BS-7857-3-1997 ISO-13722-1996.pdf

BRITISH STANDARD BS 7857-3:1997 ISO 13722:1996 Methods for Microbiological examination of meat and meat products Part 3: Enumeration of Brochothrix thermosphacta Colony-count technique ICS 07.100.30 Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 10:32:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 7857-3:1997 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Board, was published under the authority of the Standards Board and comes into effect on 15 April 1997 © BSI 11-1998 ISBN 0 580 27236 2 National foreword This British Standard reproduces verbatim ISO 13722:1996 and implements it as the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on request. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, the ISO title page, pages ii to iv, pages 1 to 4, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover. Amendments issued since publication Amd. No.DateComments Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 10:32:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 7857-3:1997 © BSI 11-1998i Contents Page National forewordInside front cover Forewordii Text of ISO 137221 Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 10:32:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ii blank Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 10:32:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 10:32:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 7857-3:1997 ii© BSI 11-1998 Contents Page Forewordii 1Scope1 2Normative references1 3Definition1 4Principle1 5Dilution fluid, culture media and reagents1 6Apparatus2 7Sampling3 8Preparation of test sample3 9Procedure3 10Expression of results3 11Precision4 12Test report4 Annex A (informative) BibliographyInside back cover Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 10:32:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 7857-3:1997 © BSI 11-1998iii 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. 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. International Standard ISO 13722 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Subcommittee SC 6, Meat and meat products. Annex A of this International Standard is for information only. Descriptors: Agricultural products, food products, animal products, meat, poultry meat, meat products, tests, microbiological analysis, counting, Brochothrix thermosphacta, bacteria count methods. Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 10:32:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI iv blank Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 10:32:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 7857-3:1997 © BSI 11-19981 1 Scope This International Standard specifies a method for the enumeration of viable Brochothrix thermosphacta in all kinds of meat and meat products, including poultry, by means of a colony-count technique. 2 Normative references The following standards contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 3100-2:1988, Meat and meat products Sampling and preparation of test samples Part 2: Preparation of test samples for microbiological examination. ISO 6887:1993, Microbiology General guidance for the preparation of dilutions for microbiological examination. ISO 7218:1996, Microbiology of food and animal feeding stuffs General rules for microbiological examinations. 3 Definition For the purposes of this International Standard, the following definition applies. 3.1 brochothrix thermosphacta gram-positive bacteria which form characteristic oxidase-negative colonies on a solid selective medium streptomycin sulfate/thallium acetate/actidione (STAA) agar under the test conditions specified in this International Standard 4 Principle 4.1 Surface plating, on a solid selective culture medium contained in Petri dishes, of a specified quantity of the test sample if the initial product is liquid, or of a specified quantity of the initial suspension in the case of other products. Preparation of other plates, under the same conditions, using decimal dilutions of the test sample or of the initial suspension. 4.2 Incubation of the plates between 22 °C and 25 °C for 48 h ± 4 h. 4.3 Subjection of the colonies to a confirmation test. 4.4 From the number of colonies confirmed, calculation of the number of Brochothrix thermosphacta per millilitre or per gram of sample from colonies obtained on plates at dilution levels chosen so as to give the most reliable result (see 9.3). 5 Dilution fluid, culture media and reagents 5.1 General For current laboratory practice, see ISO 7218. 5.2 Dilution fluid For the preparation of dilutions, see ISO 6887. 5.3 Solid selective medium: Streptomycin sulfate/thallium acetate/actidione (STAA) agar (see reference 2) 5.3.1 Base medium 5.3.1.1 Composition 5.3.1.2 Preparation Dissolve the components or the dehydrated complete medium in the water by boiling. Adjust the pH so that after sterilization it is 7,0 at 25 °C. Sterilize for 15 min in an autoclave (6.1) set at 121 °C. 5.3.2 Streptomycin sulfate solution 5.3.2.1 Composition 5.3.2.2 Preparation Dissolve the streptomycin sulfate in the water. Sterilize by filtration. Peptone20,0 g Yeast extract2,0 g Glycerol15,0 g Dipotassium hydrogen phosphate (K2HPO4) 1,0 g Magnesium sulfate heptahydrate (MgSO4.7H2O) 1,0 g Agar9 g to 18 ga Water900 ml a Depending on the gel strength of the agar. Streptomycin sulfate1,0 g Water100 ml Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 10:32:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 7857-3:1997 2 © BSI 11-1998 5.3.3 Actidione solution WARNING Actidione and thallium acetate are both toxic. Take appropriate procedures to prevent contamination of the operator and the environment when using these chemicals and their solutions. 5.3.3.1 Composition 5.3.3.2 Preparation Dissolve the actidione in the water. Sterilize by filtration. 5.3.4 Thallium acetate solution 5.3.4.1 Composition 5.3.4.2 Preparation Dissolve the thallium acetate in the water. Sterilize by filtration. 5.3.5 Complete medium 5.3.5.1 Composition 5.3.5.2 Preparation Melt the base medium and cool it in a water bath (6.9) set at 47 °C. Using sterile conditions, warm an aliquot of the other liquids (5.3.2, 5.3.3 and 5.3.4) to 47 °C in the water bath. Add the stipulated volumes (5.3.5.1) of these solutions to the cooled medium, mixing well between each addition. 5.3.6 Preparation of agar plates for enumeration Pour 15 ml to 20 ml portions of the complete medium (5.3.5) into sterile Petri dishes (6.4) and allow to solidify. The plates may be stored prior to drying at between 0 °C and 5 °C for up to 1 week. Immediately before use, dry the agar plates, preferably with the lids removed and with the agar surfaces facing downwards, in the incubator (6.2) set at a temperature between 37 °C and 50 °C, until the droplets have disappeared from the surface of the medium. Do not dry them any further. The agar plates can also be dried in a drying cabinet for 30 min with half-open lids, or overnight with the lids in place. Ready-prepared agar plates are available commercially. Store and use them according to the manufacturers instructions. 5.4 Oxidase reagent 5.4.1 Composition 5.4.2 Preparation Dissolve the reagent in the cold water. The reagent shall be prepared immediately prior to use. Commercially available disks or sticks may be used. In this case, follow the manufacturers recommendations. 6 Apparatus Usual microbiological laboratory equipment and, in particular, the following. 6.1 Apparatus for dry sterilizing (oven) or wet sterilization (autoclave) See ISO 7218. 6.2 Incubator or drying cabinet, ventilated by convection, capable of operating between 37 °C ± 1 °C and 50 °C ± 1 °C. 6.3 Incubator, capable of operating between 22 °C ± 1 °C and 25 °C ± 1 °C. 6.4 Petri dishes, made of glass or plastic, of diameter 90 mm to 100 mm. 6.5 Total delivery (blow-out) pipettes, having wide openings, of nominal capacities 10 ml and 1 ml, graduated in 0,1 ml divisions. 6.6 Rubber bulbs, or any other type of safety device suitable for use with the graduated pipettes. 6.7 Culture bottles or flasks, of suitable capacities. 6.8 Spreaders (hockey-stick type) made of glass, of diameter approximately 3,5 mm and length 20 cm, bent at right angles about 3 cm from one end; the cut ends shall be made smooth by heating. Disposal sterile plastic spreaders may also be used. Actidione (cycloheximide)150 mg Water90 ml Thallium acetate250 mg Water100 ml Base medium (5.3.1)900 ml Streptomycin sulfate solution (5.3.2)50 ml Actidione solution (5.3.3)30 ml Thallium acetate solution (5.3.4)20 ml N,N,N,N-Tetramethyl-p- phenylenediamine dihydrochloride 1,0 g Distilled water100 ml Licensed Copy: London South Bank University, London South Bank University, Fri Dec 08 10:32:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 7857-3:1997 © BSI 11-19983 6.9 Water baths, capable of being maintained at 47 °C ± 2 °C. 6.10 Colony-counting equipment, consisting of an illuminated base with a dark background and a mechanical or electronic digital counter. 6.11 pH-meter, accurate to within ± 0,1 pH unit at 25 °C. 6.12 Wires, made of platinium, or rods, made of glass or plastic, approximately 3 mm in diameter. 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 ISO 3100-1. 8 Preparation of test sample Take, in accordance with the method described in ISO 3100-2, a representative test sample. Start the examination of the pretreated sample as soon as possible. It may be stored, if necessary, at a temperature between 0 °C and ± 2 °C, but for not longer than 24 h. 9 Procedure 9.1 Test portion, initial suspension and dilutions See ISO 6887 and ISO 3100-2. 9.2 Inoculation and incubation 9.2.1 Transfer, by means of a sterile pipette (6.5), 0,1 ml of the test sample if the product is liquid, or of the initial suspension in the case of other products, to each of two agar plates (5.3.6). Repeat the procedure using further decimal dilutions. 9.2.2 Carefully spread the liquid as quickly as possible over the surface of the agar plate without touching the sides of the dish with the spreader (6.8). Use a fresh sterile spreader for each plate. Leave the plates with the lids on for about 15 min at ambient temperature for the liquid to be absorbed into the agar. 9.2.3 Invert the prepared plates (9.2.2) and incubate them for 48 h ± 4 h in the incubator (6.3) set between 22 °C and 25 °C. 9.3 Counting of the colonies After the specified period of incubation (see 9.2.3), count, using the colony-counting equipment (6.10), the characteristic colonies on each dish containing 15 to 300 colonies. Characteristic colonies are shiny, round or circular colonies of diameter 0,75 mm or larger, have an off-white colour, and are oxidase negative (see 9.4). 9.4 Confirmation Pseudomonads are able to grow on STAA agar (5.3). They shall be differentiated from B. thermosphacta by performing an oxidase test as follows. Moisten a piece of filter paper with the oxidase reagent (5.4). Take a sample of the bacterial culture obtained from the STAA agar using a platinum wire or glass or plastic rod (6.12) (a nickel/chrome wire gives false positives) and deposit it on the moistened filter paper. Oxidase-positive colonies appear within 15 s as purple colonies. B. thermosphacta is oxidase negative. 10 Expression of results 10.1 Count of B. thermosphacta 10.1.1 If at least 80 % of the selected colonies are confirmed (9.4), take as the number of B. thermosphacta the number given by the count made as in 9.3. 10.1.2 In all other cases, calculate the number of B. thermosphacta from the percentage of B. thermosphacta obtained as in 9.3 which are confirmed (9.4). Round the result to a whole number of colonies. 10.2 Method of calculation 10.2.1 General case: Dishes containing between 15 and 300 characteristic colonies Retain dishes containing not more than 300 characteristic colonies at two consecutive dilutions. It is necessary that one of these dishes contains at least 15 characteristic colonies. Calculate the number