echa MICROBMONITOR2 User manual
For Detection and Enumeration of Contaminating Microbes
in Fuels, Lubricants and Water
Instructions for Use
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Important Notice
Please read the Instructions for Use carefully before testing samples.
The test kit should only be used as part of an investigatory process into contamination in petroleum products and associated water. The
test kit must be used strictly in accordance with these instructions and/or Standard Test Method IP613 or ASTM D7978 or other instructions
authorised by ECHA Microbiology Ltd. The results only relate to the portion of sample tested and not necessarily to other petroleum product
in the system. Although guidance may be sought by the user on sampling and the interpretation of results, the responsibility for carrying out
the sampling and test procedures correctly is that of the end-user and not ECHA Microbiology Ltd. The test kit is designed to detect a
recognized group of micro-organisms of industrial significance, but it is in the nature of microbiology that there may be micro-organisms
present which are not detected by the test procedure. ECHA Microbiology Ltd does not accept any liability for any decision or assessment
taken or made as a consequence of the results obtained.
ECHA Microbiology Ltd, Units 22 & 23 Willowbrook Technology Park, Llandogo Road, St Mellons, Cardiff, UK, CF3 0EF
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MICROBMONITOR2®
WHAT IS THE MICROBMONITOR2®TEST?
MicrobMonitor2 is a simple and easy to use test kit which enables the quantitative assessment of the viable microbial
content in petroleum products including liquid fuels, oils and associated water. MicrobMonitor2 enables on-site or
laboratory testing in accordance with ASTM and IP Standard Methods ASTM D7978 and IP613 - Determination of the viable
aerobic microbial content of fuels and associated water - Thixotropic Gel Culture Method. MicrobMonitor2 is
recommended in the IATA Guidance Material on Microbiological Contamination in Aircraft Fuel Tanks for monitoring
microbial contamination in aircraft fuel tanks and is listed in the Aircraft Maintenance Manuals of Airbus, Boeing, BAE
Systems and other leading aircraft manufacturers. The test is used by a wide range of military services for monitoring
contamination in aviation, marine and ground fuels and is NATO Codified (6640-99-834-3573).
MicrobMonitor2 provides a total count of microbial colony forming units (CFU). CFUs are the conventional unit of measure
for microbial contamination used in a wide range of industries. MicrobMonitor2 detects the important bacteria, yeasts and
moulds which can contaminate petroleum products including Hormoconis resinae, Aspergillus, Candida and Pseudomonas
species.
The MicrobMonitor2 test consists of a screw-capped bottle containing a thixotropic nutrient gel. A sample is added to the gel
using a sterile plastic loop or syringe (provided). The bottle is shaken and the gel liquefies and the sample, and any
microorganisms in it are dispersed. The gel is re-set into a flat layer and the bottle is incubated in the dark. The gel
contains components which sustain the growth of viable microorganisms and the sample itself contributes additional
nutrients. Viable microorganisms in the sample grow into visible spots known as “colonies”, and a reactive compound
changes the colour of these colonies to red or purple so that they can be easily seen.
The number of colonies formed is a direct estimate of the number of viable microbial particles (CFU) present in the sample. The
actual number of colonies is counted or is estimated by reference to the Test Results Chart (page 9). The volume tested can
be 0.01 ml (using a sterile loop dispenser) or between 0.1 ml and 0.5 ml (using a sterile syringe). The number of colonies formed
is considered in relation to the volume of sample added to the test and expressed as CFU/litre of fuel, or CFU/ml of oil or
water associated with fuel.
MicrobMonitor2 is available in packs of 5 and 44 tests, each containing everything that is required to perform the test:
MicrobMonitor2 test bottles
Sterile syringes*
Sterile loop dispensers*
Bottle labels
This instruction leaflet.
* The 44 pack is available with or without sterile syringes and loop dispensers.
MicrobMonitor2 has been developed and patented by ECHA Microbiology Ltd. and it is marketed worldwide by
ECHA and its distributors.
WHAT IS THE MICROBMONITOR2®TEST USED FOR?
The MicrobMonitor2 is used to test for microbial contamination in fuel and oil products, and to monitor microbial growth in
storage tanks and supply facilities. MicrobMonitor2 can be used as a routine monitoring tool, for providing assurances
about product quality, for investigation of incidents and for confirming effectiveness of measures taken to prevent or
remediate microbial growth and contamination.
This instruction leaflet provides general instructions for using MicrobMonitor2. Further technical instruction on the use of
MicrobMonitor2 in specific applications is provided in the technical application leaflets listed below (free to download
from www.echamicrobiology.com). These leaflets include advice on sampling, sample preparation, interpretation of test
results and appropriate actions when contamination is detected:
EP096 How to Interpret MicrobMonitor2 Test Results in Accordance with IATA Guidelines for Aircraft Drain Samples.
EP119 Routine Monitoring of Aviation Fuels in Supply and Distribution Facilities, Airport Depots and Into-plane
Operations with MicrobMonitor2.
EP132 Routine Monitoring of Diesel Fuel Tanks and Distribution Systems with MicrobMonitor2.
EP166 Routine Monitoring of Marine Diesel on Ships and Offshore Installations with MicrobMonitor2 .
EP157 Technical Assistance Reading Results of MicrobMonitor2.
2
Sample & test details
MICROBMONITOR2®test results
Colonies
counted
Day 1
Colonies
counted
Day 2
Colonies
Counted
Day 3
Colonies
Counted
Day 4
Colonies
Counted
Day _______
Microbial
Contamination in
sample
CFU/ litre or CFU/ ml
Sample Ref.
Date tested
Type
Location
Volume tested (ml) 0.5/0.25/0.1/0.01/other__
Incubation Temp. (°C)
Sample Ref.
Date tested
Type
Location
Volume tested (ml) 0.5/0.25/0.1/0.01/other__
Incubation Temp. (°C)
Sample Ref.
Date tested
Type
Location
Volume tested (ml) 0.5/0.25/0.1/0.01/other__
Incubation Temp. (°C)
Sample Ref.
Date tested
Type
Location
Volume tested (ml) 0.5/0.25/0.1/0.01/other__
Incubation Temp. (°C)
Sample Ref.
Date tested
Type
Location
Volume tested (ml) 0.5/0.25/0.1/0.01/other__
Incubation Temp. (°C)
11
MICROBMONITOR2®
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Table 1
Sample Recommended test
volume Measuring
device
Aviation kerosene 0.5 ml
Syringe
Other middle distillate fuels (e.g. diesel, marine gas oil, heating oil), biofuels &
gasoline 0.25 ml
Water phase from aircraft fuel tank drains 0.1 ml
Heavy and residual fuels 0.01 ml
Loop
Lubricating, hydraulic and other oils 0.01 ml
Water phase from fuel storage tanks 0.01 ml
PREPARATION FOR TESTING
Sampling considerations
Sampling should be conducted in a clean and consistent manner which prevents introduction of background contamination
into the sample. Once fuel samples have been taken, any microbes present will tend to slowly die and it is important to
test samples as soon as possible. If the test is carried out more than 24 h after sampling, it is possible that the results will not
reflect the viable microbial content present at the time of sampling. To avoid this, part or all of the MicrobMonitor2 test
procedure can be conducted at the sampling location.
Sample type and recommended test volumes
MicrobMonitor2 can be used to test a wide variety of petroleum products and associated water. The volume of sample
which should be tested will depend on the type of sample. Refer to the table below to determine the appropriate
volume of sample to test and which measuring device to use.
The volume of sample tested can be changed in order to increase or decrease the detection level of the test. However,
testing volumes greater than those recommended above may in some circumstances lead to underestimation of microbial
numbers.
Determining whether to test fuel/oil or water phase
Depending on the system sampled and the sampling location, samples may contain fuel/oil and/or free water phase.
MicrobMonitor2 can be used to test either fuel/oil or associated water. Water phase will usually contain far more
microorganisms than are present in fuel or oil phase and therefore a smaller volume is usually tested. Because free water
phase may not always be recovered in samples, to ensure consistency when conducting regular microbiological
monitoring, it is usually recommended that fuel or oil phase from above any free water is tested. However, in some cases
it is recommended to additionally test water phase (e.g. samples from aircraft fuel tanks, and when investigating
suspected microbial growth in facilities). Further information is provided in the relevant technical guidance documents
listed on page 2.
To determine if the sample contains free water hold it up to the light and visually examine it. It may help to gently swirl the
sample to create a vortex. In order to enable access to the sample with the measuring devices provided, it may be
necessary to transfer some sample, after shaking, to a smaller clean container.
Preparing the MICROBMONITOR2®test bottles
If the MicrobMonitor2 test bottles have been stored refrigerated, allow them to equilibrate to ambient temperature before
they are used. Remove the seal from the cap of the bottle before using each MicrobMonitor2 test. Avoid prolonged
exposure of the MicrobMonitor2 test bottles to direct sunlight or other bright light during preparation, incubation and
examination of tests.
Cleanliness during testing
MicrobMonitor2 tests can be conducted on-site, in an office or in a laboratory. However, precautions should be taken to
ensure testing is conducted in a reasonably clean area to avoid contamination of the sample and the test by
microorganisms other than those in the test sample. Wash hands before and after testing. During testing wear clean, nitrile,
vinyl or polythene gloves. Avoid touching areas of the syringe and loop dispenser which come into direct contact with the
sample and avoid touching the inside of the MicrobMonitor2 test bottle cap and neck when dispensing sample.
DISPOSAL
Wear gloves and avoid touching the inside of the bottle or cap and wash hands after handling any MicrobMonitor2 test
bottles showing microbial growth. Before disposal, disinfect all MicrobMonitor2 test bottles which show growth by
immersion of the opened bottles in a strong disinfectant solution (e.g. household bleach) overnight or by incineration or by
using chlorine release tablets (available separately).
Decontaminated tests, unused tests or those showing no microbial growth can be disposed of as normal waste in
accordance with local waste regulations.
STORAGE AND SHELF LIFE
The shelf life of MicrobMonitor2 is 1 year from the date of production. The expiry date is printed on the batch label of each
MicrobMonitor2 box. Store MicrobMonitor2 tests between 2 to 22°C, in the dark. The expiry date applies only to product
stored as stipulated. If refrigerated (2 - 8°C) the MicrobMonitor2 test will usually still be useable for up to a year beyond the
designated expiry date. Do not store MicrobMonitor2 frozen. Exposure to freezing temperatures during transport is
acceptable. Avoid exposure of MicrobMonitor2 tests to light during storage and use. A slightly pink discolouration in the
MicrobMonitor2 gel may develop over time during storage but this will not affect the performance of the test.
ALSO AVAILABLE FROM ECHA
For use with MICROBMONITOR2®
The following items can assist in performing the MicrobMonitor2 test procedure:
MicrobMonitor Sampling Kit (ECHA16/SB) - A sterile, polypropylene bottle for sampling fuel systems and which
enables easy separation of water phase from fuel phase; includes alcohol wipes for decontaminating sample
points.
Incubator (ECHA14/IN) - enables optimal, consistent incubation temperature of MicrobMonitor2 tests; holds 12
MicrobMonitor2 test bottles; available battery or mains (110/230 Volt) operated.
MicrobMonitor Bumpers (ECHA16/TB) - soft, rubber-like, hemispheres which can be stuck to a table top and enable
easy breaking and shaking of MicrobMonitor2 gel.
Chlorine Release tablets (ECHA21/CP) - to disinfect MicrobMonitor2 test bottles showing microbial growth for easy
disposal.
Sterile swabs (ECHA15/SW) - swabs for sampling surface contamination (e.g. tank surfaces, filter cartridges etc.).
Long, sterile bulb pipettes (ECHA07/PP/EL23) - to enable water to be removed from the bottom of samples.
Other Test Kits & Ancillary Items
SigTests®: Sig Sulphide (ECHA02/SS) — simple test kit for Sulphate Reducing Bacteria (SRB) which can cause serious
corrosion and sulphide spoilage in fuel, oil and water systems. Can be used on-site or in the laboratory.
Biocide Rapide (ECHA01/BR) — simple rapid test kit for assessing presence and approximate concentration of
commonly used fuel and oil biocides.
Any level and bottom sampling devices (ECHA23) — for safe sampling from fuel and oil storage tanks in
compliance with industry standards.
For our full range of test kits and ancillaries see www.echamicrobiology.com.
Technical support and services
ECHA Microbiology Ltd. provides a full technical service to support our product range. ECHA also offers laboratory testing,
training courses, consultancy and on-site attendance for audit and investigation of microbial contamination and corrosion
associated with petroleum products and petroleum, marine, aviation and other industrial facilities.
MICROBMONITOR2®
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A.1 Aviation kerosene, other middle distillate fuels and gasoline.
A.1.1 Immediately prior to testing, shake the sample for 30 seconds. If fuel samples contain
visible free water, the sample should be allowed to stand for 12 minutes to enable water to
settle from the sample to be tested. For consistency, it may be advisable to apply this
settling time for all fuel samples even if free water is not observed.
A.1.2 Remove the cap of the MicrobMonitor2 test bottle taking care not to touch the inside
and place it on a clean surface.
A.1.3 Open the peel pack containing a syringe at the handle end and remove the
syringe taking care not to touch the lower barrel and nozzle.
A.1.4 Using the syringe, draw fuel from approximately 3 cm below the surface of the
sample. If there is less than 6 cm depth of fuel phase, draw sample from approximately
halfway down the fuel phase. Avoid the transfer of visible interfacial particulate, water
droplets or emulsion in the aliquot to be tested.
A.1.5 Draw fuel into the syringe until it is more than half full and then, with the syringe nozzle
uppermost, press the plunger to expel air. Expel surplus fuel until the end of the plunger
aligns with the mark for the required volume of sample (see table 1).
A.1.6 Insert the tip of the syringe into the neck of the MicrobMonitor2 test bottle and
dispense the sample by fully depressing the syringe plunger.
A.1.7 Replace the cap on the MicrobMonitor2 bottle and tighten. Record the sample
details and test date on the label provided and stick it on a narrow side of the bottle.
Proceed to step B.
TEST PROCEDURE
A. Add sample to the MICROBMONITOR2®test bottle.
Follow the appropriate steps below to add distillate fuel or biofuel (A.1), heavy/residual fuel or oil (A.2), water (A.3)
or surfaces deposits (A.4) to the MicrobMonitor2 test bottle.
MICROBMONITOR2®Test Results Chart
Appearance after incubation Number of colonies in
test bottle
(counted or estimated) Volume tested Microbial Content of
Sample
None
0.5 ml of fuel (syringe) <2000 CFU per litre
0.25 ml of fuel (syringe) <4000 CFU per litre
0.1 ml of water (syringe) <10 CFU per ml
0.01 ml of water or oil
(loop dispenser) <100 CFU per ml
10 Colonies
If possible, count the exact
number of colonies and
calculate the actual number
of CFU present in the sample
0.5 ml of fuel (syringe) 2 x 104CFU per litre
0.25 ml of fuel (syringe) 4 x 104CFU per litre
0.1 ml of water (syringe) 100CFU per ml
0.01 ml of water or oil
(loop dispenser) 1000 CFU per ml
100 Colonies
If possible, count the exact
number of colonies and
calculate the actual number
of CFU present in the sample
or
Estimate 100 colonies if
result is similar to picture
0.5 ml of fuel (syringe) c. 105CFU per litre
0.25 ml of fuel (syringe) c. 105CFU per litre
0.1 ml of water (syringe) c. 103CFU per ml
0.01 ml of water or oil
(loop dispenser) c. 104CFU per ml
Estimate 1000 colonies if
result is similar to picture
0.5 ml of fuel (syringe) c. 106CFU per litre
0.25 ml of fuel (syringe) c. 106CFU per litre
0.1 ml of water (syringe) c.104CFU per ml
0.01 ml of water or oil
(loop dispenser) c.105CFU per ml
Estimate 10,000 or more
colonies if result similar to
picture
0.5 ml of fuel (syringe) c.107CFU per litre or
above
0.25 ml of fuel (syringe) c.107CFU per litre or
above
0.1 ml of water (syringe) c.105CFU per ml or above
0.01 ml of water or oil
(loop dispenser) c.106CFU per ml or above
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A.2 Heavy and residual fuels and oils.
A.2.1 Immediately prior to testing, shake the sample for 30 seconds.
A.2.2 Remove the cap of the MicrobMonitor2test bottle and place it on a clean surface.
A.2.3 Open the peel pack containing a loop dispenser and remove it taking care not to touch the loop end and the
lower part of the handle.
A.2.4 Immerse the loop end into the fuel/oil to approximately 3 cm below the surface of the sample or, if there is less than 6
cm depth of fuel/oil phase, to halfway down the fuel/oil phase. Remove the loop from the sample and allow surplus fuel/oil
to drain off; ensure a film of fuel/oil is trapped within the loop.
A.2.5 Insert the loop into the neck of the MicrobMonitor2 test bottle and stab it into the gel and agitate it briefly to transfer
the sample.
A.2.6 Replace the cap on the MicrobMonitor2 bottle and tighten. Record the sample details and test date on the label
provided and stick it on a narrow side of the bottle. Proceed to step B.
A.3 Water phase associated with fuel/oil.
A.3.1 If a test of water phase is required, allow the sample to stand until free water has settled to the bottom of the sample. It
is recommended the water phase test is conducted after any fuel/oil phase test. The test procedure is essentially the same as
described above but an appropriate technique for separating/removing water from the sample needs to be used.
A.3.2 As appropriate for the volume of sample being tested (see table 1) use the loop dispenser or syringe to measure the
required volume of water.
a) To test 0.1 ml of water using the syringe;
The water phase can be drawn directly from the bottom of the sample and added to the MicrobMonitor2 test bottle. To
enable easy access to the water it may be necessary to first decant off some fuel from the sample and/or transfer the water
phase to a separate small, sterile container using a syringe or a long sterile pipette (supplied separately). Mix gently by
swirling but avoid mixing the fuel and water layers together.
Open the peel pack containing a syringe at the handle end and remove the syringe taking care not to touch the lower
barrel and nozzle. Insert the barrel of the syringe into the sample so that the nozzle is in the water phase. Draw water into
the syringe, remove from the sample and then, with the syringe nozzle uppermost, expel air. Expel surplus water until the end
of the plunger aligns with the 0.1 ml mark. Insert the tip of the syringe into the neck of the MicrobMonitor2 test bottle and
dispense the sample by fully depressing the syringe plunger. Replace the cap on the MicrobMonitor2 bottle and tighten.
Record the sample details and test date on the label provided and stick it on a narrow side of the bottle. Proceed to step
B.
b) To test 0.01 ml of water using the loop dispenser;
A syringe can be used to draw water from the bottom of the sample and then a drop of water placed into the loop and
added to the MicrobMonitor2 test bottle. Alternatively, transfer the water phase to a separate small, sterile container using a
syringe or a long sterile pipette (supplied separately). Avoid transfer of fuel. Invert the container with the separated water
three times to mix.
Remove the cap of the MicrobMonitor2 test bottle and place it on a clean surface. Open the peel pack containing a loop
dispenser and remove it taking care not to touch the loop end and the lower part of the handle. Immerse the loop end
into the separated water or fill the loop with a drop of water using a syringe. Allow surplus water to drain away but ensure the
circle of the loop remains filled with water; note, if a residue of fuel is present this can impair the ability of the loop to fill with
water. Insert the loop into the neck of the MicrobMonitor2 test bottle and stab it into the gel and agitate it briefly to transfer
the sample. Replace the cap on the MicrobMonitor2 bottle and tighten. Record the sample details and test date on the
label provided and stick it on a narrow side of the bottle. Proceed to step B.
Note The MicrobMonitor Sampling Kit (supplied separately) is a sample bottle which has a side siphon and water collection
chamber to enable easy separation of water from fuel samples.
a) Results of test of fuel phase sample:
If 0.5 ml of fuel has been tested, multiply the number of colonies counted or estimated by 2000 to give the number of
microbial CFU per litre of fuel. If there are no colonies after the specified incubation time there are less than 2000
microbial CFU per litre of fuel.
If 0.25 ml of fuel has been tested, multiply the number of colonies counted or estimated by 4000 to give the number of
microbial CFU per litre of fuel. If there are no colonies after the specified incubation time there are less than 4000
microbial CFU per litre of fuel.
If a different volume of fuel has been tested, then a calculation can be made as follows:
Number of microbial CFU per litre = Number of colonies counted or estimated x 1000
Volume of fuel tested (ml)
b) Results of test of water phase or oil:
If 0.1 ml of water has been tested, multiply the number of colonies counted or estimated by 10 to give the number of
microbial CFU per ml of water. If there are no colonies after the specified incubation time there are less than 10 microbial
CFU per ml of water.
If 0.01 ml of water or oil has been tested, multiply the number of colonies counted or estimated by 100 to give the number
of microbial CFU per ml of water or oil. If there are no colonies after the specified incubation time there are less than 100
microbial CFU per ml of water or oil.
If a different volume of water or oil has been tested, then a calculation can be made as follows:
Number of microbial CFU per ml = Number of colonies counted or estimated
Volume of water or oil tested (ml)
Note
If the number of colonies is too numerous to count and a sample volume of 0.5 ml or 0.25 ml of fuel or 0.1 ml or 0.01 ml
of water or oil was tested, the Test Results Chart can be used directly to determine the approximate number of
microbial CFU per litre of fuel or per ml of water or oil.
E Calculate the number of microbial CFU in the sample.
For tests of fuel, numbers of microbes are conventionally expressed as number of CFU per litre.
For tests of water phase or oil, numbers of microbes are conventionally expressed as number of CFU per ml.
INTERPRETATION OF TEST RESULTS
There are no universally accepted limits or specifications for microbiological contamination in fuels or oils. The number of
microbial CFU which define moderate and heavy contamination levels will depend on a number of factors including the
fuel type and intended use, the sampling location, whether the sample is taken from supply and distribution facilities or
from point of end use, the intended fuel storage time and specific operational circumstances. Some guidance limits are
shown in Table 2 below. These limits are intended to provide early indication that microbial growth is occurring in the
facility sampled. Higher levels of contamination will often be required before the onset of operational problems or any
detrimental effect on fuel fitness for use. For further information see the technical guidance leaflets listed on page 2.
Table 2
Sample Moderate Heavy Comment
Fuel from aircraft fuel tank drain 4000 to 20,000 CFU / litre >20,000 CFU / litre IATA
Contamination
Limits
Water from aircraft fuel tank drain 1000 to 10,000 CFU / ml >10,000 CFU / ml
Bulk representative fuel sample from
supply & distribution 4000 to 20,000 CFU / litre >20,000 CFU / ml e.g. Aviation fuel,
diesel, marine gas
oil
Fuel from bottom / drain of supply &
distribution tank / filter 10,000 to 100,000 CFU / litre >100,000 CFU / litre
Water phase from bottom / drain of
supply & distribution fuel tank / filter 100,000 to 1,000,000 CFU / ml >1,000,000 CFU / ml
Lubricating / hydraulic oil in circulation 100 to 10,000 CFU / ml >10,000 CFU / ml
Lubricating / hydraulic oil sump 1000 to 10,000 CFU / ml >10,000 CFU / ml
MICROBMONITOR2®
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A.4 Testing surfaces.
A.4.1 MicrobMonitor2can be used to test for microbial contamination on surfaces, e.g. to test deposits on tank surfaces or
filters. Sterile swabs (available separately) should be used to remove surface contamination and transfer this to a
MicrobMonitor2test bottle.
A.4.2 Open the peel pack containing a swab at the handle end and remove the swab, taking care not to touch the
cotton end of the swab and the handle near the cotton end.
A.4.3 Rub the surface to be tested using the cotton end of the swab, rotating the swab handle so that all parts of the
cotton end contact the surface. If possible swab a known area of surface.
A.4.4 Insert the cotton end of the swab into the neck of the MicrobMonitor2 test bottle and stab it into the gel and agitate
for about 15 seconds and then remove and discard.
A.4.5 Replace the cap on the MicrobMonitor2 bottle and tighten. Record the sample details and test date on the label
provided and stick it on a narrow side of the bottle. Proceed to step B.
7
6
B.1 Loosen and break up the gel in the MicrobMonitor2bottle containing the dispensed
sample, by tapping the bottle firmly in the palm of your hand or on a rubber bung or
MicrobMonitor “Bumper” (available separately).
B.2 Shake the bottle vigorously for approximately 30 seconds to liquefy the gel and
disperse the sample. The test bottle can be tapped repeatedly for 30 seconds on a
MicrobMonitor “Bumper” to assist with shaking. After shaking, the gel should be slightly
viscous but free of lumps and have a uniform consistency and clarity. The presence of
bubbles in the gel is normal and will not affect the test.
B.3 Stop shaking the test bottle abruptly so that the gel collects in the bottom of the
bottle. Proceed immediately to B4.
B.4 Tap the bottle in the palm of the hand until the gel forms a flat layer on one of
the larger flat sides. Ensure that a uniform layer reaching all corners is obtained.
Notes on transport of tests
It is preferable to conduct testing on-site to avoid errors due to changes in the microbial content of samples during transport. If there are
likely to be delays of more than 24 hours in transporting samples to a testing facility, these errors can be avoided by conducting the first
stages of the MicrobMonitor2 test procedure (up to and including step B) on-site at the sampling location and then returning the
MicrobMonitor2test to the testing facility to complete the incubation (step C). The test bottle should be kept flat during transport, should
not be agitated excessively and should be returned to the testing facility within 4 days. See note on incubation of tests (step C)
concerning appropriate adjustment of the incubation time.
Alternatively, MicrobMonitor2 test bottles can be transported to the test facility after sample has been added (step A) before shaking; the
test can then be shaken (step B) at the test facility. Incubation (step C) should be commenced within 6 hours of the sample being added
to the test bottle (or within 2 days if tests are kept cool (2 to 8°C)). If tests are transported to a test facility before shaking, it is not necessary
to keep them flat during transport and moderate agitation will not affect the test result.
B Shake to disperse sample in the MICROBMONITOR2®gel.
Transfer the MicrobMonitor2 test bottle to a warm, dark location or incubator to maintain a
nominal temperature of 25°C ± 3°C. In normal circumstances the gel should be incubated
for 4 days. Avoid exposure to light during incubation.
The gel will set firmly after a few hours. Keep the gel on the lower surface of the bottle and
avoid excessive agitation or prolonged tilting of the bottle during incubation, examination
and any transportation.
Notes on incubation of tests.
Occasional temperature fluctuations (e.g. overnight) below the specified temperature range should not affect the number of colonies
which develop and will not critically affect the test result, but microbial colonies may take longer to become visible and thus an extension of
the specified incubation time should be applied. If the temperature falls below the specified range during incubation, the incubation time
should be extended by a time equivalent to the total time the temperature is estimated to have fallen below the specified range. If the
incubation temperature falls below the specified range for a period or periods totaling 4 days or more, microbial contamination will be
underestimated and the result should be considered invalid. The temperature should not be allowed to drop below 4°C during incubation.
Incubation of the culture medium above the specified temperature range may prejudice the ability to detect some types of microorganism.
However, where microbial contamination occurs in tanks or systems where the contents have a higher temperature than 25°C, the
contaminating microorganisms will often have a preference for growth at higher temperatures; in such cases it may be appropriate to
incubate the test at a temperature similar to the system sampled.
When using MicrobMonitor2 to test for compliance with a specific industry standard (e.g. the IATA Contamination Limits shown in table 2 on
page 8), if the test result indicates contamination is in the highest category (e.g. Heavy) before 4 days, it is generally acceptable to read
and report the result without further incubation.
C Incubate the MICROBMONITOR2®test.
D Examine the MICROBMONITOR2®test.
D.1 If possible examine the test daily during incubation. As a minimum, examine it on at
least one occasion in the first 3 days and again on the final day of incubation. To examine
the test, hold it against a light background and count the number of all visible purple
colonies. A hand magnifying lens may help you to identify and count small colonies. All
purple colonies in all parts of the test bottle should be counted, including any which are in
gel which is not part of the flat layer. Once a colony is counted do not count it again
even if it grows larger; it is the number of colonies that is important, not their size. It is
recommended that colonies are marked with a marker pen on the bottle to ensure that
they are not counted twice. Ignore any air bubbles which may form in the gel.
Notes on examining tests
Colonies are usually circular but may have irregular edges. Different types of microorganisms can grow at different rates in the gel culture
medium and therefore the colonies may be of different sizes. At the recommended incubation temperature, colonies of bacteria and
yeasts usually develop within 1 to 2 days and remain quite small. Moulds develop more slowly but eventually produce large colonies which
may have a powdery or "fuzzy" appearance. Generally, the more colonies there are in the test bottle, the smaller the colonies will be.
Ignore any microbial colonies which develop after the specified incubation period is complete (allowing for any extension applied due to
temperature falling below the specified range). The test may change appearance on prolonged incubation but this should be disregarded.
Colonies will tend to become visible more quickly in samples with a higher viable microbial content.
Occasionally, some antioxidant additives in fuels may interfere with the growth indicating compound in the test and produce a uniform light
peach or orange colour in the gel (usually within 12 hours). This colour change will not interfere with the growth of any microorganisms and
in most cases microbial colonies can be counted or estimated ignoring the background colour. In exceptional cases the interference may
be so strong that users may find it difficult to distinguish colour interference from an estimated count of 10,000 colonies. In such cases the
fuel should be retested using a smaller test volume (e.g. 0.01 ml) so that the interference effect is diluted out; if the original result was
genuinely due to heavy microbial contamination it would normally be expected that the retest will show a discernible number of red/purple
colonies and the background colour will be less intense.
Some bacteria can, on prolonged incubation, spread through the gel producing a large 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 are still small (e.g. after 1 or 2
days incubation) they are more easily counted. The centre of each streak or patch should be counted as a single colony. Continue
incubation and count any new colonies which develop.
See technical leaflet EP157 Technical Assistance Reading Results of MicrobMonitor2 for further information on reading and interpreting tests
with unusual or atypical appearance.
D.2 It is usually possible to count up to about 250 colonies. If the number of colonies is too
numerous to count, visually compare the test to the Test Results Chart (see page 9)
holding the test against a white background. The chart provides an estimate of the
colony count.
MICROBMONITOR2®
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A.4 Testing surfaces.
A.4.1 MicrobMonitor2can be used to test for microbial contamination on surfaces, e.g. to test deposits on tank surfaces or
filters. Sterile swabs (available separately) should be used to remove surface contamination and transfer this to a
MicrobMonitor2test bottle.
A.4.2 Open the peel pack containing a swab at the handle end and remove the swab, taking care not to touch the
cotton end of the swab and the handle near the cotton end.
A.4.3 Rub the surface to be tested using the cotton end of the swab, rotating the swab handle so that all parts of the
cotton end contact the surface. If possible swab a known area of surface.
A.4.4 Insert the cotton end of the swab into the neck of the MicrobMonitor2 test bottle and stab it into the gel and agitate
for about 15 seconds and then remove and discard.
A.4.5 Replace the cap on the MicrobMonitor2 bottle and tighten. Record the sample details and test date on the label
provided and stick it on a narrow side of the bottle. Proceed to step B.
7
6
B.1 Loosen and break up the gel in the MicrobMonitor2bottle containing the dispensed
sample, by tapping the bottle firmly in the palm of your hand or on a rubber bung or
MicrobMonitor “Bumper” (available separately).
B.2 Shake the bottle vigorously for approximately 30 seconds to liquefy the gel and
disperse the sample. The test bottle can be tapped repeatedly for 30 seconds on a
MicrobMonitor “Bumper” to assist with shaking. After shaking, the gel should be slightly
viscous but free of lumps and have a uniform consistency and clarity. The presence of
bubbles in the gel is normal and will not affect the test.
B.3 Stop shaking the test bottle abruptly so that the gel collects in the bottom of the
bottle. Proceed immediately to B4.
B.4 Tap the bottle in the palm of the hand until the gel forms a flat layer on one of
the larger flat sides. Ensure that a uniform layer reaching all corners is obtained.
Notes on transport of tests
It is preferable to conduct testing on-site to avoid errors due to changes in the microbial content of samples during transport. If there are
likely to be delays of more than 24 hours in transporting samples to a testing facility, these errors can be avoided by conducting the first
stages of the MicrobMonitor2 test procedure (up to and including step B) on-site at the sampling location and then returning the
MicrobMonitor2test to the testing facility to complete the incubation (step C). The test bottle should be kept flat during transport, should
not be agitated excessively and should be returned to the testing facility within 4 days. See note on incubation of tests (step C)
concerning appropriate adjustment of the incubation time.
Alternatively, MicrobMonitor2 test bottles can be transported to the test facility after sample has been added (step A) before shaking; the
test can then be shaken (step B) at the test facility. Incubation (step C) should be commenced within 6 hours of the sample being added
to the test bottle (or within 2 days if tests are kept cool (2 to 8°C)). If tests are transported to a test facility before shaking, it is not necessary
to keep them flat during transport and moderate agitation will not affect the test result.
B Shake to disperse sample in the MICROBMONITOR2®gel.
Transfer the MicrobMonitor2 test bottle to a warm, dark location or incubator to maintain a
nominal temperature of 25°C ± 3°C. In normal circumstances the gel should be incubated
for 4 days. Avoid exposure to light during incubation.
The gel will set firmly after a few hours. Keep the gel on the lower surface of the bottle and
avoid excessive agitation or prolonged tilting of the bottle during incubation, examination
and any transportation.
Notes on incubation of tests.
Occasional temperature fluctuations (e.g. overnight) below the specified temperature range should not affect the number of colonies
which develop and will not critically affect the test result, but microbial colonies may take longer to become visible and thus an extension of
the specified incubation time should be applied. If the temperature falls below the specified range during incubation, the incubation time
should be extended by a time equivalent to the total time the temperature is estimated to have fallen below the specified range. If the
incubation temperature falls below the specified range for a period or periods totaling 4 days or more, microbial contamination will be
underestimated and the result should be considered invalid. The temperature should not be allowed to drop below 4°C during incubation.
Incubation of the culture medium above the specified temperature range may prejudice the ability to detect some types of microorganism.
However, where microbial contamination occurs in tanks or systems where the contents have a higher temperature than 25°C, the
contaminating microorganisms will often have a preference for growth at higher temperatures; in such cases it may be appropriate to
incubate the test at a temperature similar to the system sampled.
When using MicrobMonitor2 to test for compliance with a specific industry standard (e.g. the IATA Contamination Limits shown in table 2 on
page 8), if the test result indicates contamination is in the highest category (e.g. Heavy) before 4 days, it is generally acceptable to read
and report the result without further incubation.
C Incubate the MICROBMONITOR2®test.
D Examine the MICROBMONITOR2®test.
D.1 If possible examine the test daily during incubation. As a minimum, examine it on at
least one occasion in the first 3 days and again on the final day of incubation. To examine
the test, hold it against a light background and count the number of all visible purple
colonies. A hand magnifying lens may help you to identify and count small colonies. All
purple colonies in all parts of the test bottle should be counted, including any which are in
gel which is not part of the flat layer. Once a colony is counted do not count it again
even if it grows larger; it is the number of colonies that is important, not their size. It is
recommended that colonies are marked with a marker pen on the bottle to ensure that
they are not counted twice. Ignore any air bubbles which may form in the gel.
Notes on examining tests
Colonies are usually circular but may have irregular edges. Different types of microorganisms can grow at different rates in the gel culture
medium and therefore the colonies may be of different sizes. At the recommended incubation temperature, colonies of bacteria and
yeasts usually develop within 1 to 2 days and remain quite small. Moulds develop more slowly but eventually produce large colonies which
may have a powdery or "fuzzy" appearance. Generally, the more colonies there are in the test bottle, the smaller the colonies will be.
Ignore any microbial colonies which develop after the specified incubation period is complete (allowing for any extension applied due to
temperature falling below the specified range). The test may change appearance on prolonged incubation but this should be disregarded.
Colonies will tend to become visible more quickly in samples with a higher viable microbial content.
Occasionally, some antioxidant additives in fuels may interfere with the growth indicating compound in the test and produce a uniform light
peach or orange colour in the gel (usually within 12 hours). This colour change will not interfere with the growth of any microorganisms and
in most cases microbial colonies can be counted or estimated ignoring the background colour. In exceptional cases the interference may
be so strong that users may find it difficult to distinguish colour interference from an estimated count of 10,000 colonies. In such cases the
fuel should be retested using a smaller test volume (e.g. 0.01 ml) so that the interference effect is diluted out; if the original result was
genuinely due to heavy microbial contamination it would normally be expected that the retest will show a discernible number of red/purple
colonies and the background colour will be less intense.
Some bacteria can, on prolonged incubation, spread through the gel producing a large 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 are still small (e.g. after 1 or 2
days incubation) they are more easily counted. The centre of each streak or patch should be counted as a single colony. Continue
incubation and count any new colonies which develop.
See technical leaflet EP157 Technical Assistance Reading Results of MicrobMonitor2 for further information on reading and interpreting tests
with unusual or atypical appearance.
D.2 It is usually possible to count up to about 250 colonies. If the number of colonies is too
numerous to count, visually compare the test to the Test Results Chart (see page 9)
holding the test against a white background. The chart provides an estimate of the
colony count.
MICROBMONITOR2®
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5
8
A.2 Heavy and residual fuels and oils.
A.2.1 Immediately prior to testing, shake the sample for 30 seconds.
A.2.2 Remove the cap of the MicrobMonitor2test bottle and place it on a clean surface.
A.2.3 Open the peel pack containing a loop dispenser and remove it taking care not to touch the loop end and the
lower part of the handle.
A.2.4 Immerse the loop end into the fuel/oil to approximately 3 cm below the surface of the sample or, if there is less than 6
cm depth of fuel/oil phase, to halfway down the fuel/oil phase. Remove the loop from the sample and allow surplus fuel/oil
to drain off; ensure a film of fuel/oil is trapped within the loop.
A.2.5 Insert the loop into the neck of the MicrobMonitor2 test bottle and stab it into the gel and agitate it briefly to transfer
the sample.
A.2.6 Replace the cap on the MicrobMonitor2 bottle and tighten. Record the sample details and test date on the label
provided and stick it on a narrow side of the bottle. Proceed to step B.
A.3 Water phase associated with fuel/oil.
A.3.1 If a test of water phase is required, allow the sample to stand until free water has settled to the bottom of the sample. It
is recommended the water phase test is conducted after any fuel/oil phase test. The test procedure is essentially the same as
described above but an appropriate technique for separating/removing water from the sample needs to be used.
A.3.2 As appropriate for the volume of sample being tested (see table 1) use the loop dispenser or syringe to measure the
required volume of water.
a) To test 0.1 ml of water using the syringe;
The water phase can be drawn directly from the bottom of the sample and added to the MicrobMonitor2 test bottle. To
enable easy access to the water it may be necessary to first decant off some fuel from the sample and/or transfer the water
phase to a separate small, sterile container using a syringe or a long sterile pipette (supplied separately). Mix gently by
swirling but avoid mixing the fuel and water layers together.
Open the peel pack containing a syringe at the handle end and remove the syringe taking care not to touch the lower
barrel and nozzle. Insert the barrel of the syringe into the sample so that the nozzle is in the water phase. Draw water into
the syringe, remove from the sample and then, with the syringe nozzle uppermost, expel air. Expel surplus water until the end
of the plunger aligns with the 0.1 ml mark. Insert the tip of the syringe into the neck of the MicrobMonitor2 test bottle and
dispense the sample by fully depressing the syringe plunger. Replace the cap on the MicrobMonitor2 bottle and tighten.
Record the sample details and test date on the label provided and stick it on a narrow side of the bottle. Proceed to step
B.
b) To test 0.01 ml of water using the loop dispenser;
A syringe can be used to draw water from the bottom of the sample and then a drop of water placed into the loop and
added to the MicrobMonitor2 test bottle. Alternatively, transfer the water phase to a separate small, sterile container using a
syringe or a long sterile pipette (supplied separately). Avoid transfer of fuel. Invert the container with the separated water
three times to mix.
Remove the cap of the MicrobMonitor2 test bottle and place it on a clean surface. Open the peel pack containing a loop
dispenser and remove it taking care not to touch the loop end and the lower part of the handle. Immerse the loop end
into the separated water or fill the loop with a drop of water using a syringe. Allow surplus water to drain away but ensure the
circle of the loop remains filled with water; note, if a residue of fuel is present this can impair the ability of the loop to fill with
water. Insert the loop into the neck of the MicrobMonitor2 test bottle and stab it into the gel and agitate it briefly to transfer
the sample. Replace the cap on the MicrobMonitor2 bottle and tighten. Record the sample details and test date on the
label provided and stick it on a narrow side of the bottle. Proceed to step B.
Note The MicrobMonitor Sampling Kit (supplied separately) is a sample bottle which has a side siphon and water collection
chamber to enable easy separation of water from fuel samples.
a) Results of test of fuel phase sample:
If 0.5 ml of fuel has been tested, multiply the number of colonies counted or estimated by 2000 to give the number of
microbial CFU per litre of fuel. If there are no colonies after the specified incubation time there are less than 2000
microbial CFU per litre of fuel.
If 0.25 ml of fuel has been tested, multiply the number of colonies counted or estimated by 4000 to give the number of
microbial CFU per litre of fuel. If there are no colonies after the specified incubation time there are less than 4000
microbial CFU per litre of fuel.
If a different volume of fuel has been tested, then a calculation can be made as follows:
Number of microbial CFU per litre = Number of colonies counted or estimated x 1000
Volume of fuel tested (ml)
b) Results of test of water phase or oil:
If 0.1 ml of water has been tested, multiply the number of colonies counted or estimated by 10 to give the number of
microbial CFU per ml of water. If there are no colonies after the specified incubation time there are less than 10 microbial
CFU per ml of water.
If 0.01 ml of water or oil has been tested, multiply the number of colonies counted or estimated by 100 to give the number
of microbial CFU per ml of water or oil. If there are no colonies after the specified incubation time there are less than 100
microbial CFU per ml of water or oil.
If a different volume of water or oil has been tested, then a calculation can be made as follows:
Number of microbial CFU per ml = Number of colonies counted or estimated
Volume of water or oil tested (ml)
Note
If the number of colonies is too numerous to count and a sample volume of 0.5 ml or 0.25 ml of fuel or 0.1 ml or 0.01 ml
of water or oil was tested, the Test Results Chart can be used directly to determine the approximate number of
microbial CFU per litre of fuel or per ml of water or oil.
E Calculate the number of microbial CFU in the sample.
For tests of fuel, numbers of microbes are conventionally expressed as number of CFU per litre.
For tests of water phase or oil, numbers of microbes are conventionally expressed as number of CFU per ml.
INTERPRETATION OF TEST RESULTS
There are no universally accepted limits or specifications for microbiological contamination in fuels or oils. The number of
microbial CFU which define moderate and heavy contamination levels will depend on a number of factors including the
fuel type and intended use, the sampling location, whether the sample is taken from supply and distribution facilities or
from point of end use, the intended fuel storage time and specific operational circumstances. Some guidance limits are
shown in Table 2 below. These limits are intended to provide early indication that microbial growth is occurring in the
facility sampled. Higher levels of contamination will often be required before the onset of operational problems or any
detrimental effect on fuel fitness for use. For further information see the technical guidance leaflets listed on page 2.
Table 2
Sample Moderate Heavy Comment
Fuel from aircraft fuel tank drain 4000 to 20,000 CFU / litre >20,000 CFU / litre IATA
Contamination
Limits
Water from aircraft fuel tank drain 1000 to 10,000 CFU / ml >10,000 CFU / ml
Bulk representative fuel sample from
supply & distribution 4000 to 20,000 CFU / litre >20,000 CFU / ml e.g. Aviation fuel,
diesel, marine gas
oil
Fuel from bottom / drain of supply &
distribution tank / filter 10,000 to 100,000 CFU / litre >100,000 CFU / litre
Water phase from bottom / drain of
supply & distribution fuel tank / filter 100,000 to 1,000,000 CFU / ml >1,000,000 CFU / ml
Lubricating / hydraulic oil in circulation 100 to 10,000 CFU / ml >10,000 CFU / ml
Lubricating / hydraulic oil sump 1000 to 10,000 CFU / ml >10,000 CFU / ml
MICROBMONITOR2®
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4 9
A.1 Aviation kerosene, other middle distillate fuels and gasoline.
A.1.1 Immediately prior to testing, shake the sample for 30 seconds. If fuel samples contain
visible free water, the sample should be allowed to stand for 12 minutes to enable water to
settle from the sample to be tested. For consistency, it may be advisable to apply this
settling time for all fuel samples even if free water is not observed.
A.1.2 Remove the cap of the MicrobMonitor2 test bottle taking care not to touch the inside
and place it on a clean surface.
A.1.3 Open the peel pack containing a syringe at the handle end and remove the
syringe taking care not to touch the lower barrel and nozzle.
A.1.4 Using the syringe, draw fuel from approximately 3 cm below the surface of the
sample. If there is less than 6 cm depth of fuel phase, draw sample from approximately
halfway down the fuel phase. Avoid the transfer of visible interfacial particulate, water
droplets or emulsion in the aliquot to be tested.
A.1.5 Draw fuel into the syringe until it is more than half full and then, with the syringe nozzle
uppermost, press the plunger to expel air. Expel surplus fuel until the end of the plunger
aligns with the mark for the required volume of sample (see table 1).
A.1.6 Insert the tip of the syringe into the neck of the MicrobMonitor2 test bottle and
dispense the sample by fully depressing the syringe plunger.
A.1.7 Replace the cap on the MicrobMonitor2 bottle and tighten. Record the sample
details and test date on the label provided and stick it on a narrow side of the bottle.
Proceed to step B.
TEST PROCEDURE
A. Add sample to the MICROBMONITOR2®test bottle.
Follow the appropriate steps below to add distillate fuel or biofuel (A.1), heavy/residual fuel or oil (A.2), water (A.3)
or surfaces deposits (A.4) to the MicrobMonitor2 test bottle.
MICROBMONITOR2®Test Results Chart
Appearance after incubation Number of colonies in
test bottle
(counted or estimated) Volume tested Microbial Content of
Sample
None
0.5 ml of fuel (syringe) <2000 CFU per litre
0.25 ml of fuel (syringe) <4000 CFU per litre
0.1 ml of water (syringe) <10 CFU per ml
0.01 ml of water or oil
(loop dispenser) <100 CFU per ml
10 Colonies
If possible, count the exact
number of colonies and
calculate the actual number
of CFU present in the sample
0.5 ml of fuel (syringe) 2 x 104CFU per litre
0.25 ml of fuel (syringe) 4 x 104CFU per litre
0.1 ml of water (syringe) 100CFU per ml
0.01 ml of water or oil
(loop dispenser) 1000 CFU per ml
100 Colonies
If possible, count the exact
number of colonies and
calculate the actual number
of CFU present in the sample
or
Estimate 100 colonies if
result is similar to picture
0.5 ml of fuel (syringe) c. 105CFU per litre
0.25 ml of fuel (syringe) c. 105CFU per litre
0.1 ml of water (syringe) c. 103CFU per ml
0.01 ml of water or oil
(loop dispenser) c. 104CFU per ml
Estimate 1000 colonies if
result is similar to picture
0.5 ml of fuel (syringe) c. 106CFU per litre
0.25 ml of fuel (syringe) c. 106CFU per litre
0.1 ml of water (syringe) c.104CFU per ml
0.01 ml of water or oil
(loop dispenser) c.105CFU per ml
Estimate 10,000 or more
colonies if result similar to
picture
0.5 ml of fuel (syringe) c.107CFU per litre or
above
0.25 ml of fuel (syringe) c.107CFU per litre or
above
0.1 ml of water (syringe) c.105CFU per ml or above
0.01 ml of water or oil
(loop dispenser) c.106CFU per ml or above
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3
10
Table 1
Sample Recommended test
volume Measuring
device
Aviation kerosene 0.5 ml
Syringe
Other middle distillate fuels (e.g. diesel, marine gas oil, heating oil), biofuels &
gasoline 0.25 ml
Water phase from aircraft fuel tank drains 0.1 ml
Heavy and residual fuels 0.01 ml
Loop
Lubricating, hydraulic and other oils 0.01 ml
Water phase from fuel storage tanks 0.01 ml
PREPARATION FOR TESTING
Sampling considerations
Sampling should be conducted in a clean and consistent manner which prevents introduction of background contamination
into the sample. Once fuel samples have been taken, any microbes present will tend to slowly die and it is important to
test samples as soon as possible. If the test is carried out more than 24 h after sampling, it is possible that the results will not
reflect the viable microbial content present at the time of sampling. To avoid this, part or all of the MicrobMonitor2 test
procedure can be conducted at the sampling location.
Sample type and recommended test volumes
MicrobMonitor2 can be used to test a wide variety of petroleum products and associated water. The volume of sample
which should be tested will depend on the type of sample. Refer to the table below to determine the appropriate
volume of sample to test and which measuring device to use.
The volume of sample tested can be changed in order to increase or decrease the detection level of the test. However,
testing volumes greater than those recommended above may in some circumstances lead to underestimation of microbial
numbers.
Determining whether to test fuel/oil or water phase
Depending on the system sampled and the sampling location, samples may contain fuel/oil and/or free water phase.
MicrobMonitor2 can be used to test either fuel/oil or associated water. Water phase will usually contain far more
microorganisms than are present in fuel or oil phase and therefore a smaller volume is usually tested. Because free water
phase may not always be recovered in samples, to ensure consistency when conducting regular microbiological
monitoring, it is usually recommended that fuel or oil phase from above any free water is tested. However, in some cases
it is recommended to additionally test water phase (e.g. samples from aircraft fuel tanks, and when investigating
suspected microbial growth in facilities). Further information is provided in the relevant technical guidance documents
listed on page 2.
To determine if the sample contains free water hold it up to the light and visually examine it. It may help to gently swirl the
sample to create a vortex. In order to enable access to the sample with the measuring devices provided, it may be
necessary to transfer some sample, after shaking, to a smaller clean container.
Preparing the MICROBMONITOR2®test bottles
If the MicrobMonitor2 test bottles have been stored refrigerated, allow them to equilibrate to ambient temperature before
they are used. Remove the seal from the cap of the bottle before using each MicrobMonitor2 test. Avoid prolonged
exposure of the MicrobMonitor2 test bottles to direct sunlight or other bright light during preparation, incubation and
examination of tests.
Cleanliness during testing
MicrobMonitor2 tests can be conducted on-site, in an office or in a laboratory. However, precautions should be taken to
ensure testing is conducted in a reasonably clean area to avoid contamination of the sample and the test by
microorganisms other than those in the test sample. Wash hands before and after testing. During testing wear clean, nitrile,
vinyl or polythene gloves. Avoid touching areas of the syringe and loop dispenser which come into direct contact with the
sample and avoid touching the inside of the MicrobMonitor2 test bottle cap and neck when dispensing sample.
DISPOSAL
Wear gloves and avoid touching the inside of the bottle or cap and wash hands after handling any MicrobMonitor2 test
bottles showing microbial growth. Before disposal, disinfect all MicrobMonitor2 test bottles which show growth by
immersion of the opened bottles in a strong disinfectant solution (e.g. household bleach) overnight or by incineration or by
using chlorine release tablets (available separately).
Decontaminated tests, unused tests or those showing no microbial growth can be disposed of as normal waste in
accordance with local waste regulations.
STORAGE AND SHELF LIFE
The shelf life of MicrobMonitor2 is 1 year from the date of production. The expiry date is printed on the batch label of each
MicrobMonitor2 box. Store MicrobMonitor2 tests between 2 to 22°C, in the dark. The expiry date applies only to product
stored as stipulated. If refrigerated (2 - 8°C) the MicrobMonitor2 test will usually still be useable for up to a year beyond the
designated expiry date. Do not store MicrobMonitor2 frozen. Exposure to freezing temperatures during transport is
acceptable. Avoid exposure of MicrobMonitor2 tests to light during storage and use. A slightly pink discolouration in the
MicrobMonitor2 gel may develop over time during storage but this will not affect the performance of the test.
ALSO AVAILABLE FROM ECHA
For use with MICROBMONITOR2®
The following items can assist in performing the MicrobMonitor2 test procedure:
MicrobMonitor Sampling Kit (ECHA16/SB) - A sterile, polypropylene bottle for sampling fuel systems and which
enables easy separation of water phase from fuel phase; includes alcohol wipes for decontaminating sample
points.
Incubator (ECHA14/IN) - enables optimal, consistent incubation temperature of MicrobMonitor2 tests; holds 12
MicrobMonitor2 test bottles; available battery or mains (110/230 Volt) operated.
MicrobMonitor Bumpers (ECHA16/TB) - soft, rubber-like, hemispheres which can be stuck to a table top and enable
easy breaking and shaking of MicrobMonitor2 gel.
Chlorine Release tablets (ECHA21/CP) - to disinfect MicrobMonitor2 test bottles showing microbial growth for easy
disposal.
Sterile swabs (ECHA15/SW) - swabs for sampling surface contamination (e.g. tank surfaces, filter cartridges etc.).
Long, sterile bulb pipettes (ECHA07/PP/EL23) - to enable water to be removed from the bottom of samples.
Other Test Kits & Ancillary Items
SigTests®: Sig Sulphide (ECHA02/SS) — simple test kit for Sulphate Reducing Bacteria (SRB) which can cause serious
corrosion and sulphide spoilage in fuel, oil and water systems. Can be used on-site or in the laboratory.
Biocide Rapide (ECHA01/BR) — simple rapid test kit for assessing presence and approximate concentration of
commonly used fuel and oil biocides.
Any level and bottom sampling devices (ECHA23) — for safe sampling from fuel and oil storage tanks in
compliance with industry standards.
For our full range of test kits and ancillaries see www.echamicrobiology.com.
Technical support and services
ECHA Microbiology Ltd. provides a full technical service to support our product range. ECHA also offers laboratory testing,
training courses, consultancy and on-site attendance for audit and investigation of microbial contamination and corrosion
associated with petroleum products and petroleum, marine, aviation and other industrial facilities.
MICROBMONITOR2®
TRUSTED INNOVATIVE ACCURATE DEPENDABLE
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WHAT IS THE MICROBMONITOR2®TEST?
MicrobMonitor2 is a simple and easy to use test kit which enables the quantitative assessment of the viable microbial
content in petroleum products including liquid fuels, oils and associated water. MicrobMonitor2 enables on-site or
laboratory testing in accordance with ASTM and IP Standard Methods ASTM D7978 and IP613 - Determination of the viable
aerobic microbial content of fuels and associated water - Thixotropic Gel Culture Method. MicrobMonitor2 is
recommended in the IATA Guidance Material on Microbiological Contamination in Aircraft Fuel Tanks for monitoring
microbial contamination in aircraft fuel tanks and is listed in the Aircraft Maintenance Manuals of Airbus, Boeing, BAE
Systems and other leading aircraft manufacturers. The test is used by a wide range of military services for monitoring
contamination in aviation, marine and ground fuels and is NATO Codified (6640-99-834-3573).
MicrobMonitor2 provides a total count of microbial colony forming units (CFU). CFUs are the conventional unit of measure
for microbial contamination used in a wide range of industries. MicrobMonitor2 detects the important bacteria, yeasts and
moulds which can contaminate petroleum products including Hormoconis resinae, Aspergillus, Candida and Pseudomonas
species.
The MicrobMonitor2 test consists of a screw-capped bottle containing a thixotropic nutrient gel. A sample is added to the gel
using a sterile plastic loop or syringe (provided). The bottle is shaken and the gel liquefies and the sample, and any
microorganisms in it are dispersed. The gel is re-set into a flat layer and the bottle is incubated in the dark. The gel
contains components which sustain the growth of viable microorganisms and the sample itself contributes additional
nutrients. Viable microorganisms in the sample grow into visible spots known as “colonies”, and a reactive compound
changes the colour of these colonies to red or purple so that they can be easily seen.
The number of colonies formed is a direct estimate of the number of viable microbial particles (CFU) present in the sample. The
actual number of colonies is counted or is estimated by reference to the Test Results Chart (page 9). The volume tested can
be 0.01 ml (using a sterile loop dispenser) or between 0.1 ml and 0.5 ml (using a sterile syringe). The number of colonies formed
is considered in relation to the volume of sample added to the test and expressed as CFU/litre of fuel, or CFU/ml of oil or
water associated with fuel.
MicrobMonitor2 is available in packs of 5 and 44 tests, each containing everything that is required to perform the test:
MicrobMonitor2 test bottles
Sterile syringes*
Sterile loop dispensers*
Bottle labels
This instruction leaflet.
* The 44 pack is available with or without sterile syringes and loop dispensers.
MicrobMonitor2 has been developed and patented by ECHA Microbiology Ltd. and it is marketed worldwide by
ECHA and its distributors.
WHAT IS THE MICROBMONITOR2®TEST USED FOR?
The MicrobMonitor2 is used to test for microbial contamination in fuel and oil products, and to monitor microbial growth in
storage tanks and supply facilities. MicrobMonitor2 can be used as a routine monitoring tool, for providing assurances
about product quality, for investigation of incidents and for confirming effectiveness of measures taken to prevent or
remediate microbial growth and contamination.
This instruction leaflet provides general instructions for using MicrobMonitor2. Further technical instruction on the use of
MicrobMonitor2 in specific applications is provided in the technical application leaflets listed below (free to download
from www.echamicrobiology.com). These leaflets include advice on sampling, sample preparation, interpretation of test
results and appropriate actions when contamination is detected:
EP096 How to Interpret MicrobMonitor2 Test Results in Accordance with IATA Guidelines for Aircraft Drain Samples.
EP119 Routine Monitoring of Aviation Fuels in Supply and Distribution Facilities, Airport Depots and Into-plane
Operations with MicrobMonitor2.
EP132 Routine Monitoring of Diesel Fuel Tanks and Distribution Systems with MicrobMonitor2.
EP166 Routine Monitoring of Marine Diesel on Ships and Offshore Installations with MicrobMonitor2 .
EP157 Technical Assistance Reading Results of MicrobMonitor2.
2
Sample & test details
MICROBMONITOR2®test results
Colonies
counted
Day 1
Colonies
counted
Day 2
Colonies
Counted
Day 3
Colonies
Counted
Day 4
Colonies
Counted
Day _______
Microbial
Contamination in
sample
CFU/ litre or CFU/ ml
Sample Ref.
Date tested
Type
Location
Volume tested (ml) 0.5/0.25/0.1/0.01/other__
Incubation Temp. (°C)
Sample Ref.
Date tested
Type
Location
Volume tested (ml) 0.5/0.25/0.1/0.01/other__
Incubation Temp. (°C)
Sample Ref.
Date tested
Type
Location
Volume tested (ml) 0.5/0.25/0.1/0.01/other__
Incubation Temp. (°C)
Sample Ref.
Date tested
Type
Location
Volume tested (ml) 0.5/0.25/0.1/0.01/other__
Incubation Temp. (°C)
Sample Ref.
Date tested
Type
Location
Volume tested (ml) 0.5/0.25/0.1/0.01/other__
Incubation Temp. (°C)
11
MICROBMONITOR2®
TRUSTED INNOVATIVE ACCURATE DEPENDABLE
+ + +Results that count
For Detection and Enumeration of Contaminating Microbes
in Fuels, Lubricants and Water
Instructions for Use
TRUSTED INNOVATIVE ACCURATE DEPENDABLE
+ + +
12
Important Notice
Please read the Instructions for Use carefully before testing samples.
The test kit should only be used as part of an investigatory process into contamination in petroleum products and associated water. The
test kit must be used strictly in accordance with these instructions and/or Standard Test Method IP613 or ASTM D7978 or other instructions
authorised by ECHA Microbiology Ltd. The results only relate to the portion of sample tested and not necessarily to other petroleum product
in the system. Although guidance may be sought by the user on sampling and the interpretation of results, the responsibility for carrying out
the sampling and test procedures correctly is that of the end-user and not ECHA Microbiology Ltd. The test kit is designed to detect a
recognized group of micro-organisms of industrial significance, but it is in the nature of microbiology that there may be micro-organisms
present which are not detected by the test procedure. ECHA Microbiology Ltd does not accept any liability for any decision or assessment
taken or made as a consequence of the results obtained.
ECHA Microbiology Ltd, Units 22 & 23 Willowbrook Technology Park, Llandogo Road, St Mellons, Cardiff, UK, CF3 0EF
Tel: + 44 (0) 2920 365930 Fax: +44 (0) 2920 361195 sales@echamicrobiology.com
TRUSTED INNOVATIVE ACCURATE DEPENDABLE
+ + +
EP066.021216
MICROBMONITOR2®
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