Halma Wagtech Palintest Potatest Go User manual

Potatest™Go
Rapid Response Portable
Water Quality Laboratory
ZI PTW 10005

2
Who We Are
Over the last 20 years the Wagtech™name has become synonymous with water testing
in the most extreme circumstances and remote locations.
Developed for a range of applications, from long term surveillance to rapid response
testing in an emergency, the Wagtech™kits provide a robust solution to testing key
water quality parameters in the field.
Acquired by Palintest™in 2011, the manufacture and support of the Wagtech™portable
water quality laboratory range has now been integrated into the Palintest™product family.
urther information regarding the Wagtech
™
product range can be found at: www.palintest.com

3
Contents
1
2
3
4
5
6
7
8
9
10
11
12
Kit Layout 4
Introduction 6
Preparation 9
Taking a Sample 15
Sample Processing - Membrane Filtration 16
Incubation and Incubator Operation 20
Microbiological Results 31
Water Safety Kit (WSK) 32
Appendix 1 - Field Testing Checklist - Hints 37
Appendix 2 - Troubleshooting 38
Appendix 3 - Technical Specifications 39
Appendix 4 - Reagents and Consumables 40
Chapter Page
13 Appendix 5 - Microbiological Quick Start uide 42

Potatest™Water Test Kit, Including Water Safety Kit
Fig 1. Potatest™Water test kit with water safety kit removed from lid and opened to
show contents. Coloured circles indicate the chapter colour in which their use is explained.
1Kit Layout
28
26 27 29 23 33
24 25
22124202
10
32
8
15
17
16
2
14
19
31
3
1
9
30
34
7
11
13
18
65
21
3
4

5
1
Kit Layout
Preparing the
Media/Petri Dishes
Membrane Lauryl Sulphate Broth (MLSB)
unction Equipment
1
Media Measuring Device (MMD)
2
Absorbent Pads
3
Pad Dispenser
4
Petri Dishes (in incubator)
5
Petri Dish Rack
6
Membrane iltration Membrane Filtration Unit
7
Hand Vacuum Pump
8
Pasteur Pipettes 1mL, 5 pack
9
Forceps
10
47mm Membrane Filters
11
Incubation and
Incubator Operation
Incubator
12
Mains Power Supply
13
Battery
14
Battery Leads
15
Car Charging Lead
16
DC Socket
17
DC Cable
18
Incubator Power Cable
19
Microbiological Results Hand Lens
20
Physico-Chemical Testing Water Safety Kit (WSK)
21
Conductivity Solution, Mid Range 60mL
22
PH7 Buffer Solution, 60mL
23
Chlorine Testing Contour Comparator and accessories
24
DPD 1&3 Reagents
25
Pocket Conductivity Meter
27
pH Testing Pocket pH Sensor
26
Turbidity Testing Jackson Turbidity Tubes
28
Other Items Dilution Tube - Sample Bottle Inside
29
250mL Beaker
30
De-ion Pack
31
Instruction Manual
33
Work Surface
34
Pen
32

6
2Introduction
2.0 Introduction
The Wagtech PotatestTM is a portable water quality test kit. It has been designed primarily to
test the microbiological quality of drinking water; assessing whether or not there has
been faecal contamination of a water source. It allows the end user to test directly for Total
or aecal Coliforms as well as the critical indicators of microbiological quality: Turbidity,
pH and ree and Total Chlorine.
The Wagtech PotatestTM conforms to advice given by the World Health Organisation
(WHO) for the field based testing of microbiological water quality. The parameters measured
for, and techniques/procedures used are based on accepted laboratory methods and are
adapted for use in demanding field conditions.
More information on the “WHO uidelines for Drinking Water Quality” can be
found at www.palintest.com
As with all the kits in the Wagtech range, ease of use is integral to the design. The Wagtech
PotatestTMis suitable for use by technicians of all skill levels and this manual provides the
essential information required to conduct rapid water quality testing in the field.
This instruction manual is also available in rench, Spanish and Mandarin.
Additional advice and training is available upon request. Contact us directly at
[email protected] or via your local representative.
2.1 Before You Use Your Kit
2.1.1 Microbiological
Analysis of Drinking Water
Drinking water contaminated by faecal
matter may contain pathogenic (disease
causing) organisms and represent a risk
to public health.
It is impractical to attempt to isolate specific
pathogens because they are present in
relatively small numbers compared with
other types of microorganisms. Moreover,
there are many types of pathogen and each
requires a unique microbiological isolation
technique. The accepted approach is to
analyse for indicator organisms that inhabit
the gut in large numbers and are excreted in
human/animal faeces. The presence of these
indicator organisms in water is evidence of
faecal contamination and therefore a risk
that pathogens are present. If indicator
organisms are present in large numbers,
the contamination is considered to be
recent and/or severe.
The group of indicator bacteria tested for with
the PotatestTM are called Coliforms; more
specifically the focus is on the enumeration of
Thermotolerant Coliforms (sometimes called
aecal Coliforms). These are bacteria that
originate from faecal sources. However the
PotatestTM is also capable of testing for
Total Coliforms by simply selecting a different
incubation temperature.
Thermotolerant Coliforms or aecal Coliforms
are used in water microbiological testing
to denote coliform organisms which grow
at 44 or 44.5°C and ferment lactose to
produce acid and gas.
In practice, some organisms with these
characteristics may not be of faecal origin
and the term Thermotolerant Coliforms is
therefore more correct and is becoming
more commonly used. Nevertheless the
presence of Thermotolerant Coliforms nearly
always indicates faecal contamination.
Usually, more than 95% of Thermotolerant
Coliforms isolated from water are the gut
organism Escherichia coli (E. coli), the
presence of which is definitive proof
of faecal contamination.
As a result, it is often unnecessary to
undertake further testing to confirm the
specific presence of E. coli.

7
Total Coliforms refers to a large group of
Gram-negative, rod-shaped bacteria that share
several characteristics. The group includes
Thermotolerant Coliforms and bacteria of
faecal origin, as well as some bacteria that
may be isolated from environmental sources.
Thus the presence of Total Coliforms may or
may not indicate faecal contamination. In
extreme cases a high count for the Total
Coliform group may be associated with a
low, or even zero, count for Thermotolerant
Coliforms. Such a result would not necessarily
indicate the presence of faecal contamination.
It may be caused by entry of soil or organic
matter into the water or by conditions suitable
for the growth of other types of coliform
bacteria. Generally, Total Coliforms are grown
in or on a medium containing lactose at a
temperature of 35 or 37°C.
Carrying out microbiological analysis of
this sort therefore presents certain risks as
it is highly likely that you will be handling
equipment and materials that are potentially
contaminated with harmful pathogens. This
is especially relevant for the filter membranes,
absorbent pads and petri dishes that are
used in the test. or these reasons, general
hygiene and aseptic procedures are of
paramount importance and extra care
must be taken when working in the field.
2.1.2 Overview of the Procedure
for the Microbiological Analysis
of Drinking Water
The Wagtech kits use classical laboratory
techniques and equipment that have been
adapted for use in the field. They conform
fully with guidelines issued by WHO on
accepted field-based methods for the
microbiological analysis of drinking water.
The testing of water samples for coliform
bacteria uses a method called Membrane
Filtration.
In simple terms the process is as follows:
A known volume of sample water (100ml
or less for highly contaminated samples) is
filtered using a specific piece of apparatus
called the Membrane Filtration Unit (MFU).
2
Introduction
Membrane Filtration Method
Water sample - filtered
through a membrane filter
INCUBATOR
MFU
Vacuum
Remove membrane
filter and place
Visual inspection
Incubation at 37/44ºC
on an absorbent pad
wetted with liquid
nutrient media
on a nutrient
pad wetted with
sterile water

8
2Introduction
A vacuum hand pump attached to the M U
creates suction that pulls the sample water
through a sterile membrane filter that sits
in place in the M U.
This membrane filter has small pores in it
that allow the water to pass through easily
but any bacteria present in the water are
trapped on the surface of the filter membrane.
This filter is then removed and placed carefully
onto an absorbent pad that sits in the base
of a sterile petri dish.
The absorbent pad has been soaked in
a liquid culture medium which provides
nutrients for bacteria to grow whilst at
the same time inhibiting growth of any
non-target bacteria.
The petri dish is then placed in the portable
incubator included in the PotatestTM kits.
The temperature can be set to either 37 or
44ºC, allowing the user to carry out tests for
Total or aecal (Thermotolerant) Coliforms.
The petri dishes are incubated for a minimum
of 14 hours at optimum growth temperatures.
During this period the Coliform bacteria will
multiply rapidly to form colonies that are
visible to the naked eye.
Coliforms are identified by their ability to
cause a colour change in the growth media
when incubated. They will show up as
yellow in colour against the red/pink
background of the media.
The yellow colonies are counted and the
results expressed as Colony orming Units
per 100ml of water - C U/100ml (assuming
sample size was 100ml).
Useful to Know
• Always wash your hands before
carrying out your microbiological
analysis, and again after handling
potentially contaminated materials
• Never eat or drink while carrying
out a microbiological test
• Never smoke while carrying out a
microbiological test
• Do not directly touch any colonies
in the petri dish
• Always hold the petri dishes by
the sides and keep the lid on
whenever possible
• Try to ensure that your workspace is
clean and tidy - disinfect if possible
(methanol)
• Ensure all open wounds are
covered adequately
• Always sterilise the materials from the
test before disposal and do not dispose
of potentially contaminated materials
directly into the environment

9
3.1 Aseptic Procedure
There are specific techniques and methods
that actively assist the user in trying to keep
things clean and sterile. These are known
as aseptic procedures or techniques.
This applies most importantly to the following
items in the Wagtech test kit:
Membrane Filtration Unit:
The internal surface of the sample cup, the
internal surface of the funnel and the filter
base/bronze disc must be sterile before a
microbiological test is carried out (section 3.2).
Petri Dishes:
The internal surfaces of the petri dishes will
come into direct contact with the growth
media during the microbiological test. They
must be free from bacteria when the test
begins. They can be sterilised in a variety
of different ways (section 3.3).
Absorbent Pads:
The absorbent growth pads provide a
platform for the liquid growth media and
filter membrane which sit in the petri
dish. There are specific instructions as to
how they must be handled during the
microbiological test (section 3.4).
Culture/ rowth Media:
When preparing the media used for the test
you must ensure that the water used to
hydrate the powdered media is sterile. All the
vessels used to prepare the media and into
which the media is dispensed must also be
sterilised in a specific manner (section 3.5).
Membrane Filters/Tweezers:
The membrane filters which capture the bacteria
during the filtration process are supplied
pre-sterilised. They must be handled using
sterile tweezers and never with your fingers.
3.0 Preparation
Every effort should be made to keep the kit and all its components clean and free from
contamination. At all times you should work in a manner that limits the chance of cross
contamination of your samples.
3
Preparation
Useful to Know
• ethanol is highly flammable and is
classed as ‘Dangerous’ for shipping
purposes. For this reason it is not
included as standard in the kit
•
ethanol can be supplied separately from
the kits but the freight and associated
hazard charges may be expensive
• As an alcohol it can be challenging
to find methanol in certain countries.
Pharmacies, laboratories and hospitals
are all possible local sources
•
When methanol burns in the low oxygen
conditions present in the sample cup of
the FU, a gas called formaldehyde is
produced. This gas acts as a powerful
disinfectant and ensures a complete
sterilisation of the entire apparatus
•
ONLY ethanol can be used to sterilise the
FU in the field. Ethanol or ethylated
Spirits are not acceptable as they do not
produce formaldehyde when ignited
• To ensure that the FU is always ready
for use it is a good idea to sterilise it
after each analysis has been performed.
The FU must however be kept in sterile
condition until the next time it is used

10
3Preparation
3.2 Sterilisation of the
Membrane Filtration Unit
Upon receipt of your water test kit the
Membrane Filtration Unit (MFU) must
be washed thoroughly and then dried with
a clean cloth or paper towel.
Prior to use, the M U must also be sterilised.
This is to reduce the risk of cross contamination
of the water sample. It should be re-sterilised
each time a new sample of water is analysed.
Sterilisation in the field can be difficult. A
simple way of carrying out this sterilisation is
with the use of Methanol (Methyl Alcohol).
Items Required:
• Membrane iltration Unit
• Methanol (Methyl Alcohol)
- not supplied with kit
• Cigarette Lighter/Matches
- not supplied with kit
• Paper Towels - not supplied with kit
• Plastic 1ml Pasteur Pipette
Procedure:
1
Using the Pasteur pipette add approximately
1ml of methanol into the stainless steel
sampling cup.
2Swirl the methanol around the inside
of the sampling cup, coating as much
of the internal surface as possible.
3Holding the sampling cup facing away
from you, use a lighter/match to ignite
the methanol. The methanol will ignite
instantly. Always exercise caution during
this step. The methanol will burn with a
pale blue flame; in bright sunlight it can
be
difficult to
see this flame.
However the
heat
produced
should confirm
it has ignited.
4Place the sampling cup base down on a
flat surface while the methanol burns.
5Assemble the filter funnel and silicone
rubber base components of the M U.
Ensure that the filter funnel is inserted
in the rubber base in the correct position
for sterilisation (see below), which will
leave a small gap between the bottom
of the filter funnel and the silicone base.
Once the flame has virtually extinguished,
invert the filter funnel and silicone rubber
base components of the M U and insert
into sampling cup as shown below.
6
Under these conditions, formaldehyde gas is
produced, which has excellent bactericidal
properties. With the M U in this position,
formaldehyde gas can penetrate all
contact areas, ensuring optimal coverage.
7Leave for 15 minutes to ensure the
formaldehyde gas carries out a complete
sterilisation.
8Remove filter funnel and base assembly
from the sampling cup.
9Pour any residual solution in the
sampling cup away and re-insert the
filter funnel and base assembly.
The Membrane iltration Unit is now
sterile and ready for use. It should be
kept in the kit case until required.
IMPORTANT: This sterilisation procedure
should be repeated each time a new
water sample is to be analysed.

11
3.3 Sterilisation of the
Aluminium Petri Dishes
Your Wagtech test kit is supplied as standard
with a set of aluminium petri dishes. These
are used during the microbiological analysis.
They are used to hold an absorbent pad
soaked in growth media.
A set of 20 petri dishes is supplied with each
incubator complete with rack, which is used
to lower them into the incubator and hold
them in place during the incubation cycle.
The petri dishes are 50mm in diameter
and are designed to accommodate the
47mm diameter absorbent pads and filter
membranes used in the microbiological test.
They are manufactured from aluminium
so that they can be re-sterilised and used
again and again.
Before use it is important that these petri
dishes are sterile. They can be sterilised in
a number of different ways:
• Autoclave/Pressure Cooker
@ 121ºC for 15 minutes
• Immersion in a pan of clean boiling water
for 15 minutes then allow to air-dry
• Heating in conventional oven at a
temperature of greater than 180ºC
for 30 minutes
3.4 Preparation of the Aluminium
Petri Dishes/Absorbent Pads
1It is recommended that the absorbent
pad dispenser is sterilised before use.
Add a few drops of methanol to the
contact area, wipe with a clean cloth
and place on work surface to dry.
2Attach the absorbent pad cartridge to
the dispenser, locking it into place.
3Remove the lid
of the petri dish
and dispense a
pad into the dish
by sliding back
the grooved lever
using your thumb.
4Once the pad is
safely in the petri
dish, immediately
replace the petri
dish lid. Take care
when handling the petri dish. Never
touch the internal surfaces of the petri
dish, always hold the dish by the side.
5Repeat the procedure
until the required
number of petri
dishes are prepared
and store the dishes
in the petri dish rack.
Place the rack in the
incubator for safe
storage until ready
for use in the field. The media is added
to the absorbent pads when on site at
the time of sample processing.
3.5 Media Preparation Procedure
The PotatestTM is supplied
with five Media Measuring
Devices (MMD). MMDs are
pre-sterilised polypropylene
containers. The blue screw
cap lid features an integrated
spoon/spatula.
The MMD is used to prepare
the culture media in small
quantities for optimum ease of use in the field.
Each MMD can hold 25ml of liquid media,
enough for ten microbiological tests.
The MMD allows end-users to prepare sufficient
media for daily requirements and removes
the problems associated with preparing
and storing large amounts of liquid media.
The MMDs can be re-sterilised after each
use, without affecting product performance.
3
Preparation

12
3Preparation
3.6 Preparing Culture Media
in the Field using the Media
Measuring Device (MMD)
Items Required:
• 38.1g Membrane Lauryl
Sulphate Broth (MLSB)
• Media Measuring Device (MMD)
• 100ml distilled or clean water
• pH Meter
• Pressure Cooker/Steriliser/Autoclave
• Heat Source
1If no distilled water is available choose
the cleanest water available e.g. rainwater,
filtered water, or, if necessary, stand raw
water in a container overnight. Do not
use water which has been chlorinated.
Boil the water for at least ten minutes,
cover and allow it to cool down.
2If using raw water then it may be necessary
to filter 100ml of this water through the
membrane filter using the membrane
filtration unit (M U) - see Section 7 M U
Operation. If the water is turbid, this step
may need to be repeated. Prepare as
much filtered water as you require.
3Use the pH Meter to check the pH of
water to be used to make up the liquid
media. To make up MLSB the water should
be pH 7.2 - 7.6. pH must be corrected or
an alternative source of water will need
to be found if the value is outside of the
ideal range. Adjust the pH by using dilute
sodium hydroxide solution (increases pH)
or dilute hydrochloric acid (reduces pH).
4Boil the distilled/clean water for 10
minutes and then allow it to cool.
5Ensure that the MMD are sterile before
beginning. MMDs can be sterilised upright
using an autoclave. Screw on the lid so
that it is secure but not completely air
tight to prevent high pressure developing.
Take care not to place the MMD directly
onto the base of the autoclave. Sterilise at
121°C for 10 minutes. Alternatively, place
MMD in a pressure cooker and maintain
steam at pressure for 15 minutes.
6Remove the MMD, allow to cool.
7Once cool, unscrew the blue lid/spoon of
the MMD. Take ten level spoonfuls of media
from the 38.1g container of the MLSB and
add to the MMD. Always hold via the
lid and do not touch the spoon itself.
8ill the MMD with sterilised water to the
lower lip and screw the lid on tightly.
9Shake the MMD
to aid dissolving
of the powdered
MLSB. Once
dissolved a
bright red/pink
liquid will be
produced.
10 Ideally, to minimise the risk of
contamination, the MMD containing the
liquid MLSB should now be sterilised. Undo
the lid slightly so that it is secure but slightly
loose and sterilise again as in Step 5.
11 Upon completion, remove the MMD and
allow to cool. Tighten the tops securely
and store in a cool, dark place until
ready to use.

13
3.7 Tyndallisation of MMDs
In the event an autoclave or pressure cooker
is not available then the sterilisation of the
MMD containing the liquid MLSB media can
be carried out using a pan of boiling water.
This is a process called Tyndallisation.
When liquids are heated up to 100°C,
the heat will kill the bacterial cells but
the bacterial spores may survive.
Tyndallisation essentially consists of boiling
the substance for 15 minutes for three days
in a row. On the second day most of the
spores that survived the first day will have
germinated into bacterial cells. These cells
will be killed by the second day’s heating.
The third day kills bacterial cells from late-
germinating spores. During the waiting
periods over the three days, the substance
being sterilised is kept in warm, moist
conditions that are conducive to germination
of the spores. When the environment is
conducive to the formation of cells from
spores, the formation of spores from cells
does not occur.
The Tyndallisation procedure can be
summarised as follows:
•DAY 1: Place the MMD containing the
MLSB into a pan or pot of boiling water,
use a rack or stand if possible and try to
prevent the MMD coming into contact
with the base of the pan
• Boil for 15 minutes
• Leave the MLSB to cool and then stand
at room temperature for 24 hours
•DAY 2: Once again heat the MLSB
in a pan of boiling water for a further
15 minutes
• Leave the MLSB to cool and then stand
at room temperature for 24 hours
•DAY 3: Repeat the immersion in boiling
water for 15 minutes for a third and
final time
• The MLSB media should now be sterile
3.8 Storage of Liquid Media
The sterile MLSB should be stable for up
to 6 months if stored in a refrigerator. If no
refrigerator is available the media can be
stored for up to 3 months if kept in a dark
place away from extremes of heat and
moisture. However, if there are any signs of
contamination e.g. yellowing, cloudiness etc.,
then it has become contaminated. It should
be discarded and under no circumstances
used in a microbiological test.
3.9 Pre-Prepared Media
It is possible to use pre-prepared growth media
rather than prepare your own as described
previously. The main advantages these
options offer are that they save time and
reduce the amount of equipment required.
However it should be noted that they are
generally more expensive in terms of cost
per test, and their shelf-life is shorter.
This makes shipping to, and then using in,
remote locations more problematic. Some
of the pre-prepared media also have
specific storage requirements.
The most commonly used pre-prepared
media options are:
Ampoules
Sterile Ampoules containing 2ml of
dissolved media. Available in different
varieties for the testing of a wide range of
microorganisms. Simply unscrew the cap,
pour the media onto the absorbent pad
and discard the empty ampoule.
NutriDisks
or single use, a Nutridisk consists of a
sterile plastic petri dish which includes an
absorbent pad impregnated with dehydrated
growth media. This is re-hydrated using
sterile distilled water before use in the
microbiological test.
The Nutridisks are available in different
varieties for the testing of a wide range
of microorganisms.
3
Preparation

14
3Preparation
Nutridisks are the option most commonly
used with the Wagtech Test kits. Nutridisks
are larger than the standard aluminium
petri dishes supplied in the kit but are still
designed to fit into the standard petri dish
rack. This allows up to 7 Nutridisks to be
incubated at the same time.
To use the Nutridisks they are moistened
with 3.0-3.5ml of sterile distilled water.
They are then ready to use immediately.
An excess ring of water surrounding the
pad should be visible.
All Nutridisk types are supplied with
the appropriate membrane filters, which
are also pre-sterilised and individually
packaged. The membrane filters are
tailored to meet the special requirements
of microbial detection and are available
with 47mm or 50mm diameters.
See Appendix 4: Reagents & Consumables
Useful to Know
• Growth or culture media is a
substance designed to support the
growth of microorganisms (bacteria).
The media is a vital part of the
microbiological water quality test
• There are different types of media for
growing different types of bacteria.
The Wagtech kits use embrane
Lauryl Sulphate Broth ( LSB) as
the growth media
•
LSB is a differential media which means
it can distinguish one microorganism
type from another growing on the
same media. It uses the biochemical
characteristics of a microorganism
growing in the presence of specific
nutrients or indicators (such as Phenol
Red) added to the medium to visibly
indicate the defining characteristics
of a specific microorganism
• LSB is the growth medium for Coliform
bacteria and Escherichia coli (E.coli). It
feeds coliform bacteria but inhibits the
growth of any non-target organisms
that may be present in the sample
• The LSB in the kit is supplied as a
fine powder. This increases its shelf
life and makes transport and shipping
easier. Preferably it should be stored
in a dark environment away from
extremes of heat and moisture
• Typically in its powder form the shelf
life of the LSB is 12 months
•
In order to be used in the microbiological
test the LSB has to be prepared
in liquid form. When water is added
to powdered LSB a deep red liquid
is formed
• Coliforms are identified by their ability
to cause a colour change in the growth
media when incubated. LSB contains
lactose as the major carbon source,
which during incubation is degraded
to acid by E. coli and coliform bacteria;
this is indicated by a change of the
colony colour from red/pink to yellow

15
4.1 Collecting the Sample for Analysis
Samples can be collected using the sterilised
sampling cup of the Membrane Filtration
Unit (MFU).
It is supplied
with a cord
to allow the
sampling cup
to be lowered
into a water
course, well or
storage tank.
IMPORTANT: Always rinse the sterile
sampling cup with some of your sample
water before taking the final sample. This
eliminates any residual methanol left over
from the sterilisation process.
Care must be taken not to introduce floating
matter or material from the edge of the water
course into the water sample. It may be
preferable to attach the sampling cable to the
sterilised sampling cup and take the sample
from a bridge or other overhanging location.
Alternatively, the cup may be cast into the
water from the edge and pulled slowly and
carefully back towards the operator.
Alternatively, any suitable sterile container
or sample bottle can be used in place of
the sampling cup.
When sampling from a river or stream take
the sample as near as possible to the main
flow and not too close to the edge where
the water may be still and unrepresentative
of the sample as a whole.
When sampling from a tap or outlet that
provides water for a consumer remove
any tap attachment. Clean the tap/outlet
with a dry cloth before allowing to run for
1 minute prior to sample collection.
Once collected, the sample must be processed
immediately or as quickly as possible. The use
of a portable field test kit makes this possible.
However, if the delay between sample collection
and analysis is between 2 and 6 hours, chill
the sample rapidly to about 4°C with ice
blocks in an insulated container/cool bag.
Resuscitate the sample prior to full incubation
using the automated routine contained
within the Wagtech incubator software.
Even if the sample is kept cold, the
maximum sample storage time is 6 hours.
Analysis of samples not stored under these
conditions or processed after a period of 6
hours are unlikely to reflect the bacteriological
conditions at the time of sampling.
If chlorinated water samples are being
collected, sodium thiosulphate (not supplied
with kit) should be added to the sample
bottles to neutralise chlorine.
4
Taking a Sample
4.0 Taking A Sample
The optimum volume of sample is that which will allow the most accurate enumeration of bacteria.
The technique of membrane filtration is unsuitable for natural waters containing very
high levels of suspended material, sludges and sediments, all of which could block the
filter before an adequate volume of water has been filtered.
or potable or treated water samples the number of faecal coliform bacteria should ideally
be zero in 100ml, indicating a microbiologically safe (or more accurately LOW RISK) water
supply. The preferred sample volume is 100ml.
or raw source waters and partially treated waters, including those derived from ground
water, it is sometimes useful to reduce the sample volume to obtain faecal coliform counts
in the optimum range. This may be a reduction of the sample size to 50ml, or even 10ml in
more contaminated water sources. To aid this, the filter funnel of the membrane filtration
unit has two internal graduations at 50ml and 100ml.
100ml
50ml

16
5Sample Processing - Membrane Filtration
Ensure that at all times you work on the
work-surface included with the kit. Keep this
area as clean as possible and before
starting, wipe it down with a paper towel
and a few drops of methanol.
1Loosen the filter funnel and remove from
the rubber base support. Invert the filter
funnel and place it down on the clean
work-surface. Also place the blue rubber
base support down on the work-surface.
Ensure that these items are only ever
placed onto the clean work-surface.
2Sterilise the forceps
by passing them
from side to side
through a flame
from a lighter and
allow to cool. Take
care not to heat
for too long as this
will cause sooty
deposits to form.
3Remove a sterile, individually wrapped
membrane filter.
4Peel back the transparent outer wrapper
and use the sterile forceps to separate
the white, gridded membrane filter from
its blue backing paper and remove from
the outer wrapper. Only ever grip the
membrane filters at their edge.
5Place the membrane filter directly onto the
bronze filter support disc housed in the blue
rubber base; ensure the gridded side is
face-up. If the membrane tears or becomes
contaminated, discard and use a fresh one.
• Membrane iltration Unit
• Pistol Grip Hand Vacuum Pump
• orceps/Tweezers
•
47mm Membrane ilters (0.45 µ m pore size)
• MMD containing Liquid MLSB Media
• Petri Dishes containing Absorbent Pad
• Methanol (not supplied)
• Lighter/Matches (not supplied)
5.0 Membrane Filtration of the Sample
Items Required:

17
6Lock the membrane filter in place by
pushing the filter funnel firmly into
position in the blue rubber base. Take
care to not touch the internal surface
of the filter funnel with your hand.
7Ensure the filter funnel is aligned correctly
in the ‘Filtration’ position, indicated by
the graphic on the side of the filter funnel.
8Pour the water sample into the filter
funnel up to the 100ml graduation
(or less if using a smaller sample).
9
Discard the excess water from the sampling
cup then insert the filter funnel/base
assembly into position in the sampling cup.
Care is needed to prevent sample spillage
from the filter funnel when assembling.
NOTE: If the sample is not to be collected
in the sampling cup part of the membrane
filtration unit, then the filter funnel/base
assembly can be inserted into position
in the sampling cup straight away.
10 Connect the hand vacuum pump
and silicone tubing to the M U.
11 Use the pump to create a vacuum and
commence the filtration. The sample
level in the filter funnel will fall rapidly.
Do not pump too many times so as to
avoid drawing excess air through the
membrane filter. The water passes through
the pores in the membrane filter and
is collected in the sampling cup. Any
bacteria in the sample are collected on
the surface of the membrane filter.
5
Sample Processing - Membrane Filtration

18
5Sample Processing - Membrane Filtration
12 When all of the sample has been filtered,
detach the vacuum pump and remove
the filter funnel from the rubber base.
The membrane filter is now ready to
be removed and placed in the petri
dish containing the absorbent pad and
growth media.
13 Remove one of the sterile petri dishes
previously prepared from the rack and place
onto the work-surface. (Prepared petri
dishes should contain an absorbent pad,
if not, follow the procedure in Section 3.3).
14 Taking care to only handle the petri dish
by the sides, remove the lid and place it
on the work surface.
15 Take an MMD containing liquid MLSB
media. Shake well then remove the blue
screw lid and place it, lid-down, onto
the work-surface.
16 Lift the petri dish containing the
absorbent pad and hold between
thumb and forefinger.
17 Take the MMD containing the liquid
MLSB and carefully pour the media onto
the absorbent pad in the petri dish in a
single, decisive pouring motion. Always
ensure that the petri dish is raised from
the surface when pouring the media.
Never use the plastic Pasteur pipettes
to dispense media onto the pads.
18 Ensure the pad is well saturated with a
small excess of MLSB visible at the edges.
This should equate to between 2.5 and
3.0mls. If too much media is dispensed,
simply pour the excess away. Place the
petri dish back down on the work surface.

19
19 Use the sterile forceps to remove the
membrane filter from the filtration unit.
20 Starting at the far edge of the petri
dish, use a rolling motion to place the
membrane filter on top of the absorbent
pad. This will prevent air being trapped
between the pad and membrane filter.
21 Replace the petri dish lid and label with
sample number, place, date, time, etc.
to identify in the rack.
22 Place the petri dish into the petri dish rack
and repeat the process for all samples.
Place the filled rack into the incubator for
safe storage ready to start the incubation.
Ensure that the incubator is in the upright
position at all times to avoid leakage of
nutrient broth from petri dishes, paying
particular attention when transporting.
5.1 Resuscitation Period
It is important to note that when the last
sample has been processed, a resuscitation
period of between one and four hours must
be observed before incubation commences.
Environmental exposure can cause coliforms
to become physiologically stressed. The
resuscitation period allows coliforms to
recover before culturing.
With this in mind it is essential to plan
testing throughout the day, particularly if
visiting multiple sample sites. Try to conduct
all sample processing within a three
hour window. This ensures a maximum
resuscitation period of four hours.
The resuscitation period is especially relevant
for water samples where the environmental
exposure is due to chlorination.
The incubator operating software includes the
capacity to have an initial resuscitation period
as part of the standard incubation cycle.
5.2 Incubation Time
Incubate the samples for 18 hours at the
desired temperature. Two preset incubation
profiles are available on the Wagtech Incubator:
• To test for Total Coliforms
incubate at 37ºC for 18 hours
• To test for Thermotolerant Coliforms
incubate at 44ºC for 18 hours
5
Sample Processing - Membrane Filtration
Useful to Know
• It is preferable to run the incubator
in-situ in the case
• Ensure the incubator lid is closed
• If powering from the battery only,
ensure the case lid is closed to
minimise power consumption
• Do not place the kit directly on
the floor during incubation
• Do not incubate outdoors during
periods of cold temperature
• Ensure that the petri dish rack is full
during incubation, using empty dishes,
to allow even heat distribution
• After switching on the incubator and
selecting the desired temperature, allow
a few minutes for the set-point to be
reached and for temperature to stabilise.
The incubator will show ‘warming up’
for a period of 30 minutes

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6Incubation and Incubator Operation
6.1 Incubator Power Supply
The incubator can be powered
in a variety of ways:
• Mains electricity 100-240V AC,
via the mains adapter/charger unit
• 12V DC rechargeable battery
(included) - sealed lead acid
• An external battery (12V DC) e.g. via
the vehicle cigarette lighter attachment
6.1.1 Using the Incubator via
the Mains Adapter/Charger
• If mains electricity supply is available, this
method of operation is recommended
• Connect the cable of the mains adapter
to the incubator via the socket on the
back of the case Aor left hand side of
the incubator lid B
• When connected to the mains power
supply the incubator will show ‘Charging’
with the battery icon (incubator off)
• Whenever possible ensure that the 12V
battery is also connected to the incubator
to provide charging/trickle charging.
Connect the red and black cable connectors
to the correct terminals on the battery and
plug the cable into the right hand side of
the incubator lid. This is advisable in areas
where the mains supply may not be reliable.
In the event mains power fails, the battery
automatically provides power, thus
continuing the incubation cycle
• Turn on the incubator by pressing the
POWER button briefly
6.1.2 Powering the Incubator via the
12V DC Rechargeable Battery Only
• The incubator can also be powered
solely by the 12V DC battery and a fully
charged battery can provide up to five
full incubation cycles
•
Connect the red and black cable connectors
to the correct terminals on the battery and
plug the other end into
the right hand side of
the incubator lid
6.0 Operation of Incubator
The PotatestTMincubator is a high performance field incubator designed to deliver reliable
Total and Thermotolerant Coliform results in even the most extreme circumstances. Providing
up to 5 cycles of incubation under standard conditions when battery powered, the incubator is
simple to operate and provides performance data throughout the incubation cycle.
A
B