1、BRITISH STANDARD BS5763-5: 1981 Incorporating Amendment No. 1 Methods for Microbiological examination of food and animal feeding stuffs Part 5: Enumeration of micro-organisms Colony count at30C (surface plate technique) UDC614.31:636.084+664:579.672BS5763-5:1981 This British Standard, having beenpre
2、pared under the directionof the Food and Agriculture Standards Committee,was published underthe authority of the Executive Board and comes intoeffect on 31 December1981 BSI03-1999 The following BSI references relate to the work on this standard: Committee reference FAC/21 Draft for comment80/53484 D
3、C ISBN 0 580 12391 X Cooperating organizations The Food and Agriculture Standards Committee, under whose direction this British Standard was prepared, consists of representatives from the following: Agricultural Co-operation and Marketing Services Agricultural Research Council, Meat Research Institu
4、te British Food Manufacturing Industries Research Association* British Industrial Biological Research Association Ltd. Campden Food Preservation Research Association* Consumer Standards Advisory Committee of BSI Department of Agriculture and Fisheries for Scotland Department of Agriculture (Governme
5、nt of Northern Ireland)* Department of Industry (Laboratory of the Government Chemist)* Flour Milling and Baking Research Association* Food Manufacturers Federation Incorporated* Grain and Feed Trade Association Ltd. Institute of Brewing Local Authorities Co-ordinating Body of Trading Standards Mini
6、stry of Agriculture, Fisheries and Food* National Farmers Union National Farmers Union of Scotland Tobacco Advisory Council The organizations marked with an asterisk in the above list, together with the following, were directly represented on the Technical Committee entrusted with the preparation of
7、 this British Standard: Agricultural Research Council, Food Research Institute Brewing Research Foundation British Poultry Federation Limited Department of Health and Social Security Fruit and Vegetable Canners Association Joint Committee of the Milk Marketing Board and the Dairy Trade Federation Ov
8、erseas Development Administration Tropical Products Institute Public Health Laboratory Service Society for Applied Bacteriology UK Association of Frozen Food Producers United Kingdom Dairy Association University of Reading Amendments issued since publication Amd. No. Date of issue Comments 8161 Marc
9、h 1994 Indicated by a sideline in the marginBS 5763-5:1981 BSI 03-1999 i Contents Page Cooperating organizations Inside front cover Foreword ii 1 Scope and field of application 1 2 References 1 3 Definition 1 4 Principle 1 5 Sampling 1 6 Apparatus 1 7 Culture media and dilution fluid 2 8 Preparation
10、 of the test sample 2 9 Procedure 2 10 Expression of results 3 11 Test report 3 Publications referred to Inside back coverBS5763-5:1981 ii BSI 03-1999 Foreword This Part of BS5763 has been prepared under the direction of the Food and Agriculture Standards Committee in the light of limitations in the
11、 applicability of BS5763-1 which is identical with ISO4833:1991 published by the International Organization for Standardization (ISO). It has been published in order to provide a general reference method appropriate for suitably prepared samples from a range of foods and animal feeding stuffs for wh
12、ich methods are not given in existing British Standards, and for consideration by bodies preparing microbiological methods of test for application to food or to animal feeding stuffs. When sufficient experience has been gained in the application of this method, which will be effected by its being ca
13、lled up in appropriate new and revised British Standards, this experience will be taken into account in future revisions of this Part of this standard. The surface plating technique, which is described in this Part of this standard, is likely to be particularly suitable in the case of meat, fish and
14、 poultry. This Part of this British Standard also includes the method described by Farmiloeetal. (1954) for the calculation of a weighted average count using plates from more than one dilution. The procedure reduces the standard error of the count as compared with a simple arithmetic mean of colonie
15、s growing on replicate plates at one dilution. 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 obligat
16、ions. Summary of pages This document comprises a front cover, an inside front cover, pagesi andii, pages1 to4, 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 theinsi
17、de front cover.BS 5763-5:1981 BSI 03-1999 1 1 Scope and field of application This Part of BS5763 describes a general reference method for the enumeration of micro-organisms present in products intended for human consumption or feeding of animals, by counting the colonies growing on a solid medium af
18、ter incubating aerobically at30 C. A limitation on the applicability of this standard is imposed by the susceptibility of the method to a large degree of variability. A note at the end of clause10 gives information that takes account of this and assists in the interpretation of the results. 2 Refere
19、nces The titles of the publications referred to in this standard are listed on the inside back cover. 3 Definition For this standard, the following definition applies. micro-organisms bacteria, yeasts and moulds growing aerobically at30 C, under the conditions specified 4 Principle 4.1 Preparation o
20、f two plates, using a specified culture medium, and surface inoculating the test portion if the product is liquid, or using the specified quantity of an initial suspension in the case of other products. Inoculation of other pairs of plates, under the same conditions, using decimal dilutions of the t
21、est portion or of the initial suspension. 4.2 Aerobic incubation of the plates at30 C for72h. 4.3 Calculation of the number of micro-organisms per millilitre or per gram of sample from the number of colonies obtained on selected plates (see10.1). 5 Sampling Obtain a laboratory sample in accordance w
22、ith the appropriate British Standard applicable to the product concerned. NOTEIf no such standard exists carry out the sampling in such a way that the laboratory sample is suitable for microbiological analysis and with the agreement of the parties concerned. 6 Apparatus 6.1 Usual microbiological lab
23、oratory equipment. 6.2 Apparatus for dry sterilization (oven) or wet sterilization (autoclave). Apparatus that will enter into contact with the culture media, the dilution fluid or the sample, except for that which is supplied sterile (particularly plastics apparatus), shall be sterilized either a)
24、by being kept at170 C to175 C for not less than1h in an oven; or b) by being kept at121 1 C for not less than20min in an autoclave. NOTEApparatus in accordance with BS2646-1 is suitable. 6.3 Incubator, capable of being controlled at30 1 C. 6.4 Petri dishes, made of glass or plastics, with a diameter
25、 of90mm to100mm. NOTEDishes in accordance with BS611-2 are suitable. 6.5 Total delivery pipettes (blow-out pipettes), having a nominal capacity of1ml, subdivided in0.1ml and with an outflow opening of diameter2mm to3mm. NOTEPipettes in accordance with BS700-3 are suitable. 6.6 Water bath, or similar
26、 apparatus, capable of being controlled at45 0.5 C. 6.7 Water bath or steamer, capable of being maintained at the temperature of boiling water. 6.8 Colony counting equipment, consisting of an illuminated base with a dark background fitted with a magnifying lens to be used at a magnification of1.5 di
27、ameters, and a mechanical or electronic digital counter. 6.9 pH meter, with an accuracy of calibration of 0.2pH units at25 C. NOTEpH meters in accordance with BS3145 are suitable. 6.10 Test tubes, 18mm 180mm, or flasks or bottles of suitable capacity (see7.3 and7.4). 6.11 Drying cabinet or incubator
28、, for drying the surface of agar plates, maintained at a temperature not exceeding55 C. 6.12 Spreaders, made from nichrome wire mounted in a handle and bent so that a length of approximately30mm is at right angles to the handle. The wire can be sterilized by flaming. NOTEAn alternative is a glass sp
29、reader (hockeystick) made from glass rod approximately3.5mm in diameter and about200mm long, bent at right angles30mm from one end and with the cut ends made smooth by heating.BS5763-5:1981 2 BSI 03-1999 7 Culture media and dilution fluid 7.1 Basic materials. In order to improve the reproducibility
30、of the results, it is recommended that, for the preparation of culture media, dehydrated basic components or complete dehydrated media should be used. The manufacturers instructions shall be rigorously followed. The chemicals used shall be of analytical quality. The water used shall be distilled or
31、deionized, and shall be free from substances that might inhibit growth of micro-organisms under the test conditions. NOTEWater in accordance with the requirements of grade3 of BS3978 is suitable. If the media and dilution fluid are not used immediately, they shall be kept in the dark at a temperatur
32、e between0 C and+ 5 C, and in conditions that prevent any change in their composition. The media and dilution fluid shall not be kept for longer than1 month. Plates prepared in advance shall not be kept longer than1 day at room temperature or1 month in a refrigerator at0 C to+ 5 C. 7.2 Dilution flui
33、d (diluent). A peptone based dilution fluid composed of the following shall be used. Dilution bottles or tubes shall be prepared using appropriate volumes of the diluent. 7.3 Plate count medium 7.3.1 Composition. The plate count medium shall be composed of the following: 7.3.2 Preparation of the cul
34、ture medium 7.3.2.1 Dissolve the components or the dehydrated complete medium in the water by boiling. If necessary adjust the pH checking with the pH meter(6.9), so that after sterilization it is7.0 at25 C. 7.3.2.2 Dispense the medium into test tubes(6.10), in quantities of15ml per tube, or into fl
35、asks or bottles of capacity not greater than500ml, in quantities of approximately half the volume of the container. 7.3.2.3 Sterilize in an autoclave at121 C for20min. If the medium is to be used immediately (see7.4), cool it to45 C in the water bath(6.6). If it is not to be used immediately pour th
36、e medium into bottles or flasks, stopper, cool and store at0 C to+ 5 C (see7.1). 7.3.2.4 Before use completely melt stored medium in a steamer or boiling water bath(6.7), then cool it to45 C in the water bath(6.6). 7.4 Preparation of the agar plates. To sterile Petri dishes(6.4) add about15ml of the
37、 melted culture medium (see7.3.2.4) at a temperature of approximately45 C and allow to solidify. Immediately before use, dry the plates. NOTEThe preferred method is with the lids off and the agar surface downwards in an oven or incubator(6.11) at a temperature not exceeding55 C. 8 Preparation of the
38、 test sample Prepare the test sample from the laboratory sample in accordance with the appropriate British Standard dealing with the product concerned. NOTEIf a British Standard is not available, it is recommended that agreement be reached on this subject by the parties concerned. 9 Procedure 9.1 Te
39、st portion, initial suspension and dilutions. Measure the test portion from the test sample (see clause8). Prepare the initial suspension and the dilutions using dilution fluid (7.2) in accordance with the appropriate British Standard applicable to the product concerned. NOTEIn the absence of such a
40、 standard, suitable techniques are given in BS5763-6. 9.2 Inoculation 9.2.1 Take two agar plates(7.4). Using a sterile pipette(6.5), transfer to each plate0.1ml of the test sample, if liquid, or0.1ml of the initial suspension in the case of other products. Peptone 1.0 g Sodium chloride (NaCl) 8.5 g
41、Water 1000 ml tryptone a 5.0 g dehydrated yeast extract 2.5 g anhydrous D-glucose (anhydrous dextrose) 1.0 g agar in powder or flake form 9 g to 18 g b water 1000 ml a This term is used at present only by certain producers of media. Any other casein digest giving comparable results may be used. b Ac
42、cording to the directions of the manufacturer.BS 5763-5:1981 BSI 03-1999 3 9.2.2 Take two other agar plates(7.4). Using another sterile pipette, transfer to each plate0.1ml of the 1/10dilution of liquid products or0.1ml of the dilution 1/10 of the initial suspension of other products. Repeat the pro
43、cedure described in the preceding paragraph with the other dilutions. 9.2.3 Carefully spread the inoculum as soon as possible on the surface of each agar plate using a spreader(6.12), cover and leave the plates for about15min on the bench for absorption of liquid to take place. Use a sterile spreade
44、r for each plate. 9.3 Incubation. Invert the inoculated plates and place them in the incubator(6.3) at30 C. Leave them for72 3h. 9.4 Counting. After the specified period of incubation (see9.3), count the colonies on each plate using the colony counting equipment(6.8). Reject any plates in which more
45、 than half of the surface of the plate is overgrown. Otherwise count the colonies in half of the plate that is clear and multiply by two. 10 Expression of results 10.1 Method of calculation 10.1.1 Calculate the weighted mean count from the number of colonies (see9.4) on replicate plates at two succe
46、ssive decimal dilutions (preferably those having not more than300 colonies) according to the following formula. 1) weighted mean count For examples see10.1.4. 10.1.2 Retain only two significant figures, as follows: a) if the calculated weighted mean count is less than100, round it to the nearest mul
47、tiple of5; b) if the number is greater than100 and ends in a5, round it to the nearest multiple of20; c) if the number is greater than100 and does not end in a5, round it to the nearest multiple of10. 10.1.3 If either plate of a pair at any dilution is uncountable, e.g.because it is completely overg
48、rown, the results from all remaining plates can be used by making f aor f b= 1 see10.1.4, exampleb). 10.1.4 Examples a) dilution10 2 : 378 (plate1)296 (plate2) dilution10 3 : 40 (plate1)28 (plate2) weighted mean count 337 10 3= 3.4 10 5per gram b) dilution10 2 : 166 (plate1) plate2spoiled dilution10
49、 3 : 20 (plate1)18 (plate2) weighted mean count 170 10 3= 1.7 10 5per gram 10.1.5 If the initial suspension fails to produce colonies report the results as less than10 2micro-organisms per gram or10 per ml liquid. 10.1.6 If the initial suspension produces counts of less than30 colonies per plate report the result as less than3.0 10 3micro-organisms per gram or3.0 10 2per ml liquid. NOTEFor statistical reasons alone, in95% of cases the confidence limits of this method vary from 12% to 37% Cowell and Morisetti J.
