EN 17716:2024
(Main)Plant biostimulants - Determination of Escherichia coli
Plant biostimulants - Determination of Escherichia coli
This document gives guidance for the detection and identification of the specified microorganism Escherichia coli in technical and formulated plant biostimulants, both in liquid and solid states, and also the horizontal method for the enumeration of ß-glucuronidase-positive E. coli in plant biostimulants (both in liquid and solid states).
The qualitative method described in this document is based on the detection of E. coli in a non-selective liquid medium (enrichment broth), followed by isolation on a selective agar. Other methods can be appropriate, depending on the level of detection required.
NOTE 1 For the detection of E. coli, subcultures can be performed on non-selective culture media followed by suitable identification steps (e.g. using identification kits).
The quantitative method described in this document uses a colony-count technique at 44 °C ± 1 °C on a solid medium containing a chromogenic ingredient for detection of the enzyme ß-glucuronidase.
NOTE 2 Strains of E. coli which do not grow at 44 °C ± 1 °C and, in particular, those that are ß-glucuronidase negative, such as E. coli O157, will not be detected.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants, and where the following category Plant Biostimulants is the highest % in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.
Pflanzen-Biostimulanzien - Bestimmung von Escherichia coli
Dieses Dokument enthält Leitlinien für den Nachweis und die Identifizierung des festgelegten Mikro-organismus Escherichia coli in technischen und formulierten Pflanzen-Biostimulanzien, sowohl in flüssigem als auch in festem Zustand, sowie das horizontale Verfahren für die Zählung von β Glucuronidase-positiven E. coli in Pflanzen-Biostimulanzien (sowohl in flüssigem als auch in festem Zustand).
Das in diesem Dokument beschriebene qualitative Verfahren basiert auf dem Nachweis von E. coli in einem nicht-selektiven flüssigen Medium (Anreicherungsbouillon), gefolgt von der Isolierung auf einem selektiven Agar. Es können auch andere Methoden geeignet sein, abhängig vom erforderlichen Nachweisgrad.
ANMERKUNG 1 Für den Nachweis von E. coli können Subkulturen auf nicht-selektiven Nährmedien angelegt werden, gefolgt von geeigneten Identifizierungsschritten (z. B. unter Verwendung von Identifizierungskits).
Das in diesem Dokument beschriebene quantitative Verfahren verwendet ein Koloniezählverfahren bei (44 ± 1) °C auf einem festen Medium, das einen chromogenen Bestandteil zum Nachweis des Enzyms β Glucuronidase enthält.
ANMERKUNG 2 Stämme von E. coli, die bei (44 ± 1) °C nicht wachsen, und insbesondere solche, die β Glucuronidase-negativ sind, wie z. B. E. coli O157, werden nicht erkannt.
Dieses Dokument ist für die Mischungen von Düngeprodukten anwendbar, in denen eine Mischung aus mindestens zwei der folgenden Kategorien von Komponenten-EU Düngeprodukten besteht: Düngemittel, Kalkdünger, Bodenverbesserungsmittel, Kultursubstrate, Pflanzen-Biostimulanzien und wenn die folgende Kategorie Pflanzen-Biostimulanzien prozentual den höchsten Massen- oder Volumenanteil in der Mischung bzw., bei flüssiger Form, den höchsten Trockenmasseanteil aufweist. Wenn Pflanzen-Biostimulanzien nicht den höchsten prozentualen Anteil in der Mischung ausmachen, ist die für den höchsten prozentualen Anteil geltende Europäische Norm anwendbar. Falls eine Düngemittelmischung aus Komponenten gleicher Menge besteht oder falls die für die Mischung verwendeten Komponenten-EU Düngeprodukte identische Formulierungen haben, entscheidet der Anwender, welche Norm angewendet wird.
Biostimulants des végétaux - Détermination des Escherichia coli
Le présent document donne des lignes directrices pour la détection et l’identification du microorganisme spécifié Escherichia coli dans les produits techniques et formulés à usage biostimulant des végétaux, aussi bien à l’état liquide qu’à l’état solide, ainsi que la méthode horizontale pour le dénombrement des E. coli ß-glucuronidase-positive dans les biostimulants des végétaux (aussi bien à l’état liquide qu’à l’état solide).
La méthode qualitative décrite dans le présent document repose sur la détection des E. coli dans un milieu liquide non sélectif (bouillon d’enrichissement), suivie de l’isolement sur une gélose sélective. D’autres méthodes peuvent être appropriées, en fonction du niveau de détection requis.
NOTE 1 Pour la détection des E. coli, des subcultures peuvent être effectuées sur des milieux de culture non sélectifs, suivies d’étapes d’identification appropriées (par exemple, à l’aide de kits d’identification).
La méthode quantitative décrite dans le présent document utilise une technique de dénombrement des colonies à 44 °C ± 1 °C sur un milieu solide contenant un ingrédient chromogène pour la détection de l’enzyme ß-glucuronidase.
NOTE 2 Certaines souches d’E. coli qui ne poussent pas à 44 °C ± 1 °C et, en particulier, celles qui sont ß-glucuronidase-négative, telles que les E. coli O157, ne seront pas mises en évidence.
Le présent document est applicable aux mélanges de fertilisants constitués d’au moins deux composants appartenant aux catégories de fertilisants UE suivantes : engrais, amendements minéraux basiques, amendements du sol, supports de culture, biostimulants des végétaux et, dans lesquels la catégorie Biostimulants des végétaux représente le % le plus élevé du mélange en masse ou en volume, ou dans le cas d’un liquide, en masse sèche. Si les biostimulants des végétaux ne représentent pas le % le plus élevé du mélange, la Norme européenne relative au composant représentant le % le plus élevé du mélange s’applique. Dans le cas où un mélange de fertilisants contient des composants en quantité égale ou dans le cas où les composants des fertilisants UE utilisés pour le mélange ont des formulations identiques, l’utilisateur décide quelle norme appliquer.
Rastlinski biostimulanti - Določanje Escherichia coli
Ta dokument podaja smernice za ugotavljanje prisotnosti in identifikacijo navedenega mikroorganizma Escherichia coli v tehničnih in formuliranih biostimulantih, tako v tekočem kot v trdnem stanju, ter tudi horizontalno metodo za štetje E. coli, pozitivne na ß-glukuronidazo, v rastlinskih biostimulantih (tako v tekočem kot v trdnem stanju).
Kvalitativna metoda, opisana v tem dokumentu, temelji na ugotavljanju prisotnosti mikroorganizma E. coli v neselektivnem tekočem gojišču (obogatitveni bujon), ki mu sledi izolacija v selektivnem agarskem gojišču. Ustrezne so lahko tudi druge metode, odvisno od zahtevane ravni ugotavljanja prisotnosti.
OPOMBA 1: Za namen ugotavljanja prisotnosti mikroorganizma E. coli je mogoče precepljene kulture vzgojiti v neselektivnem gojišču kultur, čemur sledijo ustrezni koraki prepoznavanja (npr. uporaba kompletov za prepoznavanje).
Kvantitativna metoda, opisana v tem dokumentu, uporablja tehniko štetja kolonij pri temperaturi 44 °C ± 1 °C v trdnem gojišču, ki vsebuje kromogeno sestavino za določanje encima ß-glukuronidaze.
OPOMBA 2: Sevi E. coli, ki ne rastejo pri temperaturi 44 °C ± 1 °C, in zlasti tisti, ki so negativni na ß-glukuronidazo, kot je E. coli O157, ne bodo zaznani.
Ta dokument se uporablja za mešanice sredstev za gnojenje, ki vsebujejo najmanj dve od naslednjih kategorij sredstev za gnojenje EU: gnojila, sredstva za apnenje, izboljševalci tal, rastni substrati in rastlinski biostimulanti, pri čemer rastlinski biostimulanti predstavljajo največji masni ali prostorninski odstotek mešanice ali, v primeru tekoče oblike, odstotek suhe mase. Če rastlinski biostimulanti ne predstavljajo največjega odstotka v mešanici, se uporablja evropski standard za največji odstotek mešanice. Če vsebuje mešanica sredstev za gnojenje sestavine v enakih količinah ali če imajo posamezna sredstva za gnojenje EU, uporabljena za mešanico, enake formulacije1, standard za uporabo izbere uporabnik.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2025
Nadomešča:
SIST-TS CEN/TS 17716:2023
Rastlinski biostimulanti - Določanje Escherichia coli
Plant biostimulants - Determination of Escherichia coli
Pflanzen-Biostimulanzien - Bestimmung von Escherichia coli
Biostimulants des végétaux - Détermination des Escherichia coli
Ta slovenski standard je istoveten z: EN 17716:2024
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 17716
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 65.080 Supersedes CEN/TS 17716:2022
English Version
Plant biostimulants - Determination of Escherichia coli
Biostimulants des végétaux - Détermination des Pflanzen-Biostimulanzien - Bestimmung von
Escherichia coli Escherichia coli
This European Standard was approved by CEN on 26 August 2024.
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-CENELEC 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-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N
EUROPÄISCHES KOMITEE FÜR NORMUN G
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17716:2024 E
worldwide for CEN national Members.
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 7
4 Principle . 7
4.1 Qualitative method . 7
4.2 Quantitative method . 7
5 Diluent and culture media . 8
5.1 General . 8
5.2 Broth and culture media in the qualitative method . 8
5.2.1 Enrichment broth . 8
5.2.2 Selective culture media: Tryptone-Bile-Glucuronic agar (TBX) for isolation of E. coli 8
5.3 Diluent and culture media in the quantitative method . 8
5.3.1 Diluent . 8
5.3.2 Culture media: Tryptone-Bile-Glucuronic agar (TBX) . 8
6 Apparatus and glassware . 8
7 Handling of plant biostimulants and sampling . 8
8 Procedure. 9
8.1 General . 9
8.2 Qualitative method . 9
8.2.1 General . 9
8.2.2 Incubation of the inoculated enrichment broth. 9
8.2.3 Detection and identification of E. coli . 9
8.3 Quantitative method . 10
8.3.1 Test portion and initial suspension . 10
8.3.2 Serial dilutions . 10
8.3.3 Inoculation (surface plate method) and incubation . 10
8.3.4 Counting the colony-forming units . 11
9 Expression of results . 11
9.1 Expression of results in the qualitative test . 11
9.2 Expression of results in the quantitative test . 11
10 Test report . 12
Annex A (normative) Composition and preparation of culture media and reagents . 13
A.1 General . 13
A.2 Enrichment broth . 13
A.3 Other enrichment broth . 14
A.4 Selective agar for isolation . 14
A.5 Other selective agar for isolation . 15
A.6 Selective agar for confirmation . 16
Annex B (normative) Neutralization of the antimicrobial properties of the product . 17
B.1 General . 17
B.2 Suitability of the detection method . 17
B.3 Interpretation of suitability test results . 18
Annex C (informative) Interlaboratory study . 19
C.1 Materials used in the interlaboratory study . 19
C.2 Results of the interlaboratory study . 20
Annex ZA (informative) Relationship of this European Standard and the essential
requirements of Regulation (EU) 2019/1009 making available on the market of EU
fertilising products aimed to be covered . 22
Bibliography . 23
European foreword
This document (EN 17716:2024) has been prepared by Technical Committee CEN/TC 455 “Plant
biostimulants”, the secretariat of which is held by AFNOR.
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 May 2025, and conflicting national standards shall be
withdrawn at the latest by May 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes CEN/TS 17716:2022.
— the Introduction has been updated and Table 1 has been removed;
— normative references have been updated;
— in Clause 3, new terms and definitions have been added and others have been revised;
— Annexes A, B and C have been revised.
— Annex ZA has been added.
This document has been prepared under a standardization request addressed to CEN by the European
Commission. The Standing Committee of the EFTA States subsequently approves these requests for its
Member States.
For the relationship with EU Legislation, see informative Annex ZA, which is an integral part of this
document.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the United
Kingdom.
Introduction
The European Committee for Standardization (CEN) was requested by the European Commission (EC) to
draft European Standards or European Standardization deliverables to support the implementation of
Regulation (EU) 2019/1009 of 5 June 2019 [1] laying down rules on the making available on the market
of EU fertilising products (“FPR” or “Fertilising Products Regulation”).
This standardization request, presented as SR M/564 and relevant amendments, also contributes to the
Communication on “Innovating for Sustainable Growth: A Bio economy for Europe”. The interest in plant
biostimulants has increased significantly in Europe as a valuable tool to use in agriculture.
Standardization was identified as having an important role in order to promote the use of biostimulants.
The work of CEN/TC 455 seeks to improve the reliability of the supply chain, thereby improving the
confidence of farmers, industry, and consumers in biostimulants, and will promote and support
commercialisation of the European biostimulant industry.
WARNING — Persons using this document should be familiar with normal laboratory practice. This
document 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 document be
carried out by suitably trained staff.
1 Scope
This document gives guidance for the detection and identification of the specified microorganism
Escherichia coli in technical and formulated plant biostimulants, both in liquid and solid states, and also
the horizontal method for the enumeration of ß-glucuronidase-positive E. coli in plant biostimulants
(both in liquid and solid states).
The qualitative method described in this document is based on the detection of E. coli in a non-selective
liquid medium (enrichment broth), followed by isolation on a selective agar. Other methods can be
appropriate, depending on the level of detection required.
NOTE 1 For the detection of E. coli, subcultures can be performed on non-selective culture media followed by
suitable identification steps (e.g. using identification kits).
The quantitative method described in this document uses a colony-count technique at 44 °C ± 1 °C on a
solid medium containing a chromogenic ingredient for detection of the enzyme ß-glucuronidase.
NOTE 2 Strains of E. coli which do not grow at 44 °C ± 1 °C and, in particular, those that are ß-glucuronidase
negative, such as E. coli O157, will not be detected.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of
the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers,
Growing Media, Plant Biostimulants, and where the following category Plant Biostimulants is the highest
% in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not
the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend
of fertilizing products is composed of components in equal quantity or in case the component EU
fertilising products used for the blend have identical formulations , the user decides which standard to
apply.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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.
EN 17708:2024, Plant biostimulants — Preparation of sample for microbial analysis
EN 17714:2024, Plant biostimulants — Determination of microrganims concentration
EN 17702-1:2024, Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
EN 17724:2024, Plant biostimulants — Terminology
EN ISO 21148:2017, Cosmetics — Microbiology — General instructions for microbiological examination
(ISO 21148:2017)
An example of such a blend is a product with 2 claimed functions consisting of a non-microbial plant biostimulant
and an organic fertilizer composed of 1kg/kg of plant biostimulant from seaweed
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 17724:2024 and the following
apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
3.1
Escherichia coli
Gram-negative rod, motile, smooth colonies, member of Enterobacteriaceae
Note 1 to entry: The main characteristics for identification are catalase positive, oxidase negative, fermentation
of lactose, production of indole, growth on selective agar containing bile salts with characteristic colonies.
Note 2 to entry: E. coli can be isolated from moist environmental sources (air, water, soil) and is a faecal
contamination indicator.
3.2
enrichment broth
non-selective liquid medium containing suitable neutralizers (according to Annex B) and/or dispersing
agents and demonstrated to be suitable for the plant biostimulant under test
3.3
ß-glucuronidase-positive E. coli
bacteria which form typical blue to blue-green colony on Tryptone-Bile-Glucuronide agar (TBX) after
incubation at 44 °C ± 1 °C for 24 hours
4 Principle
4.1 Qualitative method
The first step of the qualitative procedure is to perform an enrichment by using a non-selective broth
medium to increase the number of microorganisms without the risk of inhibition by the selective
ingredients that are present in selective/differential growth media.
The second step of the test (isolation) is performed on a selective medium followed by identification tests.
The presence or absence of E. coli per gram or per millilitre of sample is calculated
(according to Clause 9).
4.2 Quantitative method
In the quantitative method, duplicate plates of TBX are inoculated with the specified quantity of the test
sample (if the product is liquid) or the initial suspension.
Under the same conditions, using decimal dilutions of the test sample or of the initial suspension, two
plates per dilution shall be inoculated.
The dishes are incubated for 18 h to 24 h at 44 °C ± 1 °C then examined to detect the presence of colonies
which, from their characteristics, are considered to be ß-glucuronidase-positive E. coli.
The number of colony-forming units (CFU) of ß-glucuronidase-positive E. coli per gram or per millilitre
of sample is calculated (according to Clause 9).
5 Diluent and culture media
5.1 General
The following diluents and culture media are suitable for the detection of E. coli and the enumeration of
ß-glucuronidase-positive E. coli according to the proper procedure. Other diluents and culture media may
be used if they have been demonstrated to be suitable for use.
Diluents and culture media should be prepared using the descriptions provided or from
reagents/dehydrated culture media, according to the instructions from the manufacturer. The
instructions provided by the supplier of the media/reagents should be followed for storage conditions,
expiry date and use.
NOTE Ready-to-use diluents and media may be used when their composition and/or growth yields are
comparable to those of the formulae given in the present document.
5.2 Broth and culture media in the qualitative method
5.2.1 Enrichment broth
The enrichment broth shall be used in the qualitative method to disperse the sample and to increase the
initial microbial population. A non-exhaustive list and recipes of the possible enrichment broths are given
in Annex A.
5.2.2 Selective culture media: Tryptone-Bile-Glucuronic agar (TBX) for isolation of E. coli
The selective agar shall be used in the qualitative method for the isolation and identification of E. coli. The
list and recipes of the selective agar are given in Annex A.
5.3 Diluent and culture media in the quantitative method
5.3.1 Diluent
The list and recipes of the usable diluent in the preparation of the initial suspension and further decimal
dilution is not part of the method specified in this document: EN 17708:2024 shall be used.
5.3.2 Culture media: Tryptone-Bile-Glucuronic agar (TBX)
The list and recipes of the culture medium that shall be used in the inoculation by plating technique of
the initial suspension and further decimal dilutions are given in Annex A.
6 Apparatus and glassware
The laboratory equipment, apparatus and glassware typical of microbiological laboratory according to
EN 17708:2024 shall be used.
7 Handling of plant biostimulants and sampling
The laboratory shall receive a sample which is truly representative and has not been damaged or changed
during transport or storage.
Sampling is not part of the method specified in this document: EN 17702-1:2024 shall be used.
If necessary, the sample to be tested should be equilibrated at room temperature before starting the
analysis.
8 Procedure
8.1 General
According to the aim of the analysis, one of the following described methods shall be performed.
The qualitative method (according to 8.2) shall be followed to evaluate the presence or absence of E. coli
in at least 1 g or 1 ml of the sample under test.
The quantitative method (according to 8.3) shall be followed to determine the number of ß-
glucuronidase-positive E. coli in terms of CFU per g or per ml of the sample under test.
8.2 Qualitative method
8.2.1 General
The preparation of the initial suspension is not part of the method specified in this document:
EN 17708:2024 shall be used.
Record S, the exact mass or exact volume of the sample used in the preparation of the initial suspension.
The intial suspension shall be made using the enrichment broth described in Clause A.2. Another
enrichment broth may be used (described in Clause A.3).
10 ml of the well mixed initial suspension shall be sampled and incubated (see 8.2.2).
8.2.2 Incubation of the inoculated enrichment broth
The initial suspension prepared in broth (8.2.1) shall be incubated at 32,5 °C ± 2,5 °C for at least 20 h
(maximum 72 h).
For samples with suspected contamination, the initial suspension (8.2.1) should be incubated at
44 °C ± 1°C.
8.2.3 Detection and identification of E. coli
8.2.3.1 Isolation
Using a sterile loop (10 µl), an aliquot of the incubated enrichment broth (8.2.2) shall be streaked onto
the surface of TBX agar to obtain isolated colonies.
Invert the Petri dish and then incubate at 44 °C ± 1°C for 18 h to 24 h. The total incubation time shall not
be longer than 24 h. Check for characteristic colonies (see Clause 3.3).
If the presence of stressed cells is suspected, incubate for an initial period of 4 h at 37 °C ± 1°C, and then
raise the incubation temperature to 44 °C ± 1°C for 18 h to 24 h. The incubation temperature shall not
exceed 45 °C.
8.2.3.2 Identification of E. coli
8.2.3.2.1 General
In case of doubts about the morphological characteristic grown colonies, proceed to the following tests
for these suspect colonies isolated on the selective agar. The presence of E. coli may be confirmed by other
suitable, cultural and biochemical tests.
8.2.3.2.2 Gram stain
Perform the test specified in EN ISO 21148:2017. Check for Gram-negative rods (bacilli).
8.2.3.2.3 Culture on levine eosin-methylene blue agar (EMB agar)
The suspect isolated colonies grown on TBX agar shall be inoculated onto the surface of the EMB agar
(described in A.6.1). Invert the Petri dish and then incubate at 32,5 °C ± 2,5 °C for at least 24 h (maximum
48 h).
For samples with suspected contamination, the Petri dish should be incubated at 44 °C ± 1°C.
The characteristic colonies shall be checked (described in Table A.2).
8.3 Quantitative method
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