1、CEN ENL264bL-2 93 m 3404589 0042975 b9T m EUROPEAN STANDARD EN 26461-2 NORME EUROPENNE EUROPISCHE NORM January 1993 UM: 628.U.3 : 620.1 : 543.39 : 579.852.13 Descriptois: Water, quality, water tests, microbiological analysis, micro-organisms, sulfite reducing bacteria, clostridium, filtration analys
2、is English version Water quality - Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) - Part 2 : Method by membrane filtration (IS0 6461/2 : 1986) Qualit de leau - Recherche et dnombrement des spores de micro-organismes anarobies sulfito-rducteurs (clostridia) - Parti
3、e 2 : Mthode par filtration sur membrane (IS0 6461/2 : 1986) Wasserbeschaffenheit - Nachweis und Zhlung der Sporen sulfitreduzierender Anaerobier (Clostridien) - %il 2 : Membranfiltrationsverfahren (IS0 6461/2 : 1986) This European Standard was approved by CEN on 1993-01-20. CEN members are bound to
4、 comply with the CENKENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Central
5、 Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the
6、 official versions. CEN members are the national standards bodies of Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standardization Comit Europe
7、n de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels O 1993 Copyright reserved to CEN members Ref. No. EN 26461-2 : 1993 E Foreword This European Standard is the endorsement of IS0 6461-2. Endorsement of IS0 6461-2 was recommended by lkchnical Co
8、mmittee CEN/TC 230 Water analysis under whose competence this European Standard will henceforth fall. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by July 1993, and conflicting national standards
9、shall be withdrawn at the latest by July 1993. The standard was approved and in accordance with the CENKENELEC Internal Regulations, the folowing countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg,
10、 Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, United Kingdom. CEN EN+2b4bL-2 93 3404589 0042977 462 Page 3 EN 26461-2 : 1992 Water quality - Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) - Part 2: Method by membrane filtration O Introduction The spo
11、res of sulfite-reducing anaerobes (clostridia) are wide- spread in the environment. They are present in human and animal faecal matter, in waste water and in soil. Unlike Escherichla cdi and other coliform organisms, the spores sur- vive in water for long periods as they are more resistant than vege
12、tative forms to the action of chemical and physical factors. They may thus give an indication of remote or intermittent pollution. They may even be resistant to chlorination at levels which are normally used for the treatment of water, and they are thus useful for control purposes. IS0 6461 consists
13、 of the following parts : Part 1 : Method by enrichment in a liquid medium. Part 2: Method by membrane filtration. 1 Scope This part of IS0 6461 specifies a method for the detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) by membrane filtration. 2 Field of applicatio
14、n The method can be applied to all types of water, except when a large amount of particulate material is liable to be retained by the membrane. . 3 References IS0 3696, Water for laboratory use - Specfications. IS0 5667, Water quality - Sampling - Part 2: Guidance on sampling techniques. Part3: Guid
15、ance on the preservation and handing of samples. IS0 7704, Water quality - Evaluation of membrane fiters used for microbiological anelyses. ISO8199, Water qualiiy - General guidance for microbiological examination by enumeration of micro- organisms on culture media. ) 4 Definition For the purpose of
16、 this part of IS0 6461, the following defi- nition applies. clostridia : Sulfite-reducing, spore-forming, anaerobic micro- organisms which belong to the Bacillaceae family and the genus Clostridium. 5 Principle The detection of spores of sulfite-reducing anaerobes (clostridia) in a specified volume
17、of a water sample requires the following steps. 5.1 Selection of spores Selection of spores in the sample by applying heat for a period of time sufficient to destroy vegetative bacteria. 5.2 Membrane filtration and culture Filtration of the water sample through a membrane filter having a pore size s
18、uch that bacterial spores (0.2 pm) are retained in or on the membrane filter. Placing of the filter on a specialized selective culture medium (sulfite-iron-agar), followed by incubation at 37 f 1 OC for 20 f 4 h and 44 k 4 h, and counting of any black colonies. 6 Culture media and reagents 6.1 Basic
19、 materials In order to improve the reproducibility of the results, it is recommended that, for the preparation of the diluents and culture media, dehydrated basic components or complete dehydrated media be used. Similarly, commercially prepared reagents may also be used. The manufacturers instructio
20、ns shall be rigorously followed. 1) At present at the stage of draft 1 CEN ENI2b4bl-2 93 = Page 4 EN 26461-2 : 1992 The chemical products used for the preparation of the culture media and the reagents shall be of recognized analytical quality. The water used shall be distilled or deionized water, fr
21、ee from substances that might inhibit the growth of micro-organisms under the test conditions (see IS0 3696). Measurements of pH shall be made using a pH meter, measurements being referred to a temperature of 25 OC. If the prepared culture media are not used immediately, they shall, unless otherwise
22、 stated, be stored in the dark at ap- proximately 4 OC, for no longer than 1 month. 6.2 Sulfite-iron-agar 6.2.1 Basal medium (nutrient agar) Composition Meat extract 3g Peptone 10 g Sodium chloride (NaCl) 59 Agar 15 g Water 1 O00 ml Prepamtion Steam to dissolve, make up to 1 litre with water, and ad
23、just the pH to 7,6 i 0,l with 1 mol/l sodium hydroxide solution. Sterilize at 121 f 1 OC in the autoclave for 20 min. Store in the refrigerator after solidifying. 6.2.2 Sodium sulfite (Na$303) solution. Dissolve 10 g of sodium sulfite in 100 ml of water. It is advisable to prepare a fresh solution e
24、very two weeks. 6.2.3 Iron(ll1 sulfate (FeSO4) solution. Dissolve 8 g of crystallized iron(ll) sulfate in 100 ml of water. 6.2.4 Complete medium Immediately before use, melt the basal medium (6.2.1) and to each 18 ml volume add 1 ml of sodium sulfite solution (6.2.2) and five drops of iron(ll) sulfa
25、te solution (6.2.3). Add 1 ml of the sodium sulfite solution and 5 drops of the iron(l1) sulfate solution to the agar tubes just before the embed- ding procedure (see 9.3). 6.3. Tryptose-sulfite-agar (alternative medium) Composition Tryptose Soytone Yeast extract Sodium metabisulfite Ammonium irodll
26、l) citrate Water 3404589 0042978 3T9 = Prepamtion Steam to dissolve, and adjust the pH to 7,6 f 0.1 at 25 OC. Distribute into test tubes in volumes of 18 mi. Sterilize the medium for 15 min at 121 i 1 OC. Store in the refigerator at 4 to 5 OC. Discard unused medium 2 weeks after preparation. 7 Appar
27、atus and glassware Usual microbiological laboratory equipment, and 7.1 conical flask), of capacity 2 litres. 7.2 Test tubes, 160 mm x 16 mm. 7.3 Graduated pipettes, of capacity 10 ml, graduated in divisions of 0.1 ml. 7.4 Volumetric pipettes, of capacity 10 ml. Glass flasks (Erlenmeyer flask, round-
28、bottom flask, or 7.5 Jars, of capacity 1 litre. 7.6 Steamer. 7.7 Water bath. 7.8 Membrane filtration app ratus. 7.9 Sterile membrane filters, of nominal pore size 0.2 Mm. NOTE - The quality of membrane filters may vary according to brand or even from batch to batch. It is therefore advisable to chec
29、k the qual- ity on a regular basis according to IC0 7704. 7.10 Incubator, capable of being maintained at 37 f 1 OC. 7.11 Petri dishes. 8 Sampling Refer to IS0 5667/2 and IS0 8199 for sampling techniques. 9 Procedure 9.1 Treatment of samples Refer to IS0 566713 for guidance on the preservation and ha
30、ndling of samples, and to IS0 8199. 9.2 Selection of spores (technique) Before the test, the sample of water should be heated in a water bath at 75 f 5 OC for 15 min from the time it reaches that temperature. A similar bottle containing the same volume CEN EN*2b4bL-2 73 m 3404589 O042779 235 m Page
31、5 EN 26461-2 : 1992 of water as the test sample should be used periodically as a control in order to check the heating time required. The temperature of the water in the control bottle can be constantly recorded by thermometer. 44 f 4 h. If an anaerobic jar or an anaerobic incubator is used, the mem
32、brane filter may be placed on the surface of the agar face upwards. 9.4 Interpretation 9.3 Inoculation and incubation Refer to IS0 T704 for a general description of the membrane filtration technique. According to the instructions in that document, filter a suitable volume of water. For drinking wate
33、r, spring and well water, mineral waters, sea-water, and surface water, less polluted with clostridia, filter 100 ml; for highly polluted water or sewage use smaller volumes. Volumes of water smaller than 10 ml should be mixed with 10 to 100 ml of sterile water or diluent. Adjust the dilutions so th
34、at any resultant black colonies are well separated and can be easily counted. After filtration, remove the membrane with sterile forceps and place it face downwards on the bottom of a Petri dish, ensuring that no air bubbles are trapped under the filter. Then carefully pour 18 ml of the liquefied co
35、mplete culture medium, previous- ly cooled to about 50 OC, over the membrane while holding it still with sterile forceps. After this layer of medium has set, in- cubate anaerobically or under other conditions which ensure anaerobiosis at a temperature of 37 f 1 OC for 20 f 4 h and Count all black co
36、lonies after incubation for 20 f 4 h and 44f4h. 10 Expression of results Express the results in accordance with IS0 8199. 11 Test report The test report shall state the method used and express the results as the number of sulfite-reducing anaerobes (clostridia) per volume of sample. The 44 i 4 h cou
37、nt should normally be reported. If this is not possible, the count at 20 f 4 h should be reported as approximate only. The test report shall also mention any operating details not specified in this part of IS0 6461, or regarded as optional, together with details of any incidents likely to have influenced the results. The test report shall include all the information necessary for the complete identification of the sample.
