ASTM D6974-03
(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
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 D 5259.
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 D 6469.
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 the 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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An American National Standard
Designation: D 6974 – 03
Standard Practice for
Enumeration of Viable Bacteria and Fungi in Liquid Fuels—
Filtration and Culture Procedures
This standard is issued under the fixed designation D 6974; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope D 1193 Specification for Reagent Water
D 4057 Practice for Manual Sampling of Petroleum and
1.1 This practice covers a membrane filter (MF) procedure
Petroleum Products
for the detection and enumeration of Heterotrophic bacteria
D 4175 Terminology Relating to Petroleum, Petroleum
(HPC) and fungi in liquid fuels with kinematic viscosities #24
2 -1
Products, and Lubricants
mm ·s at ambient temperature.
D 5259 Test Method for Isolation and Enumeration of
1.2 This quantitative practice is drawn largely from IP
Enterococci from Water by the Membrane Filter Procedure
Method 385 and Test Method D 5259.
D 6426 Test Method for Determining Filterability of Distil-
1.3 This test may be performed either in the field or in the
late Fuel Oils
laboratory.
D 6469 Guide for Microbial Contamination in Fuels and
1.4 The ability of individual microbes to form colonies on
Fuel Systems
specific growth media depends on the taxonomy and physi-
E 1326 Guide for Evaluating Nonconventional Microbio-
ological state of the microbes to be enumerated, the chemistry
logical Tests Used for Enumerating Bacteria
of the growth medium, and incubation conditions. Conse-
F 1094 Test Methods for Microbiological Monitoring of
quently, test results should not be interpreted as absolute
Water Used for Processing Electronic and Microelectronic
values. Rather they should be used as part of a diagnostic or
Devices by Direct Pressure Tap Sampling Valve and by the
condition monitoring effort that includes other test parameters,
Pressurized Plastic Bag Method
in accordance with Guide D 6469.
2.2 Energy Institute Standards:
1.5 This practice offers alternative options for delivering
IP 385 Viable aerobic microbial content of fuels and fuel
fuel sample microbes to the filter membrane, volumes or
components boiling below 90°C—Filtration and culture
dilutions filtered, growth media used to cultivate fuel-borne
method
microbes, and incubation temperatures. This flexibility is
offered to facilitate diagnostic efforts. When this practice is
3. Terminology
used as part of a condition monitoring program, a single
3.1 Definitions—For definition of terms used in this method
procedure should be used consistently.
refer to Terminologies D 1129 and D 4175, and Guide D 6469.
1.6 The values stated in SI units are to be regarded as the
3.1.1 aseptic, adj—sterile, free from viable microbiological
standard.
contamination.
1.7 This standard does not purport to address all of the
3.2 Acronyms:
safety concerns, if any, associated with its use. It is the
3.2.1 CFU—colony forming unit
responsibility of the user of this standard to establish appro-
3.2.2 HPC—heterotrophic plate count
priate safety and health practices and determine the applica-
3.2.3 MF—membrane filter
bility of regulatory limitations prior to use.
3.2.4 MEA—malt extract agar
2. Referenced Documents 3.2.5 TNTC—too numerous to count
3.2.6 TSA—tryptone soy agar
2.1 ASTM Standards:
3.3 Symbols:
D 1129 Terminology Relating to Water
-1
3.3.1 N—number of CFU · L
3.3.2 CC—number of colonies on membrane filter
This practice is under the jurisdiction of ASTM Committee D02 on Petroleum
3.3.3 V—sample volume filtered, mL
Products and Lubricants and is the direct responsibility of Subcommittee D02.14 on
Stability and Cleanliness of Liquid Fuels.
Current edition approved Nov. 1, 2003. Published December 2003.
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 Available from Energy Institute, 61 New Cavendish St., London, WIG 7AR,
the ASTM website. U.K.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D6974–03
4. Summary of Practice observations. This results in an underestimation of the numbers
of viable microbes in the original fuel sample.
4.1 Any free water present in a fuel sample is removed by
settling in a separatory funnel. After the water has been
7. Apparatus
removed, a known volume of the remaining fuel is filtered
through a membrane filter aseptically by one of three methods.
7.1 Separatory Funnels, glass, nominal capacity 500 mL.
4.2 The filter membrane retains microbes present in the fuel.
7.2 Measuring Cylinders, glass, nominal capacity 100 mL
Filter replicate fuel samples through fresh membranes to
and1L.
permit replicate testing, growth on alternative nutrient media,
7.3 Pipettes, glass or sterile disposable plastic, nominal
or both.
capacity 10 mL, or adjustable volume pipette and sterile
4.3 After filtration, place each membrane on one of two
disposable plastic tips.
types of agar growth media, incubate at a designated tempera-
7.4 Membrane Filter, mixed esters of cellulose, presteril-
ture for three days, and examine for the presence of CFU.
4.4 Incubate the filter media on agar for two more days, then ized, preferably gridded, 47 mm diameter, nominal pore size
reexamine. 0.45 μm.
4.5 Count the colonies manually or by electronic counter.
NOTE 1—While the recommended filter material is mixed esters of
4.5.1 If practical, identify colonies on each agar medium,
cellulose, the selection of membrane material will depend on individual
based on colony color, morphology, and microscopic exami-
preference and fuel type.
nation.
7.5 Filtration Unit, one of:
4.5.2 Convert bacterial and fungal colony counts to CFU
per litre of fuel.
7.5.1 Unit, as described in Test Method D 6426, with
pre-sterilized in-line filter housing, or
5. Significance and Use
7.5.2 Hypodermic Syringe, sterile, 100 mL, with pre-
5.1 Biodeteriogenic microbes infecting fuel systems typi-
sterilized in-line filter housing, or
cally are most abundant within slime accumulations on system
7.5.3 Filter Holder Assembly, single or manifold, glass,
surfaces or at the fuel-water interface (Guide D 6469). How-
stainless steel, or polypropylene, pre-sterilized.
ever, it is often impractical to obtain samples from these
NOTE 2—If the vacuum filtration option (7.5.3) is chosen, a vacuum
locations within fuel systems. Although the numbers of viable
source, not more than -66 kPa will also be needed.
bacteria and fungi recovered from fuel-phase samples are
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
7.7 Filter Flask, of sufficient capacity to receive the entire
the most readily available indicators of fuel and fuel system
sample being filtered plus washings.
microbial contamination.
7.8 Petri Dishes, disposable plastic or glass, nominal diam-
5.2 Growth Medium Selectivity—Guide E 1326 discusses
eter $50 mm.
the limitations of growth medium selection. Any medium
selected will favor colony formation by some species and
NOTE 3—Pre-poured Petri dishes, containing the growth media de-
suppress colony formation by others. As noted in 6.3, physical,
scribed below are available commercially and may be substituted for the
chemical and physiological variables can affect viable cell
dishes listed here.
enumeration test results.
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.
Methods D 5259 and F 1094), the test sensitivity can be
7.10 Water Bath, capable of maintaining a temperature of 47
adjusted for the population density expected in the sample.
6 2°C and receiving 500 mL bottles. Water bath capacity
5.4 Enumeration data should be used as part of diagnostic
should be sufficient to accommodate at least one bottle of each
efforts or routine condition monitoring programs. Enumeration
type of agar growth medium used.
data should not be used as fuel quality criteria.
7.11 Glass Bottles, screw cap with gas-tight closures, 500
6. Interferences mL nominal capacity.
6.1 High non-biological particulate loads (sediment) can 7.12 Culture Tubes, glass, 16 by 125 mm, screw cap.
clog the membrane and prevent filtration.
7.13 Autoclave, with capacity to hold 500 mL glass bottles
6.2 Each CFU is assumed to originate from a single micro-
upright.
bial cell. In reality, microbes often form aggregates which
NOTE 4—Items 7.10-7.13 are not needed if using commercially pre-
appear as a single colony. Consequently, viable count data are
pared Petri dishes, as indicated in Note 3.
likely to underestimate the total number of viable organisms in
the original sample.
8. Reagents and Materials
6.3 The metabolic state of individual microbes may be
affected by numerous physical-chemical variables in the fuel. 8.1 Purity of Reagents—Reagent grade chemicals shall be
used in all tests. Unless otherwise indicated, it is intended that
Injured cells or cells that have relatively long generation times
may not form colonies within the time allotted for test all reagents conform to the specifications of the Committee on
D6974–03
inhibit growth of bacteria but not yeast and molds has been validated.
Analytical Reagents of the American Chemical Society where
such specifications are available.
8.9 Ringer’s Solution, One-Quarter Strength:
8.2 The agar used in preparation of culture media shall be of
8.9.1 Composition/Litre:
microbiological grade. Whenever possible, use commercial
Sodium chloride 2.25 g
culture media.
Potassium chloride 0.105 g
Calcium chloride 0.12 g
8.3 Water Purity—Unless otherwise indicated, references to
Sodium bicarbonate 0.05 g
water shall be understood to mean reagent water as defined by
Water 1 L
Type III of Specification D 1193.
8.9.2 Preparation—Dissolve salts in 1 L of water and
8.4 Chlortetracycline, 0.1 % (w/v) aqueous. Dissolve 0.1 g
dispense 10 mL portions into screw capped culture tubes
chlortetracycline in water and dilute to 100 mL. Sterilize by
(7.12). Sterilize by autoclaving at 121°C for 15 min.
passing through a 0.2 μm filter.
8.5 Detergent Solution 0.1 % (v/v)—Dissolve 10 mL of
NOTE 8—One-quarter strength Ringer’s salts are available in tablet
polyoxyethylene (20) sorbitan monooleate in 990 mL water. form from various manufacturers.
Sterilize, either by passing through a 0.2 μm membrane filter
8.10 Sodium Hydroxide, 10 % (w/v) aqueous. Dissolve 10 g
into a sterile vessel, or autoclaving at 121°C for 15 min.
NaOH in water and dilute to 100 mL.
-1
8.6 Hydrochloric Acid, 1 mol HCl · L .
8.11 Tryptone Soy Agar (TSA):
8.7 Lactic Acid, 10 % (w/v) aqueous. Dissolve 10 g of lactic
8.11.1 Composition/Litre:
acid in water and dilute to 100 mL. Sterilize by passing through
Tryptone 15 g
a 0.2 μm filter.
Soy protein 5 g
Sodium chloride 5 g
8.8 Malt Extract Agar (MEA):
Agar 15 g
8.8.1 Composition/Litre:
Water 1 L
Malt Extract 30 g
8.11.2 Preparation—Suspend the dry ingredients in 1L of
Mycological Peptone 5 g
Agar 15 g
water and boil to dissolve. Dispense 250 mL portions into 500
Water 1 L
mL glass screw-cap bottles (7.11). Sterilize by autoclaving at
8.8.2 Preparation—Suspend the malt extract, mycological 121 6 2°C for 10 min. Cool and maintain the sterilized agar in
peptone and agar in 1 L of water and boil to dissolve. Adjust a water bath (7.10) at 47 6 2°C. Draw a sample and test the
-1
the pH to 5.4 6 0.2 using either 1 mL · L hydrochloric acid pH. If the pH � 7.3 6 0.3, reject the batch and make a fresh
(8.6) or sodium hydroxide 10 % w/v (8.10). Dispense 250 mL mixture. Make agar plates of the medium by pouring sufficient
portions into 500 mL glass screw-cap bottles (7.11). Sterilize TSA into sterile petri dishes to give a layer approximately 4
by autoclaving at 121 6 2°C for 10 min. Cool and maintain the mm thick. Allow to cool and set.
sterilized agar in a water bath (7.10) at 47 6 2°C. Optionally,
NOTE 9—TSA is available from various manufacturers in dehydrated
after the agar has cooled to 47 6 2°C, add 1 mL of a 1.0 %
form and in pre-poured plates.
aqueous solution of chlorotetracycline (filter sterilized by
NOTE 10—Alternative media to TSA may be used, providing the ability
passing through a 0.2 μm filter, see 8.4) per 100 mL MEA and
of any alternative medium to support comparable growth of bacteria that
mix by shaking. If the medium is required at pH 3.5, add 10 % are likely to be encountered in test samples can be demonstrated.
lactic acid (filter sterilized by passing through a 0.2 μm filter,
9. Procedure
see 8.7) to adjust pH. Once acidified, the MEA shall not be
9.1 Sampling:
reheated. Make agar plates of the medium by pouring sufficient
9.1.1 Samples shall be drawn in accordance with Practice
MEA into sterile petri dishes to give a layer approximately 4
D 4057 as amplified by Hill.
mm thick. Allow to cool and set.
9.1.1.1 To reduce the risk of accidental contamination,
NOTE 5—MEA is available from various manufacturers in dehydrated
samples intended for viable microbial enumeration shall not be
form and in pre-poured plates with and without added antibiotic, either of
used for other tests until after they are no longer needed for
which may be used. When sterilizing MEA prepared from commercial
enumeration testing.
dehydrated media, follow the manufacturer’s instructions for sterilization.
9.1.1.2 It may not be possible to use aseptic technique under
Avoid overheating.
NOTE 6—Alternative media to MEA may be used, providing the ability
field conditions. To reduce risk of cross-contaminating
of any alternative medium to support comparable growth of yeast and
samples, sampling devices shall be rinsed with 70 % alcohol
molds that are likely to be encountered in test samples can be demon-
(ethanol, methanol, or isopropanol) to disinfect sample contact
strated.
surfaces before samples are drawn. All samples and devices
NOTE 7—Alternative antibiotics may be used providing their ability to
should be handled in such manner as to minimize the likeli-
hood of introducing microbial contaminants into the sample.
9.1.1.3 Microbial contaminant populations are dynamic.
“Reagent Chemicals, American Chemical Society Specifications,” American
Microbes within the sample may proliferate or die during the
Chemical Society, Washington DC. For suggestions on the testi
...
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