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1、 1 AS 5013.282009 Standards Australia Australian Standard Food microbiology Method 28: Examination of specific products Liquid milks and creams PREFACE This Standard was prepared by the Standards Australia Committee FT-024, Food Products and Subcommittee FT-024-01, Food Microbiology to supersede AS
2、1766.3.131994. The objective of this revision is to update the references and to transfer the procedure to AS 5013 series. It also incorporates minor technical variations on the apparatus used in the test technique. METHOD 1 SCOPE This Standard sets out microbiological methods for the examination of
3、 liquid milk and liquid milk products such as homogenized and modified milks, buttermilks and creams. NOTE: The methods do not apply to cultured milk or cream products, or ultra-heat-treated (UHT) milks and creams. 2 REFERENCED DOCUMENTS The following documents are referred to in this Standard: AS 1
4、166 Milk and milk productsGuidance on sampling 2300 Methods of chemical and physical testing for the dairying industry 2300.1.10 Method 1.10: General methods and principlesDetermination of phosphatase activity 5013 Food microbiology (series) 5013.11.1 Method 11.1: Microbiology of food and animal fee
5、ding stuffsPreparation of test samples, initial suspension and decimal dilutions for microbiological examinationGeneral rules for the preparation of the initial suspension and decimal dilutions AS 5013.282009 AS 5013.282009 2 Standards Australia www.standards.org.au 3 DILUENTS, CULTURE MEDIA AND REA
6、GENTS 3.1 General The diluents, culture media and reagents specified in this Clause shall be made up according to the formulations given in AS 5013 series. 3.2 Peptone salt solution 3.3 Trisodium citrate dihydrate (Na3C3H5O(COO)3.2H2O) solution 2% 4 APPARATUS The required apparatus is specified in t
7、he relevant methods in AS 1166, AS 5013 and AS 2300 referred to in this Standard. 5 SAMPLES 5.1 Laboratory samples It is assumed that the laboratory sample has been obtained and delivered to the laboratory according to the procedures set out in AS 1166. NOTE: The size of the laboratory sample taken
8、in accordance with AS 1166 may be adjusted in order to provide a sufficient quantity for the required tests. 5.2 Storage and laboratory samples Laboratory samples shall be stored at a temperature not exceeding 4C, but without freezing, until tested. Testing shall commence within 24 h of sampling. 6
9、PREPARATION OF TEST SAMPLES NOTE: For samples of pasteurized milk and cream to be prepared for phosphatase testing (see Clause 9), special instructions for preparation and mixing are given in AS 2300.1.10. These instructions should be followed in place of those given below. 6.1 Observation of physic
10、al condition and preparation for mixing Observe the physical condition of the laboratory sample at the time of testing and record any abnormality. For cream samples only (except for phosphatase testing; see Note above), warm the sample to about 45C, but not above. Proceed without delay to mixing (Cl
11、ause 6.2) and the preparation of dilutions. The total time at 45C shall not exceed 10 min. 6.2 Mixing of sample 6.2.1 Liquid cream, liquid milks and liquid milk products Mix the sample well. If the first dilution is in a bottle, mix the contents by shaking 25 times in about 12 s through an arc of 30
12、 cm. If the head space is insufficient for efficient mixing, aseptically pour the contents into a larger container for further mixing. 6.2.2 Thick or coagulated cream Proceed directly to Clause 6.3. 6.3 Opening of containers The procedure shall be as follows: (a) Thoroughly clean the top of the cont
13、ainer, treat with 70% ethanol (w/v) or 80% ethanol (v/v) and allow to dry. (b) Open the closure aseptically, using scissors if necessary. 3 AS 5013.282009 www.standards.org.au Standards Australia 6.4 Preparation of dilutions for liquid milks and liquid milk products only The procedure shall be as fo
14、llows: (a) Prepare dilutions of the sample as described in AS 5013.11.1, using peptone salt solution (3.2) or other diluent as specified in the AS 5013 series. (b) Mix thoroughly and, using peptone salt solution (3.2), prepare tenfold dilutions as described in the AS 5013 series. 6.5 Preparation of
15、dilutions for cream samples only 6.5.1 Liquid cream The procedure shall be as follows: (a) Weigh 10.0 0.1 g of sample. (b) Dilute with 90 mL of peptone salt solution (3.2) prewarmed to about 45C. (c) Mix thoroughly and, using prewarmed peptone salt solution (3.2), prepare further tenfold dilutions a
16、s described in the AS 5013 series. 6.5.2 Thickened or coagulated cream The procedure shall be as follows: (a) Mix thoroughly by gently stirring with a spoon or spatula. (b) Weigh 10.0 0.1 g of sample. (c) Dilute with 90 mL of 2% trisodium citrate solution prewarmed to about 45C. (d) Mix thoroughly a
17、nd, using prewarmed peptone salt solution (3.2), prepare further tenfold dilutions as described in the AS 5013 series. 7 GENERAL TESTS 7.1 Direct microscopic count Proceed as described in Appendix A. If necessary, dilute the cream 1:10 with water of approved quality (see AS 5013) before preparing th
18、e smear. 7.2 Specific microorganisms Use the relevant procedures described in AS 5013 to examine for specific groups of microorganisms. NOTE: See Clause 8 below for thermoduric bacteria. 7.3 Test for penicillin Use the disc assay method for penicillin*. 8 COUNT OF THERMODURIC BACTERIA IN RAW MILK AN
19、D CREAM 8.1 Laboratory pasteurization Pasteurize the sample in accordance with the following procedure and at the same time use a pilot tube containing milk or cream in which a thermometer is immersed to monitor the temperature of the sample under test. * AS 1766.3.111991, Food microbiology, Method
20、3.11: Examination of specific productsDairy products Test for penicillin. AS 5013.282009 4 Standards Australia www.standards.org.au Completely immerse a screw-capped glass test tube or glass McCartney bottle holding 10 mL of sample, in water maintained at 63.5 0.5C in a thermostatically controlled w
21、ater bath fitted with a suitable agitator. The temperature of the sample shall reach 63C within 5 min. After a further 30 min, remove the tube or bottle from the water bath and immediately cool to 5C or below in iced water. In placing the container in the cooling water, avoid wetting the closure but
22、 ensure that the level of the water is above the level of the milk or cream. Upon removal from the iced water, wipe the tube or bottle with a tissue. 8.2 Colony count Use the pour plate method described in AS 5013, using plate count agar and incubating at 30 1C for 72 2 h. 9 PHOSPHATASE TEST FOR PAS
23、TEURIZED MILK AND CREAM Carry out the phosphatase test in accordance with AS 2300.1.10, directly on the chilled, undiluted sample. NOTE: After pasteurization, reactivation of phosphatase in cream can occur. Care should therefore be exercised in the interpretation of results. 10 TEST REPORT The follo
24、wing information shall be reported: (a) All details necessary for the complete identification of the sample. (b) Reference to this Australian Standard, i.e. AS 5013.28. (c) Date of testing. (d) Any abnormality observed in the physical condition of the container or the product. (e) Results of the tes
25、ts. (f) Any circumstance or conditions that may have influenced the results. 5 AS 5013.282009 www.standards.org.au Standards Australia APPENDIX A PROCEDURE FOR DIRECT MICROSCOPIC COUNT (Normative) A1 SCOPE This Appendix sets out a method for counting the number of microbial cells and clumps in liqui
26、d milks and cultured milk products by microscopic examination of a stained film, viz. by a procedure not having a separate defatting step and which may be used on raw milk and similar products with low fat content in order to obtain results more rapidly. NOTE: The degree of accuracy in estimating mi
27、crobial numbers by this method depends on the number of microorganisms present in the milk. It is generally recognized that the degree of reproducibility by one operator decreases with counts of less than 500 000 per millilitre of milk, even when the technique is followed exactly. Factors contributi
28、ng to this poor reproducibility within and between operators are inaccuracies in measuring volumes, area of smear, inadequate fixing and staining, poor microscopy, irregular distribution of microorganisms, small number of fields actually counted in proportion to fields present in the smear, and eye
29、fatigue. A2 DEFINITIONS For the purpose of this Standard, the definitions below apply. A2.1 Microbial clump count Microbial count based on groups of microorganisms which appear to be the same type: cell of like kind within 5 m of a group are deemed to belong to that group, regardless of closeness to
30、 each other, cells of different types are deemed to be separate clumps. A2.2 Individual count Microbial count of all individual cells, both within groups and in isolation. A3 REAGENTS A3.1 Methylene blue staining solution (modification of Newman Lampert Stain) Add, gradually, 0.5 g of methylene blue
31、 chloride (certified grade) to a mixture of 56 mL 95 percent ethanol and 40 mL xylene (technical grade) in a stoppered 200 mL flask. Dissolve by swirling the flask. Stand overnight (12 h to 24 h) under refrigeration (0C to 4C). Filter through fine paper* and add 4 mL glacial acetic acid to filtrate.
32、 Store in a tightly closed bottle in a cool dark place. WARNING: PREPARE THE STAIN UNDER AN EXHAUST HOOD TO AVOID INHALATION OF TOXIC SOLVENT FUMES. NOTE: As this dye has limited shelf life, discard the solution if it becomes contaminated or shows signs of deterioration. A4 APPARATUS A4.1 Pipette or
33、 micropipettor Calibrated to deliver 0.01 mL. * Whatman No. 42 or equivalent has been found to be satisfactory. AS 5013.282009 6 Standards Australia www.standards.org.au A4.2 Microscope Provided with an oil immersion objective, with field diameter in the range 146 m to 206 m. A4.3 Microscope slides
34、Clean, unscratched, and treated to remove grease. A4.4 Guide plate (template) To outline an area of 1 cm2 when positioned underneath a microscope slide. A4.5 Suitable rake A5 PREPARATION OF SAMPLE The milk/cultured dairy products shall be mixed before testing by shaking the container 25 times in abo
35、ut 12 s through an arc of 30 cm. If the head space is insufficient for efficient mixing, aseptically pour the contents into a larger sterile container for further mixing. A6 METHOD WITHOUT DEFATTING WARNING: CARRY OUT THE PROCEDURE IN A FUME CUPBOARD TO AVOID INHALATION OF TOXIC SOLVENT FUMES. A6.1
36、Preparation of smear for microscopic examination The procedure shall be as follows: (a) Thoroughly rinse the pipette or micropipettor with the mixed sample and discharge to waste. (b) Deliver the 0.01 mL of sample onto a clean microscope slide, making sure that the surface of the slide is horizontal
37、. Spread immediately over the outlined area of 1 cm2, using a cool rake. (c) Dry the film at 40C to 45C in a dust-free atmosphere, in a horizontal position. A6.2 Staining The procedure shall be as follows: (a) Flood the slide with the methylene blue staining solution (see Paragraph A3.1) and leave f
38、or 2 min. (b) Drain the slide of excess solution by resting the edge on absorbent paper. (c) Dry the slide, using a warm air draught if available. (d) Rinse the slide by dipping into three successive lots of warm tap water at 35C to 45C. Drain and air dry. NOTE: Fresh rinse water should be used for
39、each slide. A7 MICROSCOPIC EXAMINATION OF STAINED SMEARS A7.1 Mounting the slide The slide shall be mounted in the microscope (see Paragraph A4.2). A7.2 Number of fields to be counted The number of fields to be counted shall be determined by conducting a preliminary microscopic survey of the mounted
40、 smear. Table A1 specifies the number of fields to be counted according to the number of organisms per field. 7 AS 5013.282009 www.standards.org.au Standards Australia TABLE A1 NUMBER OF FIELDS TO BE COUNTED FROM SMEAR Average number of organisms per field Number of fields to be counted 03 46 712 64
41、 32 16 1325 2650 51100 8 4 2 Over 100 1 A7.3 Selection of fields A7.3.1 General Having determined the number of fields to be counted (see Paragraph A7.2), select fields at approximately equidistant intervals over the smear. Do not consciously select the field to be counted while looking down the mic
42、roscope as this may introduce a subjective bias. A7.3.2 One field Select at about the centre of the smear. A7.3.3 Two fields Select the fields at the quarter distances from the ends of one of the diagonal lines across the smear. A7.3.4 Four fields Select the fields at four points midway between the
43、centre and the four corners. A7.3.5 Eight fields Select three rows of two or three fields at equidistant intervals between the north and south sides. A7.4 Counting For each selected field, the number of microbial clumps and number of microbial cells shall be counted. A8 CALCULATIONS A8.1 Microscope
44、field area The area of the field (mm2) is determined as follows: (a) Place a stage micrometer under the microscope, and using the oil immersion lens, determine the diameter of the field. The field diameter should be in the range 0.146 to 0.206 (146 m to 206 m). (b) Calculate the area of one field us
45、ing the formula Area = 3.14 R2 (where R is the radius, or half the diameter, in mm). (c) Recalculate the field area after serving of the microscope or if the lens system is changed. AS 5013.282009 8 Standards Australia www.standards.org.au A8.2 Microscope factor (MF) The microscope factor is the rec
46、iprocal of the actual fraction of sample seen in one microscope field, and is expressed to two significant figures. The microscope factor is calculated from the following equation: smillimetre squarein area, field Microsope smillimetresquareinsmear,ofArea MF = . . . A8(2) A8.3 Microscopic count Calc
47、ulate the average number of microbial clumps and the average number of microbial cells per field. Using the microscope factor, in conjunction with the averages obtained and sample volume, calculate the number of clumps and of individual bacteria per millilitre from the following equation: smillilitr
48、einsample,ofVolume fieldpernumberAverageMF mLper count cMicroscopi = . . . A8(3) 9 AS 5013.282009 NOTES AS 5013.282009 10 NOTES 11 AS 5013.282009 NOTES AS 5013.282009 12 This Australian Standard was prepared by Committee FT-024, Food Products. It was approved on behalf of the Council of Standards Au
49、stralia on 5 June 2009 and published on 14 July 2009. The following are represented on Committee FT-024: Australian Chamber of Commerce and Industry Australian Food and Grocery Council Australian Institute of Food Science and Technology Consumers Federation of Australia Department of Agriculture, Fisheries and Forestry (Commonwealth) Department of Primary Industries, Vic. Horticulture Australia Meat and Livestock Australia National Association of Testi