1、 Reference number ISO 17995:2005(E) ISO 2005INTERNATIONAL STANDARD ISO 17995 First edition 2005-06-15 Water quality Detection and enumeration of thermotolerant Campylobacter species Qualit de leau Recherche et dnombrement despces thermotolrantes du genre Campylobacter ISO 17995:2005(E) PDF disclaime
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6、equester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2005 All rights reservedISO 17995:2005(E) ISO 2005 All rights reserved iii Contents Page Foreword iv Introduction . iv
7、 1 Scope 1 2 Normative references 1 3 Terms and definitions .1 4 Principle2 5 Apparatus .2 6 Culture media and reagents .3 7 Sampling, transport and storage .3 8 Procedure .3 9 Quality assurance6 10 Expression of results 6 11 Test report 6 Annex A (informative) Flow diagram of method 7 Annex B (info
8、rmative) Semiquantitative analysis 8 Annex C (normative) Culture media and reagents 9 Annex D (informative) Supplementary tests for further confirmation .14 ISO 17995:2005(E) iv ISO 2005 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of
9、 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. Int
10、ernational 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. International Standards are drafted in accordance with t
11、he rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval
12、by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 17995 was prepared by Technical Committee I
13、SO/TC 147, Water quality, Subcommittee SC 4, Microbiological methods. ISO 17995:2005(E) ISO 2005 All rights reserved v Introduction Campylobacter jejuni subsp. jejuni and Campylobacter coli are common causes of intestinal infections in humans. Campylobacter upsaliensis may be of like importance. Cam
14、pylobacter lari is less frequently associated with human infections. The vehicles for campylobacter infections are usually food, farm animals, pets and person-to-person contact, but water is also important. INTERNATIONAL STANDARD ISO 17995:2005(E) ISO 2005 All rights reserved 1 Water quality Detecti
15、on and enumeration of thermotolerant Campylobacter species WARNING Persons using this International Standard should be familiar with normal laboratory practice. This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user
16、 to establish appropriate safety and health practices and to ensure compliance with any national regulatory conditions. IMPORTANT It is absolutely essential that tests conducted in accordance with this International Standard be carried out by suitably trained staff. 1 Scope This International Standa
17、rd specifies a method for the detection and semiquantitative enumeration of thermotolerant Campylobacter species. The method can be applied to all kinds of filterable waters. NOTE 1 The method can also be used as a presence/absence test for Campylobacter species in a specified sample volume. NOTE 2
18、A more quantitative result can be obtained using a most probable number (MPN) set-up (see ISO 8199). 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the lat
19、est edition of the referenced document (including any amendments) applies. ISO 3696:1987, Water for analytical laboratory use Specification and test methods ISO 8199, Water quality General guidance on the enumeration of micro-organisms by culture ISO 19458, Water quality Sampling for microbiological
20、 analysis 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 thermotolerant Campylobacter species bacteria retained on filters during the filtration described in 8.2, multiplying during the selective enrichment described in 8.3, forming typical
21、colonies during incubation at elevated temperatures on the selective medium described in 8.4, forming no visible colonies during incubation in air under the conditions specified in 8.6, being highly motile, slender rods with spiral morphology and a motility characterized by darting or corkscrew- lik
22、e movements. NOTE 1 Thermotolerant Campylobacter species of relevance in human infections include Campylobacter jejuni subsp. jejuni (hereafter referred to as C. jejuni), C. coli, C. lari and possibly C. upsaliensis. ISO 17995:2005(E) 2 ISO 2005 All rights reservedNOTE 2 Thermotolerant Campylobacter
23、 species are Gram-negative, oxidase-positive and catalase-positive (strains of C. upsaliensis are reported to be catalase-negative or weakly positive) curved or spiral-shaped rods with a characteristic darting, often rotating, motility. In older cultures, coccoid forms occur. NOTE 3 Campylobacter sp
24、ecies require enriched media for optimum growth. They are very sensitive to toxic oxygen derivatives like peroxides and superoxide anions which can arise in media exposed to light and oxygen. They are microaerophilic and prefer an atmosphere containing approximately 5 % oxygen and approximately 10 %
25、 carbon dioxide (CO 2). Some Campylobacter species may require an atmosphere containing hydrogen. NOTE 4 Most C. jejuni, C. coli and C. lari grow at temperatures between 32 C and 45 C, but some strains will not grow below 35 C. Other strains may not grow above 43 C. NOTE 5 C. upsaliensis may not gro
26、w under the conditions described in this International Standard. 4 Principle The sample is filtered through membrane filters with a pore size of 0,45 m. The filters are transferred to selective enrichment broths and incubated for (44 4) h at (37 1) C in a microaerobic environment. Following incubati
27、on, inoculums from each broth are streaked onto a solid medium, modified charcoal cefoperazone desoxycholate agar (mCCDA), and incubated for (44 4) h at (41,5 1) C in a microaerobic environment. Colonies resembling campylobacters are tested for growth aerobically and, if negative, examined by micros
28、copy. If necessary, further biochemical reactions are performed. See flow diagram in Annex A. If typical campylobacters are found, the sample is positive for thermotolerant campylobacters. The result is given as the semiquantitative estimate per volume of sample (see Annex B). Non-filterable waters
29、can be analysed by direct inoculation of sample into enrichment broths. The ratio of sample to enrichment broth shall be 10 % or less. NOTE When sufficient numbers of campylobacters are present, the sample can be streaked directly onto a solid medium, mCCDA, without prior selective enrichment. 5 App
30、aratus 5.1 Incubators, thermostatically controlled at (37 1) C and at (41,5 1) C. 5.2 Equipment for membrane filtration, as specified in ISO 8199. 5.3 Membrane filters: Sterile membrane filters made of cellulose ester with a diameter of 45 mm to 50 mm and a pore size of 0,45 m. Similar filters with
31、a pore size of 0,22 m are recommended for sterilization of supplements. 5.4 Equipment for microaerobic incubation: Jars able to maintain a modified atmosphere during incubation, fitted with valves for outlet and inlet of gases; vacuum pump; monitor for gas composition; and a suitable source of nitro
32、gen, oxygen, carbon dioxide and preferably also hydrogen. NOTE Commercially available equipment (like ANOXOMAT 1) ) can reproducibly deliver the modified atmosphere to the jars. 1) ANOXOMAT is a trade name. This information is given for the convenience of users of this International Standard and doe
33、s not constitute an endorsement by ISO of this product. ISO 17995:2005(E) ISO 2005 All rights reserved 3 Alternatively, gas-generating pouches can be used if they are able to maintain an atmosphere with approximately 5 % oxygen, approximately 10 % carbon dioxide and preferably also approximately 10
34、% hydrogen. 5.5 Microscope, preferably with phase contrast. 5.6 Bottles, 150 ml to 250 ml, with airtight screw caps for the selective enrichments. 5.7 Vented Petri dishes, sterile, 9 cm. 5.8 Usual laboratory equipment. 6 Culture media and reagents All ingredients and chemicals shall be of recognized
35、 quality, “for microbiology” or better. Water used shall be distilled or of like quality, as specified for ISO 3696:1987, grade 3. Follow the instructions given in Annex C. Use of commercially available dehydrated substrates is encouraged, provided they comply with the descriptions in Annex C. They
36、shall be prepared in accordance with the manufacturers instructions. Other grades of chemicals may be used provided they can be shown to lead to the same results. 7 Sampling, transport and storage In addition to the instructions given in ISO 19458, be aware that campylobacters are very sensitive to
37、adverse conditions. Keep samples cool (3 2) C and in the dark until the filtrations have been done. Avoid unnecessary mixing with air. Filter the samples as soon as possible after collection. Store for a maximum of 30 h prior to analysis. NOTE Campylobacters survive well in clean water at (3 2) C. A
38、t higher temperatures or in other media, they may quickly deteriorate. 8 Procedure 8.1 General Parallel to samples, run a positive control spiked in sample material or in sterile water through all the steps of the procedure to demonstrate the proper functioning of the apparatus, culture media and pr
39、ocedure, and to facilitate recognition of campylobacters (8.5 to 8.6). Parts of each sample shall be enriched in the highly selective Preston broth (C.1.1) and parts in the less selective Bolton broth (C.1.2). NOTE Preston broth may be too selective to allow the recovery of some strains of C. coli.
40、Bolton broth may not be selective enough to counteract the growth of non-campylobacters in some samples. If the available sample size is limited, the most appropriate enrichment broth should be used. For waters with an expected high background count, it is more appropriate to use Preston broth, and
41、for clean waters or where the background count is likely to be low Bolton broth is more appropriate. NOTE 2 The amount of sample to be analysed varies with the sample material and the scope of the investigation. In Annex B, sample volumes for the analyses of drinking water are proposed. ISO 17995:20
42、05(E) 4 ISO 2005 All rights reserved8.2 Membrane filtration Filter the appropriate volumes of sample through sterile membrane filters (5.3). Avoid unnecessary exposure to air. With turbid samples, it may be necessary to use two or more filters for the largest sample volumes to counteract clogging. A
43、ll these filters are transferred to the same portion of enrichment broth. The use of a “filter aid” will facilitate the filtration of turbid samples. For a short description of filter aids, see ISO 8199. 8.3 Enrichment Preheat enrichment broths (C.1.1, C.1.2) to 20 C to 30 C before inoculation. Tran
44、sfer the filters (see 8.2) to bottles with 100 ml of enrichment broth immediately after filtration. Put the inoculated broths in jars (see 5.4). Leave the caps off or loosely placed on the inoculated broths during incubation to allow the modified atmosphere to reach the broths. Apply the modified at
45、mosphere (see 5.4) and incubate at (37 1) C for (44 4) h. NOTE 1 100 ml volumes of enrichment broth are used to make sure that the contaminating bacteria present on the filters are diluted sufficiently to avoid their inhibition of the growth of campylobacters during the enrichment. NOTE 2 Some labor
46、atories report successful isolation of campylobacters without the use of a modified atmosphere, closed bottles or tubes with only a small headspace of air being used instead. This procedure may need careful standardization to avoid false negative results due to sub-optimum conditions during enrichme
47、nt. NOTE 3 The Preston campylobacter-selective supplement (C.1.1.2) contains antibiotics (polymyxin B and rifampicin) known to be rather toxic towards C. coli and towards sub-lethally injured C. jejuni. Accordingly, pre-enrichment for 4 h in Preston broth without the selective supplement prior to th
48、e enrichment in the complete Preston broth (C.1.1) has been found by some laboratories to increase the recovery of campylobacters from waters with low numbers of other microorganisms. The Bolton broth selective supplement does not include antibiotics known to be toxic towards campylobacters. NOTE 4
49、Thermotolerant campylobacters may die or grow too slowly if the incubation temperature is below 36 C. 8.4 Plating on solid, selective medium After incubation, remove the broths carefully from the jars to avoid resuspension of sedimented material like contaminating bacteria. NOTE 1 When high numbers of campylobacters are expected, a rapid result is obtained by subculture after only (21 3) h of enrichment. With a sterile loop, transfer approximately 10 l of enrichment culture onto the surface
