Plant biostimulants - Determination of microorganisms' concentration

This document specifies general rules to determine the concentration of microorganisms present in plant biostimulant products.
The method is applicable to microbial plant biostimulants for verifying that the concentration of microorganisms does not exceed the respective limits outlined in the EU Regulation on Fertilising Products [1].
This horizontal method might not be appropriate in every detail for certain products. In this case, it is necessary to refer to the methodology of specific determination and quantification of the microorganisms.

Pflanzen-Biostimulanzien - Bestimmung der Konzentration von Mikroorganismen

Dieses Dokument legt allgemeine Regeln für die Bestimmung der Konzentration von Mikroorganismen in Biostimulansprodukten fest.
Das Verfahren ist auf mikrobielle Pflanzen-Biostimulanzien anwendbar, und zwar, um zu verifizieren, dass die Konzentration von Mikroorganismen die entsprechenden Grenzwerte der EU Düngeprodukte-Verordnung [1] nicht überschreitet.
Dieses horizontale Verfahren ist für bestimmte Produkte nicht unbedingt bis ins Detail geeignet. In diesem Fall ist es notwendig, auf die Methodik der spezifischen Bestimmung und Quantifizierung der Mikro-organismen zurückzugreifen.

Biostimulants des végétaux - Détermination de la concentration en microorganismes

Le présent document spécifie les règles générales pour déterminer la concentration en microorganismes présents dans les produits biostimulants des végétaux.
La méthode est applicable aux biostimulants des végétaux microbiens pour vérifier que la concentration en microorganismes ne dépasse pas les limites correspondantes indiquées dans le Règlement UE sur les fertilisants [1].
Cette méthode horizontale pourrait ne pas être appropriée dans chaque détail pour certains produits. Dans ce cas, il est nécessaire de se référer à la méthodologie de détermination et de quantification spécifique des microorganismes.

Rastlinski biostimulanti - Določevanje koncentracije mikroorganizmov

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SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17714:2021
01-november-2021
Rastlinski biostimulansi - Določevanje koncentracije mikroorganizmov
Plant biostimulants - Determination of microorganisms' concentration
Biostimulanzien für die pflanzliche Anwendung - Bestimmung der Konzentration von
Mikroorganismen
Ta slovenski standard je istoveten z: FprCEN/TS 17714
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17714:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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kSIST-TS FprCEN/TS 17714:2021
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kSIST-TS FprCEN/TS 17714:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17714
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
September 2021
ICS 65.080
English Version
Plant biostimulants - Determination of microorganisms'
concentration
Biostimulanzien für die pflanzliche Anwendung -
Bestimmung der Konzentration von Mikroorganismen

This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee

CEN/TC 455.

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, Turkey and

United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change

without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17714:2021 E

worldwide for CEN national Members.
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kSIST-TS FprCEN/TS 17714:2021
FprCEN/TS 17714:2021 (E)
Contents Page

European foreword ............................................................................................................................................ 3

Introduction .......................................................................................................................................................... 4

1 Scope .......................................................................................................................................................... 6

2 Normative references .......................................................................................................................... 6

3 Terms and definitions ......................................................................................................................... 6

4 Principe of the method ....................................................................................................................... 7

5 Sampling ................................................................................................................................................... 7

6 Preparation of sample for microbial analysis ............................................................................ 7

7 Method for enumeration of microorganism ............................................................................... 7

7.1 General...................................................................................................................................................... 7

7.2 Enumeration using a solid media.................................................................................................... 8

7.2.1 General...................................................................................................................................................... 8

7.2.2 Number of Petri dishes per dilution .............................................................................................. 8

7.2.3 Pour plate techniques ......................................................................................................................... 8

7.2.4 Surface inoculation .............................................................................................................................. 8

7.2.5 Incubation................................................................................................................................................ 9

7.2.6 Calculation and expression of results obtained with solid media .................................... 10

7.3 Enumeration and quantification using a liquid medium ..................................................... 17

7.3.1 Principle ................................................................................................................................................. 17

7.3.2 Inoculation ............................................................................................................................................ 17

7.3.3 Choice of inoculation system .......................................................................................................... 17

8 Expression of results ......................................................................................................................... 19

9 Test report ............................................................................................................................................. 19

10 Quality assurance ............................................................................................................................... 19

Bibliography ....................................................................................................................................................... 20

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FprCEN/TS 17714:2021 (E)
European foreword

This document (FprCEN/TS 17714:2021) has been prepared by Technical Committee CEN/TC 455 “Plant

biostimulants”, the secretariat of which is held by AFNOR.
This document is currently submitted to the Vote on TS.

This document has been prepared under a mandate M/564 given to CEN by the European Commission

and the European Free Trade Association, and supports essential requirements of EU Directive(s).

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FprCEN/TS 17714:2021 (E)
Introduction

This document was prepared by the experts of CEN/TC 455 ‘Plant Biostimulants’. 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 laying down rules on the making available on the market of

EU fertilising products (“FPR” or “Fertilising Products Regulation”). This request, presented as SR M/564,

also contributes to the Communication on “Innovating for Sustainable Growth: A Bio economy for

Europe”. The Working Group 5 “Labelling and denominations”, was created to develop a work program

as part of this Request. The technical committee CEN/TC 455 ‘Plant Biostimulants’ was established to

carry out the work program that will prepare a series of standards. The interest in 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.

Biostimulants used in agriculture can be applied in multiple ways: on soil, on plant, as seed treatment,

etc. A microbial plant biostimulant consists of a microorganism or a consortium of microorganisms, as

referred to in Component Material Category 7 of Annex II of the EU Fertilizing Products Regulation.

This document is applicable to all biostimulants in agriculture.

The Table 1 below summarizes many of the agro-ecological principles and the role played by

biostimulants.
Table 1 — Agro-ecological principles and the role played by biostimulants
Increase biodiversity
By improving soil microorganism quality/quantity
Reinforce biological regulation and interactions
By reinforcing plant-microorganism interactions
- symbiotic exchanges i.e. mycorrhize
- symbiotic exchanges i.e. rhizobiaciae/fava
- secretions mimicking plant hormones (i.e. trichoderma)
By regulating plant physiological processes
- for ex growth, metabolism, plant development…
Improve biogeochemical cycles
- improve absorption of nutritional elements
- improve bioavailability of nutritional elements in the soil
- stimulate degradation of organic matter

This document defines the general rules for the determination of microorganism concentration in a

sample of biostimulant product.

The specific concentrations of microorganisms required in specific standard methods take precedence

over the general rules listed in this document.
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WARNING — Person 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.
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1 Scope

This document was provided to define general rules for determine microorganism concentration present

in plant biostimulant products.

The method is applicable to microbial plant biostimulants for verifying that the concentration of

microorganisms does not exceed the respective limits outlined in the EU Regulation on Fertilising

Products [1].

This horizontal method might not be appropriate in very detail for certain products. In this case, it is

necessary to refer to the methodology of specific determination and quantification of the

microorganisms.
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.

FprCEN/TS 17708, Plant biostimulants — Preparation of sample for microbial analysis

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

• IEC Electropedia: available at https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
3.1
plant biostimulant

product stimulating plant nutrition processes independently of the product's nutrient content with the

sole aim of improving one or more of the following characteristics of the plant or the plant rhizosphere:

a) nutrient use efficiency;
b) tolerance to abiotic stress;
c) crop quality traits;
d) availability of confined nutrients in soil or rhizosphere
3.2
microorganism
entity of microscopic size, encompassing bacteria, fungi, protozoa and viruses
[SOURCE: EN ISO 11139:2018, 3.176]
3.3
colony

localized visible accumulation of microbial mass (such as prokaryotes, bacteria, micromycetes, yeast and

fungi) or organisms (such as Dreissena species) developed on or in a solid nutrient medium from a viable

particle or organism
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FprCEN/TS 17714:2021 (E)

NOTE 1 to entry: Frequently, micro colonies from nearby viable particles, before becoming visible, fuse into

one macro colony. The number of visible colonies is, therefore, usually and underestimate of the number of viable

particles.
[ISO 6107-6:2021, [3], modified]
3.4
product

portion of an identified plant biostimulant product received in the laboratory for testing

3.5
sample

portion of the product (3.4) (at least 1 g or 1 mL) that is used in the test to prepare the initial suspension

3.6
initial suspension

suspension (or solution) of the sample (3.5) in a defined volume of an appropriate diluent

4 Principe of the method

The method of determining the concentration of microorganisms has been developed to provide a

general method for enumeration of microorganisms present in the plant biostimulant product. The

results are expressed as the number of active units by volume or weight, or in any other way relevant to

the microorganism, for example colony formation units per gram or the UPM for Mycorhize.

5 Sampling

Sampling is not part of the method specified in this document (see FprCEN/TS 17702-1). If there is no

specific International or European Standard, it is recommended that the parties concerned come to an

agreement on this subject.

It is important that the laboratory receives a sample which is representative and has not been damaged

or changed during transport or storage.
6 Preparation of sample for microbial analysis

Prepare the test sample from the laboratory sample in accordance with FprCEN/TS 17708. If there is no

specific International or European Standard, it is recommended that the parties concerned come to an

agreement on this subject.
7 Method for enumeration of microorganism
7.1 General

When assessing the microbiological quality of biostimulants products, it is often not enough to know only

which microorganisms are present. In most cases, the quantitative aspect is equally important, which

brings about the need to enumerate microorganisms. This may be achieved in various ways: through

direct examination (microscopy), by inoculating solid or liquid media. However, this document only

covers enumeration using solid and liquid media.

Enumeration on solid media is based on the capacity of many microorganisms to produce colonies in or

on agar media that can be recognized as such with the naked eye or with the aid of a simple magnifying

glass.
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If the level of bacteria is expected to be very low (less than 10 colonies in or on a plate at the lowest

dilution), enumeration using liquid media is recommended (e.g. MPN) to improve statistical reliability of

the results.
7.2 Enumeration using a solid media

The different steps for enumeration using a solid media (number of petri dishes/dilution, Pour plate

techniques, surface inoculation, Incubation) are described in the different chapters below and/or specific

standard for microorganism detection/determination.
7.2.1 General

Petri dishes should be labelled with the sample number, dilution, date and any other desired information.

Dilutions should be selected to ensure that plates containing the appropriate number of colonies are

obtained (see 7.3.1) and to overcome any possible inhibitory properties.

Use a separate sterile pipette for transfers from each dilution, except if working from the highest dilution

to the lowest dilution.
7.2.2 Number of Petri dishes per dilution

For enumeration techniques in biostimulant(s) product, one plate per dilution shall be used with at least

two successive dilutions. Two plates per dilution may also be used to improve reliability.

If only one dilution is used, then two plates of this dilution shall be used to improve reliability of the

results.

For laboratories that do not operate under quality assurance principles, two plates per dilution shall be

used to improve reliability of the results.
7.2.3 Pour plate techniques

Withdraw the defined volumes of the dilution to be examined, touching the tip of the pipette against the

side of the tube to remove excess liquid adhering to the outside. Lift the sterile Petri dish lid just high

enough to insert the pipette, then dispense the contents.

After removing tempered agar medium from the water bath, blot the bottle dry with a clean towel to

prevent water from contaminating the plates. Avoid spilling the medium on the outside of the container

or on the inside of the plate lid when pouring. To avoid contamination of the media, hold the bottle in a

near horizontal position.

Also avoid setting down the bottle between pouring steps. Pour molten agar medium at 44 °C to 47 °C

into each Petri dish (generally 18 ml to 20 ml of agar in 90 mm Petri dishes and 45 ml to 50 ml in 140 mm

Petri dishes, to obtain at least 3 mm thickness) within 15 min of inoculation (to avoid aggregation of

colonies). Avoid pouring the molten medium directly on to the inoculum. Immediately mix the molten

medium and the inoculum carefully so as to obtain a homogeneous distribution of the microorganisms

within the medium, e.g. by gently moving the dish backwards and forwards, from side to side and in a

circular direction. Allow to cool and solidify by placing the Petri dish on a cool horizontal surface (the

solidification time of the agar shall not exceed 10 min).
7.2.4 Surface inoculation
7.2.4.1 General

Methods of plating designed to produce only surface colonies on agar plates have certain advantages. The

morphology of surface colonies is easily observed, improving the analyst’s ability to distinguish between

different types of colony.
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Use pre-poured plates, of at least 3 mm thickness of the agar medium, that are level and free from air

bubbles and surface moisture.

To facilitate uniform spreading, the surface of solidified agar should be dried in accordance with

EN ISO 11133:2014 or as specified in the relevant International Standard so that the inoculum is

absorbed within 15 min.
7.2.4.2 Spreading-spatula method

Using a sterile pipette, transfer the inoculum (usually 0,1 ml or 0,5 ml) of the liquid test sample or of the

initial suspension in the case of other samples to the agar plate (90 mm or 140 mm in diameter,

respectively). Repeat this step for the next decimal dilution (the colonies to be counted will then be

−1 −2

present in a dilution step of 10 in the case of liquid sample material and 10 in the case of other sample

material) and, if necessary, repeat for further decimal dilutions.

The limit of enumeration can be lowered by a factor of 10 by inoculating 1,0 ml of the test sample if liquid,

or 1,0 ml of the initial suspension for other products, either on the surface of one large agar plate

(140 mm) or on the surface of three small agar plates (90 mm). In both cases, if only one dilution is used,

prepare duplicates by using two large plates or six small ones.

Using a spreading spatula made of glass, plastic or steel (for example made from a glass rod and shaped

like a hockey stick about 3,5 mm in diameter and 20 cm long, bent at right angles at about 3 cm from one

end and flattened at the ends by heating), spread the inoculum as quickly as possible evenly over the agar

surface without touching the side walls of the Petri dish. Allow the inoculum to absorb with the lids in

place for about 15 min at room temperature.

In certain cases (as stated in the relevant International Standard), the inoculum may be deposited on a

membrane then spread as described previously.
7.2.5 Incubation

Unless otherwise stated in specific standards, invert dishes once they have been inoculated, and place

them quickly in the incubator set at the appropriate temperature. If excessive dehydration occurs (e.g. at

55 °C or in the event of strong air circulation), wrap the dishes loosely in plastic bags prior to incubation

or use any similar system of equivalent efficiency.

During the incubation period, minor variations in the incubation temperature may be unavoidable and

acceptable, for example during the usual operations of loading or unloading the incubator, but it is

important that these periods are kept to a minimum. The duration of these variations should be

monitored to ensure that they do not have a significant effect on the result.

It may sometimes be useful to laboratory operations to refrigerate inoculated dishes before incubation

for no more than 24 h. If this is done, the laboratory shall ensure that this practice does not affect the

resulting counts.

Generally, Petri dishes should be stacked no more than six high for aerobic incubation and should be

separated from each other and from the incubator walls by at least 25 mm. However, higher stacks with

less spacing may be acceptable in incubators fitted with air circulation systems; in this case, the

temperature distribution should be verified. After incubation, the dishes should normally be examined

immediately. They may, however, be stored, unless otherwise specified in specific standards, for up to 48

h in a refrigerator. Refrigerated storage is only acceptable if it has been shown to have no effect on the

numbers, appearance or the subsequent confirmation of the colonies. With certain media containing

indicator dyes, refrigerated plates should be allowed to equilibrate at room temperature before

examining, to ensure that the correct colour is regained.
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7.2.6 Calculation and expression of results obtained with solid media
7.2.6.1 Counting of colonies

Following the period of incubation stated in the specific standard, count the colonies (total colonies,

typical colonies or presumed suspect colonies) for each dish containing up to and including 300 colonies

(or any other number stated in the specific standard).

In this subclause, the cases dealt with correspond to the following general cases:

— inoculation of one 90 mm-diameter Petri dish per dilution, and at least two successive dilutions are

performed;
— maximum countable number for the total colonies present: 300 per dish;

— maximum total number of colonies (typical and atypical) present on a dish when counting typical or

presumptive colonies: preferably 300 per dish;
— maximum countable number for typical or presumptive colonies: 150 per dish;

— number of presumptive colonies inoculated for identification or confirmation in each dish retained:

in general 5.
These figures shall be defined in the specific standards.

The methods of calculation defined in the following are for the cases which occur most frequently when

tests are carried out in accordance with good laboratory practice. Special cases may occasionally occur

(e.g. the number of colonies in two dishes with the same dilution may show significant discrepancy or the

ratio of the dilution factors used for two successive dilutions may be very different) and it is therefore

necessary for the counting results obtained to be examined and interpreted by a qualified microbiologist

and, if necessary, rejected.

7.2.6.2 Method of calculation: general case (counting of total colonies or typical colonies)

For a result to be valid, it is generally considered necessary to count the colonies on at least one dish

containing at least 10 colonies [total colonies, typical colonies or colonies complying with identification

or confirmation criteria (see 7.2.6.3).

Calculate the number N of microorganisms present in the test sample as a weighted mean from two

successive dilutions using Formula (1):
N= (1)
Vd ××1,1
where

ƩC is the sum of the colonies counted on the two dishes retained from two successive dilutions, at

least one of which contains a minimum of 10 colonies;
V is the volume of inoculum placed in each dish, in millilitres;

d is the dilution corresponding to the first dilution retained [d = 1 when the undiluted liquid

product (test sample) is retained].
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kSIST-TS FprCEN/TS 17714:2021
FprCEN/TS 17714:2021 (E)

If more than one dilution is used, the ratio between the colony count of dilution d and the colony count

of dilution d is expected to be 10 %. The upper and lower limits should be specified by the laboratory

for the colony count of dilution d

EXAMPLE If colony count of dilution d1 is 250, the colony count of dilution d2 should not be less than 13

(5,2 %) and not be more than 39 (15,6 %).

Round off the calculated result to two significant figures. When doing 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 preferably as a number between 1,0 and 9,9 multiplied by the appropriate power of

10, or a whole number with two significant figures.

Report the result as the number N of microorganisms per millilitre (liquid products) or per gram (other

products).
7.2.6.3 Method of calculation: Case after identification or confirmation
7.2.6.3.1 General

When the method used requires identification or confirmation, a given number A (generally 5) of

presumptive colonies is identified from each of the dishes retained for the colony counting. After

identification or confirmation, calculate, for each of the dishes, the number a of colonies complying with

identification or confirmation criteria, using Formula (2):
a= xC (2)
where

b is the number of colonies complying with identification or confirmation criteria among the

identified colonies A;
C is the total number of presumptive colonies counted on the dish.

Round off the calculated result to the nearest whole number. When doing this, if the first figure after the

decimal point is less than 5, do not modify the preceding figure; if the first figure after the decimal point

is greater than or equal to 5, increase the preceding figure by one unit.

Calculate the number N, N or N ' of identified or confirmed microorganisms present in the test sample

by replacing C by a in the formula given in 7.2.6.2 or by replacing C by a in the formula given in

∑ ∑
7.2.6.4.1 and 7.2.6.5.3. Round off the result as specified in 7.2.6.2.

Express the result as specified in 7.2.6.2, 7.2.6.4.1 and 7.2.6.5.3, respectively.

7.2.6.3.2 Example
Counting has produced the following results:
— at the first dilution retained (10 ): 66 colonies;
— at the second dilution retained (10 ): 4 colonies.
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Testing of selected colonies was carried out:

— of the 66 colonies, 8 colonies were tested, 6 of which complied with the criteria, hence a = 50;

— of the 4 colonies, all 4 complied with the criteria; hence a = 4.
∑ a 50+ 4 54
N 49 090
−−33
V x 1,1 xd
11 xx ,11 0 1,11 x 0

Rounding off the result as specified in 7.2.6.2, the number of microorganisms is 49 000 or 4,9 × 10 per

millilitre or per gram of product.
7.2.6.4 Method of calculation: estimated counts

7.2.6.4.1 Case of two dishes (test sample or initial suspension or first dilution) contains less than

10 colonies

Requirements and recommendations of 7.2.6.4.1 concerning the lower limit of determination are

applicable.

If each of the two dishes from the test sample (liquid products), or from the initial suspension (other

products), or from the first dilution inoculated or retained, contains less than 10 colonies (total colonies,

typical colonies or colonies complying with identification or confirmation criteria) and the set of the two

dishes contains at least 4 colonies, calculate the estimated number NE of microorganisms present in the

test sample as an arithmetical mean of the colonies counted on the two dishes using Formula (3):

∑ C
N= (3)
where
ΣC is the sum of the colonies counted on the two dishes;
V is the volume of inoculum applied to each dish, in millilitres;
n is the number of dishes retained (in this case, n = 2);

d is the dilution factor of the initial suspension or of the first dilution inoculated or retained

[d = 1 when the undiluted liquid product (test sample) is used].
Round off the result as recommended in 7.2.6.2.
Express the result as follows:

— estimated number N of microorganisms per millilitre (liquid products) or per gram (other

products).
====
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7.2.6.4.2 Example
Counting has produced the following results:
— at the first dilution (10 ) retained, 8 and 9 colonies were counted
8+ 9 17
N 850
0,02
1 xx 2 10

By rounding off the result as recommended in D.3, the estimated number N of microorganisms is 850 or

8,5 × 10 per millilitre or per gram of product.

7.2.6.4.3 Case of two dishes (test sample or initial suspension or first dilution) contains no

colonies

If the two dishes from the test sample (liquid products) or from the initial suspension (other products)

or from the first dilution inoculated or retained, do n
...

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