ASTM D6974-09(2013)e2
(Practice)Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
SIGNIFICANCE AND USE
5.1 Biodeteriogenic microbes infecting fuel systems typically are most abundant within slime accumulations on system surfaces or at the fuel-water interface (Guide D6469). However, it is often impractical to obtain samples from these locations within fuel systems. Although the numbers of viable bacteria and fungi recovered from fuel-phase samples are likely to be several orders of magnitude smaller than those found in water-phase samples, fuel-phase organisms are often the most readily available indicators of fuel and fuel system microbial contamination.
5.2 Growth Medium Selectivity—Guide E1326 discusses the limitations of growth medium selection. Any medium selected will favor colony formation by some species and suppress colony formation by others. As noted in 6.3, physical, chemical and physiological variables can affect viable cell enumeration test results. Test Method D7463 provides a non-culture means of quantifying microbial biomass in fuels and fuel associated water.
5.3 Since a wide range of sample sizes, or dilutions thereof, can be analyzed by the membrane filter technique (Test Methods D5259 and F1094), the test sensitivity can be adjusted for the population density expected in the sample.
5.4 Enumeration data should be used as part of diagnostic efforts or routine condition monitoring programs. Enumeration data should not be used as fuel quality criteria.
SCOPE
1.1 This practice covers a membrane filter (MF) procedure for the detection and enumeration of Heterotrophic bacteria (HPC) and fungi in liquid fuels with kinematic viscosities ≤24 mm2 · s-1 at ambient temperature.
1.2 This quantitative practice is drawn largely from IP Method 385 and Test Method D5259.
1.3 This test may be performed either in the field or in the laboratory.
1.4 The ability of individual microbes to form colonies on specific growth media depends on the taxonomy and physiological state of the microbes to be enumerated, the chemistry of the growth medium, and incubation conditions. Consequently, test results should not be interpreted as absolute values. Rather they should be used as part of a diagnostic or condition monitoring effort that includes other test parameters, in accordance with Guide D6469.
1.5 This practice offers alternative options for delivering fuel sample microbes to the filter membrane, volumes or dilutions filtered, growth media used to cultivate fuel-borne microbes, and incubation temperatures. This flexibility is offered to facilitate diagnostic efforts. When this practice is used as part of a condition monitoring program, a single procedure should be used consistently.
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.7 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 appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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´2
Designation: D6974 − 09(Reapproved 2013)
Standard Practice for
Enumeration of Viable Bacteria and Fungi in Liquid Fuels—
Filtration and Culture Procedures
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.
ε NOTE—Subsections 8.4 and 8.8.2 were editorially corrected in August 2014.
ε NOTE—References in Section 9 and the title of IP 385 were editorially corrected in January 2015.
1. Scope 2. Referenced Documents
1.1 This practice covers a membrane filter (MF) procedure 2.1 ASTM Standards:
for the detection and enumeration of Heterotrophic bacteria D1129 Terminology Relating to Water
(HPC) and fungi in liquid fuels with kinematic viscosities D1193 Specification for Reagent Water
2 -1
≤24 mm ·s 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
1.4 The ability of individual microbes to form colonies on D6469 GuideforMicrobialContaminationinFuelsandFuel
Systems
specific growth media depends on the taxonomy and physi-
ological state of the microbes to be enumerated, the chemistry D7463 Test Method forAdenosineTriphosphate (ATP) Con-
tent of Microorganisms in Fuel, Fuel/Water Mixtures, and
of the growth medium, and incubation conditions.
Consequently, test results should not be interpreted as absolute Fuel Associated Water
D7464 Practice for Manual Sampling of Liquid Fuels, As-
values. Rather they should be used as part of a diagnostic or
condition monitoring effort that includes other test parameters, sociated Materials and Fuel System Components for
Microbiological Testing
in accordance with Guide D6469.
E1326 GuideforEvaluatingNonconventionalMicrobiologi-
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:
procedure should be used consistently.
IP 385 Determination of the Viable Aerobic Microbial Con-
1.6 The values stated in SI units are to be regarded as
tent of Fuels and Fuel Components Boiling Below
standard. No other units of measurement are included in this
390°C—Filtration and Culture Method
standard.
1.7 This standard does not purport to address all of the 3. Terminology
safety concerns, if any, associated with its use. It is the
3.1 Definitions:
responsibility of the user of this standard to establish appro-
3.1.1 For definition of terms used in this method refer to
priate safety and health practices and determine the applica-
Terminologies D1129 and D4175, and Guide D6469.
bility of regulatory limitations prior to use.
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, Liquid Fuels, and Lubricantsand is the direct responsibility of Subcom- contactASTM Customer Service at service@astm.org. ForAnnual Book ofASTM
mittee D02.14 on 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.
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/D6974-09R13E02. U.K., http://www.energyinst.org.uk.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
´2
D6974 − 09 (2013)
3.1.2 aseptic, adj—sterile, free from viable microbiological 5.3 Since a wide range of sample sizes, or dilutions thereof,
contamination. can be analyzed by the membrane filter technique (Test
MethodsD5259andF1094),thetestsensitivitycanbeadjusted
3.2 Acronyms:
for the population density expected in the sample.
3.2.1 CFU—colony forming unit
5.4 Enumeration data should be used as part of diagnostic
3.2.2 HPC—heterotrophic plate count
efforts or routine condition monitoring programs. Enumeration
3.2.3 MF—membrane filter
data should not be used as fuel quality criteria.
3.2.4 MEA—malt extract agar
6. Interferences
3.2.5 TNTC—too numerous to count
6.1 High non-biological particulate loads (sediment) can
3.2.6 TSA—tryptone soy agar
clog the membrane and prevent filtration.
3.3 Symbols:
-1 6.2 Each CFU is assumed to originate from a single micro-
3.3.1 N—number of CFU · L
bial cell. In reality, microbes often form aggregates which
3.3.2 CC—number of colonies on membrane filter
appear as a single colony. Consequently, viable count data are
3.3.3 V—sample volume filtered, mL
likely to underestimate the total number of viable organisms in
the original sample.
4. Summary of Practice
6.3 The metabolic state of individual microbes may be
4.1 Any free water present in a fuel sample is removed by
affected by numerous physical-chemical variables in the fuel.
settling in a separatory funnel. After the water has been
Injured cells or cells that have relatively long generation times
removed, a known volume of the remaining fuel is filtered
may not form colonies within the time allotted for test
through a membrane filter aseptically by one of three methods.
observations.Thisresultsinanunderestimationofthenumbers
of viable microbes in the original fuel sample.
4.2 Thefiltermembraneretainsmicrobespresentinthefuel.
Filter replicate fuel samples through fresh membranes to
7. Apparatus
permit replicate testing, growth on alternative nutrient media,
7.1 Separatory Funnels, glass, nominal capacity 500 mL.
or both.
7.2 Measuring Cylinders, glass, nominal capacity 100 mL
4.3 After filtration, place each membrane on one of two
and1L.
types of agar growth media, incubate at a designated tempera-
ture for three days, and examine for the presence of CFU.
7.3 Pipettes, glass or sterile disposable plastic, nominal
capacity 10 mL, or adjustable volume pipette and sterile
4.4 Incubatethefiltermediaonagarfortwomoredays,then
disposable plastic tips.
reexamine.
7.4 Membrane Filter, mixed esters of cellulose,
4.5 Count the colonies manually or by electronic counter.
presterilized, preferably gridded, 47 mm diameter, nominal
4.5.1 If practical, identify colonies on each agar medium,
pore size 0.45 µm.
based on colony color, morphology, and microscopic exami-
NOTE 1—While the recommended filter material is mixed esters of
nation.
cellulose, the selection of membrane material will depend on individual
4.5.2 Convert bacterial and fungal colony counts to CFU
preference and fuel type.
per litre of fuel.
7.5 Filtration Unit, one of:
7.5.1 Unit, as described in Test Method D6426, with pre-
5. Significance and Use
sterilized in-line filter housing, or
5.1 Biodeteriogenic microbes infecting fuel systems typi-
7.5.2 Hypodermic Syringe, sterile, 100 mL, 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).
7.5.3 Filter Holder Assembly, single or manifold, glass,
However, it is often impractical to obtain samples from these
stainless steel, or polypropylene, pre-sterilized.
locations within fuel systems. Although the numbers of viable
NOTE 2—If the vacuum filtration option (7.5.3) is chosen, a vacuum
bacteria and fungi recovered from fuel-phase samples are
source, not more than -66 kPa will also be needed.
likely to be several orders of magnitude smaller than those
7.6 Forceps, blunt tipped.
found in water-phase samples, fuel-phase organisms are often
the most readily available indicators of fuel and fuel system
7.7 Filter Flask, of sufficient capacity to receive the entire
microbial contamination.
sample being filtered plus washings.
5.2 Growth Medium Selectivity—Guide E1326 discusses the
7.8 Petri Dishes, disposable plastic or glass, nominal diam-
limitations of growth medium selection.Any medium selected
eter ≥50 mm.
will favor colony formation by some species and suppress
NOTE 3—Pre-poured Petri dishes, containing the growth media de-
scribed below are available commercially and may be substituted for the
colonyformationbyothers.Asnotedin6.3,physical,chemical
dishes listed here.
and physiological variables can affect viable cell enumeration
test results. Test Method D7463 provides a non-culture means 7.9 Incubator,capableofmaintainingatemperatureof25 6
of quantifying microbial biomass in fuels and fuel associated 2°C or any other temperature (within the range–ambient to
water. 60°C), as appropriate.
´2
D6974 − 09 (2013)
7.10 WaterBath,capableofmaintainingatemperatureof47 portions into 500 mL glass screw-cap bottles (7.11). Sterilize
6 2°C and receiving 500 mL bottles. Water bath capacity byautoclavingat121 62°Cfor10min.Coolandmaintainthe
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 0.1 %
aqueous solution of chlorotetracycline (filter sterilized by
7.11 Glass Bottles, screw cap with gas-tight closures,
passing through a 0.2 µm filter, see 8.4) per 100 mL MEAand
500 mL nominal capacity.
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,
see 8.7) to adjust pH. Once acidified, the MEA shall not be
7.13 Autoclave, with capacity to hold 500 mL glass bottles
upright. reheated.Makeagarplatesofthemediumbypouringsufficient
NOTE 4—Items 7.10 – 7.13 are not needed if using commercially MEA into sterile petri dishes to give a layer approximately
prepared Petri dishes, as indicated in Note 3.
4 mm thick. Allow to cool and set.
NOTE 5—MEA is available from various manufacturers in dehydrated
8. Reagents and Materials
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
dehydrated media, follow the manufacturer’s instructions for sterilization.
used in all tests. Unless otherwise indicated, it is intended that
Avoid overheating.
all reagents conform to the specifications of the Committee on
NOTE 6—Alternative media to MEAmay 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.
molds that are likely to be encountered in test samples can be demon-
strated.
8.2 Theagarusedinpreparationofculturemediashallbeof
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 Chlorotetracycline, 0.1 % (w/v) aqueous. Dissolve 0.1 g
Sodium bicarbonate 0.05 g
chlorotetracycline in water and dilute to 100 mL. Sterilize by Water 1 L
passing through a 0.2 µm filter.
8.9.2 Preparation—Dissolve salts in 1 L of water and
dispense 10 mL portions into screw capped culture tubes
8.5 Detergent Solution 0.1 % (v⁄v)—Dissolve 10 mL of
(7.12). Sterilize by autoclaving at 121°C for 15 min.
polyoxyethylene (20) sorbitan monooleate in 990 mL water.
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.
-1
8.6 Hydrochloric Acid, 1 mol HCl · L .
8.10 Sodium Hydroxide, 10 % (w/v) aqueous. Dissolve 10 g
NaOH in water and dilute to 100 mL.
8.7 LacticAcid, 10 % (w/v) aqueous. Dissolve 10 g of lactic
acidinwateranddiluteto100mL.Sterilizebypassingthrough 8.11 Tryptone Soy Agar (TSA):
a 0.2 µm filter.
8.11.1 Composition/Litre:
Tryptone 15 g
8.8 Malt Extract Agar (MEA):
Soy protein 5 g
8.8.1 Composition/Litre:
Sodium chloride 5 g
Agar 15 g
Malt Extract 30 g
Water 1 L
Mycological Peptone 5 g
Agar 15 g
8.11.2 Preparation—Suspend the dry ingredients in 1 L of
Water 1 L
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
-1
the pH to 5.4 6 0.2 using either 1 mL · L hydrochloric acid
a water bath (7.10)at47 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
TSA into sterile petri dishes to give a layer approximately
Reagent Chemicals, American Chemical Society Specifications, American
4 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 toTSAmay be used, providing the ability
MD.
of any alternative medium to support comparable growth of bacteria that
The sole source of supply of Tween 80 known to the commit
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
´2 ´2
Designation: D6974 − 09 (Reapproved 2013) D6974 − 09 (Reapproved 2013)
Standard Practice for
Enumeration of Viable Bacteria and Fungi in Liquid Fuels—
Filtration and Culture Procedures
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.
ε NOTE—Subsections 8.4 and 8.8.2 were editorially corrected in August 2014.
ε NOTE—References in Section 9 and the title of IP 385 were editorially corrected in January 2015.
1. Scope
1.1 This practice covers a membrane filter (MF) procedure for the detection and enumeration of Heterotrophic bacteria (HPC)
2 -1
and fungi in liquid fuels with kinematic viscosities ≤24 mm · s at ambient temperature.
1.2 This quantitative practice is drawn largely from IP Method 385 and Test Method D5259.
1.3 This test may be performed either in the field or in the laboratory.
1.4 The ability of individual microbes to form colonies on specific growth media depends on the taxonomy and physiological
state of the microbes to be enumerated, the chemistry of the growth medium, and incubation conditions. Consequently, test results
should not be interpreted as absolute values. Rather they should be used as part of a diagnostic or condition monitoring effort that
includes other test parameters, in accordance with Guide D6469.
1.5 This practice offers alternative options for delivering fuel sample microbes to the filter membrane, volumes or dilutions
filtered, growth media used to cultivate fuel-borne microbes, and incubation temperatures. This flexibility is offered to facilitate
diagnostic efforts. When this practice is used as part of a condition monitoring program, a single procedure should be used
consistently.
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.7 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 appropriate safety and health practices and determine the applicability of regulatory
limitations prior to use.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D4175 Terminology Relating to Petroleum, Petroleum Products, and Lubricants
D5259 Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
D6426 Test Method for Determining Filterability of Middle Distillate Fuel Oils
D6469 Guide for Microbial Contamination in Fuels and Fuel Systems
D7463 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel
Associated Water
D7464 Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological
Testing
E1326 Guide for Evaluating Nonconventional Microbiological Tests Used for Enumerating Bacteria
F1094 Test Methods for Microbiological Monitoring of Water Used for Processing Electron and Microelectronic Devices by
Direct Pressure Tap Sampling Valve and by the Presterilized Plastic Bag Method
This practice is under the jurisdiction of ASTM Committee D02 on Petroleum Products, Liquid Fuels, and Lubricantsand is the direct responsibility of Subcommittee
D02.14 on Stability and Cleanliness of Liquid Fuels.
Current edition approved May 1, 2013. Published August 2013. Originally approved in 2003. Last previous edition approved in 2009 as D6974 – 09. DOI:
10.1520/D6974-09R13E01.10.1520/D6974-09R13E02.
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.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
´2
D6974 − 09 (2013)
2.2 Energy Institute Standards:
IP 385 Viable aerobic microbial content of fuels and fuel components boiling below 90°C—Filtration and culture methodDe-
termination of the Viable Aerobic Microbial Content of Fuels and Fuel Components Boiling Below 390°C—Filtration and
Culture Method
3. Terminology
3.1 Definitions:
3.1.1 For definition of terms used in this method refer to Terminologies D1129 and D4175, and Guide D6469.
3.1.2 aseptic, adj—sterile, free from viable microbiological contamination.
3.2 Acronyms:
3.2.1 CFU—colony forming unit
3.2.2 HPC—heterotrophic plate count
3.2.3 MF—membrane filter
3.2.4 MEA—malt extract agar
3.2.5 TNTC—too numerous to count
3.2.6 TSA—tryptone soy agar
3.3 Symbols:
-1
3.3.1 N—number of CFU · L
3.3.2 CC—number of colonies on membrane filter
3.3.3 V—sample volume filtered, mL
4. Summary of Practice
4.1 Any free water present in a fuel sample is removed by settling in a separatory funnel. After the water has been removed,
a known volume of the remaining fuel is filtered through a membrane filter aseptically by one of three methods.
4.2 The filter membrane retains microbes present in the fuel. Filter replicate fuel samples through fresh membranes to permit
replicate testing, growth on alternative nutrient media, or both.
4.3 After filtration, place each membrane on one of two types of agar growth media, incubate at a designated temperature for
three days, and examine for the presence of CFU.
4.4 Incubate the filter media on agar for two more days, then reexamine.
4.5 Count the colonies manually or by electronic counter.
4.5.1 If practical, identify colonies on each agar medium, based on colony color, morphology, and microscopic examination.
4.5.2 Convert bacterial and fungal colony counts to CFU per litre of fuel.
5. Significance and Use
5.1 Biodeteriogenic microbes infecting fuel systems typically are most abundant within slime accumulations on system surfaces
or at the fuel-water interface (Guide D6469). However, it is often impractical to obtain samples from these locations within fuel
systems. Although the numbers of viable bacteria and fungi recovered from fuel-phase samples are likely to be several orders of
magnitude smaller than those found in water-phase samples, fuel-phase organisms are often the most readily available indicators
of fuel and fuel system microbial contamination.
5.2 Growth Medium Selectivity—Guide E1326 discusses the limitations of growth medium selection. Any medium selected will
favor colony formation by some species and suppress colony formation by others. As noted in 6.3, physical, chemical and
physiological variables can affect viable cell enumeration test results. Test Method D7463 provides a non-culture means of
quantifying microbial biomass in fuels and fuel associated water.
5.3 Since a wide range of sample sizes, or dilutions thereof, can be analyzed by the membrane filter technique (Test Methods
D5259 and F1094), the test sensitivity can be adjusted for the population density expected in the sample.
5.4 Enumeration data should be used as part of diagnostic efforts or routine condition monitoring programs. Enumeration data
should not be used as fuel quality criteria.
6. Interferences
6.1 High non-biological particulate loads (sediment) can clog the membrane and prevent filtration.
Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR, U.K., http://www.energyinst.org.uk.
´2
D6974 − 09 (2013)
6.2 Each CFU is assumed to originate from a single microbial cell. In reality, microbes often form aggregates which appear as
a single colony. Consequently, viable count data are likely to underestimate the total number of viable organisms in the original
sample.
6.3 The metabolic state of individual microbes may be affected by numerous physical-chemical variables in the fuel. Injured
cells or cells that have relatively long generation times may not form colonies within the time allotted for test observations. This
results in an underestimation of the numbers of viable microbes in the original fuel sample.
7. Apparatus
7.1 Separatory Funnels, glass, nominal capacity 500 mL.
7.2 Measuring Cylinders, glass, nominal capacity 100 mL and 1 L.
7.3 Pipettes, glass or sterile disposable plastic, nominal capacity 10 mL, or adjustable volume pipette and sterile disposable
plastic tips.
7.4 Membrane Filter, mixed esters of cellulose, presterilized, preferably gridded, 47 mm diameter, nominal pore size 0.45 μm.
NOTE 1—While the recommended filter material is mixed esters of cellulose, the selection of membrane material will depend on individual preference
and fuel type.
7.5 Filtration Unit, one of:
7.5.1 Unit, as described in Test Method D6426, with pre-sterilized in-line filter housing, or
7.5.2 Hypodermic Syringe, sterile, 100 mL, with pre-sterilized in-line filter housing, or
7.5.3 Filter Holder Assembly, single or manifold, glass, stainless steel, or polypropylene, pre-sterilized.
NOTE 2—If the vacuum filtration option (7.5.3) is chosen, a vacuum source, not more than -66 kPa will also be needed.
7.6 Forceps, blunt tipped.
7.7 Filter Flask, of sufficient capacity to receive the entire sample being filtered plus washings.
7.8 Petri Dishes, disposable plastic or glass, nominal diameter ≥50 mm.
NOTE 3—Pre-poured Petri dishes, containing the growth media described below are available commercially and may be substituted for the dishes listed
here.
7.9 Incubator, capable of maintaining a temperature of 25 6 2°C or any other temperature (within the range–ambient to 60°C),
as appropriate.
7.10 Water Bath, capable of maintaining a temperature of 47 6 2°C and receiving 500 mL bottles. Water bath capacity should
be sufficient to accommodate at least one bottle of each type of agar growth medium used.
7.11 Glass Bottles, screw cap with gas-tight closures, 500 mL nominal capacity.
7.12 Culture Tubes, glass, 16 by 125 mm, screw cap.
7.13 Autoclave, with capacity to hold 500 mL glass bottles upright.
NOTE 4—Items 7.10 – 7.13 are not needed if using commercially prepared Petri dishes, as indicated in Note 3.
8. Reagents and Materials
8.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society where such
specifications are available.
8.2 The agar used in preparation of culture media shall be of microbiological grade. Whenever possible, use commercial culture
media.
8.3 Water Purity—Unless otherwise indicated, references to water shall be understood to mean reagent water as defined by Type
III of Specification D1193.
8.4 Chlorotetracycline, 0.1 % (w/v) aqueous. Dissolve 0.1 g chlorotetracycline in water and dilute to 100 mL. Sterilize by
passing through a 0.2 μm filter.
8.5 Detergent Solution 0.1 % (v ⁄v)—Dissolve 10 mL of polyoxyethylene (20) sorbitan monooleate in 990 mL water. Sterilize,
either by passing through a 0.2 μm membrane filter into a sterile vessel, or autoclaving at 121°C for 15 min.
Reagent Chemicals, American Chemical Society Specifications, American Chemical Society, Washington, DC. For Suggestions on the testing of reagents not listed by
the American Chemical Society, see Annual Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National
Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.
The sole source of supply of Tween 80 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 comments will receive careful consideration at a meeting of the responsible
technical committee, which you may attend.
´2
D6974 − 09 (2013)
-1
8.6 Hydrochloric Acid, 1 mol HCl · L .
8.7 Lactic Acid, 10 % (w/v) aqueous. Dissolve 10 g of lactic acid in water and dilute to 100 mL. Sterilize by passing through
a 0.2 μm filter.
8.8 Malt Extract Agar (MEA):
8.8.1 Composition/Litre:
Malt Extract 30 g
Mycological Peptone 5 g
Agar 15 g
Water 1 L
8.8.2 Preparation—Suspend the malt extract, mycological peptone and agar in 1 L of water and boil to dissolve. Adjust the pH
-1
to 5.4 6 0.2 using either 1 mL · L hydrochloric acid (8.6) or sodium hydroxide 10 % w/v (8.10). Dispense 250 mL portions into
500 mL glass screw-cap bottles (7.11). Sterilize by autoclaving at 121 6 2°C for 10 min. Cool and maintain the sterilized agar
in a water bath (7.10) at 47 6 2°C. Optionally, after the agar has cooled to 47 6 2°C, add 1 mL of a 0.1 % aqueous solution of
chlorotetracycline (filter sterilized by 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 % lactic acid (filter sterilized by passing through a 0.2 μm filter, see 8.7) to adjust pH. Once acidified,
the MEA shall not be reheated. Make agar plates of the medium by pouring sufficient MEA into sterile petri dishes to give a layer
approximately 4 mm thick. Allow to cool and set.
NOTE 5—MEA is available from various manufacturers in dehydrated form and in pre-poured plates with and without added antibiotic, either of which
may be used. When sterilizing MEA prepared from commercial dehydrated media, follow the manufacturer’s instructions for sterilization. Avoid
overheating.
NOTE 6—Alternative media to MEA may be used, providing the ability of any alternative medium to support comparable growth of yeast and molds
that are likely to be encountered in test samples can be demonstrated.
NOTE 7—Alternative antibiotics may be used providing their ability to inhibit growth of bacteria but not yeast and molds has been validated.
8.9 Ringer’s Solution, One-Quarter Strength:
8.9.1 Composition/Litre:
Sodium chloride 2.25 g
Potassium chloride 0.105 g
Calcium chloride 0.12 g
Sodium bicarbonate 0.05 g
Water 1 L
8.9.2 Preparation—Dissolve salts in 1 L of water and dispense 10 mL portions into screw capped culture tubes (7.12). Sterilize
by autoclaving at 121°C for 15 min.
NOTE 8—One-quarter strength Ringer’s salts are available in tablet form from various manufacturers.
8.10 Sodium Hydroxide, 10 % (w
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