ASTM D6974-09(2013)
(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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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.
1. Scope 2. Referenced Documents
2.1 ASTM Standards:
1.1 This practice covers a membrane filter (MF) procedure
D1129 Terminology Relating to Water
for the detection and enumeration of Heterotrophic bacteria
D1193 Specification for Reagent Water
(HPC) and fungi in liquid fuels with kinematic viscosities ≤24
2 -1
D4175 Terminology Relating to Petroleum, Petroleum
mm ·s at ambient temperature.
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
D6426 Test Method for Determining Filterability of Middle
1.3 This test may be performed either in the field or in the
Distillate Fuel Oils
laboratory.
D6469 GuideforMicrobialContaminationinFuelsandFuel
1.4 The ability of individual microbes to form colonies on
Systems
specific growth media depends on the taxonomy and physi-
D7463 Test Method forAdenosineTriphosphate (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 GuideforEvaluatingNonconventionalMicrobiologi-
cal Tests Used for Enumerating Bacteria
1.5 This practice offers alternative options for delivering
F1094 Test Methods for Microbiological Monitoring of
fuel sample microbes to the filter membrane, volumes or
Water Used for Processing Electron and Microelectronic
dilutions filtered, growth media used to cultivate fuel-borne
Devices by Direct Pressure Tap Sampling Valve and by
microbes, and incubation temperatures. This flexibility is
the Presterilized Plastic Bag Method
offered to facilitate diagnostic efforts. When this practice is
2.2 Energy Institute Standards:
used as part of a condition monitoring program, a single
IP 385 Viable aerobic microbial content of fuels and fuel
procedure should be used consistently.
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 ofASTM 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 contactASTM Customer Service at service@astm.org. ForAnnual Book ofASTM
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/D6974-09R13. U.K., http://www.energyinst.org.uk.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6974 − 09 (2013)
3.2 Acronyms: MethodsD5259andF1094),thetestsensitivitycanbeadjusted
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
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
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.
-1
3.3.1 N—number of CFU · L
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
observations.Thisresultsinanunderestimationofthenumbers
4.2 Thefiltermembraneretainsmicrobespresentinthefuel.
of viable microbes in the original fuel sample.
Filter replicate fuel samples through fresh membranes to
permit replicate testing, growth on alternative nutrient media,
7. Apparatus
or both.
7.1 Separatory Funnels, glass, nominal capacity 500 mL.
4.3 After filtration, place each membrane on one of two
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.
and1L.
4.4 Incubatethefiltermediaonagarfortwomoredays,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
NOTE 2—If the vacuum filtration option (7.5.3) is chosen, a vacuum
found in water-phase samples, fuel-phase organisms are often
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-
colonyformationbyothers.Asnotedin6.3,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,capableofmaintainingatemperatureof25 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.
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 1.0 %
aqueous solution of chlorotetracycline (filter sterilized by
7.11 Glass Bottles, screw cap with gas-tight closures, 500
passing through a 0.2 µm filter, see 8.4) per 100 mL MEAand
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 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.
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 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 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.Dissolve10goflactic
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 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 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 committee at this time is
are likely to be encountered in test samples can be demonstrated.
SigmaAldrich Co., St. Louis, MO 63178, http://www.sigmaaldrich.com. If you are
aware of alternative suppliers, please provide this information to ASTM Interna
...
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