1、This standard, togethmi with DIN ENSEC! Par? 11 April la98 edition, supw- sedes DIM 3411 7, November 19636 edition. Ametsdmeints In cmpariSw71 with BlNl3841fl krt 7, Wccclember 192% editioni, detailh af tke methml (prucedluw and evalwatiotiorr ) haue ben changed. Tihis EurapeaniStan dlefediorr arrd
2、enumerationi ofi tk spores; af sulfite-raiucirrg anmrabes (alostridia); method by membrsrre-filtration was rmomrnendii by CEN/TecImic Switzevianldarrd Whirted Mirrgdom. 1 scope Piige4- EFd 2-64H-2:7993 Measurerneiaits af pH hais be Made using a pti tmtef, measurements. b. 44: +: 4 KG. If arr arraemb
3、icijas on am amawmbicrinmIixmr is usab, the memixme fil Nachweis und Zhlung der Sporen sulfitreduzierender Anaerobier (Clostridien); Membranfiltrationsverfahren (IS0 6461 -2 : 1986) DIN EN 26461 part 2 IS0 6461 -2. This standard, together with DIN EN 26461 Part 1, April 1993 edition, super- sedes DI
4、N 38411 Part 7, November 1986 edition. European Standard EN 26 461 -2 : 1993 has the status of a DIN Standard. A comma is used as the decimal rnarkef This standard is part of the series Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung; mikrobiologische Verfahren (Gruppe K) (
5、German standard methods for the examination of water, waste water and sludge; microbiological methods (group K). National foreword This standard has been published in accordance with a decision taken by CEN/TC 230 to adopt, without alteration, International Standard IS0 6461 -2 as a European Standar
6、d. The responsible German body involved in the preparation of this standard was the Normenausschu Wasserivesen (Water Practice Standards Committee). Expert assistance and specialized laboratories will be required to perform the analysis described in this standard. Depending on the objective of the a
7、nalysis, a check shall be made in each individual case on whether and to what extent additional boundary conditions will have to be specified. NOTE: Standard methods published as DIN Standards are obtainable from Beuth Veriag GmbH, either individ- ually or grouped in volumes. The standard methods in
8、cluded in the loose-leaf publication entitled Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlarnmuntersuchung will continue to be published by VCH Verlagsgesellschaft, Weinheim. The standard methods relating to the Wasserhaushaltsgesetz (German Water Management Act) itself and all the reg
9、ulations on waste water that have been issued to date are included in DIN-Taschenbuch (DIN Handbook) 230. Standard methods or draft standards bearing the group title German standard methods for the examination of water, waste water and sludge are classified under the following categories (main title
10、s): General information (group A) (DIN 38402) Physical and physicochemical parameters (group C) (DIN 38404) Anions (group D) (DIN 38405) Cations (group E) (DIN 38406) Substance group analysis (group F) (DIN 38407) Gaseous constituents (group G) (DIN 38408) (DIN 38409) Biological-ecological methods o
11、f analysis (group M) (DIN 3841 O) Microbiological methods (group K) (DIN 3841 1) Bio-assays (group L) (DIN 38412) Individual constituents (group P) (DIN 38413) Sludge and sediments (group S) (DIN 38414) Bio-assays with microorganisms (group T) (DIN 38515) Information on Parts of these standards that
12、 have already been published can be obtained from the offices of the Normen- ausschu Wassetwesen, telephone (O 30) 26 O1 -24 23, or from Beufh Ver/ag GmbH, D-10772 Berlin. DIN IS0 3696 is the standard corresponding to International Standard IS0 3696 referred to in clause 3. , Parameters characterizi
13、ng effects and substances (group H) Continued overleaf. EN comprises 5 pages. Beuth Verlag GmbH, Berlin, has the exclusive right of sale for German Standards (DIN-Normen). 09.93 DIN EN 26 461 Part 2 Engl. Price group 6 Sales No. 1106 Page 2 DIN EN 26 461 Part 2: 1993 Standard referred to (and not in
14、cluded in References) DIN IS0 3696 Water for laboratory use; requirements and testing Previous edition DIN 38411 Part 7 11.86. Amendments In comparison with DIN 38411 Part 7, November 1986 edition, details of the method (procedure and evaluation ) have been changed. international Patent Classificati
15、on B O1 D 061 /O0 C12N001/02 C12Q001/06 C 12 R O01 /145 G O1 N 033118 EUROPEAN STANDARD EUROPISCHE NORM NORME EUROPENNE EN 26 461 -2 January 1993 UDC 628.1 /.3 : 620.1 : 543.39 : 579.852.1 3 Descriptors: Water, quality, water analysis, microbiological analysis, microorganisms, sulfite-reducing bacte
16、ria, clostridium, membrane filtration. English version Water quality Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) Method by membrane filtration (IS0 6461 -2: 1986) Qualit de leau; recherche et dnombre- ment de micro-organismes anarobies sulfito-rducteurs (clostr
17、idia); mthode par filtration sur membrane filtrationsverfahren Wasserbeschaffenheit; Nachweis und Zhlung der Sporen sulfitreduzierender Anaerobier (Clostridien); Membran- (IS0 6461 -2 : 1986) (IS0 6461-2:1986) This European Standard was approved by CEN on 1993-01-20 and is identical to the IS0 Stand
18、ard as referred to. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national stand- ard without any alteration. Up-to-date lists and bibliographical references concerning such national standard
19、s may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A ver- sion in any other language made by translation under the responsibility of a CEN member into its own language and notified to t
20、he Central Secretariat has the same status as the 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 Eu
21、ropean Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart 36, 8-1050 Brussels O 1993. Copyright reserved to all CEN members. Ref. No. EN 26461-2:1993 E Page 2 EN 26 461 -2 : 1993 Foreword This European Standard is the endo
22、rsement of International Standard IS0 6461 -2: 1986. Endorsement of IS0 6461 -2 Water quality; detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia); method by membrane filtration was recommended by CEN/Technical Committee 230 ?Water analysis? under whose competence this
23、 European Standard will henceforth fall. In the countries bound to implement this European Standard, a national standard identical to this European Standard shall be published, and conflicting national standards withdrawn, by July 1993 at the latest In accordance with the CEN/CENELEC Internal Regula
24、tions, the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. Endorsement notice The text of the Internation
25、al Standard IS0 6461 -2: 1986 was approved by CEN as a European Standard without any modifi- cation. Page 3 EN 26 461 -2 : 1993 O Introduction The spores of sulfie-reducing anaerobes (clostridia) are wide- spread in the environment. They are present in human and animal faecal matter, in waste water
26、and in soil. Unlike Escherichk coli and other coliform organisms, the spores sur- vive in water for long periods as they are more resistant than vegetative forms to the action of chemical and physical factors. They may thus give an indication of remote or intermittent pollution. They may even be res
27、istant to chlorination at levels which are normally used for the treatment of water, and they are thus useful for control purposes. IS0 6461 consists of the following parts : Part 1 : Method by enrichment in a liquid medium. Part 2: Method by membrane filtration. 1 Scope Ths part of IS0 6461 specifi
28、es a method for the detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) by membrane filtration. 2 Field of application 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 Refere
29、nces IS0 3696. Water for laboratory use - SpecXcations. IS0 5667, Water qualiw - Samphng - Part 2: Guidance on sampling techniques. Part31 Guidance on the preservation and handling of samples. IS0 7W, Water 9udity - Evaluation of membrane filters used for microbiological anaEyses. IS0 8199, Water qu
30、ality - General guidance for microbiological examination by enumemtion of micro- organisms on culture media. ) 4 Definition For the purpose of this patt of IS0 6461, the following defi- nition applies. clostridia : Sulfite-reducing, spore-forming, anaerobic micro- organisms which belong to the Bacil
31、laceae family and the genus Clostridium. 5 Principle The detection of spores of sulfite-reducing anaerobes (clostridia) in a specified volume 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
32、destroy vegetative bacteria. 5.2 Membrane filtration and culture Filtration of the water sample through a membrane filter having a pore size such that bacterial spores (0,2 pm) are retained in or on the membrane filter. Placing of the filter on a specialized selective culture medium sulfie-iron-agar
33、), followed by incubation at 37 i: 1 OC for 20 i 4 h and 44 i 4 h. and counting of any black colonies. 6 Culture media and reagents 6.1 Basic materials In order to improve the reproducibility of the results, it is recommended that, for the preparation of the diluents and culture media, dehydrated ba
34、sic components or complete dehydrated media be used. Similarly, commercially prepared reagents may ab be used. The manufacturers instructions shall be rigorously followed. 1) At present at the stage of draft. Page 4 EN 26461-2:1993 The chemical products used for the preparation of the culture media
35、and the reagents shall be of recognized analytical quality. The water used shall be distilled or deionized water, free 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 refe
36、rred to a temperature of 25 OC. If the prepared culture media are not used immediately, they shall, unless otherwise stated, be stored in the dark at ap- proximately 4 OC, for no longer than 1 month. 6.2 Suif te-iron-agar 6.2.1 Basal medium (nutrient agar) Composition Meat extract Peptone Sodium chl
37、oride (NaCl) Agar Water Preparation Steam to dissolve, make up to 1 litre with water, and adjust the pH to 7,6 i 0,l with 1 mol/l sodium hydroxide solution. Sterilize at 121 * 1 OC in the autoclave for 20 min. Store in the refrigerator after solidfiing. 6.2.2 Sodium sulfite (Na$303) solution. Dissol
38、ve 10 g of sodium sulfite in 100 rnl of water. It is advisable to prepare a fresh solution every two weeks. 6.2.3 Ironill) sulfate (FeSO,) solution. Dissolve 8 g of crystallized iron(l1) sulfate in 100 ml of water. 6.2.4 Complete medium Immediately before use, melt the basal medium (6.2.1) and to ea
39、ch 18 ml volume add 1 ml of sodium sulfite solution (6.2.2) and five drops of iron(l1) sulfate solution (6.2.3). Add 1 mi of the sodium sulfite solution and 5 drops of the iron(1l) sulfate solution to the agar tubes just before the embed- ding procedure (see 9.3). 6.3. Tryptose-sulfite-agar (alterna
40、tive medium) Composition Tryptose Soytone Yeast extract Sodium metabisulfite Ammonium iron(ll1) citrate Water Preparation 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 refigerato
41、r at 4 to 5 OC. Discard unused medium 2 weeks after preparation. 7 Apparatus and glassware Usual microbiological laboratory equipment, and 7.1 conical flask), of capacity 2 litres. Glass flasks (Erlenmeyer flask, round-bottom flask, or 7.2 Test tubes, 160 mm x 16 mm. 7.3 divisions of 0,l mi. Graduat
42、ed pipettes, of capacity 10 ml, graduated in 7.4 Volumetric pipettes, of capacity 10 ml. 7.5 Jars. of capacity 1 litre. 7.6 Steamer. 7.7 Water bath. 7.8 Membrane filtration apparatus. 7.9 Sterile membrane filters, of nominal pore size 0,2 prn. NOTE - The quality of membrane filters may vary accordin
43、g to brand or even from batch to batch. It is therefore advisable to check the qual- ity on a reguiar basis according to IS0 7704. 7.10 Incubator, capable of being maintained at 37 t 1 C. 7.11 Petri dishes. 8 Sampling Refer to IS0 5667/2 and IS0 8199 for sampling techniques. 9 Procedure 9.1 Treatmen
44、t of samples Refer to IS0 566713 for guidance on the preservation and handling 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 + 5 OC for 15 min from the time it reaches that temperature. A similar bottle co
45、ntaining the same vOlUme Page 5 EN 26 461 - 2 : 1993 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. 9.3 Inoculation and incubation Refer to
46、 IS0 7704 for a general description of the membrane filtration technique. According to the instructions in that document, filter a suitable volume of water. For drinking water, spring and well water, mineral waters, sea-water, and surface water, less polluted with clostridia, filter 100 ml: for high
47、ly 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 that any resultant black colonies are well separated and can be easily counted. After filtration, remove the membrane with steri
48、le 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 complete culture medium, previous- ly cooled to about 50 OC, over the membrane while holding it still with sterile forceps. Afte
49、r 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 4 h and 44 f 4 h. If an anaerobic jar or an anaerobic incubator is used, the membrane filter may be placed on the surface of the agar face upwards. 9.4 Interpretation Count all black colonies after incubation for 20 f 4 h and 44 I4h. 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 sulf
