INTERNATIONAL , STANDARD

IDF 153

First edition 2002-1 1-01

Butter, fermented milks and fresh cheese - Enumeration of contaminating -

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Reference numbers IS0 13559:2002(E)

IDF 153:2002(E)

0 IS0 and IDF 2002

IS0 13559:2002(E) IDF 153:2002(E)

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Foreword

I S 0 (the lnternational Organization for Standardization) is a worldwide federation of national standards bodies ( IS0 member bodies). The work of preparing lnternational Standards is normally carried out through IS0 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. lnternational organizations, governmental and non-governmental, in liaison with ISO, also take' part in the work. IS0 collaborates closely with the International Electrotechnical Commission (IEC) on all m?tters of electrotechnical standardization.

lnternational Standards are 'drafted in accordance with the rules given in the ISOIIEC Directives, Part 3

The main task of technical committees is to prepare lnternational Standards. Draft lnternational Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an lnternational Standard requires approval by at least 75 Oh of the member bodies casting a vote.

Attention is drawn to the possibility that some of the elements of this lnternational Standard may be the subject ol patent rights. IS0 shall not be held responsible for identifying any or all such patent rights.

lnternational Standard I S 0 13559 1 IDF 153 was prepared by Technical Committee ISOrrC 34, Food products, Subcommittee SC 5, Milk and milk products, and the lnternational Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by I S 0 and IDF and separately by AOAC International.

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Foreword 8

IDF (the lnternational 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 IS0 and AOAC lnternational in the development of standard methods of analysis and sampling for milk and milk products.

Draft lnternational Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an lnternational Standard requires approval by at least 50 % of the National Committees casting a vote.

lnternational Standard IS0 13559 1 IDF 153 was prepared by Technical Committee lSO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the lnternational Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by IS0 and IDF and separately by AOAC International.

All work was carried out by the Joint ISO/IDF/AOAC Action Team, Non-Pathogen Contaminants, under the aegis of its project leader, Mr D. van den Berg (NL).

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INTERNATIONAL STANDARD IS0 13559:2002(~) IDF 153:2002(E)

Butter, fermented milks and fresh cheese - Enumeration of contaminating microorganisms - Colony-count technique at 30 "C

1 Scope I

This lnternational Standard specifies a method for the enumeration of contaminating microorganisms by means of the colony-count technique at 30 "C. The method is applicable to butter, fermented milks and fresh cheese.

2 Normative references

The following normative documents contain provisions which, through reference in this text, constitute provisions of this lnternational Standard. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this lnternational Standard are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. Members of IS0 and IEC maintain registers of currently valid lnternational Standards.

IS0 6887-1, Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - Part I: General rules for the preparation of the initial suspension and decimal dilutions

IS0 721 8, Microbiology of food and animal feeding stuffs - General rules for microbiological examinations

IS0 8261 1 IDF 122:2001, Milk and milk products - General guidance for the preparation of test samples, i~i i t ial suspensions and decimal dilutions for microbiological examination

lSO/TS 11 133-1, Microbiology of food and animal feeding stuffs - Guidelines on preparation and production of culture media - Part I: General guidelines on quality assurance for the preparation of culture media ill the laboratory

3 Term and definition

For the purposes of this lnternational Standard, the following term and definition applies.

3.1 contaminating microorganisms non-lactic acid bacteria, yeasts and moulds forming countable colonies under the conditions specified in this lnternational Standard

NOTE 1 Product-specific lactic acid bacteria will not be detected by this method

NOTE 2 In certain types of fermented milks, non-lactic acid bacteria, yeasts or moulds can be part of the microflora contributing to the desired characteristics of the product. In such cases, care should be taken when applying the method described in this lnternational Standard.

4 Principle

4.1 Poured plates are prepared using a specified culture medium, followed by surface plating of a specified quantity of an initial suspension of the product. Under the same conditions, poured plates are prepared, followed by w f a c e plating of a specified quantity of decimal dilutions of an initial suspension.

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4.2 The plates are aerobically incubated at 30 "C + 1 "C for 72 h.

4.3 The number of microorganisms per gram of test sample is calculated from the number of colonies obtained on plates chosen at dilution le,vels so as to give a significant result.

5 Diluents, culture media and reagents

5.1 General t

For current laboratory.practice, see IS0 7218.

5.2 Basic materials

See IS0 8261 1 IDF 122:2001, subclause 5.1

5.3 Diluents for general use

See IS0 8261 1 IDF 122:2001, subclause 5.2.

5.4 Diluents for special purposes

See IS0 8261 1 IDF 122:2001, subclause 5.3.

5.5 Distribution, sterilization and storage of diluents

See IS0 8261 1 IDF 122:2001, subclause 5.4.

5.6 Culture medium

5.6.1 General

All components of the culture medium shall be free from carbohydrates. The absence of carbohydrates in the culture medium used here is essential for the method described. The quality of the carbohydrate-free medium used shall be assured in accordance with lSO/TS 1 1 133-1.

5.6.2 Composition

Peptone from casein

Peptone from gelatin

Sodium chloride (NaCI)

Agar I )

Water

5.6.3 Preparation

5.6.3.1 Preparation from commercial dehydrated complete medium

F'ollow the manufacturer's instructions. Adjust the pH, if necessary, so that after sterilization it is 7,5 + 0,l at 25 "C. For fermented milks, adjust the pH, if necessary, so that after sterilization it is 8,O + 0,l at 25 "C.

1) Depending on the gel strength of the agar

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5.6.3.2 Preparation from dehydtated basic components

Dissolve and disperse by heating in the water, in the following order, the peptone from casein, the peptone from gelatin and the sodium chloride. Add the agar and heat to boiling while stirring frequently until the agar is completely dissolved, or steam for about 30 min. Filter through filter paper, if necessary. Adjust the pH so that, after sterilization, it is 7,5 +_ O,l*at 25 "C. For fermented milks, adjust the pH, if necessary, so that after sterilization it is 8,O k 0,l at 25 "C .

5.6.3.3 Distribution, sterilization and storage

Dispense the medium into test tubes (6.8), in quantities of 12 ml to 15 ml per tube, or into flasks pr bottles (6.9), in quantities of 100 ml to 150 ml. Sterilize in an autoclave set at 121 "C k 1 "C for 15 min.

If the medium is to be Used immediately, cool it in the water bath (6.5) to between 44 "C and 47 "C. If not used immediately, store it i i the dark at between 1 "C and 5 "C for no longer than 3 months. In order to avoid any delay when pouring the medium and before commencing the microbiological examination, completely melt the medium in a boiling water bath, then cool it in another water bath set at between 44 "C and 47 "C before use.

6 Apparatus

Disposable apparatus is an acceptable alternative to reusable glassware if it has suitable specifications. Reusable glassware should be capable of undergoing repeated sterilization and should be chemically inert.

Sterilize all apparatus that will come into contact with the test sample and the diluents or culture medium in accordance with IS0 8261.

Usual microbiological equipment (see IS0 7218 and IS0 8261) and, in particular, the following.

6.1 Incubator, capable of operating at 30 "C f 1 "C.

6.2 Oven or incubator, ventilated by convection, capable of operating at 50 "C f I "C , or a laminar air-flow cabinet.

6.3 Petri dishes, made of glass or plastic, of diameter 90 mm to 100 mm, or, when an inoculum of more than 0,l ml is used, dishes of diameter 140 mm.

6.4 Graduated pipettes, of nominal capacity of 1 ml k 0,02 ml or 10 ml +_ 0,2 ml.

6.5 Water baths, capable of operating at between 44 "C and 47 "C , and capable of boiling

6.6 Colony-counting equipment, consisting of an illuminated base with a dark background, fitted with a magnifying lens to be used at a magnification of 1,5x and a mechanical or electronic digital counter.

6.7 pH-meter, accurate to k 0,l pH unit at 25 "C, with readability to 0,01 units.

6.8 Test tubes. with plugs or caps. of capacity of approximately 20 ml.

6.9 Bottles or flasks, with plugs or caps, of nominal capacity 150 ml to 250 ml.

Bottles or flasks with non-toxic metal screw-caps may be used.

6.10 Sterile spreaders, with diameter of approximately 3,5 mm and length of 20 cm, bent at right angles about 3 cm from one end. The cut ends should be made smooth by heating.

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IS0 13559:2002(E) IDF 153:2002(E)

7 Sampling *

It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage.

Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in IS0 707. I

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8 Procedure -

8.1 Preparation of test portion and initial suspension

8.1.1 Gcncral

For general requirements see IS0 6887-1, and for specific requirements see IS0 8261.

Take normal aseptic precautions. The operations described in 8.1 and 8.2 shall not be carried out in direct sunlight.

8.1.2 Butter

See IS0 8261 :2001, subclause 8.2.6.

See IS0 8261 :2001. subclause 8.2.4

8.1.4 Fermented milks

See IS0 8261 :2001, subclause 8.2.9.

8.2 Further decimal dilutions

See IS0 8261,

8.3 Preparation of plates

Pour 12 ml to 15 ml of the prepared medium (5.6) into Petri dishes (6.3) and allow to solidify. Dry the plates, preferably with the lids off and the agar surface facing downwards, in an oven or incubator (6.2) set at 50 "C for 30 min. See also IS0 7218.

As an option, Petri dishes with a diameter of 140 mm may be used (6.3) when an inoculum of > 0, l ml is used

Instead of drying in an oven or incubator, the plates may also be dried in a laminar air-flow cabinet (6.2) for 30 min.

8.4 Inoculation and incubation

8.4.1 Transfer to each of two prepared plates (8.3), by means of a sterile pipette (6.4), 0,l ml of the initial suspension of the product.

8.4.2 Repeat this operation using further decimal dilutions. -,

8.4.3 Carefully spread the inoculum as quickly as possible over the surface of the plate, taking care not to touch the sides of the dish, using a sterile spreader (6.10). Use one sterile spreader for each plate. Leave the plates, with the lids on, for approximately 15 min on the bench to allow absorption of the inoculum into the plates.

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8.4.4 Invert the prepared dishes and place them in the incubator (6.1) set at 30 "C for 72 h i 2 h. Do not stack the dishes more than six high. Stacks of plates shall be separated from one another and from the walls and top of the incubator (see I S 0 7218).

8.5 C o u n t i n g of colonies *

Using the colony-counting equipment (6.6), count the colonies after incubation (8.4.4). Count the colonies characteristic for contaminating microorganisms in each dish containing not more than 150 colonies. Do not count pin-point colonies as these are not typical for contaminants.

Information as to the source of contamination may be obtained by examination of the colonies plesent. It can be valuable, therefore, to record the types of colonies present (e.g. pure or mixed, yeasts, moulds, Bacillus sp. etc.).

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9 Calculation arid expression of results

9.1 Calcu la t ion

Retain dishes containing more than 10 and less than 150 characteristic colonies at two successive dilutions

Calculate the number, N, of contaminating microorganisms per gram of test sample using the following equation:

where

Cc is the sum of characteristic colonies counted in all dishes retained;

n l is the number of dishes retained at the first dilution;

n2 is the number of dishes retained at the second dilution;

d is the dilution factor corresponding to the first dilution.

9.2 Expression of results

9.2.1 Round the result obtained in 9.1 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 in the case of an odd second figure.

EXAMPLE Round:

9.2.2 If there are only counts of less than 10, report the number of microorganisms per gram as "less than 10 x l ld" , where d is the dilution factor corresponding to the lowest dilution.

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9.2.3 If there are only counts exceeding 150, calculate an estimated count from dishes having a count nearest to 150 colonies and multiply by the reciprocal of the dilution factor corresponding to the highest dilution. Report as the "estimated number of microorganisms per gram".

9.2.4 Express the results as a number from 1 ,O to 9,9 multiplied by the appropriate power of 10.

EXAMPLE <

At the first (lo-*) dilution: 83 and 97 colonies. t

At the second (1 0-3 dilution: 13 and 10 colonies

Calculation result by using the formula:

Rounding the result to two significant figures means 9 200 or 9,2 x l o 3 contaminating microorganisms per gram of sample

10 Precision

10.1 General

See I S 0 7218 for information about the confidence limits for the estimation of small numbers of microorganisms.

NOTE No detailed precision data obtained from a collaborative study are available. However, due to the large diversity of contaminating microorganisms, depending on, for example, the type of product, topographic origin and location of production, inclusion of data from a collaborative study based on specific strains is not considered to be relevant.

10.2 Repeatability

Experience indicates that if the higher of two independent tests on the same sample frequently exceeds the lower by 30 %, the analyst should examine the procedures to determine sources of error.

11 Test report

The test report shall specify:

a) all information required for the complete identification of the sample;

b) the sampling method used, if known;

c) the test method used, with reference to this International Standard;

d) all operating details not specified in this International Standard, or regarded as optional, together with details of any incident which may have influenced the result(s);

e) the test result(s) obtained; and

f) if the repeatability has been checked, the final quoted results obtained. ..

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Bibliography

[ I ] IS0 707, Milk an< milk products - Guidance on sampling

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17.2.02 - Microbiological Methods / Chilled, Frozen, Precooked, o r Prepared Foods, and Nutmeals

AOAC Official Method 966.24 Coliform Group and Escherichia coli

in Tree Nut Meats

Microbiological Method First Action 1966 Final Action 1971

Seed 3-tube most probable number (MPN) series into lauryl sulfate tryptose broth, (b), using 1 rnL inocula of 1 : 10, 1 : 100, and 1 : 1000 dilutions, with triplicate tubes at each dilution. (For nut meats [halves

and larger pieces], begin MPN determination with 10' dilution; for nut meal, begin with lo-' dilution.) Incubate 48 h 2 h at 35OC for gas formation as evidenced by displacement of liquid in insert tube or by vigorous effervescence when tubes are shaken gently. Examine tubes for gas formation at 24 and 48 h intervals. Transfer, using 3 mm loop, from gassing tubes to BGLB, (c; omit this transfer for tree nuts), and EC broth, (g), at time gas formation is noted.

Incubate BGLB broth 48 k 2 h at 35OC. Using MPN Table 966.24A, compute MPN on basis of number of tubes of BGLB broth producing gas by end of incubation period. Report as MPN of coliforin bac terialg.

Incubate EC broth 48 k 2 h at 45.5 h 0.05OC in covered Hz0 bath. Submerge broth tubes in bath so that Hz0 level is above highest level of medium. Examine tubes for gas formation at 24 and 48 h intervals. Streak gas-positive tubes on Levine's eosin methylene blue agar plates, (d), and incubate plates 24 h 2 h at 35°C.

Pick 2 or more well-isolated typical colonies from Levine's eosin methylene blue agar plates and transfer to agar slants prepared from agar medium, (a). Incubate 18-24 h at 35OC. If typical colonies are not present, pick 2 or more colonies most likely to be E. coli. Pick 2 from every plate.

Transfer growth from plate count agar slants into following broths for identification by biochemical tests:

(a) Tryptophcrne broth.- Incubate broth, (j), 24 h 2 h at 35OC and test for indole by adding 0.2-0.3 1111~ Kovacs reagent, 967.25B(a) (see 17.9.01), to 24 h culture. Test is positive if upper layer turns red.

(b) MR-VP medium.-Incubate medium, (k), 48 h 2 h at 35OC. Aseptically transfer 1 mL culture to 13 x-

100 mm test tube to test for acetylmethylcarbinol. Add 0.6 mL 5% alcoholic a-naphthol solution (wlv), 0.2 m l KOH solution (4 + lo), and few crystals of creatine. Shake and let stand 2 h. Test is positive ir eosin pink develops. Alternatively, see 967.27D(c)(l) (see 17.9.03).

,

incubate remainder of MR-VP medium for additional 48 h and test for methyl red reaction by adding 5 drops methyl red solution to culture. Test is positive if culture turns red; negative, if yellow. (Prepare inethyl red solution by dissolving 0.1 g methyl red in 300 mL 90% alcohol and diluting to 500 mL with 1-120.) 4,

Table 966.24A: Most probable numbers (MPN) per 1 g test portion, using 3 tubes with each of 0.1, 0.01, and 0.001 g portions

d

Tablc 966.24B: Classification of biochemical typesg

(c) Koser citrate broth; 9b6.23~(1).-1ncubate 96 h at 35°C and record growth as + or -.

(d) Laziryl sulfate tryptose broth,966.23A(b).-Incubate 48 =t 2 h at 35OC. Examine tubes for gas I'ormation.

(c) Gram stain.-Perform Gram stain on 18 h agar slant (Standard Methods.for the Examination of Water and Wastewater, 18th Ed., 1992, American Public Health Association, American Water Works Association, and Water Pollution Control Federation, Washington, DC, USA). Coliform organisms will stain red (negative); Gram-positive organisms will stain blue-black.

(f) C1assjfication.-Classify biochemical types as in Table 966.24B.

Iicferences: ,JAOAC 49,270,276(1966); 51, 865, 867(1968); 58, 1 154(1975).

Revised: March 1999

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17.5.02 - Microbiological Methods / Stclplzylococcus

AOAC Official Method 975.55 Staphylococcus aureus in Foods

d

Surface Pliting Method for Isolation and Enumeration First Action 1975 Final Action 1976

[Applicable for general purpose use in testing foods expected to contain 2 10 cells of S. uwetlslg. For sillall numbers, see 987.09 (see 17.5.0 I).]

A. Apparatus

Sterile, bent glass streaking rods.-Hockey stick or hoe-shape, with fire-polished ends, 3 4 mm diameter, 15-20 cm long, with angled spreading surface 45-55 rnrn long.

B. Determination

At each dilution plated, aseptically transfer 1 mL test sample suspension, 987.09C (see 17.5.01), to triplicate plates of Baird-Parker medium, 987,09B(e)(3) (see 17.5.01), and equitably distribute the 1 inL inoculum over the triplicate plates (e.g., 0.4 mL-0.3 mL-0.3 mL). Spread inoculum over surface of agar using sterile, bent glass streaking rods. Avoid extreme edges of plate. Retain plates in upright position until inoculum is absorbed by medium (ca 10 min on properly dried plates). If inoculum is not readily absorbed, plates may be placed in incubator in upright position ca 1 h before inverting. Invert plates and incubate 4 5 4 8 h at 35-37OC. Select plates containing 20-200 colonies, unless only plates at lower dilutions (>200 colonies) have colonies with typical appearance of S. aureus, 987.093 (see 17.5.01). IS several types of colonies are observed which appear to be S. aureus, count number of colonies of each type and record counts separately. When plates at lowest dilution plated contain <20 colonies, these may be used. If plates containing >200 colonies have colonies with typical appearance of S. uureus and typical colonies do not appear at higher dilutions, use these plates for enumeration of S. aureus, but do not count nontypical colonies. Select one colony of each type counted and test for coagulase production, 987.09F (see 17.5.01). Add number of colonies on triplicate plates represented by colonies giving positive coagulase test and multiply by test sample dilution factor. Report this number as number of's. uureuslg of food tested.

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