IP 613/14

Determination of the viable aerobic microbial content

of fuels and associated water - Thixotropic Gel Culture
Method.

1 Scope method and further information is provided in the

research report RR IP 612-2013 available from the
Energy Institute.
This standard describes a procedure for use
in the field or in a laboratory to quantify viable
aerobic microorganisms present as contaminants WARNING — The use of this standard may involve
in middle distillate fuels, gasolines, biofuel blends hazardous materials, operations and equipment.
and residual fuels and associated water. The This standard does not purport to address all of
procedure quantitatively assess the viable aerobic the safety problems associated with its use. It is
microbial content as microbial colony forming the responsibility of the user of this standard to
units (cfu) and determines whether the microbial establish appropriate health and safety practices
contamination in samples drawn from fuel tanks and determine the applicability of regulatory
and systems is absent or present at light, moderate limitations prior to use.
and heavy levels.
2 Normative reference
NOTE 1 The categorisation of light, moderate and heavy
levels of contamination will depend on the fuel type, the The following referenced document is
sampling location, the facility sampled and its specific indispensable for the application of this document;
operating circumstances. The method is intended to
the edition cited applies but is subject to revision
provide a tool for assessing whether fuel storage and
distribution facilities or end user fuel tanks are subject to
by the responsible body. The test is referenced in
microbial growth and alert fuel suppliers or users to the the Normative Reference below as an example of
potential for fuel quality or operational problems and/or an on-site microbiological test for fuel.
the requirement for preventative or remedial measures.
Microbiological tests are not intended to be used to Guidelines for the investigation of the
determine compliance with absolute fuel specifications microbial content of petroleum fuels and for
or limits. The implementation of specification limits for
the implementation of avoidance and remedial
microbiological contamination in fuels is generally not
appropriate and microbial contamination levels cannot
strategies, 2nd Edition, 2008, Energy Institute,
be used alone or directly to make inferences about fuel London, ISBN 978 0 85293 524 8.
quality or fitness for use. Further guidance can be found
in the normative reference and other industry guidance 3 Terms and definitions
documents.

For the purposes of this standard, the following

NOTE 2 The test method may also be used to analyse
terms and definitions apply.
other hydrocarbon, aqueous and non aqueous liquids
and products but these applications fall outside the
scope of this method. microorganisms
Organisms that are invisible to the naked eye. In
NOTE 3 The test method detects numbers of microbial the context of this standard, microorganisms are
colony forming units (cfu), using the same detection bacteria and fungi (yeasts and moulds) which are
parameter used in the laboratory standard procedure capable of growth under aerobic conditions in the
IP 385. However, whereas IP 385 provides separate culture medium provided in the test kit.
assessment of numbers of viable aerobic bacteria
cfu and numbers of viable fungal cfu, this procedure aerobic
provides a combined total count of viable aerobic In the context of this standard, aerobic, refers
bacteria and fungal cfu. to types of microorganisms which grow in the
presence of oxygen or to conditions where
NOTE 4 A laboratory study has been conducted to sufficient oxygen is present to enable growth of
obtain data for the estimation of precision of this test aerobic microorganisms.

613.1

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 AEROBIC MICROBIAL CONTENT OF FUELS AND WATER - THIXOTROPIC GEL CULTURE, IP 613

colony encountered in liquid fuels and other petroleum

A discreet visible aggregate of microorganisms products. The gel must liquefy on shaking to
which develops when a viable microorganism, enable dispersion of sample and reset on standing
or particle containing viable microorganisms, is to enable microbial colony formation and must
introduced into a gel based nutritive culture medium contain a growth indicator to enable visualisation
and reproduces there. A period of incubation is and distinction of microbial colonies within the
necessary to allow sufficient reproduction. The prescribed incubation period. The culture medium
test described utilizes a reactive compound which shall be aseptically dispensed in 16 ml aliquots into
shortens the time for colonies to become visible sterile 60 ml capacity, rectangular, transparent
and stains them so that they appear as red or glass bottles (6.1) capped and sealed.
purple spots.
NOTE The thixotropic gel based culture medium and
colony forming unit (cfu) all the apparatus listed in clause 6 are commercially
A viable microorganism or aggregate of viable available and a list of suppliers is available from the
Energy Institute.
microorganisms, which proliferate(s) in a culture
medium to produce a visible colony.
5.2 Propane-2-ol or industrial methylated spirit
culture medium - 70 % alcohol solution in water.
Solid, semi-solid or liquid preparations which
contain nutrients which support microbial growth, 6 Apparatus
and usually other chemical agents which may inhibit
or stimulate growth by specific microorganisms or 6.1 Rectangular, sterile transparent glass
which may indicate the presence of all or specific bottles fitted with a cap and seal - capacity
microorganisms. approximately 60 ml. The bottles shall be of
suitable optical quality to enable distinction and
viable counting of microbial colonies.
Capable of growth in a culture medium.
6.2 Disposable syringe, sterile and fuel
4 Principle compatible - capacity 1 ml graduated in 0,1 ml
units.
A measured sample of fuel or water is added to
a rectangular, transparent glass bottle containing 6.3 Disposable loop dispenser, sterile and fuel
a sterile, thixotropic gel based culture medium compatible - capacity 0,01 ml.
capable of sustaining the growth of a wide range
of microorganisms encountered in liquid fuels NOTE The loop dispenser is used for dispensing
and other petroleum products. The gel liquefies 0,01 ml of water or heavy/residual fuel.
when the bottle is shaken and the sample, and
any microorganisms in it are dispersed. The gel 6.4 Pipettes with or without disposable tips,
is allowed to re-set into a flat layer on one of the sterile and fuel compatible - capacity 0,1 ml to 1
larger sides of the bottle. The bottle is incubated ml (optional).
in the dark in this position. The gel contains
components which sustain the growth of viable 6.5 Pipettes with or without disposable tips,
microorganisms and the sample itself contributes sterile and fuel compatible - capacity 0,01 ml
additional nutrients. Viable microorganisms in the (optional).
sample grow into visible colonies, and a reactive
compound changes the colour of these colonies to NOTE Pipettes or pipette tips can be purchased pre-
red or purple such that they can be easily counted sterilised or can be sterilised as described in 8.2.
or their number estimated. The number of colonies
formed is considered in relation to the volume of 6.6 Incubator capable of operating at 25 °C
sample added to the test, and expressed as cfu/l ± 3 °C. The design of the incubator shall ensure
of fuel, or cfu/ml of water associated with fuel. the culture medium is not exposed to light during
incubation (optional).
5 Reagents and materials
6.7 Marker pen, permanent with fine or
5.1 Thixotropic gel based culture medium, medium tip and capable of writing on glass
sterile. Culture medium capable of sustaining (optional).
the growth of a wide range of microorganisms

613.2

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6.8 Container, sterile, transparent, and fuel in volume are taken to enable easier visual observation
compatible – capacity 10 ml to 150 ml (optional). of the sample for water, dirt, particulates and suspected
microbial growth. However, less than 1 ml of sample is
actually required for the test.
7 Sampling and sample handling
7.3 If fuel phase is to be tested (see 9.3), the
7.1 Samples shall be taken and handled container to which sample is transferred shall be
in accordance with the Guidelines for the filled sufficiently to enable the measuring devices
Investigation of the microbial content of petroleum to be inserted to a depth of approximately 3 cm
fuels and for the implementation of avoidance and below the surface of the fuel. If water phase is
remediation strategies. also to be tested (see 9.6), the container to which
sample is transferred shall enable ready access to
NOTE The numbers of viable microorganisms in a settled water in the bottom of the sample. The
fuel sample will be highly dependent on the sampling container shall be sterile and must be compatible
location and also whether free water is present in the
with the fuel sample.
sample; test results should be interpreted accordingly.
Usually, most microbial contamination will be present
in a tank bottom or system low point, particularly at 7.4 Once fuel samples have been taken, any
any fuel water interface and in free water settled or microorganisms present will usually slowly die
suspended in the fuel. For routine monitoring, testing and it is important to test samples as soon as
low point (e.g. dead bottom or drain) samples is possible, ideally within 48 hours. Samples will
advisable as these will provide the earliest and most give increasingly less reliable results as they get
consistent indication of tank or system contamination. older.
Testing representative fuel samples (e.g. upper, middle,
and lower layer samples) can provide some indication
of the extent of contamination in bulk fuel but results 8 Apparatus preparation
will be applicable only to the specific sample at the
time of sampling; microbial contamination in bulk
8.1 If the bottles of gel culture medium have
fuel is generally unevenly distributed and levels of
contamination detected at the same sampling location
been stored refrigerated, allow them to equilibrate
will vary with time. Contamination levels in bulk fuel to ambient temperature before they are used.
in storage tanks will generally decrease with product Remove the cap seal from the neck of the bottle.
settling and increase if tank bottoms are disturbed. Avoid prolonged exposure of the culture medium
to direct sunlight or other bright light at all times.
7.2 Sampling equipment and sampling valves
shall be clean and decontaminated by rinsing 8.2 If glass pipettes or pipette tips are to be
or wiping with a 70 % alcohol solution (5.2). used for taking the test portion from the sample
Ensure alcohol evaporates before taking samples; sterilise by autoclaving at 121 ºC ± 2 ºC for
residues of alcohol may interfere with the test and 15 min or by placing in an oven at 170 ºC ± 2 ºC
cause false negative results. Alternatively, use for 1 h. The specified sterilisation time does not
commercially available alcohol wipes. Particular include additional time which may be required to
attention shall be paid to the cleanliness of allow for containers to equilibrate to the specified
sampling points as these can harbour dirt and temperature. If they are autoclaved, ensure they
microbial contamination and cause false positive are dry before use.
test results.
8.3 If an incubator (6.6) is to be used ensure
NOTE 1 Wherever possible sterile sampling containers it is operating at 25 °C ± 3 °C.
should be used but if unavailable, it is sufficient to use
clean, previously unused containers.
9 Procedure
Rinse sampling equipment such as bottom
NOTE 1 Because free water phase may not always be
samplers, bacon bombs and all level samplers and recovered in samples, for consistency when conducting
containers with fuel from the tank or system to be routine microbiological monitoring it is recommended
sampled before taking the sample for test. When that the fuel phase from above any free water phase
sampling from drain lines, take the sample as present is always tested. In some circumstances, for
soon as the contents of the drain line have been example when investigating contamination sources
flushed away, sampling the first product/water to or assessing the extent of microbial growth in a tank
come from the tank or filter vessel. or system, it may be also informative to assess the
microbial content in any free water present in the
sample. Presence of discoloured water (brown or
NOTE 2 It is recommended that samples of about 1 litre
black), a lacy interface between the fuel and water

613.3

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 AEROBIC MICROBIAL CONTENT OF FUELS AND WATER - THIXOTROPIC GEL CULTURE, IP 613

layers or soft, organic debris in the fuel or water layer Place the cap on a clean surface.
are all indications of likely microbiological activity.
Water phase will usually contain substantially higher
9.3.3 Using a syringe (6.2) or an alternative
numbers of microbial cfu than fuel phase and a different
interpretation of results is required. measuring device (6.4), draw fuel from
approximately 3 cm below the surface of the fuel
phase of the sample. If there is less than 6 cm
NOTE 2 Although accuracy and minimum detection
levels can be improved by conducting replicate tests, depth of fuel, draw sample from approximately
the procedure described is designed for both field and halfway down the fuel phase. The transfer of
laboratory use and, in the field, any increase in accuracy visible interfacial particulate, water droplets
obtained by replication will usually be sacrificed for or emulsion in the aliquot to be tested shall be
operational expediency. avoided.

NOTE 3 If microorganisms other than those in the If using the syringe (6.2), open the sterile syringe
sample are introduced into the culture medium they pack at the handle end and remove the syringe
may give rise to spurious colonies. To avoid this, the taking care not to touch the lower barrel and
test should be conducted in as clean an environment as
nozzle. Draw fuel into the syringe until it is more
practicable and care should be taken to avoid touching
surfaces of dispensing apparatus, pipettes and sample than half full and then, with the syringe nozzle
containers which come into direct contact with the uppermost, expel air. Expel fuel surplus to the
sample or culture medium. volume needed for the test (see below).

9.1 Visually inspect the sample to identify if If using the loop dispenser (6.3), for testing heavy
water is present by holding the sample up to the and residual fuels only, open the peel pack at the
light and examine it for haze or lack of clarity. handle end and remove the dispenser taking care
Then swirl the sample to produce a vortex and not to touch the loop and lower part of the shaft.
examine both the bottom of the vortex and the
bottom of the sample container for water droplets Aviation kerosene fuels
and particulate matter. Record the visual clarity The volume to test is 0,5 ml. Dispense this volume
as clear and bright or not clear and bright. Record directly into the gel culture medium bottle.
whether water droplets or particulate matter
were, or were not, observed on swirling. Other middle distillate fuels and gasoline
The volume to test is 0,25 ml. Dispense this
9.2 If the sample is not in a container suitable volume directly into the gel culture medium bottle.
for visual inspection or does not enable use of the
measuring devices to remove an aliquot for test Heavy and residual fuels
in accordance with 9.3 for a fuel phase test or The volume to test is 0,01 ml. Use the syringe
9.6 for a water phase test, the sample shall be (6.2) to dispense a drop of fuel onto the loop
transferred to a suitable, sterile container. dispenser (6.3). Allow surplus fuel to drain away
but ensure that the circle of the loop is filled, and
NOTE 1 Borosilicate glass has been found to be suitable then stab it into the gel culture medium in the
bottle and agitate briefly to transfer the sample
NOTE 2 Containers can be sterilised as described in into the gel. Alternatively, use a pipette (6.5) to
clause 8.2. Alternatively, suitable disposable, sterile dispense 0,01 ml fuel directly into the gel culture
containers are commercially available. Some incorporate medium bottle.
a mechanism for separation of fuel and water phases.
NOTE 1 The volume of sample tested can be changed
9.3 Testing the fuel phase of samples in order to increase or decrease the detection level of
the test. However, the performance of the test has not
9.3.1 Immediately prior to analysis, shake the been validated for testing fuel volumes greater than
sample vigorously by hand for approximately those stated above and ideally these should not be
exceeded.
30 s and then allow to stand for 12 min ± 1 min.
If depth of the fuel phase in the sample is less
than 6 cm then allow a settling time of 2 min per The syringe and loop dispenser and other
cm. disposable measuring devices shall be used
only once and discarded after use. If re-useable
measuring devices are used, they shall be freshly
9.3.2 Immediately before dispensing fuel sample
cleaned and re-sterilised between each use.
remove the cap of a gel culture medium bottle; do
not touch the inside of the cap or bottle neck.

613.4

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9.3.4 Once the fuel has been dispensed into the temperature within the specified range. Occasional
gel culture medium bottle, replace and tighten the temperature fluctuations (e.g. overnight) below the
cap and proceed in accordance with 9.4. specified temperature range should not affect the
number of colonies which develop and will not critically
affect the test result, but microbial colonies may
NOTE 2 The prescribed settling time will typically take longer to become visible and thus an extension
enable any suspended free water to settle out from of the specified incubation time should be applied.
the top 6 cm of sample. In some cases, for example If temperature falls below the specified range during
where fuels are wet and visibly hazy, water phase may incubation, the incubation time should be extended by
not separate readily from the fuel phase and higher a time equivalent to the total time the temperature is
microbiological counts can be expected. Depending on estimated to have fallen below the specified range. If
the test objectives, an additional settling time may be the incubation temperature falls below the specified
applied to the sample prior to testing. However, the range for a period or periods totalling 4 days or more,
sample should always be tested as soon as possible microbial contamination will be underestimated and the
and within 48 h of the sample being drawn. result should be considered invalid. The temperature
of the culture medium should not be allowed to drop
9.4 Dispersing the test portion in the gel below 4°C.
culture medium
Incubation of the culture medium above the specified
9.4.1 Loosen and break up the gel culture temperature range may prejudice the ability to detect
medium in the bottle containing the dispensed some types of microorganism. However, where
microbial contamination occurs in tanks or systems
test portion by tapping the bottle firmly on the
where the contents have a higher temperature than
palm of the hand or on a large rubber bung or 25 °C, the contaminating microorganisms will often
thick rubber mat. Taking care not to break the have a preference for growth at higher temperatures, in
glass bottle. such cases it may be considered valid to incubate the
test at the temperature of the system sampled.
9.4.2 Shake the bottle vigorously for
approximately 30 s to liquefy the gel and disperse 9.5.2 If possible examine the culture medium
the test portion. daily during the incubation period. As a minimum,
examine it on at least one occasion in the first
NOTE 1 After shaking the gel it should be slightly 3 days of incubation and again on a final day of
viscous but free of lumps and have uniform clarity. The incubation. During each observation count visible
presence of air bubbles in the gel will not affect the red or purple colonies as they develop. Once a
test. colony is counted do not count it again even if
it grows larger; it is the number of colonies that
9.4.3 Flick the bottle so that the gel collects in are required, irrespective of their size. Avoid
the bottom of the bottle. Proceed immediately to agitating or shaking the bottle during incubation
9.4.4 before the gel resets. and examination.

9.4.4 Tap the bottle in the palm of the hand NOTE 2 It is recommended colonies are marked with
until the gel forms a flat layer on one of the larger a permanent marker pen (6.7) on the glass of the gel
flat bottle sides. Ensure a uniform layer of gel culture medium bottle to ensure that they are not
reaching all corners of the bottle is obtained. counted twice.

9.5 Incubating and examining the gel culture It is usually possible to count up to about 250
medium colonies. If the number of colonies is greater than
100, although a reasonable colony count can
usually be obtained, the colony count will exceed
9.5.1 Transfer the gel culture medium bottle to a
the range currently defined for quantitative
warm, dark location or incubator (6.6) to nominally
assessment of microbial content and consequently
maintain a temperature of 25 °C ± 3 °C, see
accuracy and precision may be low. If colonies
Note 1. Keep the gel on the lowest surface of the
cannot be counted, estimate the approximate
bottle. The gel will set firm after a few hours. It
colony count as per clause 9.5.3.
is important not to tilt the bottle or disturb the
gel during this time. In normal circumstances the
culture medium test shall be incubated for 4 days. 9.5.3 If the number of colonies is too numerous
to count, visually compare the test to the chart
NOTE 1 When conducting the test in the field it provided in Annex A, holding the test bottle
will not always be possible to maintain incubation against a white background.

613.5

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 AEROBIC MICROBIAL CONTENT OF FUELS AND WATER - THIXOTROPIC GEL CULTURE, IP 613

NOTE 3 The chart provides an estimated colony count are still small (e.g. after 1 or 2 days incubation) they
to the nearest factor of ten. The result can only be are more easily counted. The centre of each streak or
considered semi-quantitative as the number of colonies patch should be counted as a single colony. Continue
present exceeds the range considered acceptable for incubation and count any new colonies which develop.
fully quantitative assessment of microbial content.
SAFETY When examining the culture medium do
If samples have a very high viable microbial content, not open the bottles. Wash hands after handling the
individual colonies will not be distinguishable and bottles. Once the final examination of the culture
the whole gel becomes red or purple (see picture medium has been conducted the test bottle shall
in the chart in Annex A showing a test with be decontaminated by adding two disinfectant
≥10 000 colonies). In such cases the microbial pills to the test bottle and shaking vigorously for
content is equal to or exceeds the maximum 30 s or by immersing the open bottle and cap in a
detection level of the test. strong disinfectant solution. The bottle shall then
be left for 24 h before discarding in accordance
NOTE 4 Points to be considered when examining culture with local regulations.
medium for colonies:
9.6 Testing water phase of samples (optional)
Colonies are usually circular but may have irregular
edges. Different types of microorganisms can grow
at different rates in the gel culture medium and
9.6.1 If an assessment of microbial content of
therefore the colonies may be of different sizes. At free water phase is required, after completion of
the recommended incubation temperature, colonies of fuel phase analysis as described in 9.3 to 9.4,
bacteria and yeasts usually develop within 1 to 2 days allow the sample to stand until water phase has
and remain quite small. Moulds colonies develop more settled to the bottom of the sample. Use the
slowly but eventually produce large colonies which may syringe (6.2) or pipette (6.4) to remove water
have a powdery or ‘fuzzy’ appearance. Generally, the from the bottom of the sample and transfer to
more colonies there are in the test bottle, the smaller a separate small, sterile container (6.8). Avoid
the colonies will be. Colonies will tend to become visible
transferring any of the fuel with the water.
more quickly in samples with a higher viable microbial
content.
NOTE 1 To enable ready access to water phase, it may
be necessary to first decant off some fuel phase from
All red or purple colonies in all parts of the test bottle
the sample.
should be counted, including any which are in gel which
is not part of the flat layer formed as described in 9.4.4.
9.6.2 Once water has been removed from any
It can be useful to photograph tests at each time of fuel, invert the container containing the water
examination for subsequent reference when determining three times to homogenise the water prior to
final colony counts. sampling. Immediately before dispensing the
water sample remove the cap of a gel culture
Occasionally, some antioxidant additives in fuels may medium bottle, do not touch the inside of the cap
interfere with the growth indicating compound in the or bottle neck. Place the cap on a clean surface.
gel culture medium and produce a uniform light peach
or orange colour in the gel (usually within 12 h). This 9.6.3 As appropriate for the volume of water
colour change will not interfere with the growth of any
to be tested (see Note below), use the loop
microorganisms and in most cases microbial colonies
can be counted or estimated ignoring the background
dispenser (6.3), syringe (6.2) or pipette (6.4 or
colour. In exceptional cases the interference may be 6.5) to measure the required volume of water to
so strong that users may find it difficult to distinguish test.
colour interference from an estimated count of 10 000
colonies. In such cases the fuel should be retested NOTE 2 The usual volume of water to test is 0,01 ml.
using a smaller test volume (e.g. 0,01 ml) so that the The volume of water sample tested can be changed
interference effected is diluted out; if the original result in order to increase or decrease the detection level of
was genuinely due to heavy microbial contamination it the test. The test may be used to test water volumes
would normally be expected that the retest will show up to 1 ml. For example, for water present in samples
a discernable number of red/purple colonies and the from aircraft fuel tank drains it may be appropriate to
background colour will be less intense. test 0,1 ml of water to improve the detection level of
the test because under normal circumstances, water in
Some bacteria are motile and can, on prolonged aircraft tanks is clean condensate water and will have a
incubation, spread through the gel producing a large lower microbial count than water in other fuel facilities.
irregularly shaped colony, streak or patch of red or
purple colour in the gel. These bacteria usually grow
quickly and thus if tests are examined while the colonies

613.6

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 AEROBIC MICROBIAL CONTENT OF FUELS AND WATER - THIXOTROPIC GEL CULTURE, IP 613

9.6.4 If using the loop dispenser (6.3) open where

the peel pack at the handle end and remove the
dispenser taking care not to touch the loop and C Number of colonies counted or estimated
lower part of the shaft. Immerse the loop in the
V Volume of sample tested in millilitres
water and withdraw. Alternatively use a syringe
(6.2) to withdraw some of the water and dispense
a drop of water onto the loop dispenser. Allow 10.2 Water phase test portion
surplus water to drain away but ensure that the
circle of the loop is filled. Immediately stab the Calculate the number of microbial cfu/ml of water
loop into the gel culture medium and agitate to using the following equation:
transfer the test portion into the gel.
Number of microbial cfu/ml = C
9.6.5 If using the syringe (6.2) to take larger V
volumes of water, 0,1 ml to 1 ml, open the sterile
syringe pack at the handle end and remove the where
syringe taking care not to touch the lower barrel
and nozzle. Draw just over the required volume of C Number of colonies counted or estimated
water into the syringe and then, with the syringe V Volume of sample tested in millilitres
nozzle uppermost, expel air and water surplus
to the volume needed for the test. Dispense the
required measured volume directly into the gel 11 Expression of Results
culture medium bottle.
11.1 Results derived from a test where the
9.6.6 Once water has been dispensed into the number of colonies was counted
gel culture medium bottle, replace and tighten the
cap and proceed in accordance with clause 9.4 Report the result as the viable aerobic microbial
and 9.5. content in microbial colony forming units per litre
(cfu/l) of fuel or microbial colony forming units per
NOTE 3 The syringe and loop dispenser (6.2 and 6.3) millilitre (cfu/ml) of water to the nearest whole
and other disposable measuring devices should be number. If recommended test volumes are used
used once only and discarded after use. If re-useable results will be calculated to the nearest number of
measuring devices are used, they should be freshly colony forming units shown in Table 1 and shall
cleaned and re-sterilised between each use. be reported as such.

10 Calculation Table 1 Readability of test for recommended

sample types and volume tested
Calculate the number of microbial cfu/l of fuel in
accordance with clause 10.1 and/or the number Sample type Volume Report result
of microbial cfu/ml of water in accordance with tested (ml) to the nearest
clause 10.2.
Aviation kerosene 0,5 2 000 cfu/l
fuels
If the number of colonies is estimated by visually
comparing to the chart provided in Annex A only Other middle 0,25 4 000 cfu/l
a semi-quantitative estimate of the colony count distillate fuels and
is obtained. For commonly used test volumes (0,5 gasolines
ml, 0,25 ml and 0,01 ml), the chart provides the Heavy residual 0,01 100 000 cfu/l
corresponding approximate number of microbial fuels
cfu/l of fuel or microbial cfu/ml of water based on Water associated 0,01 100 cfu/ml
the equations shown below. with fuel

10.1 Fuel phase test portion If the number of colonies counted was greater
than 100, it shall be stated on the test report that
Calculate the number of microbial cfu/l of fuel the colony count exceeded the range currently
using the following equation: defined for quantitative assessment of microbial
content by the test method and that accuracy and
Number of microbial cfu/l = C x 1 000 precision may be low.
V

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 AEROBIC MICROBIAL CONTENT OF FUELS AND WATER - THIXOTROPIC GEL CULTURE, IP 613

Report the volume of sample tested and the Report the volume of sample tested and the
conditions of test incubation, including total conditions of test incubation, including total
incubation time and, as far as is known, incubation time and, as far as is known,
the nominal incubation temperature and any the nominal incubation temperature and any
fluctuation outside the range specified. fluctuation outside the range specified.

Report the visual appearance of the original Report the visual appearance of the original
sample prior to any separation. sample prior to any separation.

It shall be stated on the test report, if testing was It shall be stated on the test report, if testing was
commenced more than 48 h after samples were commenced more than 48 h after samples were
taken. If the test is carried out after 48 h it is taken. If the test is carried out after 48 h it is
possible that the results will not reflect the viable possible that the results will not reflect the viable
microbial content present at time of sampling. microbial content present at time of sampling.

11.2 Results derived from a test where the 12 Test report

number of colonies was estimated
The test report shall contain at least the following
If the result was derived from a test where the information,
number of colonies was estimated (see clause
9.5.3) using the chart provided in Annex A, report a) a reference to this Standard;
the result as the viable aerobic microbial content in
approximate number of microbial colony forming b) the type and complete identification of
units per litre (cfu/l) of fuel or approximate number the product tested;
microbial colony forming units per millilitre (cfu/
ml) of water to one significant figure in scientific c) the result of the test (see clause 11);
notation to the nearest power of ten, as shown
d) any deviation, by agreement or otherwise,
in the right hand column of the chart (e.g.
from the procedure specified;
approximately 106 cfu/l of fuel).
e) the date the test was commenced (i.e.
It shall be stated in the test report that the result sample added to test bottle) and completed
is a semi-quantitative estimate as colonies were (i.e. final examination after incubation);
too numerous to count.
f) the time and date the sample was taken;

g) the type of facility or equipment tested;

h) the location of the sampling point and the

sampling method.

613.8

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IMPORTANT: This document is subject to a licence agreement issued by the Energy Institute, London, UK. It may only be used in accordance with the licence terms and conditions. It must not be forwarded to, or stored, or accessed by, any unauthorised user. Enquiries: e:pubs@energyinst.org t:
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 AEROBIC MICROBIAL CONTENT OF FUELS AND WATER - THIXOTROPIC GEL CULTURE, IP 613

Annex A
(normative)
Chart for the visual estimation of the numbers of colonies
Fully quantitative range of detection
Count exact number of colonies
No. of colonies Calculation of microbial content
counted
Calculate the microbial content using the
appropriate equation for fuel or water
No colonies sample:

Number of microbial cfu/l of fuel =

C x 1 000
V
OR
Number of microbial cfu/ml of water =
Count exact number C
of colonies V
(10 shown)
Where:
C Number of colonies counted or
estimated
V Volume of sample tested in millilitres
If possible, count
exact number of
colonies
(100 shown)
Semi-quantitative range of detection
Estimate number of colonies by comparison to pictures
Approx.
No. of colonies
Volume tested microbial
estimated
content
0,25 ml or 0,5 ml fuel c. 105 cfu/l
c. 100 colonies 0,01 ml of fuel* c. 107 cfu/l
0,01 ml of water c. 104 cfu/ml

0,25 ml or 0,5 ml fuel c. 106 cfu/l

c. 1 000 colonies 0,01 ml of fuel* c. 108 cfu/l

0,01 ml of water c. 105 cfu/ml

0,25 ml or 0,5 ml fuel ≥ c. 107 cfu/l

≥ c. 10 000
0,01 ml of fuel* ≥ c. 109 cfu/l
colonies

0,01 ml of water ≥ c. 106 cfu/ml

* 0,01 ml is normally only used for heavy residual fuels

613.9

This document is issued with a single user licence to the EI registered subscriber: qa@ccicsg.com
IMPORTANT: This document is subject to a licence agreement issued by the Energy Institute, London, UK. It may only be used in accordance with the licence terms and conditions. It must not be forwarded to, or stored, or accessed by, any unauthorised user. Enquiries: e:pubs@energyinst.org t:
+44 (0)207 467 7100