EN ISO 8199:2007

SLOVENSKI STANDARD
01-december-2007
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SIST ISO 8199:1997
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Water quality - General guidance on the enumeration of micro-organisms by culture (ISO
8199:2005)
Wasserbeschaffenheit - Allgemeine Anleitung zur Zählung von Mikroorganismen durch
Kulturverfahren (ISO 8199:2005)
Qualité de l'eau - Lignes directrices générales pour le dénombrement des micro-
organismes sur milieu de culture (ISO 8199:2005)
Ta slovenski standard je istoveten z: EN ISO 8199:2007
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 8199
NORME EUROPÉENNE
EUROPÄISCHE NORM
October 2007
ICS 07.100.20
English Version
Water quality - General guidance on the enumeration of micro-
organisms by culture (ISO 8199:2005)
Qualité de l'eau - Lignes directrices générales pour le Wasserbeschaffenheit - Allgemeine Anleitung zur Zählung
dénombrement des micro-organismes sur milieu de culture von Mikroorganismen durch Kulturverfahren (ISO
(ISO 8199:2005) 8199:2005)
This European Standard was approved by CEN on 27 September 2007.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2007 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 8199:2007: E
worldwide for CEN national Members.

Foreword
The text of ISO 8199:2005 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 8199:2007 by
Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by April 2008, and conflicting national standards shall be withdrawn at the
latest by April 2008.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 8199:2005 has been approved by CEN as a EN ISO 8199:2007 without any modification.

INTERNATIONAL ISO
STANDARD 8199
Second edition
2005-06-15
Water quality — General guidance on the
enumeration of micro-organisms by
culture
Qualité de l'eau — Lignes directrices générales pour le dénombrement
des micro-organismes sur milieu de culture

Reference number
ISO 8199:2005(E)
©
ISO 2005
ISO 8199:2005(E)
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ii © ISO 2005 – All rights reserved

ISO 8199:2005(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Principle. 1
4 General. 1
5 Diluents and culture media. 2
6 Sterilization of apparatus and glassware. 5
7 Samples . 5
8 Enumeration after inoculation of test portions of the sample in (or on) solid media . 6
9 Enumeration by inoculation in liquid media . 15
Annex A (informative) Criteria for the choice of enumeration technique. 22
Annex B (informative) MPN tables. 27
Bibliography . 37

ISO 8199:2005(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 8199 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
This second edition cancels and replaces the first edition (ISO 8199:1988), which has been technically revised.

iv © ISO 2005 – All rights reserved

ISO 8199:2005(E)
Introduction
Techniques for the isolation and enumeration of micro-organisms, based on their ability to grow on specified
culture media, are an important and widely used means of assessing the microbiological quality of water. The
purpose of this guide is to gather in a single document the information common to the various enumeration
techniques so as to avoid repetition of technical details in individual standards and to facilitate the choice of
the technique most suitable for a particular problem.

INTERNATIONAL STANDARD ISO 8199:2005(E)

Water quality — General guidance on the enumeration of micro-
organisms by culture
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This standard does not purport to address all of the safety problems, if any, associated with
its use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this International
Standard be carried out by suitably trained staff.
1 Scope
This International Standard presents guidance for carrying out manipulations which are common to each
technique for the microbiological examination of water, particularly the preparation of samples, culture media
and apparatus. It also describes the various enumeration techniques available and the criteria for the choice
of a particular technique. This International Standard is mainly intended for bacteria, yeasts and moulds.
Some aspects are also applicable to viruses and parasites.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods
ISO 19458, Water quality — Sampling for microbiological analysis
3 Principle
The general principle of these techniques consists of inoculating a known volume of a water sample on or into
a culture medium (solid or liquid). It is assumed that after incubation each micro-organism present multiplies,
giving either a colony visible directly on the solid medium, or changes in observable properties of the liquid
medium. The choice of a particular method depends not only on the nature of the micro-organisms sought, but
also on the nature of the water and the reasons for the examination.
4 General
Uniformity of temperatures and (incubation) times: The following accepted ranges of temperatures and times
during incubation or storage are applied, when appropriate for the intended target organism, and unless
otherwise stated in the specific standard.
ISO 8199:2005(E)
Storage temperatures:
(− 70 ± 10) °C; (− 20 ± 5) °C; (5 ± 3) °C;
Incubation temperatures:
(22 ± 2) °C; (36 ± 2) °C;
Sterilization temperatures:
(115 ± 3) °C; (121 ± 3) °C; (170 ± 10) °C;
Incubation times:
(21 ± 3) h; (44 ± 4) h; (68 ± 4) h.
Other times and temperatures may be specified for specific methods when necessary.
The upper incubation temperature limits are very strict (they can have large influences on the growth). The
lower temperature limits may be exceeded for short periods, e.g. due to opening the door of an incubator, but
recovery shall be rapid.
Tolerances on volumes and masses: Unless otherwise stated, the accepted range of any measured value is:
stated value ± 5 %.
5 Diluents and culture media
5.1 General
5.1.1 Quality requirements
Use constituents of uniform quality and analytical-grade chemicals for the preparation of media. Other grades
of chemical may be used provided they can be shown to produce the same results. Alternatively, dehydrated
complete media or diluents may be used. Follow the manufacturer's instructions strictly.
Use glass-distilled or demineralized water which is free from substances that might affect growth of micro-
organisms under the test conditions. The water shall comply with the requirements of ISO 3696:1987, grade 3.
Unless otherwise stated, the ingredients are added to the volume of water instead of making up to a certain
volume, as is normal in the preparation of microbiological culture media.
Before use, check the quality of the media, diluents and filters, e.g. by following the procedures described in
ISO 7704, ISO/TS 11133-1 or ISO/TS 11133-2.
5.1.2 Sterilization
Dispense diluents and culture media in containers suitable for sterilization by autoclaving. For most purposes,
a temperature of (121 ± 3) °C for 15 min is adequate. However, a different time and temperature may
sometimes be required and details are given in each individual standard.
Alternatively, with thermolabile substances, removal of micro-organisms may be effected by filtration through a
filter with a pore size of 0,2 µm, specified by the manufacturer as being suitable for “sterilization”.
5.2 Diluents
The diluents given in this subclause are commonly used in water microbiology. However, the list is not
exhaustive and other appropriate diluents may be used.
After preparation, distribute each solution into bottles and sterilize, e.g. by autoclaving at (121 ± 3) °C for
15 min. Alternatively, the diluent can be aseptically distributed after sterilization. Store at room temperature or
in a refrigerator at (5 ± 3) °C for a maximum of 6 months. If a diluent shows any change from its normal
appearance, discard it.
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ISO 8199:2005(E)
5.2.1 Saline solution
Composition
Sodium chloride (NaCl) 8,5 g
Water (see 5.1.1) 1 000 ml
Preparation
Dissolve the ingredients in the water, if necessary by heating. Adjust the pH by adding sodium hydroxide
solution [c(NaOH) = 1 mol/l] or hydrochloric acid [c(HCI) = 1 mol/l] so that, after sterilization (see 5.1.2), it will
correspond to 7,0 ± 0,5 at 25 °C.
5.2.2 Peptone diluent
Composition
Enzymatic digest of casein (peptone) 1,0 g
Water (see 5.1.1) 1 000 ml
Preparation
Dissolve the ingredients in the water, if necessary by heating. Adjust the pH by adding sodium hydroxide
solution [c(NaOH) = 1 mol/l] or hydrochloric acid [c(HCl) = 1 mol/l] so that, after sterilization (see 5.1.2), it will
correspond to 7,0 ± 0,5 at 25 °C.
5.2.3 Peptone saline solution
Composition
Enzymatic digest of casein (peptone) 1,0 g
Sodium chloride (NaCl) 8,5 g
Water (see 5.1.1) 1 000 ml
Preparation
Dissolve the ingredients in the water, if necessary by heating. Adjust the pH by adding sodium hydroxide
solution [c(NaOH) = 1 mol/l] or hydrochloric acid [c(HCI) = 1 mol/l] so that, after sterilization (see 5.1.2), it will
correspond to 7,0 ± 0,5 at 25 °C.
5.2.4 Ringer's solution, quarter-strength
Composition
Sodium chloride (NaCl) 2,25 g
Potassium chloride (KCl) 0,105 g
Calcium chloride (anhydrous) (CaCl ) 0,12 g
Sodium hydrogen carbonate (NaHCO ) 0,05 g
Water (see 5.1.1) 1 000 ml
ISO 8199:2005(E)
Preparation
Dissolve the ingredients in the water, if necessary by heating. Adjust the pH by adding sodium hydroxide
solution [c(NaOH) = 1 mol/l] or hydrochloric acid [c(HCI) = 1 mol/l] so that, after sterilization (see 5.1.2), it will
correspond to 7,0 ± 0,2 at 25 °C.
5.2.5 Phosphate buffer solution
a) Phosphate solution
Composition
Potassium dihydrogen orthophosphate (KH PO ) 34 g
2 4
Water (see 5.1.1) to 1 000 ml
Preparation
Dissolve the potassium dihydrogen orthophosphate in 500 ml of the water. Adjust the pH to a value of
7,2 ± 0,2 by adding sodium hydroxide solution [c(NaOH) = 1 mol/l] or hydrochloric acid [c(HCI) = 1 mol/l]. Add
more water up to 1 000 ml. If the solution needs to be stored, sterilize it (see 5.1.2) before storage.
b) Magnesium chloride solution
Composition
Magnesium chloride (MgCI ) 38 g
Water (see 5.1.1) 1 000 ml
Alternatively, an equivalent mass (99 g) of magnesium sulfate (MgSO .7H O) may be used. If the solution
4 2
needs to be stored, sterilize it (see 5.1.2) before storage.
Preparation
Dissolve the magnesium chloride in the water.
c) Final solution
Composition
Phosphate solution (a) 1,25 ml
Magnesium chloride solution (b) 5,0 ml
Water (see 5.1.1) 1 000 ml
Preparation
Add the phosphate solution (a) and the magnesium chloride solution (b) to the water, dispense in convenient
volumes and sterilize (see 5.1.2). The final pH should correspond to 7,0 ± 0,2 at 25 °C.
5.3 Culture media
Once a bottle of dehydrated medium (chemical) is opened, date the container and indicate a maximum
storage time.
4 © ISO 2005 – All rights reserved

ISO 8199:2005(E)
In general, most media after sterilization in sealed containers may be stored satisfactorily for several months
at room temperature provided they are kept in the dark and remain sealed. Media dispensed aseptically may
be stored at (5 ± 3) °C for up to 1 month, or longer if approved. Before use, inspect them carefully for
contamination, excessive evaporation, or other evidence of deterioration. Most reagents are best kept at
(5 ± 3) °C. Use culture media supplied pre-poured in accordance with the manufacturer's instructions.
Pre-cool the medium to 45 °C to 50 °C if heat-sensitive supplements need to be added after autoclaving.
6 Sterilization of apparatus and glassware
Sterilize apparatus and glassware which is not supplied sterile by one of the following methods:
a) in an oven, at (170 ± 10) °C for at least 1 h (excluding pre-heating time);
b) in an autoclave, at (121 ± 3) °C for at least 15 min.
If membrane filters are not obtained sterile, sterilize them before use in accordance with the manufacturer's
instructions.
7 Samples
7.1 Sampling
Take samples in accordance with ISO 19458.
7.2 Preparation of test sample
Before examination, mix the sample thoroughly by vigorous agitation to achieve uniform distribution of micro-
organisms and, depending on the nature of the water and the microbial content anticipated, make any
dilutions necessary at this stage.
In the case of plate counts, decimal dilutions can be used. For membrane filtration (with a smaller surface
area), smaller dilution steps are recommended.
For ten-fold dilutions, measure 90 ml or 9 ml of the diluent into sterile dilution bottles or tubes. Alternatively,
volumes of diluent, pre-sterilized in screwcapped bottles, can be used. One or more ten-fold dilutions are
made by transferring one volume of water sample to nine volumes of diluent. Mix the solution thoroughly (with
a fresh pipette or by mechanical means) and transfer one volume of this dilution to another nine volumes of
diluent. These steps are repeated as many times as required. Prepare sufficient volumes of each dilution for
all the tests to be carried out on the sample.
For dilutions other than ten-fold, the volume of diluent to volume of sample shall be adjusted accordingly.
Various approaches can be taken, i.e. 3- or 4-fold dilution series, or decimal dilution series of which both 10 ml
and 30 ml volumes are filtered. Four-fold dilutions can be made as described above for ten-fold dilutions,
except that, in this case, one volume of water sample is mixed with three volumes of diluent.
If the concentration of the target organism is expected to be high, hundred-fold dilution steps can be used.
For general guidance on the preparation of ten-fold dilutions, see ISO 6887-1.
ISO 8199:2005(E)
8 Enumeration after inoculation of test portions of the sample in (or on) solid media
8.1 Principle
A test portion of the water sample, or a dilution, is inoculated either directly or concentrated on a membrane
on the surface of a specified solid culture medium or in a molten medium so that, on incubation, micro-
organisms form colonies either on or in the medium.
For practical purposes, each colony is considered to have originated from a single micro-organism or a clump
of micro-organisms present in the test portion at the moment of inoculation. Taking into account the volume of
the test portion and the number of colonies formed, the result can therefore be expressed as a number of
colony-forming units (cfu) or colony-forming particles (cfp) in a given volume of the sample, e.g. 1 ml or 100 ml.
8.2 Procedures
8.2.1 General
Three main procedures may be used for the inoculation of solid media.
a) Pour plate technique.
The test portion is mixed with the medium, which has previously been melted and cooled to a temperature
close to that of solidification; after incubation, the colonies that develop within and on the surface of the
medium are counted.
b) Spread plate technique.
The test portion is spread over the dry surface of an agar medium and, after incubation, colonies that develop
on its surface are counted.
c) Membrane filtration technique.
The test portion is passed through a membrane filter, which retains the micro-organisms sought; the
membrane is then placed on an agar medium or on an absorbent pad impregnated with liquid or rehydrated
medium. On incubation, colonies form on the surface of the membrane. Alternatively, for certain organisms,
such as anaerobes, the membrane may be placed face downwards in a Petri dish and overlaid with molten
agar medium.
8.2.2 Choice of technique
The choice of technique depends on several factors, including the physical and chemical characteristics of the
water as well as the nature of the micro-organisms sought (see Annex A, Clause A.3), their probable
concentration, the effective recovery of stressed or (sub-lethally) injured micro-organisms, the test precision
and the sensitivity required. Indications are given in 8.2.3.1, 8.2.4.1 and 8.2.5.1 of the volumes of water
samples that can be used for each technique and the limits of detection are discussed in Clause A.2. The
accuracy of the techniques is also discussed in Clause A.2. The requirements of regulations may also
influence the choice of technique to be used by indicating, for example, the precision desired or whether the
presence or absence of an organism in a specified test volume will be sufficient.
8.2.3 Pour plate technique
8.2.3.1 Test portion
The volume of the test portion of the sample, or of a dilution of the sample, can vary between 0,1 ml and 5 ml,
depending on the size of the Petri dish and the volume of culture medium used. The dilution should be chosen
so that the expected number of typical colonies formed on plates of diameter 90 mm to 100 mm is between
about 10 and 150. The total number of colonies on the plate (typical and non-typical) should be less than 300
6 © ISO 2005 – All rights reserved

ISO 8199:2005(E)
(see ISO 7218). Note, however, that the total number of countable colonies depends on the size of the
colonies and the number may have to be reduced for large colonies.
8.2.3.2 Inoculation
Melt the medium required in boiling water or by any other suitable process (e.g. an appropriate air incubator, a
steam flow-through autoclave or a microwave oven, if the heating time/temperature combination has been
validated for the medium preparation). Avoid over-heating and remove the medium as soon as it has been
melted. Place the molten medium in a water bath at (45 ± 1) °C for sufficient time so that the medium will
equilibrate to this temperature. It is preferable not to keep a medium molten for more than 4 h. Do not melt
agar media more than once.
Prepare and mark the Petri dishes required. Make any dilutions necessary in accordance with 7.2. Distribute,
after thorough mixing, the test portions into the dishes.
Remove each tube or flask of melted medium in turn from the water bath, dry the outside of the tube or flask
and flame the neck. Add the medium to each Petri dish without delay, avoiding the test portion to minimize
heat shock, and mix carefully so as to obtain a uniform distribution of micro-organisms. Generally, 15 ml of
medium is used for a test portion of 1 ml or 2 ml; for larger test portions, adjust the concentration of the
medium accordingly. Leave the plates to cool on a horizontal surface in order to allow the agar to solidify. As
soon as the agar has solidified, incubate the plates in accordance with 8.2.6.
NOTE Agar preparator pourer-stacker systems can be useful in laboratories analysing large numbers of samples.
8.2.4 Spread plate technique
8.2.4.1 Test portion
For a Petri dish of between 90 mm and 100 mm diameter, the volume of the test portion of the sample, or of a
dilution of the sample, should be 0,1 ml to 0,5 ml. Choose the dilution so that the expected number of typical
colonies formed lies between about 10 and 150. The total number of colonies on the plate (typical and non-
typical) should be less than 200. Note, however, that the total number of countable colonies depends on the
size of the colonies and the number may have to be reduced for large colonies.
8.2.4.2 Inoculation
Prepare and mark plates, each containing about 15 ml of culture medium. Dry the surface of the medium
before use (if necessary). Pipette the test portion on to the surface of the medium and spread over the surface
with a sterile rod or mechanical device. After absorption of the inoculum, incubate the plates in accordance
with 8.2.6.
For the drying of the plates the following points are of importance:
 The degree of humidity in culture media is important because optimum growth of bacteria will depend on
the humidity conditions in and on the medium. Extensive humidity loss may lead, for example, to an
increase in the concentrations of inhibitors in selective culture media and a reduction in the water activity
at the surface of the medium.
 When bacteria that do not spread rapidly are cultured, and the plates look dry after acclimatization, the
circumstances are such that drying is not always necessary. In that case, drying can be omitted, as it only
increases the likelihood of contamination and unnecessary humidity loss.
 Select the temperature and drying time so that the likelihood of contamination is kept as low as possible
and heating will not negatively affect the quality of the culture medium. The drying time will depend on the
degree to which condensation is present in the Petri dish, but shall be kept as short as possible.
In order to avoid contamination, and if the plates are not dried in a laminar-flow cabinet, plates shall always be
dried with the surface of the culture medium to be inoculated turned downwards.
ISO 8199:2005(E)
In practice, the plates will be dried by placing them with the agar surfaces downwards and with half-open lids
in an incubator set at a temperature between 25 °C and 50 °C. Dry the plates until the droplets have
disappeared from the surface of the lids. Do not dry any further. The agar plates can also be dried (in the
same way as described above) in a laminar-flow safety cabinet (at room temperature) for 30 min to 60 min, or
overnight at room temperature with the lids in place.
8.2.5 Membrane filtration technique
8.2.5.1 Test portion
The maximum volume of the test portion depends on the filterability of the water sample and on the
membranes used. This technique is suitable for waters that contain little particulate or colloidal (e.g. iron)
matter in suspension, for example drinking water. With membranes of mean pore size 0,45 µm, it may be
possible to filter several litres of such water through a single membrane, and so achieve a high level of test
sensitivity. For some organisms (like Legionella), however, filtration through a membrane with a mean pore
size of 0,2 µm may be necessary.
A test volume of the sample or a dilution of it should be chosen so that the expected number of typical
colonies formed on a membrane of 47 mm to 50 mm in diameter is between about 10 and 100. The total
number of colonies on the filter (typical and non-typical) should be less than about 200. Note, however, that
the total number of countable colonies depends on the size of the colonies and the number may have to be
reduced for large colonies.
8.2.5.2 Filtration
Connect the sterile filtration apparatus to a source of vacuum. Place a sterile membrane filter, grid-side
upwards, on the porous disc of the filter base, with only the outer part of the membrane filter being grasped by
flat-ended sterile forceps. Position the sterile funnel securely on the filter base. Pipette or pour one of the
following into the funnel (with the vacuum stopcock turned off):
a) a known volume of the sample, or dilution of it, carefully mixed (at least 10 ml);
b) the contents of a flask or bottle containing the test portion and sufficient diluent to bring the total volume
to at least 10 ml;
c) at least 10 ml of diluent, to which the test portion, measured with a pipette, is added directly and mixed
with the pipette.
Open the stopcock and apply sufficient vacuum (about 70 kPa) to filter the water through the membrane.
Close the stopcock as soon as the sample has been filtered. It may be advisable to rinse the funnel by filtering
one to three 10 ml to 30 ml portions of sterile diluent while the filter is still in place.
8.2.5.3 Transfer of membrane
Remove the funnel (make sure the stopcock is closed before doing this) and transfer the membrane with
sterile forceps to one of the following, ensuring that no air bubbles are trapped between the membrane and
the medium:
a) an agar medium in a Petri dish;
b) a sterile absorbent pad previously saturated with a liquid medium, or a dehydrated medium pad
reconstituted with sterile water, in a Petri dish (to avoid any confluent growth, pour off any excess medium,
preferably before placing the membrane on the pad);
c) a Petri dish, or on to a little agar medium in a Petri dish, subsequently overlaying the membrane with
molten agar medium at (45 ± 1) °C.
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ISO 8199:2005(E)
For different volumes of the same sample, the funnel may be re-used without disinfection provided that the
smallest volumes and/or the most diluted sample are filtered first. To filter another sample, either use a
separate sterile apparatus or disinfect the funnel, for example by flaming. Alternatively, follow the
manufacturer's instructions for disinfection.
8.2.6 Incubation
Invert the inoculated plates and place them either in an incubator or in a water-tight container in a water bath.
If necessary, pack the plates in plastic (bags) to prevent desiccation of the medium (e.g. when a fan-assisted
incubator is used). Plates containing membranes on absorbent pads should be placed (not inverted) in an air-
or water-tight container to prevent desiccation of the medium, if necessary. Do not make higher stacks than
6 Petri dishes to ensure that all plates reach incubation temperature rapidly.
Choose the duration and temperature of incubation by consulting a standard method, as they depend on the
micro-organism, or groups of micro-organisms, sought.
Incubation may be carried out in two stages, as follows:
Pre-incubation of variable duration, for example 2 h to 4 h at a lower temperature (e.g. 25 °C to 30 °C), can be
used to permit resuscitation of stressed organisms, followed by a longer period of incubation at the usual
temperature for the organism sought. The change may be effected either by transfer to another incubator or
water bath, or by using apparatus in which the temperature is automatically changed after a given time. Ideally,
place all the plates in the incubator or water bath at the same time.
Alternatively, the membranes may be incubated for a limited period (e.g. 2 h or 4 h) on a resuscitation medium
and then transferred to another medium, which is usually selective, for further incubation.
8.3 Enumeration
8.3.1 General
Examine the plates or membranes immediately after incubation. If this is not possible, they may be kept at
(5 ± 3) °C for short periods (e.g. a few days) provided that this does not affect the numbers, appearance or
subsequent confirmation of the colonies. If the plates or membranes are stored, validate the storage period as
being acceptable for the method and sample type.
8.3.2 Colonies to be counted and confirmed
For “total” counts on a non-selective medium, all the colonies are counted. With selective and differential
media, only those colonies that show the characteristic appearance of the organism sought are counted.
Magnification may be used (unless otherwise stated) when colony size is small and/or when it is otherwise
difficult to differentiate colonies from other particles. In general, it is unusual for such counts to indicate the
organisms belonging only to one taxonomic group, but in practice this discrepancy may be accepted and the
results can be expressed as presumptive. For more precise characterization, confirmatory tests are necessary.
It may be impracticable, however, if there are numerous colonies, to confirm the identity of all of them. In such
instances, examine all typical colonies from a sub-area of the plate or membrane (see also 8.4.3).
8.4 Calculation of results
8.4.1 Principle
A large part of this subclause has been taken from ISO 7218:1996/Amd 1:2001.
The methods of calculation defined below take account of the cases that occur most frequently when tests are
carried out in accordance with the methods of good practice of a laboratory. Rare, special cases may occur
(for example, significant discrepancy between the number of colonies on two plates with the same dilution, or
ISO 8199:2005(E)
a very different ratio to that of the dilution factor between the plates of two successive dilutions), and it is
therefore necessary that results obtained from counting be examined, interpreted or even refused by a
qualified microbiologist.
8.4.2 General case
The calculation of results given in this subclause can be used in cases when the total number of colonies on
the plates is between 10 and 200 (spread plate and membrane filtration techniques) or between 10 and 300
(pour plate technique), and where the number of typical colonies is between 10 and 150 (spread plate and
pour plate techniques) or between 10 and 100 (membrane filtration technique).
Since each colony is assumed to have arisen from one micro-organism or from a single aggregate of micro-
organisms, the result is expressed as the number of colony-forming units (cfu) or colony-forming particles (cfp)
in a specified reference volume of the sample (generally 100 ml or 1 ml) by Equation (1):
Z
CV=× (1)
ss
V
tot
where
C is the estimate of the number of cfu or cfp in the reference volume V of the sample;
s s
Z is the sum of colonies counted on plates or on membranes derived from dilutions d , d , ., d , or
1 2 i
derived from separate volumes of the test portion (sample or dilution);
V is the reference volume chosen to express the concentration of the micro-organisms in the sample;
s
V is the calculated total volume of original sample included in the plates enumerated. V is either the
tot tot
sum of the separate volumes from the test portion (sample or dilution) or calculated by Equation (2):
V = (n V d ) + (n V d ) + . + (nVd) (2)
tot 1 1 1 2 2 2 i i i
where
V is the calculated total volume of original sample included in the plates enumerated;
tot
n , n , ., n is the number of plates counted for dilution d , d , ., d ;
1 2 i 1 2 i
V , V , ., V is the test volume used with dilution d , d , ., d ;
1 2 i 1 2 i
d , d , ., d is the dilution used for the test portion volume V , V , ., V (d = 1 for an undiluted
1 2 i 1 2 i
sample, d = 0,1 for a ten-fold dilution, etc.).
NOTE 1 The final result thus obtained is a function of the weighted average of the counts from each plate.
Unless otherwise stated, round off the calculated results to two significant figures, when reporting final results.
In order to do this, if the third figure is less than 5 do not modify the preceding figure; if the third figure is
greater than or equal to 5, increase the preceding figure by one unit.
Express the result as a number preferably between 1,0 and 9,9 multiplied by the appropriate power of 10, or a
whole number with two significant figures.
10 © ISO 2005 – All rights reserved

ISO 8199:2005(E)
An example of the calculation for the pour or spread plate technique (enumerated in duplicate) is as follows:
EXAMPLE 1
If the volume of the test solution used (V ) is 1 ml, and the following counts are obtained at the respective dilutions:
i
Dilution Counts
−2
81 colonies and 97 colonies
−3
9 colonies and 15 colonies
then:
Z = 81 + 97 + 9 + 15 = 202
V = (2 × 1 × 0,01) + (2 × 1 × 0,001) = 0,022

tot
and if V is per ml:
s
C=×1= 9 182
s
0,022
then, after rounding off: C = 9,2 × 10 cfu/ml (or cfp/ml).
s
An example of the calculation for the membrane filtration technique (enumerated in singular) is as follows:
EXAMPLE 2
Test volume (V )
Counts
i
100 ml 82 colonies
10 ml 11 colonies
then:
Z = 82 + 11 = 93
V = (1 × 100 × 1) + (1 × 10 × 1) = 110

tot
and if V is per 100 ml:
s
C=×100= 84 cfu/100 ml (or cfp/100 ml).
s
It is desirable to report the results of such colony counts together with the 95 % confidence limits, which may
be derived by application of the following equations (see Reference [5]):
When Z W 20, the final result with 95 % confidence interval (CI) is:
1)
  
ZZ±22Z Z
CV±=95 % CI ×= ± ×V
  
ss s
  
VVV
tot tot tot
  
where the symbols are as defined above.
ISO 8199:2005(E)
2)
When Z < 20, the effect of the increasing skew (asymmetry) of the Poisson distribution on the confidence
limits is approximately taken into account by adding the small corrections included in the following
equation:

ZZ+±22 +1
Upper and lower 95 % CI=×V

s

V
tot

NOTE 2 The last equation gives a mean and 95 % CI even for Z = 0, which may be misleading.
NOTE 3 The number “2” (before the square root) in the equations is a rounding-off of 1,96 (≅ 2).
NOTE 4 The 95 % CI calculations assume a Poisson distribution in all cases.
8.4.3 Case after identification or confirmation
When the method used requires identification or confirmation, all presumed suspect colonies should be
inoculated from each of the plates retained for the counting of colonies. When this is impracticable, all typical
colonies (n) should be examined from a sub-area of the plate or membrane (n may vary from plate to plate).
The main reason for recommending the confirmation of all presumptive colonies from a certain area instead of
a random selection is that laboratories may not be able to isolate an objective random subset of the suspect
colonies. If a reliable random sample is obtainable, then the minimum and at the same time sufficient average
number for routine monitoring is to confirm about n = 5 colonies per plate. The recommended practice is to
isolate all colonies when the number is between 1 and 5. Thereafter, an average of n = 5 is appropriate. (If the
number of presumptive colonies is 6 or 7, it does not make much sense to select only a subset of n = 5.) After
identification or confirmation, calculate for each of the plates the confirmed result as the proportion of
presumptive colonies complying with the identification or confirmation criteria, using Equation (3):
k
xz=× (3)
n
where
x is the estimated number of confirmed colonies per plate;
k is the number of colonies complying with identification or confirmation criteria among the inoculated
colonies n;
n is the number of presumptive positive colonies inoculated from a plate for confirmation;
z is the total number of presumptive positive colonies counted on the plate.
Do not round off the intermediate confirmed test results x.
Calculate the number C of identified or confirmed micro-organisms present in the test sample by replacing Z
s
by X (the sum of x), using the equation given in 8.4.2. Round off and express the result as recommended in
8.4.2.
EXAMPLE
Counting has produced the following result:
Dilution Counts
−3
66 colonies and 80 colonies
−4
4 colonies and 7 colonies
12 © ISO 2005 – All rights reserved

ISO 8199:2005(E)
Confirmation of selected colonies was carried out:
of the 66 colonies, 8 colonies were tested, 6 of which complied with the criteria; hence x = ×=66 49,5;
of the 80 colonies, 9 colonies were tested, 6 of which complied with the criteria; hence x = 53,3;
of the 7 colonies, 5 colonies were tested, 4 of which complied with the criteria; hence x = 5,6;
of the 4 colonies, all 4 colonies were tested and all 4 complied with the criteria; hence x = z = 4,0.
4 4
X = 49,5 + 53,3 + 5,6 + 4,0 = 112,4
and if V is per ml:
s
X 112,4 112,4
CV=× = ×V= ×1= 51 091
ss s
V (2××1 0,001)+ (2×1× 0,000 1) 0,002 2
tot
After rounding off: C = 5,1 × 10 cfu/ml (or cfp/ml).
s
When partial (fractional) confirmation, like in the example above, is employed, the resulting confirmed results
[5]
do not follow the Poisson distribution. The equations for the 95 % CI as in 8.4.2 are not valid .
8.4.4 Special cases
8.4.4.1 All plates of the test sample containing less than 10 colonies
Counts from 20 up to the (practical) upper limit of each method are in the optimal precision range. Precision is
still acceptable for counts between 10 and 20. Precision decreases rapidly as colony numbers decrease below
10. Depending on the purpose of the test, a lower limit of determination can be defined at a count lower
than 10.
According to ISO/TR 13843, the definition of limit of deter
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