1、December 2009DEUTSCHE NORM English price group 8No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 07.100.30!$+“1560856ww
2、w.din.deDDIN ISO 18593Microbiology of food and animal feeding stuffs Horizontal methods for sampling techniques from surfaces usingcontact plates and swabs (ISO 18593:2004)English version of DIN ISO 18593:2009-12Mikrobiologie von Lebensmitteln und Futtermitteln Horizontales Verfahren fr Probenahmete
3、chniken von Oberflchen mittelsAbklatschplatten und Tupfer (ISO 18593:2004)Englische Fassung DIN ISO 18593:2009-12www.beuth.deDocument comprises pages13DIN ISO 18593:2009-12 2 Contents Page National foreword .2 National Annex NA3 Introduction .5 1 Scope 6 2 Normative references 6 3 Principle6 4 Cultu
4、re media and dilution fluid.7 5 Apparatus and glassware .7 6 Sampling techniques.8 7 Transport 9 8 Procedure .9 9 Expression of results and calculation.11 Bibliography 13 National foreword This standard has been prepared by Technical Committee ISO/TC 34 “Food products“ (Secretariat: AFNOR, France).
5、The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-01-06 AA Mikrobiologische Lebensmitteluntersuchung einschlielich Schnellverfahren. ISO 7218 r
6、eferred to in clause 2 has not been adopted as national standard. The revised version ISO 7218:2007 has been published as DIN EN ISO 7218:2007-11. The pipettes used for the drop-plate method specified in ISO 18593:2004, subclause 8.2, are piston-operated pipettes capable of delivering a volume of 50
7、 l. These pipettes are not given in subclause 5.8. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 6887-1 DIN EN ISO 68871 ISO 8261 DIN EN ISO 8261 ISO/TS 11133-1 DIN ISO/TS 11133-1 DIN ISO 18593:2009-12 3 National Annex NA (informative
8、) Bibliography DIN EN ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of the test samples, of initial suspension and of decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions DIN EN ISO 8261,
9、Milk and milk products General guidance for the preparation of test samples, initial suspensions and decimal dilutions for microbiological examination DIN ISO/TS 11133-1, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part 1: General guidelin
10、es on quality assurance for the preparation of culture media in the laboratory DIN ISO 18593:2009-12 4 This page is intentionally blank DIN ISO 18593:2009-12 5 Microbiology of food and animal feeding stuffs Horizontal methods for sampling techniques from surfaces using contact plates and swabs Intro
11、duction It can be important to determine the presence of, or the number of, viable microbes on the surfaces of utensils, work surfaces and other equipment in contact with food to estimate the level of contamination during production, or the effectiveness of cleaning and disinfecting protocols. The h
12、orizontal methods described in this International Standard include a surface contact method using contact plates and a swab method. The contact plate method is only applicable to flat surfaces, whereas the swab method can be used for all types of surfaces. For the sampling of large surfaces ( 100 cm
13、2), sterile cloths or sponges can be used. This alternative method is useful for the estimation of the microbial load of surfaces. Results are often presented as hygiene scores based on the number of colony-forming units (CFU) per square centimetre present on a test surface. DIN ISO 18593:2009-12 6
14、1 Scope This International Standard specifies horizontal methods for sampling techniques using contact plates or swabs on surfaces in the food industry environment (and food processing plants), with a view of detecting or enumerating viable microorganisms. NOTE The term “environment” means any item
15、in contact with the food product or likely to represent a contamination or recontamination source, for example, material, premises, operators. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cite
16、d 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
17、for the preparation of the initial suspension and decimal dilutions ISO 7218, Microbiology of food and animal feeding stuffs General rules for microbiological examinations 3 Principle 3.1 Because these methods are not quantitatively reliable or reproducible, results should only be used in a “trend a
18、nalysis”. 3.2 A contact plate (or dipslide) filled with a suitable agar medium is pressed against the surface to be tested. After incubation, an estimate of the surface contamination is obtained by counting the number of developed colonies. 3.3 Using the swab method, a specified area of the surface
19、to be examined is marked (e.g. using a template) and then wiped. The swab sticks are broken into a tube or bottle containing a sterile dilution fluid or neutralizing fluid and mixed by hand. If the surface is wiped with a sterile (damp) cloth or sponge, the sampling device is stored in a known volum
20、e of dilution liquid (e.g. 100 ml for 100 cm2). In the laboratory the initial suspension and, if necessary, further decimal dilutions are used to determine the number of microorganisms using the procedures described in the methods for the enumeration of the (groups of) microorganisms to be investiga
21、ted. NOTE The incubation time and temperature depend on the type of microorganisms to be detected. 3.4 For selective media, appropriate confirmatory tests can be performed. The number of colony-forming units of specific microorganisms per square centimetre or per swab is calculated from the number o
22、f (confirmed) colonies. 3.5 After sampling, the surface is cleaned and disinfected, if necessary, to avoid traces of nutrients resulting from the sampling procedure remaining on the sampled surface. DIN ISO 18593:2009-12 7 4 Culture media and dilution fluid NOTE For further information, see the rele
23、vant International Standards for the target microorganisms to be detected or enumerated. 4.1 Neutralizing liquid In general, the base for neutralizing liquid is buffered peptone water, or peptone salt, or any other appropriate diluent (such as quarter-stength Ringers solution, phosphate buffer at pH
24、 7,5, peptone solution at 1 g/l). In cases where residues of disinfectants are expected, appropriate neutralizers should be added to the dilution fluid and to the media used on the contact plates to prevent any inhibitory effect of the disinfectants on the growth of microorganisms. 5 Apparatus and g
25、lassware For general requirements, see ISO 7218. Disposable apparatus is an acceptable alternative to reusable glassware if it has similar specifications. Usual microbiological laboratory apparatus and, in particular, the following. 5.1 Contact plate, plastic dish with diameter 65 mm, filled with a
26、controlled volume of agar medium (chosen according to the target microrganisms), especially made for the sampling of surfaces. Dishes vary in diameter or area, according to the type of surface to be sampled. The agar shall form a convex meniscus with the dish. NOTE It is also possible to use any oth
27、er device (nutritive medium in a flexible or rigid container) which enables contact with the surface to be sampled, such as a dipslide (5.2). 5.2 Dipslide, synthetic slide (7 cm2to 10 cm2), one or both sides of which are covered with a layer of a solid growth medium (chosen according to the target m
28、icroorganisms). NOTE Various growth media are available according to the microorganism(s) sought. 5.3 Swab, breakable stick with cotton or synthetic material (such as alginate or rayon) swab contained in a tube or envelope. The swab shall be individually wrapped and sterilized. The material used sha
29、ll be documented free of inhibiting substances. NOTE For some types of surface, the cotton residues can contaminate the internal parts of these surfaces after sampling. 5.4 Cloth, damp, sterile woven material, free from antimicrobial substances, packed individually in sterile plastic bags, used for
30、the sampling of large surfaces (W 100 cm2). 5.5 Sponge, damp, sterile square of flat sponge, free from antimicrobial substances, packed individually in sterile plastic bags, used for the sampling of large surfaces (W 100 cm2). 5.6 Containers, such as bottles, tubes or flasks, suitable for the steril
31、ization and storage of culture media. 5.7 Cool box, insulated, capable of maintaining the samples at low temperature during transportation to the laboratory. 5.8 Graduated pipettes, having wide openings and a nominal capacity of 1 ml, graduated in 0,1 ml divisions, or automated pipettes delivering 1
32、00 l and 1 000 l. DIN ISO 18593:2009-12 8 5.9 Mixer, for mixing liquids in culture tubes. 5.10 Peristaltic homogenizer or homogenizer using horizontal shaking, with sterile plastic bags to prepare initial suspensions by peristaltic movement (peristaltic homogenizer) or vibration movement (homogenize
33、r using horizontal shaking). 5.11 Petri dishes, made of plastic, of diameter 65 mm. 5.12 pH-meter, capable of being read to the nearest 0,01 pH unit at 25 C 1 C, enabling measurements to be made which are accurate to 0,1 pH unit. 5.13 Template, made of a corrosion-resistant material (e.g. a frame of
34、 stainless steel enclosing an area of 20 cm2to 100 cm2), which is easy to clean and can be sterilized. 6 Sampling techniques 6.1 General It is important that the laboratory receive a sample which is representative of the surface tested and has not been changed during transport and storage, or by res
35、idues of disinfectants. Disinfectants are generally formulated for a disinfection contact time of 5 min to 15 min. Wait for a period in accordance with the disinfectant specification before investigating the surface with swabs or contact plates to assess the performance of the cleaning and disinfect
36、ion programme (or otherwise according to the disinfectant specification). An appropriate neutralizer for all situations cannot be prescribed. Generally, sorbitan monooleate (30 g/l) and lecithin (3 g/l) are useful to neutralize residues of absorbed disinfectants (e.g. quaternary ammonium compounds,
37、amphotericides). Sodium thiosulfate (5 g/l) is a good neutralizer for halogen-based products. In the case of peroxide-based disinfectants, catalase or peroxidase may be used as neutralizer. One unit of these enzymes catalyses the decomposition of 1 mol of hydrogen peroxide per minute at 25 C and at
38、pH 7,0 0,2. A number of disinfectant neutralizers are recommended in EN 1276 1, EN 1650 2, EN 13697 3and EN 13704 4. The components of a neutralizer which may be used in most situations is given in Table 1. Make up in a solution of peptone (1 g/l) and sodium chloride (8,5 g/l), distribute in tubes o
39、r bottles, and sterilize for 15 min at 121 C. Table 1 Neutralizer which can be used in most situations Component Conc. Sorbitan monooleate (Polysorbate 80) 30 g/l Lecithin 3 g/l Sodium thiosulfate 5 g/l L-Histidine 1 g/l Saponin 30 g/l DIN ISO 18593:2009-12 9 6.2 Contact plate method After removal f
40、rom the transport containers, press the agar surface of the contact plate or the dipslide firmly and without any lateral movement against the test surface. From the literature it is known that optimal results for contact plates are obtained with a contact time of 10 s and a pressure obtained with a
41、mass of 500 g. Close the contact plates or dipslides immediately after inoculation and put them back in the transport container. 6.3 Swab or cloth/sponge method 6.3.1 Swab method Remove a swab from the sterile wrapping and moisten the tip by immersing it in a tube containing dilution liquid. Press t
42、he tip of the swab against the wall of the tube to remove excess water. Place the tip of the swab on the surface to be investigated and streak an estimated area of 20 cm2to 100 cm2, whilst rotating the swab between thumb and forefinger in two directions at right angles to each other. Put the swab in
43、 the tube with dilution liquid and aseptically break or cut off the stick. 6.3.2 Sponge/cloth method Open the plastic bag or container containing the cloth or sponge. Remove aseptically the cloth or sponge with sterile forceps or a sterile gloved hand. Moisten the cloth or sponge with sufficient qua
44、ntity of diluent (without excess). In the case of humid surfaces, this is not necessary. Return the cloth or sponge to the plastic bag and close it in a manner that will ensure no leakage. Sample the chosen surface in two perpendicular directions, changing the face of the cloth or sponge. Place the
45、cloth or sponge in the sterile container, add the diluent and close. Add a known and sufficient volume of diluent, so that the cloth or sponge is still moist at the analysis. Alternatively, open the plastic bag containing the cloth or sponge. Gripping the sponge through the bag, pull the reversed ba
46、g over the hand. Use the sponge to collect the sample as described above and transfer the cloth or sponge to a sterile plastic bag. Close the bag in a manner that will ensure no leakage. 7 Transport Transport the samples obtained with the swab method, preferably within 4 h, to a cool box set at 1 C
47、to 4 C. Examine in the laboratory as soon as possible and not later than 24 h, as described in Clause 8. Transport the contact plates and/or dipslides, preferably within 4 h, in a way that no contamination can occur. 8 Procedure 8.1 Contact plate method Incubate the contact plates or dipslides accor
48、ding to the type of microorganisms to be enumerated (see Note to Clause 4). The contact plate method shall be not used for the specific detection of pathogenic microorganisms. DIN ISO 18593:2009-12 10 8.2 Swab method (including cloth and sponge) Thoroughly mix the contents of tubes containing swabs
49、using a mixer (5.9) for 30 s, adjusting the speed so that the wall of the tube is wetted up to a height of 2 cm to 3 cm below the top. Add to the plastic bags containing cloths or sponges an amount of dilution fluid or neutralizing fluid (4.1) depending on the size of the area investigated (e.g. 100 ml for 100 cm2). Then treat the contents of the bags in a peristaltic homogenizer (5.10) for 1 min, or a homogenizer using horizontal shaking (5.10) for 30 s. This represents the initial suspension. If high numbers of microorganisms are
