BS ISO 19344:2015

BSI Standards Publication

Milk and milk products —

Starter cultures, probiotics
and fermented products —
Quantification of lactic acid
bacteria by flow cytometry
BS ISO 19344:2015 BRITISH STANDARD

National foreword
This British Standard is the UK implementation of ISO 19344:2015.
The UK participation in its preparation was entrusted to Technical
Committee AW/5, Chemical analysis of milk and milk products.
A list of organizations represented on this committee can be
obtained on request to its secretary.
This publication does not purport to include all the necessary
provisions of a contract. Users are responsible for its correct
application.
© The British Standards Institution 2015.
Published by BSI Standards Limited 2015
ISBN 978 0 580 85108 7
ICS 67.100.10
Compliance with a British Standard cannot confer immunity from
legal obligations.
This British Standard was published under the authority of the
Standards Policy and Strategy Committee on 31 December 2015.
Amendments/corrigenda issued since publication
Date Text affected
 BS ISO 19344:2015

INTERNATIONAL ISO
STANDARD 19344
IDF
232
First edition
2015-12-15

Milk and milk products — Starter

cultures, probiotics and fermented
products — Quantification of lactic
acid bacteria by flow cytometry
Lait et produits laitiers — Cultures, probiotiques et produits fermentés
— Quantification de bactéries lactiques par cytométrie en flux

Reference numbers
ISO 19344:2015(E)
IDF 232:2015(E)

© ISO and IDF 2015

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

COPYRIGHT PROTECTED DOCUMENT

© ISO and IDF 2015, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office International Dairy Federation
Ch. de Blandonnet 8 • CP 401 Silver Building • Bd Auguste Reyers 70/B • B-1030 Brussels
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11 Tel. + 32 2 325 67 40
Fax +41 22 749 09 47 Fax + 32 2 325 67 41
copyright@iso.org info@fil-idf.org
www.iso.org www.fil-idf.org

ii  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Contents Page

Forewords...................................................................................................................................................................................................................................... iv
Introduction................................................................................................................................................................................................................................. vi
1 Scope.................................................................................................................................................................................................................................. 1
2 Normative references....................................................................................................................................................................................... 1
3 Terms and definitions...................................................................................................................................................................................... 1
4 Principle......................................................................................................................................................................................................................... 2
5 Diluents and reagents...................................................................................................................................................................................... 3
5.1 General............................................................................................................................................................................................................ 3
5.2 Peptone-salt solution.......................................................................................................................................................................... 3
5.3 Diluents and reagents for staining protocols................................................................................................................. 3
5.3.1 Protocol A................................................................................................................................................................................ 4
5.3.2 Protocol B................................................................................................................................................................................ 4
5.3.3 Protocol C................................................................................................................................................................................ 5
6 Apparatus...................................................................................................................................................................................................................... 6
7 Sampling......................................................................................................................................................................................................................... 7
8 Preparation of test sample.......................................................................................................................................................................... 7
8.1 General............................................................................................................................................................................................................ 7
8.2 Freeze-dried cultures......................................................................................................................................................................... 7
8.3 Frozen cultures........................................................................................................................................................................................ 8
8.4 Fermented milk products............................................................................................................................................................... 8
9 Procedure..................................................................................................................................................................................................................... 8
9.1 General............................................................................................................................................................................................................ 8
9.2 Staining........................................................................................................................................................................................................... 8
9.2.1 Protocol A................................................................................................................................................................................ 9
9.2.2 Protocol B................................................................................................................................................................................ 9
9.2.3 Protocol C................................................................................................................................................................................ 9
9.3 Flow cytometry analysis............................................................................................................................................................... 10
9.3.1 General................................................................................................................................................................................... 10
9.3.2 Instruments and settings........................................................................................................................................ 10
9.4 Gating............................................................................................................................................................................................................ 11
9.4.1 General................................................................................................................................................................................... 11
9.4.2 Protocol A............................................................................................................................................................................. 11
9.4.3 Protocol B............................................................................................................................................................................. 13
9.4.4 Protocol C............................................................................................................................................................................. 14
10 Calculation and expression of results...........................................................................................................................................15
11 Critical factors affecting results..........................................................................................................................................................16
12 Precision..................................................................................................................................................................................................................... 17
12.1 Interlaboratory test.......................................................................................................................................................................... 17
12.2 Repeatability........................................................................................................................................................................................... 18
12.3 Reproducibility..................................................................................................................................................................................... 18
13 Test report................................................................................................................................................................................................................. 18
Annex A (informative) Diagram of staining protocols......................................................................................................................19
Annex B (informative) Calculation example of the appropriate sample dilution................................................21
Annex C (informative) Interlaboratory test................................................................................................................................................22
Bibliography.............................................................................................................................................................................................................................. 25

© ISO and IDF 2015 – All rights reserved  iii

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Forewords
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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 5, Milk
and milk products and the International Dairy Federation (IDF). This document is being published
jointly by ISO and IDF.

iv  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

IDF (the International Dairy Federation) is a non-profit private sector organization representing the
interests of various stakeholders in dairying at the global level. IDF members are organized in National
Committees, which are national associations composed of representatives of dairy-related national
interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and
governments/food control authorities.
ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis
and sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International
Standards using the logos and reference numbers of both organizations.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. IDF shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
ISO 19344 | IDF 232 was prepared by the IDF Standing Committee on Analytical Methods for Dairy
Microorganisms and the ISO Technical Committee ISO/TC 34 on Food products, Subcommittee SC 5 on
Milk and milk products.
The work was carried out by the IDF/ISO Project Group on Quantification of Lactic Acid Bacteria by Flow
Cytometry of the Standing Committee on Analytical Methods for Dairy Microorganisms under the aegis of
its project leader, Sandra Casani (DK), Ph.D.

© ISO and IDF 2015 – All rights reserved  v

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Introduction
Quantification of lactic acid bacteria is an important factor in assessing the quality of starter cultures,
probiotics and fermented milk products. Examination of lactic acid bacteria in these products can be
done following different method principles, with plate count techniques being the most traditional and
widely used. Newer techniques include flow cytometry, which is able to determine cells as active and/or
total units. Advantages of the use of flow cytometry include low variation, differentiation between
active and total cells, and possibility of high analysis throughout. Furthermore, the quantification and
use of the fraction of active cells per total cells is a key feature and an important flow cytometry tool to
evaluate the fitness of a given cell population. This is of special relevance for certain applications such
as optimization of production process and stability assessment during shelf-life.
The International Organization for Standardization (ISO) and the International Dairy Federation
(IDF) draw attention to the fact that compliance with this document may involve the use of patents
concerning the staining of protocol C as described in this document.
Neither ISO nor IDF take position concerning the evidence, validity and scope of these patent rights.
The holder of these patent rights has ensured ISO and IDF that he/she is willing to negotiate licences
either free of charge or under reasonable and non-discriminatory terms and conditions with applicants
throughout the patented territory. In this respect, the statement of the holder of these patent rights is
registered with ISO. Information may be obtained from:
Chr. Hansen A/S
Boege Alle 10-12
2970 Hoersholm
Denmark

Attention is drawn to the possibility that some of the elements of this document may be the subject
of patent rights other than those identified above. Neither ISO nor IDF shall be held responsible for
identifying any or all such patent rights.
ISO (www.iso.org/patents) and IEC (http://patents.iec.ch) maintain online databases of patents
relevant to their standards. Users are encouraged to consult the databases for the most up-to-date
information concerning patents.

vi  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015

INTERNATIONAL STANDARD ISO 19344:2015(E)

IDF 232:2015(E)

Milk and milk products — Starter cultures, probiotics

and fermented products — Quantification of lactic acid
bacteria by flow cytometry
WARNING — The use of this International Standard may involve hazardous materials and
operations. This International Standard does not purport to address all the safety problems
associated with its use. It is the responsibility of the user of this International Standard to
establish safety and health practices and to determine the applicability of regulatory limitations
prior to use.

1 Scope
This International Standard specifies a standardized method for the quantification of active and/or
total lactic acid bacteria and probiotic strains in starter cultures used in dairy products by means of
flow cytometry. The method is also applicable to probiotics used in dairy products and to fermented
milk products such as yogurts containing primarily lactic acid bacteria.
This International Standard does not apply to taxonomical differentiation of bacteria. Due to its non-
specificity, the method may quantify other bacteria than those within the scope of this International
Standard, when present in the sample. This may lead to overestimation of the counts.
The minimum bacterial cell concentration in the sample before applying this standardized method
depends on the dilution rates used in the individual protocols. Typically 106 cells per gram or ml are
considered within the minimum range.

2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 6887-1:1999, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial
suspension and decimal dilutions for microbiological examination — Part 1: General rules for the
preparation of the initial suspension and decimal dilutions
ISO 6887-5:2010, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial
suspension and decimal dilutions for microbiological examination — Part 5: Specific rules for the
preparation of milk and milk products
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 7889 | IDF 117, Yogurt — Enumeration of characteristic microorganisms — Colony-count technique at
37 °C
ISO 15214, Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of
mesophilic lactic acid bacteria — Colony-count technique at 30 °C

3 Terms and definitions

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

© ISO and IDF 2015 – All rights reserved  1

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

3.1
lactic acid bacteria
gram-positive, non-motile, non-spore forming, catalase-negative, nitrate-reductase-negative and
cytochrome oxidase-negative bacterium that does not liquefy gelatine or produce indole
Note 1 to entry: Lactic acid bacteria have a fermentative metabolism which is mainly saccharolytic. Lactic acid is
the major end product from carbohydrate utilization.

EXAMPLE Lactic acid bacteria of importance for the dairy industry are: Streptococcus thermophilus,
Lactococcus lactis, Pediococcus, Enterococcus, Leuconostoc and Lactobacillus.

3.2
probiotic strains
probiotic strains are live microorganisms which, when administered in adequate amounts, are intended
to confer a health benefit to the host
EXAMPLE Probiotic strains of importance are: Bifidobacterium animalis, Lactobacillus casei, Lactobacillus
paracasei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus plantarum and
Propionibacterium freudenreichii.

Note 1 to entry: See Reference [1].

3.3
active fluorescent units
AFU
events counted in a gate specific for scatter/fluorescence characteristics of presumed live cells, i.e. cells
stained for the specific activity indicator used in the protocol
3.4
non-active fluorescent units
n-AFU
events counted in a gate specific for scatter/fluorescence characteristics of presumed dead cells, i.e.
cells damaged to an extend that they do not stain for the specific activity indicator used in the protocol
3.5
total fluorescent units
TFU
sum of AFU and n-AFU
3.6
% active fluorescent units
% AFU
percentage ratio of AFU to TFU

4 Principle

4.1 A test portion or sample is prepared, and diluted if necessary.

4.2 Initial suspensions, and/or dilutions if needed, are stained according to one of the following three
protocols, differing on the target of fluorescent cell staining, in order to discriminate active and total
fluorescent units:

a) dual staining targeting nucleic acid with the non-permeant red-fluorescent dye propidium iodide
(PI) and intracelullar enzyme activity based on cleavage of 5(6)-carboxyfluorescein diacetate
(cFDA) mixed isomers to green-fluorescent carboxyfluorescein by intracellular esterases;

2  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

b) dual nucleic acid staining with PI and a cell-permeant green fluorescent dye, i.e. SYTO®1) 24 green
fluorescent cell-permeant nucleic acid stain;
c) single staining with the membrane-potential-sensitive cyanine dye 3,3’-diethyloxacarbocyanine
iodide (DiOC2). Wavelength of emitted light changes with metabolic activation of cells.
The choice of the staining protocol depends on the user’s preferences or possibilities.

4.3 The stained samples are analysed by means of a flow cytometer using a combination of light
scattering (LS) and detection of emitted fluorescent light. As cells pass into the flow cytometer, each cell
is counted and the fluorescence is recorded.

4.4 Gating is conducted to separate cells from noise and to differentiate AFUs and n-AFUs.

4.5 Calculation of the concentration in the original sample is a multiplication of AFUs (or TFUs) per
volume of analysed sample and the dilution factors employed in the sample preparation.

5 Diluents and reagents

5.1 General
Unless otherwise specified, use only reagents of recognized analytical grade, and distilled or deionized
water or water of equivalent purity, according to ISO 7218.
Prepare the initial suspension (common for all protocols) with the diluent as specified in 5.2.
The composition and the preparation of all the reagents used in each of the three staining protocols (A, B
and C) are specified in 5.3. An overview of the diluents and reagents per protocol is given in Table 1.

5.2 Peptone-salt solution

The composition and preparation of the peptone-salt solution is according to ISO 6887-5:2010, 5.2.1.
NOTE For the preparation of the initial suspension, and dilutions if needed, other diluents for general use
mentioned in ISO 6887-5:2010 can be used if they can be shown to lead to the same results.

5.3 Diluents and reagents for staining protocols

WARNING — Propidium iodide is a potential mutagen. Proper actions for deactivation should be
taken in case of spilling. Preparation and application of the dye solution shall be carried out in a
fume cupboard, using protective equipment and following good laboratory practices.

Table 1 — Reagents used per protocol

Reagent Protocol A Protocol B Protocol C
Diluent PBS (5.3.1.1) PBS (5.3.2.1) MRS or M17 broth
(5.3.3.1 or 5.3.3.2)
Dye solution cFDA (5.3.1.2) PI (5.3.2.2) Glucose solution (5.3.3.3.1)
PI (5.3.1.3) SYTO® 24 (5.3.2.3) DiOC2 (5.3.3.3.2)
Buffer solution (5.3.3.3.3)

1) SYTO® 24 green fluorescent cell-permeant nucleic acid stain is supplied by Life Technologies. This information
is given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of the
product named. Equivalent products may be used if they can be shown to lead to the same results.

© ISO and IDF 2015 – All rights reserved  3

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

5.3.1 Protocol A

5.3.1.1 Phosphate-buffered saline (PBS)

5.3.1.1.1 Composition

— 9 g sodium chloride (NaCl)

— 795 mg sodium hydrogenphosphate heptahydrate (Na2HPO4·7H2O)
— 144 mg potassium dihydrogen phosphate (KH2PO4)

5.3.1.1.2 Preparation

Dissolve the components (see 5.3.1.1.1) in water. Add water to a final volume of 1 000 ml. Adjust the pH
with HCl to 7,4 ± 0,05, if necessary. Distribute the solution into aliquots and sterilize in an autoclave set
at 121 °C ± 1 °C (liquid cycle) for 15 min. The diluent can be stored at cooling temperature (3 °C ± 2 °C)
for up to 6 months.

5.3.1.2 5(6)-Carboxyfluorescein diacetate (cFDA) mixed isomers solution

5.3.1.2.1 Composition

— 230 mg 5(6)-cFDA mixed isomers

— 100 ml dimethyl sulfoxide (DMSO)

5.3.1.2.2 Preparation

A 5 mmol/l solution is prepared by dissolving cFDA in DMSO at the amounts specified in 5.3.1.2.1. The
solution can be stored at −18 °C ± 2 °C, protected from light, for up to 6 months.

5.3.1.3 Propidium iodide (PI)

5.3.1.3.1 Composition

— 100 mg PI
— 100 ml ultrapure water

5.3.1.3.2 Preparation

Dissolve the PI in ultrapure water to a final concentration of 1,0 mg/ml, corresponding to approximately
1,5 mmol/l. This can be stored at 3 °C ± 2 °C, protected from light, for up to 6 months.
NOTE The concentration of the PI solution used is 0,1 % and the final concentration is 0,002 %. This is below
the potential toxicity level.

5.3.2 Protocol B

5.3.2.1 Phosphate-buffered saline (PBS)

See 5.3.1.1.

4  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

5.3.2.2 Propidium iodide (PI)

See 5.3.1.3 for the preparation of the PI solution. The PI solution shall be further diluted to 0,2 mmol/l
with water prior to use.
NOTE The concentration of the PI solution used is 0,01 % and the final concentration is 0,000 1 %. This is
below the potential toxicity level.

5.3.2.3 SYTO® 24 green fluorescent cell-permeant nucleic acid stain

The stain is a 5 mmol/l solution in DMSO. Store at −20 °C, protected from light, for up to 12 months. The
solution shall be diluted to 0,1 mmol/l with water before use.

5.3.3 Protocol C

5.3.3.1 MRS broth

The composition and the preparation are specified in ISO 15214 except for no addition of agar.

5.3.3.2 M17 broth

The composition and the preparation are specified in ISO 7889 except for no addition of agar.

5.3.3.3 Stain mixture

The stain mixture consists of 210 µl 50 % glucose solution (5.3.3.3.1), 210 µl 1,5 mmol/l DiOC2 (5.3.3.3.2)
and 50 ml buffer solution (5.3.3.3.3). The stain mixture is prepared the same day as it is used.

5.3.3.3.1 Glucose solution

5.3.3.3.1.1 Composition

— 50 g D(+)-glucose monohydrate
— 50 g water

5.3.3.3.1.2 Preparation

A 50 % glucose solution is prepared by dissolving the glucose in the water. This is aided by warming
the solution to below the boiling point. Avoid evaporation. The solution is autoclaved at 121 °C ± 1 °C for
15 min and can be stored unopened at 3 °C ± 2 °C for up to 3 months.

5.3.3.3.2 3,3’-diethyloxacarbocyanine iodide (DiOC2)

5.3.3.3.2.1 Composition

— 69 mg 3,3’-DiOC2, ≥ 98 %
— 100 ml dimethyl sulfoxide (DMSO)

5.3.3.3.2.2 Preparation

The DiOC2 staining is prepared as a 1,5 mmol/l solution by weighing DiOC2 into DMSO at the amounts
specified in 5.3.3.3.2.1. Dispense in, e.g., 1 ml tubes. Keep dark, as DiOC2 is unstable in light, at 5 °C ± 3 °C
for up to 12 months.

© ISO and IDF 2015 – All rights reserved  5

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

5.3.3.3.3 Buffer solution

5.3.3.3.3.1 Composition

— 7,6 g sodium chloride (NaCl; 130 mmol/l)

— 0,5 g sodium dihydrogenphosphate dihydrate (NaH2PO4·H2O; 3 mmol/l)
— 1,24 g sodium hydrogenphosphate dihydrate (Na2HPO4·2H2O; 7 mmol/l)
— 1 000 ml water

5.3.3.3.3.2 Preparation

Weigh and dissolve the three salts in the water at the amounts specified in 5.3.3.3.3.1. Stirring is
applied until the salts are dissolved. Adjust pH to 6,5 ± 0,05 with 2,5 mol/l HCl. The solution shall then
be filtered through a 0,22 μm filter. The mixture can be kept at 3 °C ± 2 °C for up to one week. For longer
periods, up to 6 months, storage at −20 °C is recommended.

6 Apparatus
Usual laboratory equipment and, in particular, the equipment required for the preparation of test
samples and dilutions specified in ISO 6887-5, as well as the following, shall be used.

6.1 Water bath, capable of operating at 21 °C ± 1 °C.

6.2 Analytical balance, capable of weighing to the nearest 1 mg, with readability to 0,1 mg.

6.3 pH-meter, with temperature compensation, accurate to ± 0,1 pH unit.

6.4 Incubator, heating block or equivalent, capable of operating at the temperatures specified in
Table 2.

Table 2 — Incubation temperatures required per protocol

Protocol Incubation temperatures
A 30 °C ± 1 °C and 37 °C ± 1 °C
B 37 °C ± 1 °C
C 30 °C ± 1 °C and 37 °C ± 1 °C

6.5 Flasks, bottles and test tubes, of sufficient capacity to contain the required volumes and
leave adequate head-space for mixing. The capacity depends on the staining protocol and on the flow
cytometry equipment.

6.6 Pipettes, sterile, calibrated for bacteriological use, accurate to within 2 % of the volume being
pipetted.

6.7 Vortex mixer.

6.8 Filter, sterile, with membrane filters of a pore size 0,22 μm and 25 μm.

6.9 Flow cytometer, instrument capable of detecting and counting particles or cells when passing
individually in a directed flow through a beam of excitation light. The instrument must be equipped with

6  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

a blue laser emitting at 488 nm and with light detectors (fluorescence emission and light scattering).
Further details on instrument properties and settings are given in 9.3.2.

6.10 Automated sample preparation unit, automated sample processor capable of handling liquids for
sample dilution, mixing, incubation and/or injection into the flow cytometer. This equipment is optional.

7 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling
method is given in ISO 707 | IDF 50[2].
A representative sample is collected for analysis. Unless frozen, test samples shall be cooled after
sampling to between 3 °C ± 2 °C and kept at that temperature until testing or freezing. The age of the
samples at testing and freezing and the storage conditions may influence the counting result.
The sample shall not be damaged or changed during transportation or storage. The sample shall be
homogenous and representative of the batch of product to be tested.
Commercial starter cultures and commercial dairy products shall be stored prior to testing as
recommended by the manufacturer.

8 Preparation of test sample

8.1 General
General requirements are in accordance with ISO 6887-1:1999 and ISO 6887-5:2010.
The sample shall be handled for testing in accordance with good laboratory practices.
Initial suspensions and, if needed, further dilutions of the samples to be tested, i.e. freeze-dried
cultures, frozen cultures and fermented milk products containing cultures, are prepared as specified
in 8.2 to 8.4. The appropriate dilution required to be analysed by flow cytometry depends on the initial
concentration of cells in the sample, the staining protocol and the flow cytometry equipment.

8.2 Freeze-dried cultures

Prior to preparation of the initial suspension, allow the freeze-dried sample to acclimatize to room
temperature before opening the pouch or container. Alternatively, mix the content of the original
sample thoroughly, remove the needed test portion with a sterile spatula, and transfer it to a sterile
container or pour the needed test portion into a sterile container. Allow the sample to reach ambient
temperature of 20 °C to 25 °C.
Thoroughly mix the contents of the closed container/bag by repeatedly shaking and inverting it.
Prepare an initial suspension (between 10 and 100 fold) by weighing the test sample into a suitable
sterile vessel and then adding the required amount of diluent. Alternatively, weigh the test sample
directly into the bottle with the diluent. The diluent shall be peptone-salt solution (5.2). The
temperature of the diluent shall be the same as that of the test sample in order to avoid damaging the
microorganisms by sudden changes in temperature.
For rehydration, the initial suspension is left at ambient temperature of 20 °C to 25 °C for approximately
10 min, and no longer than 45 min, shaking occasionally or continuously (e.g. using a peristaltic
homogeniser), before further dilution.
Make sure that the sample is dissolved before proceeding immediately with further testing steps. If
needed, further dilutions are prepared by thoroughly mixing a specific amount of the initial suspension
with the appropriate volume of diluent (5.2).

© ISO and IDF 2015 – All rights reserved  7

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

As an alternative to manual sample preparation, an automated sample preparation module dedicated to

sample preparation, dilution and/or injection can be used.

8.3 Frozen cultures

Prior to initiating preparation of the initial suspension, the test sample shall be thawed. This can be
done by leaving the sample at an ambient temperature of 20 °C to 25 °C until the sample has just thawed.
Alternatively, place the sample in a water bath at 21 °C ± 1 °C and keep it in the water bath until the test
portion has just thawed, see ISO 26323 | IDF 213[3]. Samples shall be tested as soon as they have been
thawed. Mix the test sample carefully after thawing.
Prepare the initial suspension, and appropriate dilution(s) if needed, as stated in 8.2. The test sample is
measured by volume or weight. The unit of the reported final result shall reflect the choice of unit for
the preparation of the test sample.

8.4 Fermented milk products

Samples from fermented milk products, e.g. yogurts, are prepared as follows.
a) Add 90 ml ± 0,1 ml of peptone-salt solution (5.2) at an ambient temperature of 20 °C to 25 °C to a
sterile bottle.
b) Mix the sample (e.g. yogurt) either by inverting and shaking or by mixing it with a sterile spatula.
c) Weigh 10 g ± 0,1 g of the sample and add it into the diluent in order to prepare dilution 10−1. The
temperature of the diluent shall be the same as that of the test sample in order to avoid damaging
the microorganisms by sudden changes in temperature.
d) Shake the bottle slowly 10 times by inverting and shaking.
e) If needed, immediately prepare the serial dilution to obtain the adequate dilution for testing.
NOTE For a final dilution of 10−4 to 10−5, there will be so little background interference in the flow cytometer
from yogurt matrices that no specific pre-treatment is needed. For yogurt samples containing particles, e.g. fruit
pieces or vanilla, the debris might be removed by filtering the final dilution through a 25 μm filter before staining.

9 Procedure

9.1 General
Following preparation of the test sample, testing includes the following steps:
— staining (9.2);
— flow cytometry analysis (9.3);
— gating (9.4);
— calculation of concentrations (Clause 10).

9.2 Staining
Initial suspensions, and/or dilutions from the test sample if needed, are processed and stained
depending on the chosen protocol as specified in 9.2.1, 9.2.2 and 9.2.3 prior to quantification by flow
cytometry. Diagrams for the three staining protocols are found in Annex A.
The individual staining protocols include dilution steps to achieve an appropriate cell concentration.
For further details on appropriate dilution see Clause 11 and for a calculation example see Annex B.

8  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

9.2.1 Protocol A

The staining principle is based on the enzymatic activity of cells. In active cells, the non-fluorescent
dye cFDA is cleaved by cellular esterase releasing the green fluorescent carboxyfluorescein (maximum
fluorescence emission at 520 nm). For a better separation of active and non-active or damaged cells,
a counterstaining with PI is performed with a fluorescence emission maximum at 620 nm (red
fluorescence).
The test sample (8.2, 8.3 or 8.4) is diluted appropriately in PBS (5.3.1.1). In the last dilution step, 870 µl
PBS and 100 µl sample are added to 10 µl cFDA (5.3.1.2; 5 mmol/l in DMSO).
The solution is mixed thoroughly using, e.g., a mechanical stirrer for 5 s and incubated 15 min in the
dark at 30 °C for mesophilic strains and 37 °C for thermophilic strains.
20 µl PI (5.3.1.3; 1,5 mmol/l in water) are added to the mixture above and the solution is mixed
thoroughly and incubated for 15 min in the dark at room temperature.
The mixture is then ready for flow cytometry analysis (9.3). Analyse the sample within 45 min after
adding the cFDA (see A.1).

9.2.2 Protocol B

The staining principle is based on a dual nucleic acid staining with cell permeant dye SYTO® 24
(fluorescence emission maximum at 515 nm) and cell impermeant dye PI (fluorescence emission
maximum at 620 nm). SYTO® 24 permeates the membrane of total cells and stains the nucleic acids
with green fluorescence. PI penetrates only bacteria with damaged membranes, causing a reduction
in SYTO® 24 green fluorescence when both dyes are present. Thus, live bacteria with intact cell
membranes fluoresce bright green (defined as active fluorescent cells), bacteria with slightly damaged
membranes exhibit both green and red fluorescence (defined as damaged cells) and bacteria with
broken membranes fluoresce red (defined as non-active fluorescent cells).
The test sample (8.2, 8.3 or 8.4) is diluted appropriately in PBS (5.3.2.1). In the last dilution step, 100 µl
of the sample is added to 880 µl of PBS (5.3.2.1) and mixed thoroughly.
10 µl PI (5.3.2.2; 0,2 mmol/l in water) and 10 µl SYTO® 24 (5.3.2.3; 0,1 mmol/l in water) are added to
the mixture. This is mixed thoroughly using, e.g., a mechanical stirrer for 5 s and incubated in the dark
for 15 min at 37 °C.
After the dual staining procedure, the sample shall be analysed by flow cytometry (9.3) immediately
(see A.2).

9.2.3 Protocol C

The staining principle is based on the membrane-potential-sensitive DiOC2, which changes emission
wavelength when active cells build up membrane potential. In all cells, the DiOC2 binds to the membrane
with a green fluorescence emission maximum at 500 nm. When cells are activated, the maximum
fluorescence emission wavelength is red-shifted. The degree of the red-shift is strain dependent.
The test sample (8.2, 8.3 or 8.4) is diluted appropriately in MRS broth (5.3.3.1) for activation of the
metabolism. The exception to this general rule is Streptococcus thermophilus which activates better in
M17 broth (5.3.3.2).
The dilution for this protocol is divided into two steps. Firstly, the sample is diluted in MRS (or M17) to
activate the cells. To ensure optimal activation, the test sample shall be diluted at least 10 times and it
shall be incubated for 30 min at 30 °C for mesophilic and 37 °C for thermophilic strains. Secondly, the
sample is diluted 25 times into the stain mixture (5.3.3.3) to reach the appropriate dilution. This is
incubated at room temperature for 30 min.
The cells are then ready to be analysed by means of flow cytometry (9.3). Flow cytometry testing shall
be completed within 30 min after the end of staining (see A.3).

© ISO and IDF 2015 – All rights reserved  9

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

9.3 Flow cytometry analysis

9.3.1 General

The stained samples are analysed by flow cytometry.

Flow cytometry is a technique for rapid quantification of cells combining light source, optics, flow
chamber, liquid sample delivery system, light detectors, electronics and software. The flow cytometer
provides a constant flow of sheath fluid through a cuvette, singularizing and preparing the cells for
analysis. The cuvette is traversed by a beam of light to illuminate flowing cells. Around the cuvette,
detectors collect scattered light in two angles [forward scatter channel (FSC) and side scatter channel
(SSC)] and fluorescence in different colours (e.g. green, yellow, red). When a cell reaches the flow
cuvette, a proportion of the light beam is scattered and cellular fluorescence markers are excited to
emit fluorescence. The detection of the scattered light and the concomitant emission of fluorescence
are referred to as an event, i.e. each cell passing through gives rise to one event, each of which shows
the state of the cell.
Whereas the detected fluorescence is linked to the state of the cell, i.e. enzyme activity, membrane
integrity and/or membrane potential, the FSC provides information on cell size and optical density. The
SSC provides information on cell morphology, reflectivity and granularity.
The recorded events per microlitre sample indicate how many cells are counted and at the same time
differentiate, based on fluorescence parameters, the cells into two categories: active fluorescent and
non-active fluorescent.

9.3.2 Instruments and settings

Flow cytometer configuration settings for the optimal functioning of the three protocols are given in
Table 3. Some of the parameters, e.g. excitation source and filters for the detectors, are important for
the proper performance of the individual protocols, whereas some are less important, e.g. event rate is
equipment dependent and varies significantly from instrument to instrument.
The flow cytometer shall be properly calibrated in accordance with manufacturer instructions. Most
flow cytometers are calibrated for accurate volumetric determination of the analysed sample for easy
calculation of the cell concentration. For instruments without calibrated volumetric determination, the
number of AFUs or TFUs in the sample is calibrated against standardized fluorescent beads, added to
the sample as an internal standard, with a known concentration.
Even for instruments with calibrated volumetric determination, the use of standard fluorescent beads
is mandatory as it greatly increases the ability to trace inaccuracies in small volume determinations and
enables verification of proper performance of the detectors. These beads with known concentration and
fluorescence intensities shall be used to demonstrate appropriate detection of relevant wavelengths
and intensities of the emitted light as well as to document calibration status. The use of standard beads
also provides the ability to make accurate comparisons of data from sample to sample, from day to day
or from laboratory to laboratory.
The flow cytometer instrument shall be operated by a trained technician and as described in the
instruction manual provided by the manufacturer. The flow cytometer should also be cleaned and
serviced regularly in accordance with the manufacturer’s instructions.

10  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Table 3 — Recommendations for flow cytometer: configuration and settings

Optical Excitation source Laser 488 nm, minimum 20 mW

configuration
Detectors Emission: minimum 2 fluorescence channels and
scatter channels
FL1: Green channel
Protocol A: 500–570 nm
Protocol B: 500–540 nm
Protocol C: 515–545 nm
FL2 or FL3: Orange or red channel
Protocol A: Orange/red > 570 nm
Protocol B: Red > 630 nm
Protocol C: Red > 650 nm
Light scattering:
Side scatter channel (SSC) and
forward scatter channel (FSC)
Fluidic Sample flow rate 15 to 120 µl per mina
configuration
Sample volume analysed 20 µl to 250 µl
Event rate Max: 20 000 events/seca
Overall analysis Triggering parameters used SSC or both FSC and SSC
parameters
Amplifier and signal conditioning Log scale
(linearity or logarithmic scale)
a Settings for these parameters depend on the instrument. Manufacturer guidelines should be followed.

9.4 Gating

9.4.1 General

Gating refers to the evaluation of FSC, SSC and fluorescence results for each of the recorded events.
Typically, these results are presented by the instrument software in histograms and/or dot plots with
optional axis. The data shall be evaluated in a systematic way to firstly separate cells from noise and
secondly differentiate AFUs and n-AFUs.
Thus, gating may be conducted in accordance with the following general recommendations.
a) Separate the cells from noise and media debris in a FSC vs. SSC plot.
b) Gate all cells and only do subsequent data plots on these.
c) Plot the intensity of the red vs. the green fluorescence to differentiate AFUs from n-AFUs.
d) If it is difficult to discriminate the two populations, it is suggested to plot a histogram of counts vs.
the ratio of red over green intensity to help the eye discriminate between cell populations. This
improves greatly the ability to discriminate cell populations.
e) If still in doubt whether a population is active or non-active, inactivate the active cells either
chemically or by heating and thereby document that the presumed live population is shifted to the
presumed dead ones. Damaged cells shall not be included in the count of active cells.

9.4.2 Protocol A

To discriminate background noise from cells, the FSC and SSC thresholds are set to exclude background
signals from cells (see Figure 1). Gating is conducted as described in 9.4.1.

© ISO and IDF 2015 – All rights reserved  11

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Key
A forward scatter channel (LS1) 1 LS1 plot (forward light scatter)
X forward scatter channel 2 LS2 plot (sideward light scatter)
Y counts 3 LS1 and LS2 dot plot showing cell population

B side scatter channel (LS2)

X side scatter channel
Y counts

C forward scatter channel vs. side scatter channel

X forward scatter channel
Y side scatter channel

NOTE The figure demonstrates the threshold to discriminate cells from noise. The cells are gated in the FSC
vs. SSC plot.

Figure 1 — Protocol A, Example (Streptococcus thermophilus)

By gating red vs. green fluorescence, two to three populations (active/damaged/non-active) can be
differentiated in the dot plot (see Figure 2).

12  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Key
A fluorescent light plot (FL) 3 (red) 1 FL 3 (detector for red fluorescence)
X FL3 2 FL 1 (detector for green fluorescence)
Y counts 3 non-active cells
4 damaged cells
B fluorescent light plot (FL) 1 (green) 5 active cells
X FL1
Y counts

C FL1 vs. FL3

X FL1
Y FL3

NOTE The figure demonstrates the threshold settings to discriminate fluorescent cells from noise. The
active cells, non-active cells and an intermediate cell population are gated in the FL red vs. FL green plot.

Figure 2 — Protocol A, Example (Streptococcus thermophilus, freeze dried)

9.4.3 Protocol B

As described in the general recommendations for gating, the parameters FSC for the size, SSC for the
structure, fluorescent light plot (FL) 1 for green fluorescence and FL 3 for red fluorescence are evaluated.
In the resulting dot plot of FL1 vs. FL3, according to the principle of the dual labelling with Syto® 24 and
PI, three different populations are identified and defined in the gates of active fluorescent cells, non-
active fluorescent cells and an intermediate population, here termed “damaged” (see Figure 3). Only the
population in the gate active will be calculated as the quantification result of AFUs for the sample.

© ISO and IDF 2015 – All rights reserved  13

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Key
X FL1 1 non-active
Y FL3 2 damaged
3 active

NOTE The figure demonstrates flow cytometry multiparameter dot plots of green fluorescence (FL1) vs. red
fluorescence (FL3)

Figure 3 — Protocol B, Example (Lactobacillus casei paracasei, freeze-dried)

9.4.4 Protocol C

Cells (active and non-active) are discriminated from background noise in the SSC vs. green fluorescence
plot. This total population of cells is then gated in the far red (> 650 nm) vs. green plot (515 nm –
545 nm) as shown in Figure 4 (left and centre).

14  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Key
A cells gated from background 1 cells gated from background
X intensity green fluorescence (515 nm – 545 nm) 2 total fluorescent cells
Y intensity side scatter light 3 red-shifted active fluorescent cells
4 red-shifted active fluorescent cells
B active cells gated from non-active cells
X intensity green fluorescence (515 nm – 545 nm)
Y intensity far red fluorescence (> 650 nm)

C ratio far red over green fluorescence

X ratio far red over green fluorescence
Y counts

Figure 4 — Protocol C, Examples of gating cells (Bifidobacterium, freeze dried)

The active cells (high intensity far red) are clearly separated from the non-active (lower intensity far
red). In more difficult situations where the borderline between active and non-active cells is not so
clear, a plot of counts vs. ratio of far red over green (Figure 4, C) gives a clear indication of the border
between active and non-active cells.
As the protocol discriminates between active and non-active based on the intensity of red fluorescence,
it is important to tune the instrument to achieve the same log intensity of red and green fluorescence
for the active cells.

10 Calculation and expression of results

Four categories of results can be obtained by following this standardized method:
a) active fluorescent units per millilitre or gram (AFU/ml or AFU/g): number of active fluorescent unit
events divided by the volume or weight of tested sample multiplied by the dilution factor;
b) non-active fluorescent units per millilitre or gram (n-AFU/ml or n-AFU/g): number of non-active
fluorescent unit events divided by the volume or weight of tested sample multiplied by the
dilution factor;
c) total fluorescent units (TFU/ml or TFU/g): sum of active and non-active units per millilitre or gram;

© ISO and IDF 2015 – All rights reserved  15

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

d) % active fluorescent units (% AFU): percentage ratio of active fluorescent units (a) to total
fluorescent units (c).
For a), b) and c), round the calculated result to two significant figures. For a three-figure number, round
the third figure to the nearest zero. If the third figure is 5, round to the figure below if the second figure
is even and to the figure above if the second figure is odd. The result shall be expressed as a number
from 1,0 to 9,9 multiplied by the appropriate power of 10.
For the calculation examples below, the following parameters have been used:
— injection volume (V) on flow cytometer: 20 µl;
— recorded AFU events (n): 2 000 events;
— recorded n-AFU events (m): 4 000 events;
— initial dilution (a): 100 fold;
— additional dilutions (b): 1 × 10 fold;
— protocol staining dilution (c): 10.
EXAMPLE 1 Calculating AFU/ml using protocol A:

AFU 1000 µl
= × (a × b × c) × n
ml V

AFU 1 000 µl
= × (100 × 10 × 10) × 2 000 = 1 × 10 9
ml 20µl
EXAMPLE 2 Calculating n-AFU/ml using protocol A:

nAFU 1000 µl
= × (a × b × c) × m
ml V

nAFU 1 000 µl
= × (100 × 10 × 10) × 4 000 = 2 × 10 9
ml 20µl
EXAMPLE 3 Calculating TFU/ml:

TFU AFU nAFU 1 × 10 9 2 × 10 9 3 × 10 9

= + = + =
ml ml ml ml ml ml
EXAMPLE 4 Calculating % AFU:

AFU 1 × 10 9
% AFU = × 100 % = × 100 % = 33, 3 %
TFU 3 × 10 9

11 Critical factors affecting results

In order to obtain valid and reliable results, the following factors should be considered carefully.
a) Homogeneity and representativeness of the sample. Inhomogeneous samples shall be replicated or,
alternatively, the sample size shall be sufficiently large to include the expected variations.

16  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

b) Appropriate dilution of the samples. Samples shall be diluted to a degree that enables detection of
cells:
1) around the optimal rate for the instrument in order to avoid shadowing of events, i.e. two
events counted as one, and to optimize the cell to noise response;
2) with a minimum of 1 000 cell events per sample. This number reflects the need to be absolutely
sure that the counted events are not noise stemming from particulate matter in the media and
that the number of counted events shall be sufficient to give robust counting statistics.
This means that the appropriate dilution depends on the flow rate through the instrument; the
volume available for the analysis; the time available for each counting; and finally the maximum
counting speed of the flow cytometer.
The appropriate dilution ensures that the maximum counting speed is not exceeded; the minimum
number of counted events is obtained within an acceptable time; and the available volume of
diluted sample is sufficient.
See Annex B for a calculation example of the appropriate dilution.
c) The flow cytometer shall be properly maintained, calibrated and operated. Special care shall be
taken so that the laser is aligned correctly as described by the manufacturer. As described in 9.3.2,
the performance of the instrument shall be optimized and verified on a routine basis by standard
fluorescent beads.
d) The excitation laser and the filter and mirror settings on the detectors shall be suitable for the
chosen protocol. The different fluorophores emit light at different wavelengths. The correct and
unambiguous detection and separation of AFUs, n-AFUs and noise depends on this.
e) The detected intensities of light will to some extent be sensitive to cell aggregation. This is most
easily seen in a plot of SSC vs. FSC, where the larger aggregations of cells will result in an elongated
distribution of cells. Thus, the trained operator will be able to conclude whether cells are not
completely separated into single cells.
f) Air bubbles shall be avoided as they distort the light in the cuvette and thus may obscure the
detected events. Typically, instruments hold an option to purge the liquid path to remove bubbles.
g) The sample matrix may contain excessive amounts of particulate matter that can be detrimental
to the proper analysis of the sample. As an example, for yogurt samples, an appropriate maximum
dilution is recommended to reduce the adverse effect of the yogurt matrix. See Annex B for a
calculation example of the appropriate maximum dilution.

12 Precision

12.1 Interlaboratory test

Details of the interlaboratory test on the precision of the method are summarized in Table C.1 and
Table C.2 (see also Reference [4]). The values for the repeatability and reproducibility were determined
by using nine commercially available starter cultures of lactic acid bacteria or probiotic strains (frozen
or freeze-dried) used in fermented milk products worldwide. Furthermore, one commercial yogurt
product containing two strains of lactic acid bacteria was also included in the interlaboratory test. For
each of the ten sample types, two samples (batches at different concentrations) were tested to obtain
the reported precision data.
The interlaboratory test was carried out in accordance with ISO 5725-1[5], ISO 5725-2[6] and
Reference [7]. The values derived from this interlaboratory test may not be applicable to concentration
ranges, cultures and matrices other than those given. The concentration ranges tested, the homogeneity
of the samples and the characteristics of the lactic acid bacteria or probiotic strains among the
products selected were representative of the worldwide market and were in accordance with
ISO 27205 | IDF 149[8].

© ISO and IDF 2015 – All rights reserved  17

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

The precision data reported has been calculated from active and total fluorescent results (log10
transformed) obtained by testing the samples with the three staining protocols in parallel.

12.2 Repeatability
The absolute difference between two individual single test results (AFU per gram or TFU per gram,
log10 transformed), obtained using the same method on identical test material in the same laboratory
by the same operator using the same equipment within a short interval of time, will not be greater, in
more than 5 % of cases, than the values given in Table 4.

12.3 Reproducibility
The absolute difference between two individual single test results (AFU per gram or TFU per gram,
log10 transformed), obtained using the same method on identical test material in different laboratories
with different operators using different equipment, will not be greater, in more than 5 % of cases, than
the values given in Table 4.

Table 4 — Repeatability limits r and reproducibility limits R collectively defined for the product
types, log10 AFU/g and log10 TFU/g
Parametersa Product typeb Mean Sr SR r R
AFU/g Frozen and freeze-dried lactic acid 11,345 0,023 0,160 0,06 0,45
bacteria or probiotic strains
Yogurt 8,868 0,030 0,279 0,08 0,76
TFU/g Frozen and freeze-dried lactic acid 11,484 0,026 0,134 0,07 0,38
bacteria or probiotic strains
Yogurt 9,185 0,040 0,421 0,10 1,17
a All precision data are expressed as decadic logarithms of AFU or TFU per gram sample.
b See Annex C for further details on strains.

13 Test report
The test report shall contain at least the following information:
a) all information necessary for complete identification of the sample;
b) the sampling method used, if known;
c) the test method used, with reference to this International Standard, i.e. ISO 19344 | IDF 232, and
the relevant flow cytometry protocol;
d) all operating details not specified in this International Standard, or regarded as optional, together
with details of any incidents which may have influenced the test result(s);
e) the test result(s) obtained, or, if the repeatability has been checked, the final quoted result obtained.

18  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Annex A
(informative)

Diagram of staining protocols

A.1 Protocol A

© ISO and IDF 2015 – All rights reserved  19

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

A.2 Protocol B

A.3 Protocol C

20  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Annex B
(informative)

Calculation example of the appropriate sample dilution

To obtain, for example, the minimum target of 1 000 events from a 1E+10/gram sample, counted in 10 s
on an instrument injecting approximately 10 µl/sec, the maximum dilution shall be as follows.
The minimum number of analysed cells equals 1 000 and the injected volume is 10 µl/sec × 10 s or
100 µl. This means that the cell concentration in the diluted sample is 1E+1/µl, equivalent to 1E+4/ml.
Therefore, the maximum dilution will be:
1E+4 /ml / 1E+10 /gram, which is equal to 1E-6 g/ ml or 1 g in 1E+6 ml.
In the high end, each flow cytometer has an upper limit of counting speed. To count, for example, a
1E+10/gram sample in 10 s on an instrument which has an upper limit of detection at 50 000/sec, the
minimum dilution shall be as follows.
The maximum number of analysed cells equals 50 000/sec × 10 s or 5E+5 cells. The injected sample
volume is still 100 µl and the cell concentration in the diluted sample is 5E+5 / 100 µl or 5E+6 /ml.
Therefore, the minimum dilution of this sample will be:
5E+6 /ml / 1E+10 /g, which is equal to 5E-4 g /ml or 1 g in 2E+3 ml.
This means that the appropriate dilution will be in the range between 2 000x and 1 000 000x.

© ISO and IDF 2015 – All rights reserved  21

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Annex C
(informative)

Interlaboratory test

An international interlaboratory test involving 15 laboratories located in five countries was carried out
in February 2014 considering commercially available starter cultures containing lactic acid bacteria,
probiotics and yogurt according to ISO 5725-1[5], ISO 5725-2[6] and Reference [7]. For each sample type,
two individual batches with different concentrations were analysed in parallel with the three different
staining protocols specified in this International Standard, i.e. six examinations per laboratory per
sample type. Each examination result was the result of duplicate analyses.
This collaborative trial was organized by Chr. Hansen A/S (Denmark), Dupont (Germany) and Danone
(France). The method, including all relevant instructions, was submitted to all participating partners.
After collection and preparation of the resulting data, statistical analyses were performed in close
co-operation with the IDF Project Group Statistics of analytical data – Interlaboratory study results.
Further details on the collaborative study can be found in Reference [4].

22  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Table C.1 — Results of the collaborative study for log10 AFU/g

Parameter Samples typesb

1 2 3 4 5 6 7 8 9 10
No. of 15 15 15 15 15 15 15 15 15 15
participants
No. of samples 2 2 2 2 2 2 2 2 2 2
Total 6 6 6 6 6 6 6 6 6 6
examinationsa
per participant
Total 90 90 90 90 90 90 90 90 90 90
examinations
per sample type
Mean value, in 8,868 10,021 11,487 11,114 11,473 11,085 11,476 11,896 11,322 12,226
log10 AFU/g
Results used to 86 83 87 86 89 89 86 88 88 83
estimate r, R
Repeatability 0,030 0,021 0,025 0,021 0,021 0,022 0,025 0,026 0,022 0,021
standard
deviation, sr, in
log10 AFU/g
Repeatability 0,080 0,068 0,063 0,066 0,060 0,058 0,055 0,052 0,050 0,051
limit, r (=2,8 sr)
Reproducibility 0,279 0,111 0,152 0,172 0,156 0,142 0,265 0,150 0,114 0,124
standard
deviation sR , in
log10 AFU/g
Reproducibility 0,755 0,613 0,542 0,439 0,326 0,316 0,286 0,311 0,334 0,348
limit, R (=2,8 sR)
a Each examination is the result of duplicate analyses with the three different staining protocols
b Description of test samples used:
Sample 1 – Commercial yogurt product containing Lactobacillus bulgaricus and Streptococcus thermophilus
Sample 2 – Commercial frozen starter culture containing Lactobacillus acidophilus
Sample 3 – Commercial freeze-dried starter culture containing Streptococcus thermophilus
Sample 4 – Commercial freeze-dried starter culture containing Lactobacillus bulgaricus
Sample 5 – Commercial freeze-dried starter culture containing Lactobacillus acidophilus
Sample 6 – Commercial freeze-dried starter culture containing Lactobacillus reuteri
Sample 7 – Commercial freeze-dried starter culture containing Lactobacillus paracasei
Sample 8 – Commercial freeze-dried starter culture containing Bifidobacterium lactis
Sample 9 – Commercial freeze-dried starter culture containing Lactococcus lactis and Leuconostoc
Sample 10 – Commercial freeze-dried starter culture containing Lactococcus lactis

© ISO and IDF 2015 – All rights reserved  23

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Table C.2 — Results of the collaborative study for log10 TFU/g

Parameter Samples typesb

1 2 3 4 5 6 7 8 9 10
No. of participants 15 15 15 15 15 15 15 15 15 15
No. of samples 2 2 2 2 2 2 2 2 2 2
Total 6 6 6 6 6 6 6 6 6 6
examinationsa
per participant
Total 90 90 90 90 90 90 90 90 90 90
examinations per
sample type
Mean value, in 9,185 10,048 11,687 11,385 11,546 11,304 11,654 11,944 11,480 12,305
log10 TFU/g
Results used to 60 83 87 85 87 89 86 86 89 85
estimate r, R
Repeatability 0,040 0,023 0,023 0,041 0,021 0,023 0,028 0,024 0,022 0,026
standard
deviation, sr, in
log10 AFU/g
Repeatability 0,102 0,106 0,095 0,094 0,076 0,075 0,062 0,054 0,062 0,068
limit, r (=2,8 sr)
Reproducibility 0,421 0,111 0,144 0,118 0,136 0,134 0,186 0,139 0,111 0,112
standard deviation
sR , in log10 AFU/g
Reproducibility 1,169 1,099 0,930 0,813 0,638 0,481 0,286 0,242 0,276 0,289
limit, R (=2,8 sR)
a Each examination is the result of duplicate analyses with the three different staining protocols
b Description of test samples used:
Sample 1 – Commercial yogurt product containing Lactobacillus bulgaricus and Streptococcus thermophilus
Sample 2 – Commercial frozen starter culture containing Lactobacillus acidophilus
Sample 3 – Commercial freeze-dried starter culture containing Streptococcus thermophilus
Sample 4 – Commercial freeze-dried starter culture containing Lactobacillus bulgaricus
Sample 5 – Commercial freeze-dried starter culture containing Lactobacillus acidophilus
Sample 6 – Commercial freeze-dried starter culture containing Lactobacillus reuteri
Sample 7 – Commercial freeze-dried starter culture containing Lactobacillus paracasei
Sample 8 – Commercial freeze-dried starter culture containing Bifidobacterium lactis
Sample 9 – Commercial freeze-dried starter culture containing Lactococcus lactis and Leuconostoc
Sample 10 – Commercial freeze-dried starter culture containing Lactococcus lactis

24  © ISO and IDF 2015 – All rights reserved

 BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

Bibliography

[1] FAO. Guidelines for the evaluation of probiotics in food. Report of a Joint FAO/WHO working
group on drafting guidelines for the evaluation of probiotics in food. London Ontario: Canada,
2002. Available at ftp://ftp.fao.org/es/esn/food/wgreport2.pdf
[2] ISO 707 | IDF 50, Milk and milk products — Guidance on sampling
[3] ISO 26323 | IDF 213, Milk products — Determination of the acidification activity of dairy cultures
by continuous pH measurement (CpH)
[4] CASANI S., K.F. HANSEN and S. CHARTIER. Interlaboratory Collaborative Study on a Flow
Cytometry Method for Lactic Acid Bacteria Quantification in starter cultures, probiotics and
fermented milk products according to ISO 19344 | IDF 232. Bull. Int. Dairy Fed. 478, 2015
[5] ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results — Part 1:
General principles and definitions
[6] ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results — Part 2: Basic
method for the determination of repeatability and reproducibility of a standard measurement method
[7] LERAY O. Guidance for the evaluation of precision characteristics of physicochemical
quantitative analytical methods for milk and milk products. Bull. Int. Dairy Fed. 453, 2012
[8] ISO 27205 | IDF 149, Fermented milk products — Bacterial starter cultures — Standard of identity

© ISO and IDF 2015 – All rights reserved  25

BS ISO 19344:2015
ISO 19344:2015(E)
IDF 232:2015(E)

ICS 67.100.10
Price based on 25 pages

© ISO and IDF 2015 – All rights reserved 

This page deliberately left blank
NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW

British Standards Institution (BSI)

BSI is the national body responsible for preparing British Standards and other
standards-related publications, information and services.
BSI is incorporated by Royal Charter. British Standards and other standardization
products are published by BSI Standards Limited.

About us Revisions
We bring together business, industry, government, consumers, innovators Our British Standards and other publications are updated by amendment or revision.
and others to shape their combined experience and expertise into standards We continually improve the quality of our products and services to benefit your
-based solutions. business. If you find an inaccuracy or ambiguity within a British Standard or other
The knowledge embodied in our standards has been carefully assembled in BSI publication please inform the Knowledge Centre.
a dependable format and refined through our open consultation process.
Organizations of all sizes and across all sectors choose standards to help Copyright
them achieve their goals. All the data, software and documentation set out in all British Standards and
other BSI publications are the property of and copyrighted by BSI, or some person
Information on standards or entity that owns copyright in the information used (such as the international
We can provide you with the knowledge that your organization needs standardization bodies) and has formally licensed such information to BSI for
to succeed. Find out more about British Standards by visiting our website at commercial publication and use. Except as permitted under the Copyright, Designs
bsigroup.com/standards or contacting our Customer Services team or and Patents Act 1988 no extract may be reproduced, stored in a retrieval system
Knowledge Centre. or transmitted in any form or by any means – electronic, photocopying, recording
or otherwise – without prior written permission from BSI. Details and advice can
Buying standards be obtained from the Copyright & Licensing Department.
You can buy and download PDF versions of BSI publications, including British
and adopted European and international standards, through our website at Useful Contacts:
bsigroup.com/shop, where hard copies can also be purchased. Customer Services
If you need international and foreign standards from other Standards Development Tel: +44 845 086 9001
Organizations, hard copies can be ordered from our Customer Services team. Email (orders): orders@bsigroup.com
Email (enquiries): cservices@bsigroup.com
Subscriptions
Subscriptions
Our range of subscription services are designed to make using standards
Tel: +44 845 086 9001
easier for you. For further information on our subscription products go to
Email: subscriptions@bsigroup.com
bsigroup.com/subscriptions.
With British Standards Online (BSOL) you’ll have instant access to over 55,000 Knowledge Centre
British and adopted European and international standards from your desktop. Tel: +44 20 8996 7004
It’s available 24/7 and is refreshed daily so you’ll always be up to date. Email: knowledgecentre@bsigroup.com
You can keep in touch with standards developments and receive substantial
Copyright & Licensing
discounts on the purchase price of standards, both in single copy and subscription
format, by becoming a BSI Subscribing Member. Tel: +44 20 8996 7070
Email: copyright@bsigroup.com
PLUS is an updating service exclusive to BSI Subscribing Members. You will
automatically receive the latest hard copy of your standards when they’re
revised or replaced.
To find out more about becoming a BSI Subscribing Member and the benefits
of membership, please visit bsigroup.com/shop.
With a Multi-User Network Licence (MUNL) you are able to host standards
publications on your intranet. Licences can cover as few or as many users as you
wish. With updates supplied as soon as they’re available, you can be sure your
documentation is current. For further information, email bsmusales@bsigroup.com.

BSI Group Headquarters

389 Chiswick High Road London W4 4AL UK