ISO 9232:2003

Yogurt — Identification of characteristic microorganisms (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus)

ISO 9232|IDF 146:2003 specifies tests for the identification of the characteristic microorganisms in yogurt on the basis of their morphological, cultural and physiological properties. It is applicable to strains isolated from yogurts in which both characteristic microorganisms are present and viable.

Yaourt — Identification des micro-organismes caractéristiques (Lactobacillus delbrueckii subsp. bulgaricus et Streptococcus thermophilus)

General Information

• Status: Published
• Publication Date: 12-Jan-2003
• ICS: 07.100.30 - Food microbiology
• Technical Committee: ISO/TC 34/SC 5 - Milk and milk products
• Drafting Committee: ISO/TC 34/SC 5 - Milk and milk products
• Current Stage: 9093 - International Standard confirmed
• Completion Date: 23-Sep-2020

Relations

• Consolidated By: ISO/IEEE 11073-10404:2010 - Health informatics — Personal health device communication — Part 10404: Device specialization — Pulse oximeter
• Effective Date: 06-Jun-2022
• Amended By: ISO 9232:2003/Amd 1:2023 - Yogurt — Identification of characteristic microorganisms (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) — Amendment 1: Inclusion of performance testing of culture media and reagents
• Effective Date: 06-Jun-2022

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 9232
IDF
146
First edition
2003-02-01


Yogurt — Identification of characteristic
microorganisms (Lactobacillus
delbrueckii subsp. bulgaricus and
Streptococcus thermophilus)
Yaourt — Identification des micro-organismes caractéristiques
(Lactobacillus delbrueckii subsp. bulgaricus et Streptococcus
thermophilus)




Reference numbers
ISO 9232:2003(E)
IDF 146:2003(E)
©
ISO and IDF 2003

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ISO 9232:2003(E)
IDF 146:2003(E)
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©  ISO and IDF 2003
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ISO 9232:2003(E)
IDF 146:2003(E)
Contents Page
Foreword. iv
Foreword. v
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle . 1
5 Culture media, diluents and reagents. 2
6 Apparatus and glassware. 5
7 Procedure. 6
7.1 Isolation of colonies . 6
7.2 Phenotypic characteristics required for identification of L. delbrueckii subsp. bulgaricus. 6
7.3 Phenotypic characteristics required for identification of S. thermophilus. 7
8 Expression of results. 8
9 Test report. 8
Annex A (normative) Main attributes tables . 9
Annex B (normative) Milk cultures of lactic acid bacteria — Determination of the contents of
lactic acid and lactate enantiomers . 11
Bibliography . 17

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ISO 9232:2003(E)
IDF 146:2003(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee has
been established has the right to be represented on that committee. International organizations, governmental
and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 9232IDF 146 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,
Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International.
It is being published jointly by ISO and IDF and separately by AOAC International.

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ISO 9232:2003(E)
IDF 146:2003(E)
Foreword
IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National
Committee in every member country. Every National Committee has the right to be represented on the IDF
Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in
the development of standard methods of analysis and sampling for milk and milk products.
Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the
National Committees for voting. Publication as an International Standard requires approval by at least 50 % of
the National Committees casting a vote.
ISO 9232IDF 146 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,
Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International.
It is being published jointly by ISO and IDF and separately by AOAC International.
All work was carried out by the Joint ISO/IDF/AOAC Action Team, Lactic acid bacteria and starters, of the
Standing Committee on Microbiological methods of analysis, under the aegis of its project leader, Prof. B.
Bianchi Salvadori (IT).

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ISO 9232:2003(E)
INTERNATIONAL STANDARD
IDF 146:2003(E)

Yogurt — Identification of characteristic microorganisms
(Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus
thermophilus)
1 Scope
This International Standard specifies tests for the identification of the characteristic microorganisms in yogurt
on the basis of their morphological, cultural and physiological properties.
It is applicable to strains isolated from yogurts in which both characteristic microorganisms are present and
viable.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 6887-1, 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 7218, Microbiology of food and animal feeding stuffs — General rules for microbiological examinations
ISO 7889IDF 117:2002, Yogurt — Enumeration of characteristic microorganisms — Colony-count technique
at 37 °C
ISO 8261IDF 122, Milk and milk products — General guidance for the preparation of test samples, initial
suspensions and decimal dilutions for microbiological examination
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
characteristic microorganisms in yogurt
Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus
4 Principle
4.1 The morphological, cultural and biochemical characteristics of L. delbrueckii subsp. bulgaricus are
determined.
4.2 The morphological, cultural and biochemical characteristics of S. thermophilus are determined.
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ISO 9232:2003(E)
IDF 146:2003(E)
5 Culture media, diluents and reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and glass-distilled or
demineralized water or water of equivalent purity. The water used for the preparation of the enzyme solutions
should be at least double glass-distilled. See also ISO 6887-1 and ISO 8261IDF 122. For other materials,
see ISO 7889IDF 117.
5.1 Culture media
Use only freshly prepared culture media which shall not be exposed to direct sunlight. If the prepared culture
media are not used immediately, they shall, unless otherwise specified, be cooled and stored at between 2 °C
and 4 °C for no longer than 1 week and under conditions which do not produce any change in their
composition. As for reagents, see storage conditions in ISO 7218.
5.1.1 Skimmed milk
5.1.1.1 Composition
Low-heat-treated, spray-dried skimmed
100 g
milk, free from growth inhibitors
Water up to 1 000 ml

5.1.1.2 Preparation
Dissolve the dried milk in the water. Distribute 10 ml portions of the obtained solution in test tubes of
16 mm × 160 mm (6.5). Sterilize in an autoclave at 110 °C ± 1 °C for 30 min or at 115 °C ± 1 °C for 20 min.
After sterilization and before use, check the sterility by incubating the test tubes in the incubator (6.1) set at
37 °C for 3 days.
5.1.2 MRS broth
5.1.2.1 Composition
Peptone 1(tryptic digest of casein) 10,00 g
Meat extract 10,00 g
Yeast extract (dried) 5,00 g
Glucose (C H O) 20,00 g
6 12 6
Tween 80 (sorbitan mono-oleate) 1,00 ml
Dipotassium hydrogen phosphate (K HPO) 2,00 g
2 4
Sodium acetate trihydrate (CH CO Na·3HO) 5,00 g
3 2 2
Diammonium citrate [C H O (NH )] 2,00 g
6 6 7 4 2
Magnesium sulfate heptahydrate (MgSO ·7HO) 0,20 g
4 2
Manganese sulfate tetrahydrate (MnSO ·4HO) 0,05 g
4 2
Water up to 1 000 ml

5.1.2.2 Preparation
Separately dissolve each component in the already boiling water. Cool in a water bath (6.8) to 50 °C. Adjust
the pH so that after sterilization it is 6,5 ± 0,2 at 25 °C ± 1 °C, by using a reagent (5.2) and checking with the
pH-meter (6.4).
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ISO 9232:2003(E)
IDF 146:2003(E)
Distribute 20 ml portions of the obtained medium in test tubes of 20 mm × 200 mm (6.5). Sterilize in an
autoclave at 121 °C ± 1 °C for 15 min.
NOTE When using commercially available MRS media, the obtained results may differ significantly from one supplier
to the other. Therefore, always check commercially MRS medium against the medium prepared as described above.
5.1.3 Basic medium for fermentation tests
5.1.3.1 Composition
Use the composition as described in 5.1.2.1 for the MRS broth, but omitting the meat extract and the glucose
component.
5.1.3.2 Preparation
Prepare the basic medium as described in 5.1.2.2 for the MRS broth, using the components described in
5.1.3.1 and adjusting the pH so that after sterilization it is 6,95 ± 0,05 instead of pH 6,5 at 25 °C ± 1 °C.
5.1.4 Culture medium for production of CO
2
5.1.4.1 Composition
Use the composition as described in 5.1.2.1 for the MRS broth, but omitting the meat extract component and
replacing the 20 g of glucose with 50 g of glucose.
5.1.4.2 Preparation
Prepare the culture medium as described in 5.1.2.2 for the MRS broth using the components described in
5.1.4.1 and adjusting the pH so that after sterilization it is 6,95 ± 0,05 instead of pH 6,5 at 25 °C ± 1 °C.
Distribute 10 ml portions instead of 20 ml (as described in 5.1.2.2) of the obtained medium in the test tubes of
16 mm × 160 mm (6.5). Sterilize in an autoclave at 121 °C ± 1 °C for 15 min.
5.1.5 Overlay agar
5.1.5.1 Composition
Bacteriological agar 20 g
Water up to 1 000 ml

5.1.5.2 Preparation
Dissolve the agar in the water. Distribute 10 ml portions in tubes of 16 mm × 160 mm (6.5). Sterilize in an
autoclave at 121 °C ± 1 °C for 15 min.
5.1.6 Litmus milk
5.1.6.1 Composition
Litmus powder 0,70 g
Skimmed milk (5.1.1) up to 1 000 ml

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ISO 9232:2003(E)
IDF 146:2003(E)
5.1.6.2 Preparation
Prepare the litmus milk as described in 5.1.1.2 for the skimmed milk, using the components as described
in 5.1.6.1.
NOTE Litmus powder or skimmed milk with litmus is commercially available.
5.1.7 M17 broth
5.1.7.1 Basic medium
5.1.7.1.1 Composition
Peptone 1 (tryptic digest of casein) 2,50 g
Peptone 2 (peptic digest of meat) 2,50 g
Peptone 3 (papain digest of soya) 5,00 g
Yeast extract (dried) 2,50 g
Meat extract 5,00 g
β-Glycerophosphate (disodium salt) (C H O PNa) 19,00 g
3 7 6 2
Magnesium sulfate heptahydrate (MgSO ·7HO) 0,25 g
4 2
Ascorbic acid (C H O) 0,50 g
6 8 6
Water up to 950 ml

5.1.7.1.2 Preparation
Separately dissolve the components in the boiling water. Cool on a water bath (6.8) to 50 °C. Adjust the pH so
that after sterilization it is 6,8 ± 0,2 at 25 °C ± 1 °C by using a reagents (5.2) and checking with the
pH-meter (6.4).
Distribute 19 ml portions of the obtained medium in test tubes of 20 mm × 200 mm (6.5). Sterilize for 15 min in
an autoclave at 121 °C ± 1 °C.
5.1.7.2 Lactose solution
5.1.7.2.1 Composition
Lactose (C H O) 10 g
12 22 11
Water up to 100 ml

5.1.7.2.2 Preparation
Dissolve the lactose in the water. Sterilize for 15 min in an autoclave at 121 °C ± 1 °C.
5.1.7.3 Complete medium
5.1.7.3.1 Composition
Lactose solution (5.1.7.2) 1 ml
Basic medium (5.1.7.1) 19 ml

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ISO 9232:2003(E)
IDF 146:2003(E)
5.1.7.3.2 Preparation
Immediately before use, add the lactose solution to the test tubes with the basic medium (5.1.7.1). Mix by
swirling.
NOTE When using commercially available M17 media, the obtained results may differ significantly from one supplier
to the other. Therefore, always check commercially M17 medium against the medium prepared as described above.
5.1.8 Culture medium for growth in presence of 6,5 % NaCl
5.1.8.1 Composition
Use the composition as described in 5.1.7.1.1 for the M17 broth, but replacing the 19 g of β-glycerophosphate
component with 65 g of sodium chloride (NaCl).
5.1.8.2 Preparation
Prepare the culture medium as described in 5.1.7.1.2 for the M17 broth but distributing 10 ml instead of 19 ml
(as described in 5.1.7.1.2) of the obtained medium in the test tubes of 16 mm × 160 mm (6.5). Sterilize for
15 min in an autoclave at 121 °C ± 1 °C.
5.2 Reagents for adjustment of pH
5.2.1 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l approximately.
5.2.2 Hydrochloric acid solution, c(HCl) = 0,1 mol/l approximately.
5.3 Reagent for staining, ethanolic solution of methylene blue, 6 g/l.
5.4 Reagent for catalase reaction, hydrogen peroxide (H O ), 1,5 % (volume fraction).
2 2
6 Apparatus and glassware
Sterilization of equipment that will come into contact with the test sample or the culture medium shall be
carried out in accordance with the requirements of ISO 8261IDF 122. The glassware shall be resistant to
repeated sterilization.
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.
6.1 Incubators, capable of operating at 10 °C ± 1 °C, at 15 °C ± 1 °C, at 37 °C ± 1 °C and at 45 °C ± 1 °C.
6.2 Test tube agitator, for example a vortex mixer.
6.3 Magnifying lens, magnification × 8 to × 10.
6.4 pH-meter, with temperature compensation, accurate to ± 0,1 pH unit at 25 °C ± 1 °C (see also
ISO 7218).
6.5 Test tubes, with rubber stoppers or caps, of diameter and length 16 mm × 160 mm and
20 mm × 200 mm, to hold the culture medium.
6.6 Graduated pipettes, for bacteriological use, sterilized and calibrated to the tip, capable of delivering
1 ml ± 0,02 ml and 10 ml ± 0,2 ml (see ISO 6887-1).
Presterilized pipettes made of synthetic materials may be used instead of glass pipettes.
6.7 Glass rod.
6.8 Water baths, capable of operating at 10 °C ± 1 °C, at between 44 °C and 47 °C, at 45 °C ± 1 °C, at
50 °C ± 1 °C, and capable of boiling.
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ISO 9232:2003(E)
IDF 146:2003(E)
7 Procedure
7.1 Isolation of colonies
Select colonies from the plates used for counting, as obtained according to ISO 7889IDF 117, by using a
magnifying lens (6.3), if needed. Inoculate the stipulated broths to obtain pure cultures after incubation.
Incubate in the incubator (6.1) set at 37 °C for 24 h, under the atmospheric conditions described in
ISO 7889IDF 117.
7.2 Phenotypic characteristics required for identification of L. delbrueckii subsp.
bulgaricus
7.2.1 Culture media
7.2.1.1 Use skimmed milk (5.1.1) and MRS broth (5.1.2) for routine cultures and physiological tests, as
described below.
7.2.1.2 For fermentation tests, use a commercially manufactured kit, having shown it is appropriate for
this purpose with the microorganisms under investigation. Follow the manufacturer's instructions precisely.
NOTE Basic medium for fermentation tests (5.1.3) is supplied with the diagnostic kit and is used to prepare the
colony inoculum.
7.2.2 Characteristics to be considered
7.2.2.1 Morphology
Use freshly prepared 24-h pure cultures grown in skimmed milk (5.1.1) in an incubator (6.1) set at 37 °C for
24 h. Stain smears of cultures with methylene blue (5.3) for a few minutes before making a microscope
examination. For shape and cell arrangement, refer to Table A.1 in Annex A. Volutin granules should be
visible within the cells.
7.2.2.2 Catalase reaction
Mix equal volumes of the MRS broth culture (see 7.1), incubated in the incubator (6.1) set at 37 °C for 18 h to
24 h, with 1,5 % hydrogen peroxide in a test tube (6.5) fitted with a rubber stopper. Prepare a non-inoculated
control broth at the same time.
Gently turn the tubes upside down once to favour mixing, and observe for bubbles of oxygen forming in the
broth at room temperature over 20 min. L. delbrueckii subsp. bulgaricus will not produce oxygen. The test is
negative if gas is seen in the control tube.
7.2.2.3 Growth in the broth at 15 °C and 45 °C
Use a drop of culture of the test strain, incubated in the MRS broth (see 7.1) in the incubator (6.1) set at 37 °C
for 18 h to 24 h, to inoculate two fresh MRS broths (5.1.2.2). One broth should previously have been brought
in a water bath (6.8) to 15 °C and the other in another water bath to 45 °C. Incubate one in an incubator (6.1)
set at 15 °C and the other in an incubator set at 45 °C for up to 7 days. Observe for turbidity. L. delbrueckii
subsp. bulgaricus does not grow at 15 °C, but does grow at 45 °C giving turbidity.
7.2.2.4 Production of CO
2
Inoculate 10 ml of culture medium (5.1.4) with 0,1 ml of an MRS broth culture of the test strain (see 7.1)
incubated in an incubator (6.1) set at 37 °C for 18 h to
...