1、 SWnpdard referred ta (and moti induiialM in Refrtrnces): DIN 180 369E Water fan laboratory. ue requirement of caapacites;25 mi and lD ml! rwpectbet+. f water as the test smpl$ shaulkd b usedi periQdiCaii. a6 a- corn, in arder ta Nachweis und Zhlung der Sporen sulfitreduzierender Anaerobier (Clostri
2、dien); Flssigkeitsanreicherung (IS0 6461 - 1 : 1986) This standard, together with DIN EN 26461 Part 2, April 1993 edition, super- sedes DIN 38411 Part 7, November 1986 edition. European Standard EN 26 461 -1 : 1993 has the status of a DIN Standard. A comma is used as the decimal marker: This standar
3、d is part of the series Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung; mikrobiologische Verfahren (Gruppe K) (German standard methods for the examination of water, waste water and sludge; microbiological methods (group K). National foreword This standard has been publishe
4、d in accordance with a decision taken by CEN/TC 230 to adopt, without alteration, International Standard IS0 6461 -1 as a European Standard. The responsible German body involved in the preparation of this standard was the Normenausschu Wasserwesen (Water Practice Standards Committee). Expert assista
5、nce and specialized laboratories will be required to perform the analysis described in this standard. Depending on the objective of the analysis, 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 metho
6、ds published as DIN Standards are obtainable from Beufh Verlag GmbH, either individ- ually or grouped in volumes. The standard methods included in the loose-leaf publication entitled Deutsche finheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung will continue to be published by VCH Verlag
7、sgeselIschaff, Weinheim. The standard methods relating to the Wasserhaushalfsgesefz (German Water Management Act) itself and all the regulations on waste water that have been issued io date are included in DIN-Taschenbuch (DIN Handbook) 230. Standard methods or draft standards bearing the group titl
8、e German standard methods for the examination of water, waste water and sludge are classified under the following categories (main titles): General information (group A) (DIN 38402) Physical and physicochemical parameters (group C) (DIN 38404) Anions (group D) (DIN 38405) Cations (group E) (DIN 3840
9、6) Substance group analysis (group F) (DIN 38407) Gaseous constituents (group G) (DIN 38408) Parameters characterizing effects and substances (group H) (DIN 38409) Biological-ecological methods of analysis (group M) (DIN 38410) Microbiological methods (group K) (DIN 3841 1) Bio-assays (group L) (DIN
10、 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 have already been published can be obtained from the offices of the Nor- menausschu Wasserwesen, telephon
11、e (O 30) 26 O1 -24 23, or from Beuth Verlag GmbH, D-10772 Berlin. DIN IS0 3696 is the standard corresponding to International Standard IS0 3696 referred to in clause 3. Continued overleaf. En comprises 5 pages. Beoih VerIag GmbH, Berlin, has the exclusive right of sale for German Standards (DIN-Norm
12、en). DIN EN 26467 Part 1 Engl. Price group 6 09.93 Sales No. 1106 Page 2 DIN EN 26461 Part 1 : 1993 Standard referred to (and not included 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
13、Part 7, November 1986 edition, details of the method (procedure and evaluation ) have been changed. International Patent Classification C12Q001/06 C 12 R O01 /145 G O1 N 033/18 EUROPEAN STANDARD NORME EUROPENNE EN 26 461 -1 EUROPISCHE NORM January 1993 UDC 628.1 /.3 620.1 : 543.39 579.852.1 3 Descri
14、ptors: Water, quality, water analysis, microbiological analysis, microorganisms, sulfite-reducing bacteria, clostridium. English version Water quality Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) Method by enrichment in a liquid medium (IS0 6461 -1 : 1986) Quali
15、t de leau; recherche et dnombre- ment de micro-organismes anarobies sulfito-rducteurs (clostridia); mthode par enrichissement dans un milieu liquide Wasserbeschaffenheit; Nachweis und Zhlung der Sporen sulfitreduzierender Anaerobier (Clostridien); Flssigkeits- anreicherung (IS0 6461 -1 : 1986) (IS0
16、6461 -1 : 1986) This European Standard was approved by GEN on 1993-01-20 and is identical to the IS0 Standard as referred to. CEN members are bound to comply with the CENICENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national stand- ard
17、 without any alteration. Up-to-date lists and bibliographical references concerning such national standards 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 lan
18、guage 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 official versions. CEN members are the national standards bodies of Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Irelan
19、d, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European 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
20、members. Ref. No. EN 26 461 -1 :1993 E Page 2 EN 26 461 - 1 : 1993 Foreword This European Standard is the endorsement of International Standard IS0 6461 -1 : 1986. Endorsement of IS0 6461 -1 Water quality; detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia); method by
21、enrichement in a liquid medium was recommended by CEN/Technical Committee 230 Water analysis under whose competence this 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
22、 conflicting national standards withdrawn, by July 1993 at the latest In accordance with the CEN/CENELEC Internal Regulations, the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Neth
23、erlands, Norway Portugal, Spain, Sweden, Switzerland and United Kingdom. Endorsement notice The text of the International Standard IS0 6461 -1 : 1986 was approved by CEN as a European Standard without any modifi- cation. O Introduction Page 3 EN26461-1:1993 4 Definition The spores of sulfite-reducin
24、g anaerobes (clostridia) are wide- spread in the environment. They are present in human and animal faecal matter, in waste water and in soil. Unlike Escherichia coli and other coliform organisms, the spores sur- vive in water for long periods as they are more resistant than vegetative forms to the a
25、ction 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 of the following par
26、ts : 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 enrichment in a liquid medium. 2 Field of application The metho
27、d is applicable to all types of water, including turbid water. 3 References IS0 3696. Water for laboratory use - Specifications. IS0 5667, Water quality - Sampling - Part 2: Guidance on sampling techniques. Fart3: Guidance on the preservation and handling of samples. IS0 8199, Water quality - Genera
28、l guidance for microbiological examination by enumeration of micro- organisms on culture media.” For the purpose of 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
29、 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 destroy vegetative bacteria
30、. 5.2 Enrichment culture Detection and enumeration of spores of sulfite-reducing anaerobes by inoculating volumes of the sample into liquid enrichment media, followed by incubation at 37 f 1 OC for 44 f 4 h in anaerobic conditions. 6 Culture media and reagents 6.1 Basic materials In order to improve
31、 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 instructions shall be rigorously followe
32、d. The chemical products used for the preparation of the culture media and the reagents shall be of recognized analytical quality. 1) At present at the stage of dreh. Page 4 EN 26 461 -1 : 1993 The water used shall be distilled or deionized water, free from substances that might inhibit the growth o
33、f 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 stated, be stored in the dark at ap proximately 4
34、 OC, for no longer than 1 month. 6.2 Culture media and diluent 6.2.1 Diluent Use one of the diluents given in IS0 8199. 6.2.2 Differential reinforced clostridial medium (DRCM) 6.2.2.1 Single strength basal medium Composition Peptone tryptic digest of meat Meat extract Yeast extract Starch Hydrated s
35、odium acetate Glucose L-Cysteine- hydrochloride Water Preparation Mix the peptone, meat extract, sodium acetate and yeast ex- tract with 800 ml of water. With the remaining 200 ml of distilled water, prepare a starch solution as follows: mix the starch in a little cold water to form a paste. Heat th
36、e rest of the water to boiling point and slowly add it to the paste with constant stirring. Then add this starch solution to the first mixture and heat to boiling point until it dissolves. Finally, add the glucose and L-cysteine hydrochloride. Dissolve. Adjust the pH to 7,l to 7,2 with 1 moll1 sodiu
37、m hydroxide. Transfer 25 mi alquots of the medium into screw-capped bot- tles of capacity 25 ml. Sterilize in the autoclave at 121 f 1 OC for 15 min. 6.2.2.2 Double strength basal medium Prepare the double strength medium as in 6.2.2.1 but reduce the volume of water by half. Transfer 10 mi and 50 ml
38、 aliquots of the medium into screw- capped bottles of capacities 25 mi and 100 ml respectively. 6.2.3 Sodium sulfite iNa$03), 4 % rn/rn) solution Dissolve 4 g of anhydrous sodium sulfite in 100 ml of water. Sterilize by filtration. Store at between 2 and 5 OC. It is advisable to prepare a fresh solu
39、tion every 14 days. 6.2.4 Iron(1ll) citrate (C6H50,Fe), 7 % (m/m) solution. Dissolve 7 g of iron(lll) citrate in 100 mi of water. Sterilize by filtration. Store at between 2 and 5 OC. It is advisable to prepare a fresh solution every 14 days. 6.2.5 Complete medium 6.2.5.1 On the day of analysis, mix
40、 equal volumes of the solu- tions of sodium sulfite (6.2.3) and ironlll) citrate (6.2.41. 6.2.5.2 Add 0,5 ml of the mixture (6.2.5.1) to each bottle of single strength medium (6.2.2.1). which has been freshly heated and cooled. 6.2.5.3 Add 0,4 ml of the mixture (6.2.5.1 to each 10 mi, and 2 ml to ea
41、ch 50 ml, of double strength medium (6.2.2.2) similarly treated. 7 Apparatus and glassware Usual microbiological laboratory equipment, and 7.1 silicate glass of capacities 200, 100 and 25 ml. Screw-cap bottles or vials and stoppers of boron 7.2 Volumetric pipettes, of capacities 10 and 1 ml. 7.3 Wat
42、er baths, thermostatically controlled. 7.4 Test tubes, 150 mm x 13 mm. 7.5 Iron wire. 7.6 Incubator, capable of being maintained at 37 f 1 OC. 8 Sampling Refer to IS0 5667/2 and IS0 8199 for sampling techniques. 9 Procedure 9.1 Treatment of samples Refer to lSO5667l3 for guidance on the preservation
43、 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 I 5 OC for 15 min from the time it reaches that temperature. A similar bottle containing the same volume Page 5 EN 26 461 - 1 : 1993 of water as
44、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 Add 50 ml of sample (9.2) to a 100 ml screw-cap bottle contain- ing 50 ml
45、 of the double strength complete medium (6.2.5.31. Add 10 ml of sample (9.2) to a series of five 25 ml screw-cap bottles containing 10 ml of double strength complete medium (6.2.5.3). Add 1 ml of sample (9.2) to a series of five 25 ml screw-cap bottles containing 25 ml of single strength complete me
46、dium (6.2.5.2). If necessary, add 1 ml of a 1 -. 10 dilution of the sample (9.2) to a series of five 25 ml screw-cap bottles containing 25 ml of single strength complete medium (6.2.5.2). In order to carry out a qualitative examination of 100 ml of drinking water or bottled water without making an M
47、PN count, use a 200 ml vial filled with a mixture of 100 ml of double strength complete medium (6.2.5.3) and 100 ml of sample (9.2). If necessary, top up all the bottles with the single strength com- plete medium (6.2.5.2) to bring the volume of liquid level with the neck and to ensure that only a v
48、ery small volume of air re- mains, then seal the bottles hermetically, or incubate under anaerobic conditions. Incubate the inoculated bottles at 37 i: 1 OC for 44 t 4 h. Large volumes of culture in hemetically sealed glass bottles may explode due to gas production. The addition of iron wire, heated
49、 to redness and placed into the medium before inocula- tion, may aid anaerobiosis. 9.4 Interpretation Bottles in which blackening is observed, as a result of the reduction of sulfite and the precipitation of ironill) sulfide, shall be regarded as positive. 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 most probable number of sulfite-reducing anaerobes (clostridia) per volume of sample. It shall al
