL pH. If the pH ≠ 7.3 6 0.3, reject the batch and make a fresh
mixture. Make agar plates of the medium by pouring sufficient
4
TSA into sterile petri dishes to give a layer approximately 4
Reagent Chemicals, American Chemical Society Specifications, American
mm thick. Allow to cool and set.
Chemical Society, Washington, DC. For Suggestions on the testing of reagents not
listed by the American Chemical Society, see Annual Standards for Laboratory NOTE 9—TSA is available from various manufacturers in dehydrated
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia form and in pre-poured plates.
and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, NOTE 10—Alternative media to TSA may be used, providing the ability
MD. of any alternative medium to support comparable growth of bacteria that
5
The sole source of supply of Tween 80 known to the committee at this time is
are likely to be encountered in test samples can be demonstrated.
Sigma Aldrich Co., St. Louis, MO 63178, http://www.sigmaaldrich.com. If you are
aware of alternative suppliers, please provide this information to ASTM Interna- 9. Procedure
tional Headquarters. Your comments will receive careful consideration at a meeting
of the responsible technical committee,1 which you may attend. 9.1 Sampling:

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 D6974 − 09 (2013)
9.1.1 Samples shall be drawn in accordance with Practice 9.3.2 Option A—Using Test Method D6426 testing device
D7464. (7.5.1).
9.1.1.1 To reduce the risk of accidental contamination, 9.3.2.1 Assemble the rig using sterile tubing and a pre-
samples intended for viable microbial enumeration shall not be assembled, sterile filter housing that contains a 0.45 µm filter
used for other tests until after they are no longer needed for (7.4).
enumeration testing. 9.3.2.2 Dispense the desired sample into a sterile reservoir
9.1.1.2 It may not be possible to use aseptic technique under flask.
field conditions. To reduce risk of cross-contaminating 9.3.2.3 Allow the Test Method D6426 testing device to
samples, sampling devices shall be rinsed with 70 % alcohol pump sample through the filter housing. Record the total
(ethanol, methanol, or isopropanol) to disinfect sample contact volume of sample filtered.
surfaces before samples are drawn. All samples and devices 9.3.2.4 Filter Detergent Wash—Wash the membrane free of
should be handled in such manner as to minimize the likeli- fuel by pumping 10 mL of sterile detergent solution (8.5)
hood of introducing microbial contaminants into the sample. through the testing device.
9.1.1.3 Microbial contaminant populations are dynamic. 9.3.2.5 Filter Rinse—Wash the membrane free of detergent
Microbes within the sample may proliferate or die during the solution by filtering three successive 10 mL portions of
interval between collection and testing. Consequently, samples one-quarter strength Ringer’s solution (8.2).
shall be processed (9.2) within 24 h after collection. 9.3.3 Option B—Using a hypodermic syringe (7.5.2).
9.1.1.4 If samples are to be processed later than 4 h after 9.3.3.1 Draw desired sample portion into syringe.
collection, store the samples either on ice, or refrigerated at >0 9.3.3.2 Affix preassembled in-line filter housing that con-
to 5°C until processed (9.1.1.3). Avoid freezing samples. tains a 0.45 µm filter (7.4) to the syringe’s Luer-lok fitting.
Chilled samples may be processed (9.2) without warming them 9.3.3.3 Apply gentle pressure to the syringe plunger and
to room temperature. dispense fuel into a graduated receiving vessel (7.2 or 7.7).
9.2 Sample Preparation: Record the volume of fuel filtered.
9.2.1 Allow sample to stand for 1 h and then examine 9.3.3.4 Filter Detergent Rinse—Remove the filter housing
visually. from the syringe carefully, taking precautions not to spill any
9.2.2 If the sample contains free water, transfer it to a sterile of the fuel that remains on the inlet side of the housing. Draw
separatory funnel (7.1); allow the fluid and sediment to settle. 10 mL of detergent solution into the syringe, reattach the filter
After they have settled out of the fuel-phase, draw off the water housing, and wash the membrane free of fuel by pumping 10
and associated particulate matter into a sterile flask. mL of sterile detergent solution (8.3) through the in-line filter
Alternatively, separate the water-phase and associated particu- housing.
late matter by pipetting them from the bottom of the sample 9.3.3.5 Filter Wash—Again remove the filter housing from
bottle. (Warning—Fuels are toxic substances and micro- the syringe carefully, and draw 30 mL of one-quarter strength
organisms may be pathogenic, allergenic, toxigenic, or some Ringer’s solution into the syringe. Reattach the filter housing,
combination thereof. The analyst shall know and observe the and wash the membrane free of detergent solution by pumping
normal good laboratory practices and safety procedures re- 30 mL of sterile one-quarter strength Ringer’s solution (8.2)
quired in a microbiology laboratory while preparing, using, and through the in-line filter housing.
disposing of cultures, reagents, and materials, and while 9.3.4 Option C—Vacuum filtration (7.5.3).
operating sterilization and other equipment and instrumenta- 9.3.4.1 Using sterile forceps (7.6), place a sterile 0.45 µm
tion.) pore-size filter membrane (7.4) onto the filter support, and
assemble the filter holder.
NOTE 11—Further analysis by microscopy and other microbiological 9.3.4.2 Dispense the desired sample into the filter reservoir.
techniques, as described in Guide D6469, can be conducted on the
water-phase and associated particulates.
Apply suction, and filter the test portion through the membrane
filter. Record the volume of sample filtered.
9.2.3 Shake the fuel-phase of the sample to distribute 9.3.4.3 Filter Detergent Rinse—Maintaining suction, wash
suspended microorganisms uniformly. the membrane filter free of fuel with a 10 mL portion of sterile
9.2.4 Sub-sample test portions of the fuel-phase either using detergent solution (8.3).
a sterile measuring cylinder (7.2) for larger volumes or sterile 9.3.4.4 Filter Rinse—Maintaining suction, wash the filter
pipette (7.3) for smaller volumes. free of detergent solution with three successive 10 mL portions
NOTE 12—To increase the likelihood of recovering 20 to 60 CFU on the of sterile one-quarter strength Ringer’s solution (8.2).
membrane filter, filter different volume portions: 200 mL fuel, 20 mL fuel,
and 2 mL fuel. Care should be taken when adding the 2 mL aliquot to
9.4 Transfer to Growth Media:
ensure even sample distribution over the membrane. Testing replicate 9.4.1 Remove the membrane filter from filter housing.
subsamples will provide a basis for estimating the combined effect of the (Warning—Fuel samples are flammable. Disinfect forceps and
fuel sample’s bioburden heterogeneity and procedural variability on the glassware by rinsing in ethanol, methanol, or isopropanol and
test results. (Caution—Do not pipette by mouth.) allowing the alcohol to evaporate. Open flames must not be
9.3 Sample Filtration: used in the vicinity of fuel samples.)
9.3.1 For Options A, B, or C, filter two test portions of equal 9.4.1.1 Carefully disassemble the filter housing, keeping the
volume, each through an unused filter (7.4), thereby providing exposed surface of the filter membrane facing up.
one filter each for bacterial and fungal enumerations. 9.4.1.2 Using a sterile forceps, remove the filter.

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 D6974 − 09 (2013)
9.4.2 Using a rolling action, apply the membrane filter, where:
grid-side up, onto the surface of the appropriate medium MEA N = number of CFU · L-1,
(8.4) or TSA (8.5) media in the petri dishes. Ensure good CC = colony count on the plate, see 9.6, and
contact between the membrane filter and the medium. V = sample volume filtered, in millilitres.
9.4.3 Use a fine point permanent marker to label the bottom 9.6.3 If multiple portions were filtered, average and com-
of each petri dish with the sample and growth medium identity. pute the standard deviation for the results of the replicate CFU
9.5 Incubation, · L-1 calculations.
9.5.1 Place the dishes in an incubator (7.13) controlled at 25 9.6.4 If no colonies appeared on the growth media let CC =
6 2°C. Invert the petri dishes containing TSA. 1, compute N and report results as <N CFU bacteria · L-1 or as
9.5.1.1 If the temperature of the system from which the <N CFU fungi · L-1, as appropriate.
sample was collected is >5°C warmer than the standard
incubation temperature, using an incubator adjusted to the 10. Report
ambient temperature may improve viable cell recovery. Al- 10.1 Report the results as CFU bacteria · L-1 and as CFU
though 25°C is suitable for most systems, a higher incubation fungi · L-1, as appropriate. If no colonies appeared on the
temperature, at or within 5°C of the temperature of the system growth media let CC = 1, compute N and report results as <N
from which the sample was collected, may be used when the CFU bacteria · L-1 or as <N CFU fungi · L-1, as appropriate.
temperature of the system sampled exceeds 30°C. Incubation
temperature shall be controlled at 62°C. 10.2 Report the incubation temperature.
9.5.2 Examine TSA and MEA dishes for the appearance of
colonies after three days and MEA again after five days 11. Precision and Bias
incubation. 11.1 Precision—The precision of the procedure in Practice
9.6 Colony Counting: D6974 cannot be determined due to the inherent taxonomic and
9.6.1 Using a counting device, determine the number of physiological variably of fuel microbial contaminants.
CFU that have developed on the membrane surface after three 11.2 Bias—Since there is no accepted reference material
days (TSA) and five days incubation (MEA). suitable for the bias in this test method, no statement on bias is
9.6.2 Calculate the CFU bacteria per litre in the sample from made.
the colony count on the TSA plate and the CFU fungi per litre
from the colony count on the MEA plate (see 9.6.1) using the 12. Keywords
following equation: 12.1 bacteria; biodegradation; biodeterioration; colony
CC 3 1000 forming unit; enumeration; fuel microbiology; fungi; mem-
N5 (1)
V brane filter technique; viable count

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