TAIEX Workshop on International

Standard Methods
for Microbiological Analysis
of Drinking Water
organised in co-operation with

Chemistry Administration (CA)

Ministry of Industry, Trade and Small & Medium Enterprises

1
Laboratory manual for the microbiological analysis
of drinking water: sterilization, sample collection,
microbiological media and storage, laboratory
facilities, equipment, materials, procedures, and
result analysis

Mrs Maria MARTINS, guas do Norte, Portugal

2
INDEX
1. Laboratory Manual for Microbiological Analysis
1.1 Sterilization
1.2 Sample collection
1.3 Microbiological media and storage
1.4 Laboratory facilities
1.5 Equipment
1.6 Materials
1.7 Procedures
1.8 Result analysis
1.9 Test Report
2. References

3
 1. Laboratory Manual for Microbiological Analysis

4. LABORATORY
1.STERILIZATION 7. PROCEDURES
FACILITIES

2. SAMPLE 8. RESULT
5. EQUIPMENT ANALYSIS
COLLECTION

3.
MICROBIOLOGICAL
6. MATERIALS 9. TEST REPORT
MEDIA AND
STORAGE

4
1.1 Sterilization

5
 1.1 Sterilization

EQUIPMENT EQUIPMENT
& MATERIAL & MATERIAL
FOR FOR
SAMPLING ANALYSIS

CONTROLLED
ENVIRONMENT
FOR SAMPLING
& ANALYSIS

6
 1.1 Sterilization

Laboratory shal have procedures for:

Sterilization of material and equipment for Sampling

Sterilization of material and equipment for Analysis

Environmental Quality Control

7
1.2 Sample
Collection
 1.2 Sample Collection

SAMPLING IS A CRUCIAL STAGE IN THE

CHARACTERIZATION OF A WATER SAMPLE

The location and manner in which a sample is collected and handled

in the field is the first step to ensuring representative and reliable
analytical data.
 1.2 Sample Collection

ALL THE SAMPLING STAGES ARE VERY IMPORTANT:

Sampling containers

Sampling procedure

Sample packing and transport

Sample preservation

Sampling Storage

Records

10
 1.2 Sample Collection

Select the correct SAMPLING CONTAINER (sterile bottle

for Microbiological parameters)

11
 1.2 Sample Collection

LABEL OF CONTAINER INCLUDE:

Sample number

Code of container
Parameters for analysis

Sampling procedure

Sampling date

12
 1.2 Sample Collection

LABEL OF REFRIGERATED BOX INCLUDE:

Sample number

Sampling point

Sampling data
Number of containers

13
 1.2 Sample Collection

EVERYDAY RECORDS (IN THE FIELD):

Date/ Hour of Samplimg

Sampling point

Residual chlorine / Temperature

Name of technician/ Signature

Ref. Equipment of measurement (chlorine/ temperature)

Special remarks ( atmospheric conditions/ problems with

sampling points/ etc.)

14
 1.2 Sample Collection

EVERYDAY RECORDS (IN THE FIELD):

15
 1.2 Sample Collection

PROCEDURE FOR SAMPLING FROM THE TAP (7 steps)

1
Sample should only be collected if
the tap presents normal
conditions of higiene and
conservation

If possible, remove all external

accesories of the tap (filters,
pipes, etc.)

16
 1.2 Sample Collection

2
Disinfect the tap, using a flame.

If a flame can not be used, clean the

tap (the external part & interior if
possible) with a cotton soaked in
ethanol 70%. After, soak the end of
the tap in ethanol 70% (2-3 min).

Disinfect your hands with ethanol/

disinfectant

17
 1.2 Sample Collection

Open the tap, with maximum flow

for
5 10 seconds.

Reduce the flow and let the water

run for 1- 5 min ( to eliminate the
ethanol and temperature of flame
heating)

Measure the temperature until a

constant value is reached (to ensure
a the flush was effective)
Register the Water
Temperature &
Equipment used
18
 1.2 Sample Collection

4
Without closing the tap collect the
sample for microbiology (sterile
container)

NOTE: do not fill the flask totally;

The flask should be opened brifely;
the stopper should be kept in your
hand; do not touch the tap, the
inside of the flask and stopper with
your fingers.

19
 1.2 Sample Collection

5
For the other parameters, wash the
flasks 2 3 times with the tap water
and fill the flasks totally, at a
constant flow (unless stated
otherwise)

20
 1.2 Sample Collection

6
If required, measure residual chlorine in the sampling point

Register the
residual chlorine

21
 1.2 Sample Collection

Store carefully the samples in a refrigerated box

Transport the samples to the Laboratory (< 8h).

The flask for temperature

Each box should & MB should be
contain, at least, 2 positioned in the center
coolers. of the box

22
 1.2 Sample Collection
The bottle for MB should not be in contact with the coolers (to
avoid thermal shock of the microorganisms). This bottle should
be put in the middle of the box, together with flask for
temperature measurement.

The temperature of samples AT ARRIVAL TO LAB, should always

be LOWER than temperature of samples at sampling point.
23
 1.2 Sample Collection

AT LABORATORY:

Read the temperature of the samples in the arrival at Laboratory

Golden rule : The temperature of samples at arrival to Lab,

should always be lower than temperature of samples at sampling
point. (for the parameters that require refrigeration)

Wash refrigerated boxes / coolers everyday

24
 1.2 Sample Collection

REJECT THE SAMPLE IF:

The sample has not been collected in the proper

container
The volume of water is smaller than required by
the analytical method

The sample is not correctly identified

The sample does not indicate sampling point, date or time of

sampling

The sample has been delivered after its maximum time for
storage
25
 1.3 Microbiological Media and
Storage

1.3 Microbiological Media and Storage

26
 1.3 Microbiological Media and Storage
ISO 11133:2014 - Microbiology of food, animal feed and
water -- Preparation, production, storage and
performance testing of culture media

27
 1.3 Microbiological Media and Storage

ISO 11133:2014

defines terms related to quality assurance of culture media;

Specifies the requirements for the preparation of culture media intended

for the microbiological analysis of food, animal feed, and samples from
the food or feed production environment as well as all kinds of water
intended for consumption or used in food production;

These requirements are applicable to all categories of culture media

prepared for use in laboratories performing microbiological analyses.

28
 1.3 Microbiological Media and Storage

ISO 11133:2014

Sets criteria and describes methods for the performance testing of

culture media. It applies to producers such as:

commercial bodies producing and/or distributing ready-to-use or

semi-finished reconstituted or dehydrated media;

non-commercial bodies supplying media to third parties;

microbiological laboratories preparing culture media for their

own use.

29
 1.3 Microbiological Media and Storage

ISO 11133:2014

Culture media meeting established performance criteria are therefore a

pre-requisite for any reliable microbiological work.

a) the acceptability of each batch of medium,

b) that the medium is "fit for purpose", and
c) that the medium can produce consistent results.

These three criteria are an essential part of internal quality control

procedures

30
 1.3 Microbiological Media and Storage

ISO 11133:2014

For reliable microbiological analysis it is essential to use culture media of

proven quality.

For all media described in standard methods it is essential to define the

minimum acceptance criteria required to ensure their reliability.

It is recommended that in the determination of the performance

characteristics of a culture medium tests are carried out that conform
with this International Standard.

31
1.4 Laboratory
Facilities

32
 1.4 Laboratory Facilities

Flow of Personal
Assembly
Material
Cleaning
Waste
Sterilization
HVAC
FACILITY
EQUIPMENT

Open
PERSONNEL MICROBIAL Vs
CONTAMINATION Closed
SOURCES PROCESS

UTILITIES
MATERIALS
Raw material
WATER Reusable resin &
Membrane filter
33
 1.4 Laboratory Facilities

According to ISO 17025:2005

Laboratory facilities for testing shall facilitate correct

performance of laboratory activities.

There shall be effective separation between neighbouring

areas in which there are incompatible activities.

Measures shall be taken to prevent cross-contamination,

interference or adverse influence.

34
 1.4 Laboratory Facilities

According to ISO 17025:2005

Access to and use of areas affecting laboratory activities shall be

controlled. The laboratory shall determine the extent of control
based on its particular circumstances.

The laboratory shall ensure that the environmental conditions are

suitable to realize the laboratory activities and do not invalidate
the results or adversely affect the required quality of any
measurement.

Particular care shall be taken when sampling and tests are

undertaken at sites other than a permanent laboratory facility.
35
 1.4 Laboratory Facilities

According to ISO 17025:2005

The technical requirements for accommodation and

environmental conditions that can affect the results of tests and
calibrations shall be documented.

Measures shall be taken to ensure good housekeeping in the

laboratory. Special procedures shall be prepared where
necessary.

36
 1.4 Laboratory Facilities

According to ISO 17025:2005

The laboratory shall establish, monitor, control (where

appropriate) and record environmental conditions as required by
the relevant specifications, methods and procedures or where
they influence the quality of the results.

Tests and calibrations shall be stopped when the environmental

conditions influence the results of the tests.

37
1.5 Equipment

38
 1.5 Equipment

MAIN LIST OF NECESSARY EQUIPMENT

Apparatus, suitable for sterilization by steam (autoclave),

according to the instructions given in ISO 8199.
Incubator, thermostatically controlled (22C, 36C, 44C, ).
pH meter, with an accuracy of 0,1 at 20 C to 25 C.
Magnetic stirrer with heating
Water bath 90 - 100 C
Precision scale
Water bath 45 1 C
Peristaltic pump
Dispensette
39
 1.5 Equipment

MAIN LIST OF NECESSARY EQUIPMENT (cont)

Biological safety cabinet

Freezer thermostatically controlled at -20 5 C
Refrigerator thermostatically controlled at 5 3 C
Microwave
Membrana filtration apparatus as specifed in ISO 8199
Ultraviolet lamp 365 nm
Quanty-Tray sealer
Quanty-Tray comparator
Colony counting equipment
Ultrapure Water Purification System
40
1.6 Materials

41
 1.6 Materials

MAIN LIST OF NECESSARY MATERIALS

Sterile filter funnels

Sterile Membrane filters, composed of cellulose esters or other

suitable material, usually about 47 mm or 50 mm in diameter, with
filtration characteristics equivalent to a rated nominal pore diameter
of 0,45 m and, preferentially, with grid lines.

Glass or plastic Petri dishes

Anaerobic jars or similar

42
 1.6 Materials

MAIN LIST OF NECESSARY MATERIALS (Cont)

System generator an atmosphere of about 90% hydrogen and

10% carbon dioxide

Tweezers for handling of the membranes

All reagentes necessary to perform the methods

Sterile Loops

43
1.7 Procedures

44
 1.7 Procedures
MICROBIOLOGY LABORATORY SHALL HAVE PROCEDURES
DOCUMENTED FOR:

Sterilization of Material and Equipment for Sampling and Analysis

Water Sampling
ISO 19458:2006, Water quality Sampling for microbiological analysis

Media Preparation and Storage

ISO 11133:2014, Microbiology of food, animal feed and water
Preparation, production, storage and performance testing of culture media

Water for Analytical Laboratory Use

ISO 3696:1987, Water for analytical laboratory use Specification and test
methods
45
 1.7 Procedures
MICROBIOLOGY LABORATORY SHALL HAVE PROCEDURES
DOCUMENTED FOR:

Microbiology Analysis

Quality Control for Environmental Conditions

ISO 14698-2:2003 Cleanrooms and associated controlled

environments -- Biocontamination control - Part 2: Evaluation and
interpretation of biocontamination data.

Quality Control for Result Analysis

ISO/TR 13843:2000 - Water quality -- Guidance on validation of

microbiological methods

46
1.8 Result Analysis

47
 1.8 Result Analysis

According to ISO 17025:2005

The laboratory shall have procedures for monitoring the validity of

activities undertaken and the quality of the laboratory output.

The resulting data shall be recorded in such a way that trends are
detectable and, where practicable, statistical techniques shall be
applied to the reviewing of the results.

This monitoring shall be planned and reviewed and shall contain

both internal and external activities as applicable.

48
 1.8 Result Analysis

According to ISO 17025:2005

Internal quality control activities are those actions that routinely

monitor the quality of the laboratory output and can include, but
not be limited to, the following:

a) regular use of certified reference materials and/or reference

material and/or quality control material;

NOTE: It is recommended to use certified reference material from

producers that meet ISO 17034. ISO Guide 33 provides guidance on
the selection and use of reference materials.

49
 1.8 Result Analysis

According to ISO 17025:2005

b) regular use of traceable instrumentation;

c) functional check of measuring and testing equipment;

d) use of check standards with control charts;).

e) periodic intermediate checks on measuring equipment;

f) replicate tests or calibrations using the same or different methods;

g) retesting or recalibration of retained items;

h) correlation of results for different characteristics of an item;

i) review of reported data by qualified laboratory personnel;

j) intra-laboratory comparisons (i.e. organized within a laboratory).

50
1.9 Test Report

51
 1.9 Test Report

According to ISO 17025:2005

a) a title (e.g. Test Report);

b) the name and address of the laboratory, and the location where
the tests were carried out, if different from the address of the
laboratory;

c) unique identification of the test report (such as the serial

number), and on each page an identification in order to ensure
that the page is recognized as a part of the test report or
calibration certificate, and a clear identification of the end of the
test report or calibration certificate;
52
 1.9 Test Report

According to ISO 17025:2005

d) the name and address of the customer;

e) identification of the method used;

f) a description of, the condition of, and unambiguous identification

of the item(s) tested;

g) the date of receipt of the test where this is critical to the validity
and application of the results;

53
 1.9 Test Report

According to ISO 17025:2005

h) the date(s) of performance of the test;

i) the date of issue of the test report;

j) reference to the sampling plan and procedures used by the

laboratory or other bodies where these are relevant to the validity or
application of the results;

54
 1.9 Test Report

According to ISO 17025:2005

k) a statement to the effect that the results relate only to the items
tested, where relevant;

l) the test results with, where appropriate, the units of measurement;

m) the name(s), function(s) and signature(s) or equivalent

identification of person(s) authorizing the test report

NOTE : It is recommended that laboratories include a statement

specifying that the test report or calibration certificate shall not be
reproduced except in full, without written approval of the
laboratory.

55
2. References

56
 2. References

ISO 3696:1987, Water for analytical laboratory use Specification and test
methods

ISO 7704:1985, Water quality Evaluation of membrane filters used for

microbiological analyses

ISO 8199:2005, Water quality General guidance on the enumeration of

micro-organisms by culture

ISO 11133:2014, Microbiology of food, animal feed and water Preparation,

production, storage and performance testing of culture media

ISO 19458:2006, Water quality Sampling for microbiological analysis

57
 References

ISO 14698-2:2003 Cleanrooms and associated controlled environments --

Biocontamination control - Part 2: Evaluation and interpretation of
biocontamination data.

ISO 17025:2005 - General requirements for the competence of testing and

calibration laboratories

ISO/TR 13843:2000 - Water quality -- Guidance on validation of

microbiological methods

Standard Methods for the Examination of Water and Wastewater 22th edition

The Microbiology of Drinking Water (2002) - Methods for the examination of

waters and associated materials (Environment Agency - DWI)

58
 Laboratory manual for the microbiological analysis of drinking
water: sterilization, sample collection, microbiological media and
storage, laboratory facilities, equipment, materials, procedures, and
result analysis

THANK YOU FOR YOUR ATTENTION!

Mrs Maria Martins, Laboratory Coordinator of guas do Norte, Portugal

maria.martins@adp.pt
59