SIST-TS CEN ISO/TS 11133-2:2004
(Main)Microbiology of food and animal feeding stuffs - Guidelines on preparation and production of culture media - Part 2: Practical guidelines on performance testing of culture media (ISO/TS 11133-2:2003)
Microbiology of food and animal feeding stuffs - Guidelines on preparation and production of culture media - Part 2: Practical guidelines on performance testing of culture media (ISO/TS 11133-2:2003)
This Technical Specification specifies criteria and methods for the performance testing of culture media. This Technical Specification applies to:
3 commercial bodies producing and/or distributing ready-to-use or semi-finished reconstituted or dehydrated media to microbiological laboratories;
3 non-commercial bodies which supply media to third parties;
3 microbiological laboratories that prepare culture media for their own use and need to evaluate the performance of these media.
Mikrobiologie von Lebensmitteln und Futtermitteln - Anleitung für die Vorbereitung und Herstellung von Nährmedien - Teil 2: Praktische Anleitung zur Leistungsprüfung von Nährmedien (ISO/TS 11133-2:2003)
Microbiologie des aliments - Guide pour la préparation et la production des milieux de culture - Partie 2: Guide général pour les essais de performance des milieux de culture (ISO/TS 11133-2:2003)
La présente Spécification technique définit des criteres et des méthodes pour les essais de performance des milieux de culture. Cette Spécification Technique s'applique :
3 aux entités commerciales qui produisent des milieux prets a l'emploi, semi-finis reconstitués ou déshydratés et/ou les distribuent aux laboratoires de microbiologie ;
3 aux entités non commerciales qui fournissent des milieux a des tiers ;
3 aux laboratoires de microbiologie qui préparent des milieux de culture pour leur propre usage et qui doivent en évaluer les performances.
Mikrobiologija živil in krme - Navodila za pripravo in izdelavo gojišč - 2. del: Praktična navodila za vrednotenje gojišč (ISO/TS 11133-2:2003)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN ISO/TS 11133-2:2004
01-maj-2004
0LNURELRORJLMDåLYLOLQNUPH1DYRGLOD]DSULSUDYRLQL]GHODYRJRMLãþGHO
3UDNWLþQDQDYRGLOD]DYUHGQRWHQMHJRMLãþ ,6276
Microbiology of food and animal feeding stuffs - Guidelines on preparation and
production of culture media - Part 2: Practical guidelines on performance testing of
culture media (ISO/TS 11133-2:2003)
Mikrobiologie von Lebensmitteln und Futtermitteln - Anleitung für die Vorbereitung und
Herstellung von Nährmedien - Teil 2: Praktische Anleitung zur Leistungsprüfung von
Nährmedien (ISO/TS 11133-2:2003)
Microbiologie des aliments - Guide pour la préparation et la production des milieux de
culture - Partie 2: Guide général pour les essais de performance des milieux de culture
(ISO/TS 11133-2:2003)
Ta slovenski standard je istoveten z: CEN ISO/TS 11133-2:2003
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST-TS CEN ISO/TS 11133-2:2004 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
---------------------- Page: 2 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
TECHNICAL SPECIFICATION
CEN ISO/TS 11133-2
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
December 2003
ICS 07.100.30
English version
Microbiology of food and animal feeding stuffs - Guidelines on
preparation and production of culture media - Part 2: Practical
guidelines on performance testing of culture media (ISO/TS
11133-2:2003)
Microbiologie des aliments - Guide pour la préparation et la Mikrobiologie von Lebensmitteln und Futtermitteln -
production des milieux de culture - Partie 2: Guide général Anleitung für die Vorbereitung und Herstellung von
pour les essais de performance des milieux de culture Nährmedien - Teil 2: Praktische Anleitung zur
(ISO/TS 11133-2:2003) Leistungsprüfung von Nährmedien (ISO/TS 11133-2:2003)
This Technical Specification (CEN ISO/TS) was approved by CEN on 8 December 2002 for provisional application.
The period of validity of this CEN ISO/TS is limited initially to three years. After two years the members of CEN will be requested to submit
their comments, particularly on the question whether the CEN iSO/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN ISO/TS in the same way as for an EN and to make the CEN ISO/TS
available. It is permissible to keep conflicting national standards in force (in parallel to the CEN ISO/TS) until the final decision about the
possible conversion of the CEN ISO/TS into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, 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
© 2003 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN ISO/TS 11133-2:2003 E
worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
CEN ISO/TS 11133-2:2003 (E)
Contents Page
Foreword. 3
Introduction . 3
1 Scope . 3
2 Normative references . 4
3 Terms and definitions. 4
4 Criteria for routine quality control. 4
5 Methods for use in performance testing of culture media . 7
6 Documentation of test results . 13
Annex A (informative) Example of card for recording test results of culture media prepared by the user
laboratory. 15
Annex B (normative) Recommended test microorganisms for commonly used culture media (giving
information on the culture medium, culture conditions, test microorganisms, culture
collection number of test organisms and the expected reactions). 16
Bibliography . 26
2
---------------------- Page: 4 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
CEN ISO/TS 11133-2:2003 (E)
Foreword
This document (CEN ISO/TS 11133-2:2003) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN, in collaboration with Technical Committee
ISO/TC 34 “Food products”.
This document "Microbiology of food and animal feeding stuffs – Guidelines on preparation and production of
culture media" consist of two parts:
Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory
Part 2: Practical guidelines on performance testing of culture media
Annex A is informative. Annex B is normative.
This document includes a Bibliography.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Specification: Austria, Belgium, Czech Republic, Denmark,
Finland, France, Germany, Greece, Hungary Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway,
Portugal, Slovakia, Spain, Sweden, Switzerland and the United Kingdom.
Introduction
It is essential to use culture media of proven quality to carry out microbiological analysis of food reliably. For all
media described in standardized methods it is essential to define the minimum acceptance criteria required to
ensure media reliability. It is recommended that in the determination of the performance characteristics of a culture
medium tests are carried out that conform with this Technical Specification. This applies to:
a) commercially prepared ready-to-use or dehydrated media;
b) culture media prepared from basic constituents in the user’s laboratory.
The establishment of widely accepted minimum performance criteria for media should lead to more consistent
quality of commercially made products and thus reduce the extent of testing necessary in the user’s laboratory.
Furthermore the minimum acceptance criteria measured by the methods defined in this Technical Specification can
be used by all microbiological laboratories to evaluate the productive, selective and/or elective properties of a
culture medium.
In the microbiological analysis of food and animal feeding stuffs the requirements of this Technical Specification
have priority in the assessment of media quality.
1 Scope
This Technical Specification sets criteria and methods for the performance testing of culture media. This Technical
Specification applies to:
commercial bodies producing and/or distributing ready-to-use or semi-finished reconstituted or dehydrated
media to microbiological laboratories;
3
---------------------- Page: 5 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
CEN ISO/TS 11133-2:2003 (E)
non-commercial bodies supplying media to third parties;
microbiological laboratories preparing culture media for their own use and evaluating the performance of these
media.
2 Normative references
This Technical Specification incorporates by dated or undated reference, provisions from other publications. These
normative references are cited at the appropriate places in the text and the publications are listed hereafter. For
dated references, subsequent amendments to or revisions of any of these publications apply to this Technical
Specification only when incorporated in it by amendment or revision. For undated references the latest edition of
the publication referred to applies (including amendments).
ENV ISO 11133-1:2000, Microbiology of food and animal feeding stuffs – Guidelines on preparation and production
of culture media – Part 1: General guidelines on quality assurance for the preparation of culture media in the
laboratory (ISO/TR 11133-1:2000).
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ENV ISO 11133-1:2000 apply.
4 Criteria for routine quality control
4.1 General quality criteria
4.1.1 Quality of culture media
The quality of culture media depends on the quality of the basic ingredients, correct formulation, quality of
preparation procedures, elimination of contaminant microbial agents and appropriate packaging and storage
conditions (see ENV ISO 11133-1).
The manufacturer or producer in the laboratory shall comply with the physico-chemical characteristics of the culture
media as specified in the corresponding standard. Furthermore, quality assessment shall ensure that the culture
medium conforms to stated recommendations, including:
distributed quantity and/or thickness;
appearance, colour and homogeneity;
gel consistency;
moisture content;
pH value;
buffering capacity;
microbial contamination.
The individual components and any nutritive or selective supplements shall also undergo suitable quality
assessment procedures.
4
---------------------- Page: 6 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
CEN ISO/TS 11133-2:2003 (E)
4.1.2 Quality of basic media components
Culture media described in the International Standards were judged satisfactory; however, due to the variability of
their quality, it may be acceptable for media manufacturers to modify the concentration of some basic biological
ingredients, as listed below:
peptones and meat or yeast extracts variable in their nutritive properties;
agar variable in its gelling properties;
buffering substances;
bile salts, bile extract and desoxycholate, antibacterial dyes, depending on their selective properties;
antibiotics depending on their activity.
4.2 Microbiological quality criteria
4.2.1 General
The microbiological performance tests shall be carried out on a sample which is representative of a batch of end
product.
4.2.2 Microbial contamination
An appropriate quantity, depending on the size of the batch of culture medium, shall be tested for microbial
contamination by incubation under appropriate conditions. Target limits for the percentage of contaminated plates
or containers of liquid medium should be established for each medium or specified by the manufacturer.
Manufacturers should draw up specifications based on media components, processing limits and type of
packaging.
NOTE 1 The samples to be tested should be at least 1 plate or tube or 1 % of plates or tubes from the beginning and 1 plate
or tube or 1 % of plates or tubes from the end of a pouring or dispensing process. The plates or tubes should be incubated for at
least 18 h at 37ºC or under the incubation conditions which are used routinely for this medium according to the specific
standard.
NOTE 2 For statistical sampling plans refer to the ISO 2859-1:1999.
4.2.3 Growth
4.2.3.1 General
To evaluate each batch of complete culture medium, nutrient components or supplements, growth shall be
appropriately assessed by either:
a) quantitative; or
b) semi-quantitative; or
c) qualitative methods.
Quantitative, semi-quantitative or qualitative evaluations shall be performed by the methods described in this
Technical Specification or by another generally accepted technique. For interpretation of the results of testing, it is
necessary to compare the amount of growth on the test medium with that on a reference medium. The use of a
specific reference medium is therefore mandatory for quantitative methods (see the specific standard or Annex B)
For semi-quantitative or qualitative methods, the use of a specific reference medium (see corresponding specific
standard or Annex B) or a culture medium giving a "positive" reaction helps to interpret results. The reference
medium must be of known good quality chosen from a recently released batch, or, if comparing long term stability,
a batch from another supplier,or a ready-to-use medium, etc.
5
---------------------- Page: 7 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
CEN ISO/TS 11133-2:2003 (E)
In addition, growth of the target strains shall be typical in appearance, size and morphology of the colonies and
growth of the non-target strains shall be partly or completely inhibited.
4.2.3.2 Productivity
Solid, semi-solid or liquid culture media shall be inoculated with an appropriate inoculum (5.2.1.1) of the working
culture of each of the defined test microorganisms using an appropriate device.
Productivity shall reach a defined minimum limit (see corresponding specific standard or Annex B).
For quantitative methods the Productivity Ratio P (1) is determined as follows:
R
N
S
P = (1)
R
N
O
where
N is the total colony count obtained on the culture medium under test (obtained from one or more plates);
s
N is the total colony count obtained on the defined reference culture medium obtained from one or more
o
plates, and shall be
NOTE The Productivity Ratio of a non selective medium is at least 0,7 for microorganisms that can grow easily on that
medium. The P of the target microorganisms on a selective medium should be at least 0,1. These values are generally
R
achievable, however less rigorous criteria can be accepted for certain combinations of media and test microorganisms (see
corresponding specific standard or Annex B)
For semi-quantitative methods, the scores of consecutive sectors of a plate inoculated by the ecometric technique
are summed to obtain the growth index G , which varies according to the culture medium. It is therefore important
I
to compare them with previous indices and/or G of a reference medium and to ensure that variations are not
I
excessive. The expected range of variations for each culture medium can also be established once sufficient
experience of the method has been gained.
Qualitative evaluations shall be carried out visually by allocating growth scores.
4.2.3.3 Selectivity
To assess selectivity quantitatively, selective culture media and a reference medium are inoculated with an
appropriate inoculum (5.2.1.2.) of the defined test microorganism using an appropriate device. Selectivity has to
reach defined values (see corresponding specific standard or Annex B).
The Selectivity Factor S (2), is calculated as follows:
F
S = D-D (2)
F O S
where
D is the highest dilution showing growth of at least 10 colonies on the reference medium;
O
D is the highest dilution showing comparable growth on the test medium.
S
S , D and D are expressed in log units.
F O S 10
-4 -3
NOTE 1 If e.g. D 10 = log 4,0 and D 10 = log 3,0 then the selectivity factor is S = 1,0.
O 10 S 10 F
NOTE 2 The S of non-target microorganisms on a selective medium should be at least 2. This value is generally achievable.
F
However, less rigorous criteria can be accepted for certain combinations of media and test microorganisms (see corresponding
specific standard or Annex B).
6
---------------------- Page: 8 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
CEN ISO/TS 11133-2:2003 (E)
For semi-quantitative and qualitative methods the growth of the non-target strain(s) shall be inhibited partly or
completely.
4.2.4 Biochemical and physiological characteristics (selectivity and specificity)
The colony morphology and the diagnostic features together with the degree of selectivity should be established in
order to obtain a complete picture of the performance of a medium.
The essential characteristics of specificity shall be defined and achieved. For differential media the quality of
biochemical / physiological characteristics of the target microorganism(s) and the degree of inhibition of non-target
microorganisms should be determined with an appropriate set of test strains.
4.2.5 Antimicrobial testing characteristics
The antimicrobial action of antibiotics depends upon their agar diffusion characteristics and any antagonistic effects
from the components present. Media for testing the presence or absence of antimicrobial substances in food
samples should conform to reference methods.
4.3 Performance evaluation and interpretation of results
A batch of culture medium performs satisfactorily if all the test microorganisms used perform according to the given
specifications. It shall be accepted if both general and microbiological quality criteria are met.
5 Methods for use in performance testing of culture media
5.1 General
Examples of quantitative, semi-quantitative and qualitative testing methods for solid culture media and liquid media
are described. In most cases in the user’s laboratory semi-quantitative and qualitative techniques will meet the
performance testing requirements of a batch of culture medium.
For special cases, e.g. evaluation of a new medium or a new manufacturer, etc., quantitative testing methods shall
be performed by the user’s laboratory.
Familiarity with general microbiological techniques is assumed and therefore the methods are not given in
exhaustive detail.
Suitable test microorganisms are listed in Annex B (see also ENV ISO 11133-1).
NOTE It is the intention in the future, that new and revised individual standards for detection or enumeration of specific
microorganisms or groups of microorganisms will describe the relevant test microorganisms to be used, together with the
acceptance criteria for each culture medium in the standard.
In liquid media the interactions leading to the successful growth of microorganisms are more complex, hence
defining performance testing methods is less straightforward than for solid media.
For the successful isolation of targeted microorganisms in a multistage method, for example detection of
Salmonella, several complex interactions take place at each growth stage. Here a control test using appropriate
samples, culture and reference materials should be set up, so that the productivity or the sensitivity, respectively, of
the whole method is demonstrated. This is in addition to demonstrating that each component medium is fit for
purpose.
5.2 Test microorganisms
The appropriate reference strains of target (productivity) and non-target (selectivity) microorganisms for each
culture medium are given in Annex B. The test microorganisms should meet the requirements given in 5.2.2 of ENV
ISO 11133-1:2000, e.g. robust, weakly growing, biochemically unreactive or injured strains, as appropriate.
7
---------------------- Page: 9 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
CEN ISO/TS 11133-2:2003 (E)
Guidance on the preservation and maintenance of reference strains is given in Annex B of CEN/TS ISO/TC 11133-
1.
5.2.1 Preparation of the working culture
Working cultures shall be prepared as a pure stationary phase culture in a non-selective broth from the reference
stock culture.
Different techniques may be used, but shall guarantee the purity of the inoculum, as well as its standardisation
which allows it to be used at a later stage.
NOTE Frozen inocula may be used if it can be shown that the microorganism can survive for the chosen period.
5.2.1.1 Working culture for productivity testing
For semi-quantitative or qualitative tests and productivity testing of target microorganisms an inoculum level is used
to obtain 10 cfu to 100 cfu per plate or tube of medium.
5.2.1.2 Working culture for selectivity testing
4 6
For selectivity testing of culture media a suspension of the non-target microorganism containing 10 cfu to 10 cfu
per ml is inoculated onto the plate or into the tube of medium.
5.2.1.3 Incubation conditions
Incubate the inoculated culture media in accordance with the conditions described in the corresponding standard
and given in the appropriate tables in Annex B.
5.3 Methods for solid culture media
5.3.1 Quantitative plating method
5.3.1.1 General
This is a general method suitable for most solid culture media. It may not be suitable for testing some types of
moulds.
5.3.1.2 Procedure
Use working cultures as described in 5.2.1.
Select an appropriate number of plates representative of each batch to be tested and ensure the surface of
each plate is adequately dried. Plates of the reference medium should be similarly prepared (see 4.4.4 of ENV
ISO 11133-1:2000).
Spread onto the surface of the test and reference plates an inoculum of the diluted working culture to give
counts that fall within the recommended limits given in 5.2.1.
NOTE 1 The modified Miles-Misra surface drop method and other dropping systems or a spiral plater may also be used.
NOTE 2 The pour plate method may also be used for culture media normally used for enumeration in this way. Incubate
plates under appropriate conditions as defined in the individual standards.
Count the colonies present on each plate or from each drop as appropriate. Assess the size and appearance
of the colonies.
8
---------------------- Page: 10 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
CEN ISO/TS 11133-2:2003 (E)
5.3.1.3 Calculation
Based on the volume spread on the plates and the dilution factor, the mean count on the medium can be
calculated. In the case of dropping methods the number of drops and their volume must be considered.
5.3.1.4 Interpretation of results
To interpret the results, the Productivity Ratio P (4.2.3.2), and where appropriate the Selectivity Factor S (4.2.3.3),
R F
should be calculated.
5.3.2 Semi-quantitative streaking method based on ecometry
5.3.2.1 General
The streaking method is suitable for performance testing of solid and liquid culture media but the method is only
semi-quantitative. Growth indices are therefore only indicative and it can only be regarded as a supplementary test
for solid culture media.
When using this method the culture media tested should be dried to the same degree and the whole procedure
shall be standardized so that results of different batches can be compared.
5.3.2.2 Procedure
Agar plates are prepared in the usual manner with about 15 ml of agar. Media normally used for the pour plate
technique, for example Plate Count Agar (PCA), may also be tested by surface plating on solidified media.
Use working cultures as described in 5.2.1.
The plates are streaked as shown in Figure 1 using a 1 ml loop. Four parallel lines are drawn with the loop at
approximately 0,5 cm intervals over sector A. Streaking is repeated for sectors B and C and terminated in
sector D with a single line. A template can be used beneath the plate to facilitate accurate streaking.
The incubation times and temperatures stated in the standard methods are used.
NOTE Only the loop, not the wire, should be dipped in the culture. The loop should be completely filled with the culture.
Excess liquid should be removed by pressing the wider part of the loop three times against the edge of the container. When
streaking plates the angle between the loop and agar surface should be 20° to 30°. The pressure of the loop on the agar surface
and the rapidity of streaking must be consistent throughout. Dipping the loop in the culture whilst foam and/or bubbles are on
the surface of the broth should be avoided.
Normally the same loop is used for streaking all sectors from A to D without flaming the loop between streaks. In
some cases where a lower growth index G is expected to demonstrate distinct differences, changing or sterilising
I
the loop between streaking sectors A and B may be appropriate.
9
---------------------- Page: 11 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
CEN ISO/TS 11133-2:2003 (E)
Figure 1 — Pattern of inoculation by modified streaking method and angle of loop
5.3.2.3 Calculation
After incubation, the appearance, colony size and intensity of growth are assessed and the growth index G
I
calculated. Each streaking line showing growth is scored with 1. The maximum score per plate is 16. The streak is
scored as 0,5 if growth only occurs along half of its length. A streak without growth or with scanty growth (less than
half the length), is scored as 0. The scores are summed to obtain the G . For example, if growth was obtained in
I
sectors A and B and in half of sector C the G would be 10.
I
5.3.2.4 Interpretation of results
The growth index G given by a target strain should be at least 6 in order to conclude that the medium is
I
acceptable. In the case of non selective media the G is normally higher.
I
In addition, growth of the target strain shall be typical, and growth of non-target strains shall be partly or completely
inhibited.
5.3.3 Qualitative streaking method
5.3.3.1 General
The method is suitable for supplementary performance testing of solid culture media.
The method is only qualitative and scores are therefore only indicative.
5.3.3.2 Procedure
Agar plates are prepared in the usual manner with about 15 ml of agar. Media normally used for the pour plate
technique, for example Plate Count Agar (PCA), may also be tested by surface plating on solidified media.
Use working cultures as described in 5.2.1.
10
---------------------- Page: 12 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
CEN ISO/TS 11133-2:2003 (E)
The test microorganisms are streaked in parallel straight lines with a 1 ml loop on the surface of the test
medium. Several test microorganisms can be streaked on the same plate without crossing.
NOTE Other standardized streaking techniques can be used.
The incubation times and temperatures stated in the standard methods are used.
5.3.3.3 Interpretation of results
The growth on the plates after incubation is assessed as:
0 corresponds to zero growth,
1 corresponds to weak growth, and
2 corresponds to good growth.
Target microorganisms shall score 2 and have typical appearance, size and colony morphology. The growth of
non-target microorganisms shall be partly or completely inhibited (0 or 1).
5.4 Methods for liquid culture media
5.4.1 General
To determine the productivity of a liquid medium an appropriate inoculum shall be used. The quantitative,
semi-quantitative and qualitative methods described below assess productivity and selectivity. The proposed
methods record the quantity of growth after appropriate incubation by plating or streaking from the liquid media
onto agar media and enumerating colony forming units (cfu) or calculating scores from the liquid medium. For
qualitative methods in liquid media the characteristic reactions are assessed visually.
5.4.2 Quantitative dilution method for target and non-target microorganisms
The method is also appropriate for evaluation of new culture media, broths or diluents.
5.4.2.1 Procedure
Select an appropriate number of tubes or 10 ml portions of each batch of liquid medium to be tested.
Inoculation of target microorganisms: Inoculate test broth and reference broth for each test organism with a
small number (e.g. 10 cfu to 100 cfu into each tube; for preparation of the inoculum see 5.2.1.) and mix.
Inoculation of non-target microorganisms: Inoculate test broth and reference broth for each test organism with
a higher number (>1000 cfu into each tube; for preparation of the inoculum see 5.2.1.) and mix.
Inoculation of target and non-target microorganisms as a mixed culture: For testing mixed cultures in selective
media inoculate test broth and reference broth with a small number of target microorganisms (e.g. 10 cfu to
100 cfu for every tube; for preparation of the inoculum see 5.2.1.) and in the same tube with a higher number
of non-target microorganisms (> 1000 cfu into each tube; for preparation of the inoculum see 5.2.1.) and mix.
Inoculation of target and non-target microorganisms in diluents and transport media: Inoculate diluents with
test microorganisms (e.g. 100 cfu to 1000 cfu into each tube; for preparation of the inoculum see 5.2.1.) and
mix.
The incubation times and temperatures stated in the standard methods are used.
Diluents should be incubated for 45 min at room temperature and then plated out. Transport media should be
incubated at an appropriate temperature and time according to normal usage and then plated out.
11
---------------------- Page: 13 ----------------------
SIST-TS CEN ISO/TS 11133-2:2004
CEN ISO/TS 11133-2:2003 (E)
Remove an aliquot volume or if necessary a dilution from each broth after the incubation step and spread to a
non-inhibitory agar plate as described in 5.3.1.
NOTE 1 The modified Miles-Misra surface drop method, other dropping systems or a spiral plater can be used to give
countable colonies on the plates.
NOTE 2 To test mixed cultures, spreading should be done when possible on non-selective agar plates which allow
differentiation of the microorganisms in the mixed culture (e.g. Plate Count Agar with MUG for counting Escherichia coli and
Salmonella spp.). When it is not possible to distinguish mixed cultures on non-selective agar, selective agar media should be
used providing that their performance has been previously tested.
5.4.2.2 Reading, calculation and interpretation
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.